Essentials of Industrial Microbiology

Essentials of
Industrial Microbiology
Basanta Rai
ESSENTIALS OF
INDUSTRIAL
MICROBIOLOGY
Basanta K. Rai
PREFACE
The present book is basically a compilation of a series of lectures on INDUSTRIAL

MICROBIOLOGY and MICROBIAL BIOCHEMISTRY I have delivered over the
years to B. Tech (Food Technology) and B. Sc. (Microbiology) respectively at
Central Campus of Technology, Dharan, Nepal.
The chapters included herein more than cover the current syllabus of Industrial
Microbiology for B. Tech (III year). Within the scope and limitation of the syllabus,
I have tried to put together information as meticulously as possible. Some
descriptions have become outdated, genetic engineering in particular. However, the
basic concept is still useful. The book contains a large number of cross-referenced
diagrams, tables and index to assist the students/readers.
Thanks are due to those authors whose books I have freely consulted. As an
acknowledgement, I have appended a short bibliography, which I hope will be
helpful to the students in finding out additional reading materials.
I am very much thankful to NAAST College, Dharan-16, Nepal for providing the
much-needed computer facility during the early stages of the work (1998!).
I am very much hopeful that the book will fulfill its intended purpose. Any criticism
for the improvement (updating) of the work will be thankfully received.
Dharan, Mar 2012 Basanta Kumar Rai

CONTENTS
PREFACE i
CONTENTS ii
CHAPTER 1
THE SCOPE OF INDUSTRIAL MICROBIOLOGY 1
1.1 Introduction 1
1.2 Industrially important groups of microorganisms 2
1.3 Desirable properties of industrial microorganisms 3
CHAPTER 2
SCREENING OF MICROORGANISMS 4
2.1 Introduction 4
2.2 Examples of screening 5
CHAPTER 3
GENERAL TECHNIQUES OF SELECTION OF MICROORGANISMS 9
3.1 Introduction 9
3.2 Pure culture techniques 10
CHAPTER 4
STRAIN IMPROVEMENT 13
4.1 Introduction 13
4.2 Mutation approach 13
4.3 Common mutagens 14
4.4 Improvement in the yield 16
4.5 Strain improvement by recombination 19
CHAPTER 5
GENETIC ENGINEERING 21
5.1 Introduction 21
5.2 Basis of gene manipulation 21
5.3 Tools needed for gene cloning 21
5.4 Technique of gene cloning 21
CHAPTER 6
PRESERVATION / MAINTENANCE OF INDUSTRIAL CULTURES 36
6.1 Introduction 36
6.2 Primary stock culture 36
6.3 Maintenance of purity of culture 40
6.5 Some culture collection centers 40
CHAPTER 7
CONCEPT OF BIOTECHNOLOGY 41
7.1 Introduction 41
7.2 Biotechnology as an interdisciplinary activity 43
7.3 Scope and importance of biotechnology 43
7.4 Potential hazards of biotechnology 45
CHAPTER 8
MICROBIAL GENETICS 47
8.1 Introduction 47
8.2 Genetic material 47
8.3 The genetic code 51
8.4 Central Dogma and Teminism 55
8.5 Teminism (Central Dogma reverse) 55
8.6 Transcription 55
8.7 Translation 65
8.8 Metabolic regulation 71
CHAPTER 9
CONCEPT OF FERMENTATION TECHNOLOGY 80
9.1 Introduction 80
9.2 Fermenter and fermentation process 80
9.3 Fermenter design 87
9.4 Aeration and agitation 91
9.5 Basic variables for monitoring fermentation 94
9.6 Fermenter scale up 95
CHAPTER 10
BASICS OF ENZYME TECHNOLOGY 96
10.1 Introduction 96
10.2 Classification and nomenclature of enzymes 97
10.3 Catalytic power of an enzyme 99
10.4 Factors affecting enzyme activity 99
10.5 Mechanism of catalysis107
10.6 Enzyme kinetics 108
10.7 General properties of protein enzymes 113
10.8 Mechanism of enzyme biosynthesis 114
10.9 The kinetics of enzyme biosynthesis 115
10.10 Enzymes in various industries 116
10.11 Production of microbial enzymes 116
10.12 Production aspects 117
10.13 Advantages of microbial enzymes 117
10.14 General production and purification methods 118
10.15 General process of enzyme recovery 120
10.16 Conversion to storage form 124
10.17 Amylase production 125
iii
10.18 Proteases 127
10.19 Immobilized enzymes 130
10.20 Enzyme engineering 137
CHAPTER 11
YEAST TECHNOLOGY 139
11.2 Taxonomic consideration 139
11.3 Reproduction in yeasts 139
11.4 Industrial categorization of yeasts 141
11.5 Food yeast 143
11.6 Bakers yeast 147
CHAPTER 12
BREWING TECHNOLOGY 161
12.2 Materials required for beer making 163
12.3 Outline of beer production 179
12.4 Production detail 181
12.5 High gravity brewing 206
12.6 Quality control 206
12.7 Some terminologies 208
12.8 Some famous beers of the world 211
12.9 Beer spoilages 211
12.10 Purification of carbon dioxide 212
CHAPTER 13
MICROBIAL PRODUCTION OF ETHANOL 215
13.2 Industrial production 216
13.3 Continuous fermentation 219
13.4 Biochemistry of ethanol fermentation 219
13.5 Biochemistry of higher alcohol production 220
13.6 Methanol 220
13.7 Distillation and rectification 221
13.8 Uses of ethanol 224
13.9 Industrial alcohol 224
13.10 Proof and proof spirit 225
13.11 Dehydrated (absolute) alcohol 225
13.12 Ethanol from cellulose 227
CHAPTER 14
WINE TECHNOLOGY 234
iv
14.2 Wine yeasts 237
14.3 Grapes 238
14.4 Production of Red Table Wine 239
14.5 Production of White Table Wine 246
14.6 Fortified and sweet wines 247
14.7 Sparkling wines 249
14.8 Wine spoilages and defects 252
CHAPTER 15
BRANDY AND WHISKEY 253
15.1 Brandy 253
15.2 Whiskey 255
CHAPTER 16
VINEGAR PRODUCTION 264
16.2 Production of distilled vinegar 264
CHAPTER 17
MICROBIAL PRODUCTION OF CHEMOTHERAPEUTIC AGENTS 272
17.2 Streptomycin 275
17.3 Penicillin 280
17.4 Tetracycline 289
CHAPTER 18
MICROBIAL PRODUCTION OF FLAVORS AND FRAGRANCE 293
18.2 Diacetyl 293
18.3 Lactones 294
18.4 Butyric acid 296
CHAPTER 19
MICROBIAL PRODUCTION OF POLYSACCHARIDES 298
19.2 General process for microbial EPS production 298
19.3 General uses of EPS 299
19.4 Xanthan gum 299
19.5 Dextran 301
19.6 Alginic acid 303
CHAPTER 20
MICROBIAL PRODUCTION OF FATS 306
20.2 Some microorganisms capable of producing lipids 309
20.3 General cultural conditions 311
v
20.4. Microbial production of PUFA-rich oil 312
20.5 Outline of biosynthesis of simple lipid in eukaryotes 313
20.6 Future prospects 313
CHAPTER 21
RIBOFLAVIN PRODUCTION BY YEAST 314
21.2 Microbial production of riboflavin 315
CHAPTER 22
MICROBIAL PRODUCTION OF ORGANIC ACIDS 319
22.1 Citric acid 319
22.2 Fumaric acid 324
22.3 Gluconic acid 325
CHAPTER 23
MICROBIAL PRODUCTION OF AMINO ACIDS 328
23.2 Production aspect 328
23.3 Production of l-glutamic acid 328
23.4 Microbial production of l-tryptophan 335
23.5 Microbial production of l-lysine 338
CHAPTER 24
YEAST ENZYMES AND MINOR PRODUCTS 343
24.1 Justifying the design of specialized equipment 343
24.2 Invertase 343
24.3 Lactase 346
24.4 Lipases 348
24.5 Yeast polygalacturonase 350
CHAPTER 25
MICROBIAL PROTEINS 352
25.2 Some commercial and semi-commercial processes 353
25.3 General considerations 353
25.4 Bacterial process 354
25.5 Fungal proteins 358
25.6 Safety aspects of scp 361
CHAPTER 26
TRADITIONAL FERMENTED FOODS AND BEVERAGES 362
26.2 Classification of fermented foods 364
26.3 Safety of fermented foods 364
vi
26.4 Miso 365
26.5 Soy sauce 367
26.6 Natto 370
26.7 Japanese sake 371
26.8 Kinema 372
26.9 Tempeh 375
26.10 Tempeh bongkrek 378
26.11 Ontjom 378
26.12 Fermented vegetables 378
26.13 Jand, nigar, and raksi 382
CHAPTER 27
MICROBIOLOGICAL ANALYSIS OF NUTRIENTS 386
27.2 Growth response 386
27.3 Metabolic response 386
27.4 Methods of microbiological assay 388
27.5 Example: assay of folic acid 389
CHAPTER 28
MICROBIAL PRODUCTION OF VITAMIN B12 AND ß-CAROTENE 391
28.2 Microbial production of vitamin B12 391
28.3 Microbial production of -carotene 393
CHAPTER 29
BIOFERTILIZERS 396
29.2 Production of rhizobium culture 398
29.3 Production of blue-green algae 399
BIBLIOGRAPHY 401
INDEX 405
vii
CHAPTER 1
THE SCOPE OF INDUSTRIAL MICROBIOLOGY
1.1 INTRODUCTION
Industrial microbiology is one of the most important areas of applied microbiology.

Basically, it deals with screening, improvement, management, and exploitation of
microorganisms for the production of various useful end products in large quantities
(commercial scale).
From industrial microbiology standpoint, microorganisms can be considered chemical

factories in miniature because they have immense capability to transform an array of
raw materials into diverse end products. The overall reaction characterizing the
industrial application of microorganisms can be summarized as:
microorganisms
Substrate (raw materials)  End products and/or Services
Microorganisms are involved in the above reaction in ways more than one. The
products and services these microorganisms are capable of generating can be limited
only by imagination. Some of the more important categories of products/services
presently available through the use of microorganisms are:
1. Microbial cell (biomass)

 Live (bakers yeast, test-cultures for microbiological assay, etc.)
 Dead or processed (yeast autolysates, single cell protein, etc.)
2. Enzymes (invertase, lipase, pectin esterase, rennin, etc.)
3. Metabolites
 Primary metabolites (metabolites needed for the growth of the
organisms themselves, e.g., ethanol, vitamins, amino acids, etc.)
 Secondary metabolites (metabolites not essential for growth but are
produced as a survival tool in response to environmental
conditions, e.g., antibiotics, polysaccharides, etc.)
4. Transformed products
 Semi-synthetic penicillins, etc.
5. Biofertilizers
 Microbial inoculants such as Rhizobium, Mycorrhiza, etc.
6. Biopesticides
 Bacillus thuringiensis against lepidoptereans
7. Waste degradation
 Sewage treatment by fermentation/digestion
1.2 INDUSTRIALLY IMPORTANT GROUPS OF MICROORGANISMS
The following is a brief treatment of some of the important groups of industrially

important microorganisms. Mushroom has not been dealt with for obvious reasons.
1.2.1 BACTERIA
They are unicellular prokaryotes that reproduce predominantly by binary fission.

Typically, the size is around 1-2 μm. The basic shapes are described as spherical,
rod-shaped, and helical (see Fig. 1.1a). Some bacteria of industrial importance and
their associated uses are:
 Streptomyces sp.— for streptomycin production

 Bacillus subtilis— for fermented soy food, protease, antibiotics
 Corynebacterium sp.— for amino acid production
 Lactobacillus sp. — for fermented dairy products, fermented vegetables
 Bacillus thuringiensis — for biopesticides
 Xanthomonas campestris —for xanthan gum
 Acetobacter sp. — for vinegar production
1.2.2 MOLDS
Molds are multicellular eukaryotes. They lack chlorophyll. They have mycelial
structure, which gives the impression of a fluffy/cottony colony (see Fig. 1.1c).
Some of the industrially important molds and their uses are:
 Penicillium chrysogenum — for penicillins

 Aspergillus oryzae — for amylase, oriental alcoholic beverages, etc.
 Mucor miehi — for fungal rennet
 Rhizopus oligosporus —for tempeh
 Aspergillus niger — for citric acid and fumaric acid
1.2.3 YEASTS
They lack chlorophyll and are therefore either saprophytes or parasites. They are
unicellular and non-motile. They are bigger than bacteria (5-20 μm) and generally
reproduce by budding (see Fig. 1.1b). Some of them produce pseudomycelia (e.g.,
Candida sp). Some of the industrially important yeasts are:
 Saccharomyces sp. — for alcoholic fermentation, bakers yeast

 Candida utilis — for feed yeast
 Cryptococcus — for feed yeast
 Ashbya gossypyii — for riboflavin (vitamin B2 ) production
2
scar bud
spiral coccoid elongate

(helical) (spherical) (rod)
(a) Bacteria (b) Yeast (d) Mold
Fig. 1.1 Typical morphology of bacteria, yeast, and mold
1.3 DESIRABLE PROPERTIES OF INDUSTRIAL MICROORGANISMS
It is difficult to generalize but the most important desirable properties an industrially

important microorganism must possess are:
(i) Rapid growth in relatively cheap and readily available substrate

(ii) Production of the desired metabolite in large quantities
(iii) Non-pathogenicity
(iv) Genetic stability
(v) Temperature tolerance
(vi) Short fermentation time
(vii) Ease of removal of cells from the fermentation broth
(viii) Amenable to genetic manipulation.
In practice, however, no single microorganism has all the above-listed properties.

The choice is therefore a compromise.
3
CHAPTER 2
SCREENING OF MICROORGANISMS
2.1 INTRODUCTION
A set of highly selective procedures that allow the detection and isolation of
microorganisms producing the desired metabolite (having desired properties)
constitutes Primary Screening. Ideally, primary screening should be rapid, inexpensive,
predictive, specific but effective for a broad range of compounds, and applicable on
a large scale. Primary screening is time-consuming and labor-intensive since a large
number of isolates have to be screened to identify a few potential ones.
However, this possibly is the most critical step since it eliminates the large bulk of
unwanted and useless isolates, which are either non-producers or producers of known
compounds. The need for the latter would become obvious in the light of the fact that
till 1987 more than 3000 different metabolites were well characterized, and every
year about 100 new ones are added to this list. Therefore, the rapid and accurate
determination of new metabolites is necessary to avoid a wasteful duplication of effort.
Some simplified examples of screening approaches are given in Table 2.1.
Table 2.1 Simplified examples of screening of microorganisms
Product Screen
Amylase Starch-Agar method
Protease Precipitation Test
Antibiotics Primary Screening: Crowded-Plate Technique
Secondary Screening: Specialized Tests
Amino acids Auxanography
Rapid and effective screening techniques have been devised for a variety of
microbial products, which utilize either a property of the product or that of its
biosynthetic pathway for the detection of desirable isolates. The initial screening is
done (ordinarily) in plates using agar media. The microorganisms thus selected are
subjected to Secondary Screening. This screening differs from the primary screening
both with respect to objective and the level of sophistication. A vast amount of
information regarding the organism as well as the metabolite is obtained here.
Several trials are done to optimize the cultural condition for maximizing the product
yield. Some of the tests done are:
1. For Microorganisms
 Classification and identification

 Cultural requirements
 Pathogenicity
 Genetic stability
 Scope for improvement, e.g., by mutation, genetic manipulation
2. For Product Yield
 Comparison with yield from known commercial strains
3. For Metabolite
 Identification of the compound

 Immediate or potential use
 Toxicity to animals
 Novelty (newness)
4. For Process
 Shake-flask culture (behavior)

 Pilot-scale culture (inoculum build-up)
 Large-scale culture (main fermentation)
Indeed, a host of techniques must be used to gather all of the above-mentioned

information. The final isolates are quite often further tested vis-à-vis strain
improvement. This final level of selection is termed Rational Screening or Selective
Screening.
2.2 EXAMPLES OF SCREENING
2.2.1 SCREENING OF AMINO ACID PRODUCERS
The most widely used technique in this case is auxanography. Auxanogram is a plate
culture in which diffusion gradients have been produced in the medium involving
one or more substances that affect the growth of the microorganisms. The test
primarily measures the ability of an isolate (the test organism) to interact with test
compounds, e.g., ability of the microorganism to metabolize different sugars, the
antibiotic activity of a test compound, etc. Auxanogram was originally developed by
Biejerinck in 1889 to determine the ability of yeasts to utilize different sugar
substrates. The method is now widely used to identify yeasts at the level of species.
5
STEPS
Preparation of first plate
1. A filter strip (1.512 cm) is put across the bottom of a Petri dish in such a
way that the two ends pass over the edge of the dish (see Fig. 2.1).
2. A paper disc of the size of the Petri dish is placed over the paper strip on
the bottom of the dish
3. Molten nutrient agar is poured on the paper disc in the dish and allowed to
solidify
4. Microbial source material, such as soil, is subjected to dilution such that
aliquots on plating will produce well-isolated colonies
5. Aliquots are plated out
Preparation of second plate
1. A minimal medium lacking the amino acid, say lysine, under consideration is
seeded with a special mutant (test organism) that cannot itself synthesize
lysine. Because of this inability, the test organism, ideally, cannot grow in
this minimal medium.
2. The seeded medium is poured on to a fresh Petri dish.
3. The plate is allowed to set.
Top view Side view
Test culture
Filter (lysine dependent)
strip Petri plate
paper Petri plate with

disc filter strip
Minimal medium
Growth
Petri plate with in poor
filter paper disc medium
Soil culture
Petri plate with
poor medium
Diffusion Growth of test culture
of lysine in minimal medium
Fig. 2.1 Auxanographic screening of lysine producers
4. Now, the agar in the first plate (prepared is step 1) is carefully and
aseptically lifted out with the help of a pair of tweezers and spatula and
placed on the surface of the second plate. Without inverting, the plate is
incubated at a suitable temperature.
6
The lysine produced by the colonies present in the upper layer can diffuse into the
lower layer of agar containing the test organism. Any growth observed in the lower
layer can now be regarded as growth stimulated by lysine diffused from the colony
just above in the first agar. The corresponding colony in first plate can now be
subcultured for further assay. Obviously, the entire work must be done aseptically.
2.2.2 SCREENING OF ANTIBIOTIC PRODUCERS FROM SOIL
A typical laboratory method for the isolation and testing of antibiotic producers
from soil sample is given in Fig. 2.2. The method is normally called Crowded Plate
method. The overall screening protocol is outlined in Fig. 2.3.
PROCEDURE
1. Take 10 g soil and prepare 10-1 dilution

2. Transfer about 2 loopfuls of sample suspension on the agar plate labeled '1'
3. Spread the suspension over the entire surface with a dally rod
4. Use the residue of the dally rod as an inoculum for the plate labeled '2'
5. Spread thoroughly as in step 3
6. Carry out the sequential transfers to 3 other properly labeled plates. Use the
methods given in steps 1 to 5
7. Incubate (inverted) all the plates at 25C for 5-7 days. Observe for the zones
of inhibition in between
8. Subculture the selected colonies from the crowded plate
Dally rod Zone of inhibition
Incubation at Subculturing
Zone of inhibition
25oC/5-7days
Growth
c c d
a b d a b
Incubation at Incubation at
30oC/3 days 30oC/3 days
Cross-streaking of Diffusion of Streaking of

antibiotic producer antibiotic test organisms
Test organisms:
a = Bacillus subtilis; b = Staphylococcus aureus; c = Escherichia coli; d = Saccharomyces cerevisiae
Fig. 2.2 Simplified protocol for the isolation of antibiotic producers from soil
7
SOIL SAMPLE
* Plating of a suitable dilution to give 300-400 colonies
* Incubation at 25oC for 5-7 days for growth
Crowded plate with colonies showing
antibiotic activity (zones of inhibition)
Subculture
Streak culture  Purification
STOCK CULTURE
Agar plates/broth/small fermenter

Quantitative and qualitative information, such as:
* Yield and spectrum
* Side effects in humans
* Molecular structure
* Nutritional requirements for the culture
* Genetic stability
* Pathogenicity
Classification of microorganisms
Strain improvement
INDUSTRIALLY IMPORTANT MICROORGANISMS
Fig. 2.3 Screening of antibiotic producer from soil
8
CHAPTER 3
GENERAL TECHNIQUES OF SELECTION OF MICROORGANISMS
3.1 INTRODUCTION
The microbial profile of our environment is as diverse as can be, both with respect to
type and number. Several selection techniques are available at present for isolating
microorganisms of our interest from any environment. Basically, such methods
function by facilitating the growth of the desired species so that the subsequent
isolation becomes easier. There are three main groups of selection methods: (i)
Chemical, (ii) Physical, and (iii) Biological. The basic strategy in all of the above
methods is to create environment conducive to physical segregation or even
encouragement of the growth of the desired species while discouraging or even
inhibiting the rest.
3.1.1 CHEMICAL METHODS
 Use of special nitrogen or carbon source (for example, cellulose medium for
isolating cellulolytic organisms)
 Use of dilute media (this has limited use in industrial microbiology)
 Use of toxic or inhibitory substances (for example media amended with
antibiotics, dyes, bile salts, etc.)
3.1.2 PHYSICAL METHODS
 Heat treatment: spore-formers can be selected by heating the sample to 80°C

for 10 min and culturing.
 Incubation temperature: selection of psychrophiles, thermophiles, etc., is
possible by this method.
 pH of the medium: this is especially useful for the isolation of yeasts/molds
and archeans.
 Cell size and motility: microorganisms can be selected based on size by using
filters of varying pore sizes.
3.1.3 BIOLOGICAL METHOD
Nature also exerts a selective force on microorganisms. Sometimes, animals can

serve as a reservoir of a given species of microorganism. For example, when sputum
organisms from a patient suffering from streptococcal pneumonia are injected into
laboratory mice, all organisms but Streptococcus pneumoniae are killed by the defense
mechanism of the mouse. The mouse thus becomes, in a sense, a biological reservoir
of pneumococci!
3.2 PURE CULTURE TECHNIQUES
Selective methods can be employed to obtain a large proportion of the

microorganisms we are interested at. It now becomes easier to carry out further
isolation works. A variety of techniques can be employed for the isolation. The
descendant of a single isolation in pure culture constitutes a strain. A strain is usually
made up of succession of cultures and is often derived from a single colony. If a
strain is derived from a single parental cell, it is termed a clone.
3.2.1 GENERAL METHODS OF ISOLATION
 Streak-plate technique (radiant, continuous, discontinuous, etc.)

 Pour-plate technique
 Spread-plate technique
 Hypheal tip technique
 Micromanipulator technique
3.2.1.1 Streak-plate technique
It is a very simple and rapid technique of isolation. In it, the sample broth is streaked
onto a dry agar surface in a series of non-overlapping streaks (see Fig. 3.1). The
process thins out the cells and at some point the cells are separated sufficiently apart
to give rise to discrete colonies.
Inoculating loop
Confluent growth
Isolated colonies
Incubation
Four-way streak
Confluent growth
Isolated colonies
Incubation
Continuous streak
Fig. 3.1 Streak-plate isolation
3.2.1.2 Pour-plate technique
It entails mixing of sample broth in a melted agar medium and plating out a suitable
dilution. The method has some limitations in that psychrophiles or organisms that
cannot withstand a temporary shock of 45-50°C cannot survive. Besides, the isolated
10
colonies remain embedded in the medium and subculturing them entails digging
through the agar. The single most important advantage in it is that it can be used for
quantitative (enumeration) purpose also. A variation of this method is to transfer 0.1ml
of the pre-diluted sample broth and pour 15-20 ml liquid agar medium over it. The
plate is rotated gently in the shape of ‘8’ to affect uniform mixing.
3.2.1.3 Spread-plate technique
It involves spreading onto the solid agar surface about 0.1 ml of the culture.
Turntables can be used for the spreading. In manual method, a loopful of culture is
transferred to the agar surface and spread uniformly with a bent glass rod (called
dally rod). To affect finer isolation, the residue in the rod is used to spread-plate yet
another fresh plate (see Fig. 3.2). In this way, 4-6 plates can be used for the
spreading. As the spreading progresses, at some point the cells will be sufficiently
apart to the affect isolation.
Dally rod
1 2 3 4
Inoculum
broth Incubation Well-isolated
colonies
Thick growth
1 2 3 4
Fig. 3.2 Spread-plate isolation
3.2.1.4 Hypheal tip method
This method is useful particularly for the isolation of molds. A small segment of
mycelia that radiate outward can be aseptically cut out and placed on a fresh medium
for growth.
3.2.1.5 Micromanipulator technique
It is used only when clones are required. With this method, an unequivocal selection
of a single cell is possible. The method uses an instrument called micromanipulator (a
high resolution microscope fitted with manipulating ancillary) and needs
considerable expertise. During manipulation, the cell is first identified. Isolation
takes place on an agar surface. A sharp needle (provided with the manipulator) is
brought close to the cell. The needle is allowed to rest on the agar surface near the
cell so that a small dent is formed. Next, the field is shifted away from the needle tip.
The dented impression made by the needle during the shifting causes the cell to
follow the course of the needle and thus gets separated. The cell is later cut out from
the agar surface and subcultured (see Fig. 3.3 for the schematic diagram).
11
Slide
Manipulating
needle 2
1 3
4
5
7
6
Target cell
Field
2
1 3
4
5
7
6
Movement of field
Fig. 3.3 Isolation by micromanipulator
12
CHAPTER 4
STRAIN IMPROVEMENT
4.1 INTRODUCTION
The initial source of an industrial microorganism is the natural environment,

but the original (wild) isolate is greatly modified in the laboratory. As a result of
this modification, progressive improvement in the yield can be anticipated. The
most dramatic example is that of penicillin produced by Penicillium chrysogenum.
Over the years, the yield has improved by 50,000 fold from the initial yield of a
mere 1-10 μg/ml. The basic objective in improving strain is to make it reliable
and efficient so that the microbial process becomes economical.
After an organism is screened, it becomes necessary to increase the product yield

from fermentation to minimize production costs. Product yield can be improved by:
 Developing a suitable medium for fermentation

 Refining the fermentation process
 Improving productivity of the strain
Generally, major improvements arise from the last approach; all fermentation
enterprises place considerable emphasis on this aspect. The techniques and
approaches used to genetically modify strains to increase the production of the
desired product, collectively, are called strain improvement or strain development. Strain
improvement is based on following 3 approaches:
1. Mutant selection (induced mutation)

2. Recombination
3. Recombinant DNA Technology (Genetic Engineering).
It is important to note here that productivity is not alone the function of large yield.
Productivity is also the function of microbial properties such as resistance to
infection, temperature tolerance, resistance to analogs, genetic stability, appropriate
flocculation characteristics, etc. Strain improvement programs therefore place due
emphasis on these aspects also.
4.2 MUTATION APPROACH
Simply stating, mutation is a stable change in a gene such that the changed condition
is stably inherited by offsprings. Mutation can occur either spontaneously or by
deliberate induction. The former method cannot be relied on, as it is terribly
inefficient. A more direct approach is to use mutagenic agents (mutagens). The
frequency of mutation can be achieved at very high levels with this method.
4.2.1 THE BASIC PROCEDURE
In this method, the cells or spores are contacted with (exposed to) a given mutagen
for a given time period. The exposure may take place on the surface of agar in a
Petri dish, in a liquid culture, etc; the process usually kills about 99% of the exposed
cells. Of the remaining that is isolated by screening (subculturing), only a small
percentage will have actually mutated. Not all the mutants will have the property as
desired, though. Those that bear the desired property are identified, selected, and
tested further using other special procedures.
4.3 COMMON MUTAGENS
The most commonly used mutagens for strain development can be classified into
two groups, viz., chemical, and radiation
4.3.1 SOME COMMON EXAMPLES OF CHEMICAL MUTAGENS
 Nitrous acid (HNO2)
Mutation in microorganisms occurs by the removal of amino group from nucleotide

base. It is carried out by suspending the cells in an acidic buffer and adding sodium
nitrite to it. For example, oxidative deamination of cytosine results in uracil (see Fig.
4.1). This change causes abnormal base pairing and consequent replication errors.
NH2 O
N HNO2 HN
O N O N
H H
Cytosine Uracil
Fig. 4.1 Mutation by nitrous acid
 Alkylating agents
Alkylating agents give relatively high mutant yields. The most common alkylating
agents are Ethyl Ethane Sulfonate (EES), Ethyl Methane Sulfonate (EMS), Diethyl
Sulfonate (DES), N-Methyl-N’-Nitro-N-Nitrosoguanidine (NTG), etc. Mutation is
carried out by suspending the cells in neutral buffer followed by addition of
mutagen. The reaction may be stopped by using sodium thiosulfate or by dilution
during plating. Alkylating agents add alkyl groups to the nitrogen in the 7th position
of the purine. This alkylation creates labile N-glycosidic bond that hydrolyzes to
leave depurinated site (see Fig. 4.2). If not repaired immediately, any base may join to
fill up the gap. This event leads to unmatched bases. NTG is the most potent of
chemical mutagens. It can produce very high mutant yields. It is also an alkylating
agent and acts primarily at the replication fork.
Mutation using NTG can be achieved by adding ~ 0.5 g NTG/liter of broth culture
and allowing the mixture to stand at 28°C for 30 min. The cells are then recovered
14
by centrifuging. The recovered cells are washed with suitable buffer and plate-
cultured for the regrowth into colonies with diagnostic features.
NH2 Alkyl grp NH

2
N N Alkylating agent O N N
7 7
9
+ 9
O N N N N
H
Adenine Depurinated base
Fig. 4.2 Action of alkylating agent
 Base analogs
Compounds such as 5-bromouracil and 2-aminopurine are base analogs of thymine

and adenine respectively. By simple analogy, according to the principle of A=T,
G≡C, 5-bromouracil should base-pair with adenine. However, due to structural
differences, 5-bromouracil pairs with guanine instead. Such a mispairing produces
abnormal hydrogen bondings and subsequent replication error. See the structure of
the analogs in Fig. 4.3.
NH2 O O
6 N 6 N 6 CH3 6 Br
N1 5 7 N1 5 7 HN1 5 HN1 5
8 8
2 4 9 2 4 9 2 4 2 4
3 3 3 3
N N H 2N N N O N O N
H H H H
6-amino purine 2-amino purine Thymine 5-bromo uracil
(adenine) (adenine analog) (5-methyl uracil) (thymine analog)
Fig. 4.3 Examples of base analogs
 Intercalating agents
These are flat molecules that can slip in (intercalate) between the base pairs in the
central stack of DNA helix. This results in the distortion of DNA structure and
consequent error in replication of DNA. Some of the common examples of such
mutagens are nitrogen mustards, proflavine, acridine orange (Fig. 4.4), etc.
Nitrogen mustards are some poly-(β-chloroethyl) amines with the general formula
RN(CH2 CH2Cl)2, where R is an alkyl, alkylamine, or alkyl chloride group.
6 5 4
7 3
8 2
N
9 10 1
Acridine orange
Fig. 4.4 Structure of acridine orange
15
4.3.2 RADIATION
Several systems of mutagenesis have been developed using X-rays, γ-rays, neutrons,
UV-rays, etc. Radiation systems are relatively convenient. If handled properly, they
may be used to produce high mutant yields. Radiations used for mutation can be
conveniently divided into two groups, ionizing radiations and UV-radiations (non-
ionizing).
 Ionizing radiations
These include X-rays, γ-rays, neutrons, etc., and are used only when chemical agents
or UV-rays are ineffective. Ionization methods work by causing chromosomal
breakage, for instance, translocation and transversion. X-rays are produced by
bombarding solids with electrons. The wavelength of the rays is typically 100-150 nm.
Gamma rays have wavelength less than 100 nm. The most common source of γ-
radiation is cobalt-60.
 UV-rays
UV-rays entering around 260-280 nm are strongly absorbed by nucleic acids and the
rays are therefore genotoxic. The rays work principally by forming dimers between two
thymines (see Fig. 4.5).
O O O O
CH3 H3C
HN CH3 HN CH3 HN NH
UV-radiation
+
O N O N O N N O
H H H H
Thymine Thymine Thymine dimer
Fig. 4.5 Formation of thymine dimers
Dimer structures bring about replication errors. However, the user must be careful
in handling UV-light, as this is mutagenic if exposed to skins and eyes. Care should
be taken not to expose the treated cells immediately to the visible light. The
exposure leads to photo-reactivation, a term referring to restoration to full activity
(viability) of the treated cells upon exposure to visible light. The visible light
reactivates a special enzyme, called PRE, which restores the DNA structure by
splitting (unlinking) the dimer. The reactivation rate is about 80%. The organism can
have other survival mechanisms also. One system uses a family of genes called uvr
genes, using which a short fragment of single-stranded DNA (containing the dimer) is
cut off and later replaced with a sound one. Similarly, dimers can also be repaired by
recombination.
4.4 IMPROVEMENT IN THE YIELD
The ability of mutants to produce large amounts of end product is based on one or
more of the following:
16
 Resistance to infection
 Improved foaming characteristics
 Change in cell wall permeability
 Development of analog resistance
 Mutation to auxotrophy
 Change to constitutivity from inducibility
Brief descriptions of some of the more important points are as follows:
4.4.1 CELL WALL PERMEABILITY
The end products of a microbial process should preferably be extracellular so that the
recovery becomes easier. Due to homeostatic system, microorganisms cannot produce
within the cells more metabolites than can be compatible (in quantity, that is). In
such cases leaky cells have been produced and successfully used. Such cells do not
experience any concentration effect within the cell because the product is
continuously secreted out in the medium. This leads to overproduction of the
desired metabolite.
4.4.2 ANALOG RESISTANCE
Organisms, from simple to developed, balance their metabolic reactions by a

mechanism called feed-back inhibition. If a specified metabolite crosses a critical limit,
the same metabolite will exert an influence on enzyme(s) several steps ahead of the
metabolic sequence that led to its formation. One consequence of this is, under
normal condition, the product we desire cannot be produced in large amounts. Thus,
feed-back inhibition works as an off-on switch of metabolic regulation. Organisms
also have another regulatory mechanism known as repression. Repression works at the
level of transcription and is therefore much slower in action. Organisms use
repression mechanism for the fine-tuning of metabolic regulation. One of the
various alternatives for overcoming this problem is to desensitize the enzyme(s)
involved in feedback inhibition. For this, the cells are treated with the analogs of
metabolite responsible for repression (for example, deoxyglucose is an analog of
glucose). The analogs are generally toxic to the organisms and most of them cannot
survive this treatment. The few cells that survive the analog treatment are called
analog resistant strains. These cells not only become immune to analogs but also
develop insensitive feedback enzyme systems. This faulty metabolic regulation leads
to uncontrolled production (overproduction) of the desired metabolite (see Fig. 4.6
also).
4.4.3 AUXOTROPHIC MUTANTS
Auxotrophic mutants are strains of microorganisms, derived by mutation, that

require one or more key factors for growth (not needed by the parent organism). The
requirement arises because the organism’s ability to synthesize the key metabolite
has been lost due to genetic alteration. The organism cannot grow unless these
metabolites are supplied externally. This loss of ability is termed auxotrophy. If the
organism is dependent on lysine, for example, it becomes a lysine auxotroph. The
17
position in the pathway where the synthesis of such a compound has been blocked is
called metabolic block (Fig. 4.6).
Metabolic block
A B C D E F G
J* H Key nutrient
(Analog of J)
I
J
End product
Fig. 4.6 Mechanism of auxotrophy

--------------------------------------------
Since E is the key nutrient, the organism cannot survive without it (and so called E-dependent
or E-auxotroph). If E can be supplied (externally) in regulated amounts the cell can be made to
run the pathway as if it were normal. However, since the pathway is blocked at E, the central
pathway diverts towards J via H. If J is the product of our interest, overproduction of J
through metabolic block between D and E is obvious. However, J cannot accumulate in
concentrations more than that is compatible. Slight excess of J causes an inhibition on
enzyme between D and H. To overcome this, the organism is again mutated for analog
resistance so that the enzyme fails to recognize J* (analog) and therefore J (main product).
This event leads to another level of overproduction of J.
Auxotrophic mutants are of immense value in industrial microbiology, particularly in

the production of primary metabolites such as amino acids. The cultured organism
can be constantly supplied with regulated amounts of the compound just following
the metabolic block. The organism carries out normal metabolic reactions but since
there is a metabolic block ahead, the compounds just preceding the block will tend
to accumulate.
4.4.3.1 Example of strain improvement by analog resistance
The selection of mutants resistant to glucose analog 2-deoxyglucose (DOG, see Fig.
4.7) has proved extremely valuable when applied to brewing yeasts. In normal
fermentations, due to catabolite repression, the yeast takes up maltose only when
about half of the glucose present in the wort has been metabolized. Mutants resistant
to the action of DOG are derepressed: they have the ability to utilize maltose and
glucose simultaneously, and have improved fermentation characteristics.
6 6
CH2OH CH2OH
5 O 5 O
4 1 4 1
2 2
3 3
Glucose 2-deoxyglucose (glucose analog)
Fig. 4.7 Glucose and glucose analog

18
4.5 STRAIN IMPROVEMENT BY RECOMBINATION
Recombination can be defined as formation of new gene combination among those

present in different strains. It is used to combine desirable alleles (one of two or more
alternative forms of genes) present in two or more strains into one to increase
product yield or to generate new products. Recombination may be based on:
 Sexual recombination
 Parasexual cycle
 Protoplast fusion
4.5.1 SEXUAL RECOMBINATION
Conjugation, mediated by sex factor, occurs in many bacteria and actinomycetes,

including Streptomyces. Conjugation leads to formation of, usually, a partial diploid in
which crossing-over produces recombinant genotypes. Recombinants are recovered
and used for genetic studies like linkage mapping. Similarly, yeasts have two mating
types: the cells of the opposite mating types fuse to form diploid heterozygous cells
(which are non-mating types). The diploid cells undergo meiosis to produce four
haploid spores, which give rise to vegetative cells.
4.5.2 PARASEXUAL CYCLE
Most industrially important fungi are asexual. However, their haploid hyphae
sometimes fuse to produce heterokaryons (cells having two distinct nuclei). Later on
the two nuclei of heterokaryons fuse and produce diploid nuclei. Occasionally,
mitotic recombination coupled with meiotic reduction yields haploid nuclei from the
diploid ones, giving rise to recombinants. In some cases, attempts have been made
to use parasexuality for strain improvement, e.g., in Penicillium chrysogenum.
4.5.3 PROTOPLAST FUSION
Protoplasts of bacteria, actinomycetes and fungi are isolated by treatment with a

variety of enzymes responsible for degrading cell walls, e.g., cellulase, pectinase, etc.
An osmoticum, usually sorbitol, is necessary for protoplast stability, and fusion is
induced by polyethylene glycol (PEG: HO(CH2CH2O)nH) treatment. Protoplast
fusion has been used to produce Cephalosporium acremonium (mold) strains with ability
to give significantly higher yields of Cephalosporin C.
Protoplasts can also be prepared by treatment of bacteria (in logarithmic growth

phase) with penicillin and then continuing culturing for several generations. The cells
are harvested, washed, and suspended in a suitable hypertonic solution until used.
Since protoplasts formed are destroyed under hypotonic conditions, the extent of
formation of protoplast is determined indirectly from the number of normal cells
surviving under hypotonic conditions.
19
4.6.3.1 Basic process of PEG-induced protoplast fusion
PEG induces reproducible and high-frequency recombination. It also has low

toxicity to most cell types. A protoplast mixture is treated with 28-50% PEG (mol
wt. 1500-6000) for 15-30 min followed by gradual washing of the protoplast to
remove PEG; protoplast fusion occurs during the washing process. The washing
medium may be alkaline (pH 9-10) and may contain a high Ca2+ concentration (50
mMol/L); this approach is a combination of PEG and high pH with Ca2+ treatment,
and is usually more effective than either treatment alone. PEG is negatively charged
and binds to cations (e.g., Ca2+) which in turn bind to the negatively charged
molecules of plasma membrane (plasmalemma). The PEG can also bind to the
cationic molecules of plasma membrane. During the washing process, PEG
molecules pull out the plasmalemma components bound to them. This disturbs
plasmalemma organization thereby leading to fusion of protoplasts located close to
each other (Fig. 4.8).
A
PEG A B PEG A B
(28-50%) dilution
+
pH 9-10
Ca2+: 50mM/L bridging heterokaryon hybrid cell
B
Fig. 4.8 Outline of PEG-induced protoplast fusion
Regeneration of the transformed cells is carried out by spread-plating the treated

cells on a suitable hypertonic agar medium (containing sodium succinate and
polyvinyl pyrrolidone) and incubating at 30-35°C. The transformants are obtained by
selecting cells with some given phenotypic characteristics.
20
CHAPTER 5
GENETIC ENGINEERING
5.1 INTRODUCTION
An array of sophisticated techniques used to isolate, manipulate, move, propagate and

express DNA is called genetic engineering. It is also called recombinant DNA technology
(rDNA technology). The science has two main applications, viz., in (i) basic research,
and (ii) applied research. Genetic engineering for commercial application is sometimes
called biotechnology. In a true sense, genetic engineering is one of the many disciplines of
biotechnology. The tendency to use the two terms interchangeably is simply an
indication of how overwhelmingly biotechnology is dependent on genetic engineering.
5.2 BASIS OF GENE MANIPULATION
The basis of gene manipulation is the gene cloning. Since the cloning is carried out at a
molecular level it is also called molecular cloning. The term cloning has several contextual
meanings. In a biological sense, a clone refers to cells with an identical genotype. The
cell multiplication is therefore asexual. In microbiology, the descendants of a single
cell are called clones. As used in genetic engineering, a clone refers to identical host cells
that carry identical recombinant DNA molecule. The objective of gene cloning is to isolate
and produce specific genes in pure form and large quantities.
5.3 TOOLS NEEDED FOR GENE CLONING
Several tools are required for gene cloning. The tools used in genetic engineering
differ from those used in classical engineering in that the former is molecular in
nature. Some of the indispensable tools are given in Table 5.1.
5.4 TECHNIQUE OF GENE CLONING
The basic steps are:
 Obtaining the DNA of our interest (foreign DNA)

 Insertion of foreign DNA into a suitable cloning vector to form a
recombinant/ hybrid/ chimeric DNA.
 Introduction of the recombinant vector into the host cell (organism).
 Selection of host cells that have been transformed (i.e., selection of clones).
 Amplification of the clones.
Table 5.1 Tools of molecular cloning
Category Tools
1 Enzymes Restriction enzymes
DNA polymerase
Reverse transcriptase
DNA ligase
2 Marker genes/Reporter genes Lac z gene, β-galactosidase gene, etc.
3 Cloning vectors Modified  phage, plasmids, phasmids
(phagemids), cosmids
4 Advanced vectors Shuttle vectors, expression vectors
5 Linkers and adaptors Linkers and adaptors
6 Substrate Foreign DNA/Passenger DNA/DNA insert
5.4.1 OBTAINING DNA OF INTEREST
The DNA to be inserted into the host cell can be obtained by 3 methods, viz., (i) from
the genome, (ii) by reverse transcription, and (iii) by chemical or enzymatic synthesis. A brief
description of the different methods is given in the following paragraphs.
5.4.1.1 Genomic DNA
Isolation of a specific gene is a daunting job. Even in a simple organism like E. coli,
a given gene accounts for <0.05% of the genome. Sorting out the specific gene is
therefore a very complex job. When the genomic DNA is absolutely necessary, one
can obtain them from genomic libraries. A genomic library is a collection of plasmid
clones or phage lysates containing recombinant DNA molecules so that the sum total
DNA inserts (noun) in this collection, ideally, represents the entire genome of the
concerned organism.
The genomic library is constructed by first extracting the total DNA and breaking it
into appropriate sizes by mechanical means, sonication, or enzymes. The fragments
are separated according to sizes in agarose gel. The fragments are collected and
inserted in cloning vectors (discussed later). This approach is called shotgun approach
because the whole genome of a cell is cloned in the form of random and
unidentified clones. See Fig. 5.1 for the outline of gene library preparation.
5.4.1.2 DNA by reverse transcription
Prokaryotic DNAs are naked (they are free from interfering structures, e.g., histones).
They also do not contain introns (non-coding DNA). The primary transcript of mRNA
in prokaryotes therefore does not undergo processing before being expressed.
Human (eukaryotic) genomes present several difficulties in constructing a gene bank.
The chromosomes exist as a complex of protein (nucleoprotein), the separation of
which is difficult. Besides, they contain introns. Introns are eukaryotic DNA
sequences widely dispersed throughout the genome. Introns are transcribed but
22
never expressed. Using genomic DNA from eukaryotes therefore presents problems
in knowing whether the selected fragment of DNA is an intron or an exon (coding
DNA). After transcription, mRNA in eukaryotes undergoes processing so that it
becomes free from intron sequences before being expressed. Such pure mRNAs can
be obtained by running the total RNA extract through poly-U sepharose or poly-T
cellulose columns. Since mRNAs have poly-A tails, they form hydrogen bond with
poly-U sepharose or poly-T cellulose of the support while the rest pass down. The
bound mRNAs are later eluted from the column.
Entire genome from donor

cells (prokaryotes)
Fragmentation
Random DNA fragments Vector DNA
Joining
Circular recombinant DNA

* Introduction into host cell
* Selection of transformants
(recombinants)
Library of clones
(Gene bank)
Fig. 5.1 Preparation of gene bank
This processed mRNA can now be used to produce exon DNA. Since the DNA
constructed thus is complementary to mRNA, the former is called complementary- or
copy DNA (cDNA) and the collection, by analogy with the gene bank, cDNA library
or cDNA bank.
The synthesis of DNA using mRNA as the template is called reverse transcription. The
enzyme catalyzing this reaction is called reverse transcriptase. This enzyme is obtained
from the RNA virus called AMV (Avian Mycoblastosis Virus). This enzyme
performs reactions similar to that of DNA polymerase and has an absolute
requirement for a primer with a free 3'-OH. See Fig. 5.2 for the outline of the
preparation of cDNA.
After the cDNA has been prepared in large amounts, rest of the protocol is similar
to that for constructing gene bank.
5.4.1.3 DNA by chemical / enzymatic synthesis
The method invokes a mental process called reverse translation which does not occur in
vivo. Before proceeding, however, the investigator must have a clear objective as to
which protein he is attempting to synthesize. Stated differently, the primary structure
of the protein to be prepared should be worked out beforehand. The corresponding
codon in mRNA is then found out from the universal codon. Finally, artificial synthesis
of DNA (complementary to mRNA) is carried out. The method is not foolproof, as
23
the problem due to degeneracy in codons cannot be overlooked. As of now, there are
instruments available (called oligonucleotide synthesizer or gene machine) that can
polymerize up to 50 nucleotides in a few days. Since the complete gene is usually
several folds longer, several such pieces are later joined in order to construct the
complete sequence.
Whatever the source of DNA, a large number of copies of DNA inserts is needed.
This can be achieved by using Polymerase Chain Reaction (PCR).
3' A A A A A 5'
mRNA
5'-T T T T T -3'
Poly-T primer
3' A A A A A 5'
5'-T T T T T -3' Reverse transcriptase
Primer
3' A A A A A 5'
5'-T T T T T 3' DNA strand
RNAse or NaOH to
hydrolyze mRNA
5'-T T T T T 3' Single-stranded
DNA polymerase DNA
3' A A A A A Spontaneously
5'- T T T T T formed single-
SI nuclease stranded loop
(for cutting the loop)
3' A A A A A 5'
5'-T T T T T 3'
Blunt-end, double-stranded cDNA
Fig. 5.2 Preparation of cDNA
5.4.2 THE POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) was developed by Kary Mullis in 1985. It is an
extremely powerful technique for gene amplification. The developer received Nobel
Prize in 1993. Fully automated machines are now available that can produce billions
of copies within an hour. The device is commonly called thermocycler (Fig. 5.3b).
Nowadays, several versions of PCR are available, e.g., anchored PCR, inverse PCR,
etc., each of which is designed for a specific purpose. The PCR is carried out in vitro.
The basic requirements for running a PCR are:
 DNA to be amplified
 Two nucleotide primers (about 20 nucleotides long). These primers are
complementary to the 3' ends of the DNA to be amplified.
 Four nucleotides (deoxy), viz., dTTP, dATP, dGTP, and dCTP.
 Heat-stable DNA polymerase
The heat-stable DNA polymerases used in PCR are from hyperthermophilic archeans.
Some of the important DNA polymerases and their source are given in Table 5.2.
24
Table 5.2 Some important DNA polymerases and their source
Enzyme Source organism

Taq Thermus aquaticus
Vent Thermococcus litoralis
Pfu Pyrococcus furiosus
Dynazyme Thermus brockianus
Dynazyme® has been claimed to be the most efficient of the above enzymes. It has a
half-life of >3 hrs at 96°C. It also has proofreading activity, strand-displacement activity,
and deep vent component. Deep vent component is responsible for separation the
G:C-rich regions that tend to form very strong loop within the single strands.
5.4.2.1 The basic procedure of PCR
At the start of the PCR, all the ingredients are added in a reaction mixture. Primers
and the deoxynucleoside triphosphates should be in large excess. The step-by-step
illustration appears in Fig. 5.3a.
Double-stranded DNA
dNTPs 5' 3'
3' 5'
DNA polymerase
Primers Reaction mixture
Denaturation
5' 3'
STEP I
3' 5'
Annealing
5' 3'
5' 5' STEP II
3' 5'
Incubation
5' 3'
5' 5' STEP III
3' 5'
Fig. 5.3a The basic steps of PCR
STEP-I:
The reaction mixture is heated to 96-98ºC to denature DNA. The DNA double helix
separates as a result of heating. The step is also called denaturation and is analogous to
the term melting used to indicate DNA unwinding.
25
SSTEP-II
TThe mixture iss cooled to 40-60ºC (the steep is called annnealing). Anneaaling permits tthe
pprimers to basse-pair with coomplementaryy 3’ ends of each of the sepaarated DNA
sstrands.
SSTEP-III
IIncubation is done
d at 70-75ººC so that thee DNA strands are replicateed as usual by the
eenzyme by eloongating the prrimers
TThe incubationn marks the completion

c off the cycle. Thhe next cycle sstarts from steep-I
wwhere the twoo DNA dupleexes are denatured. The seecond cycle reesults in 4 DN NA
dduplexes from
m two duplexes. Since the ennzyme is therm mostable it neeed not be addded
inn each cycle. Thus, at thee end of nth cycles
c 2n coppies of DNA duplexes cann be
eexpected. In programmedd automatic thermocyclerrs, 20-30 cyc ycles are usuually
aadequate. This produces 10 1 6-109 fold increase
i in thhe number oof duplexes. TThe
mmachine takes less than 5 min
m for a cyclle and 1.5-2.5 hrs is all thatt is needed too go
tthrough 20-300 cycles.
Fig. 5.3b
5 Thermoccycler (PCR machine)
m
IIn gene cloninng, only the deesired genes arre taken out from
f a large, ccomplex genome.
TThis removal is achieved withw the help of molecular scissors
s called restriction enzyymes.
TThese enzymees cut the DNA at some specific base seqquence termedd recognition sitte or
rrestriction site. The
T tailored gene
g can now w be joined to a cloning vvector (descriibed
nnext) with the help of an ennzyme DNA ligigase.
55.4.3 JOINING THE DNA

A INTO CLO
ONING VECT
TORS
TThere are sevveral types ofo restriction enzymes. Thhe microorgan anisms use thhese
eenzymes as naatural defensee mechanism.. They cut thhe foreign DN NA should thhese
eenter the host cell.
c Each of these
t enzymes has specific base recognitionn site where it can
ccut the DNA A. The DNA can be cut in many diffferent ways. Some restricttion
eenzymes cut thhe DNA from m the end. Theey are commoonly called exonnucleases. Somee of
tthem cut the internal section of the DNA D and are therefore caalled endonucleaases.
FFurther, somee cut the DNA A straight to produce
p bluntt DNA. The m most widely uused
rrestriction enzzymes in genetic engineerinng are endonucleases that pproduce a stagggered
ccut in the DN NA duplex. The
T cuts are complementaary and can aagain be brouught
226
together through base-pairing. The strands having such ends are sticky or cohesive. The
straight cuts are not cohesive and, by analogy, called blunt ends or flush ends. Some of
the examples of restriction endonucleases appear in Table 5.3.
Table 5.3 Examples of some restriction endonucleases
Enzyme Source organism Restriction (cleavage) site and pattern

5' 3'
-G G A T C C-
Bam HI Bacillus amyliquefaciens H cohesive end -C C T A G G-
-G A A T T C-
Eco RI E. coli RI cohesive end -C T T A A G-
-A A G C T T-
Hin dIII Hemophilus influenza Rd cohesive end -T T C G A A-
-C T G C A G-
Pst I Providencia stuartii 164 cohesive end
-G A G C T C-
-G T C G A C-
Sal Streptomyces albus G cohesive end
-C A G C T G-
-A G C T-
Alu Arthrobacter luteus blunt end -T C G A-
Note: The staggered cleavage sites have palindrome sequence.
5.4.3.1 Linkers
DNA molecules which have staggered cuts can easily join with another
complementary base pair but the straight/blunt ends cannot. In such cases linkers
may be artificially joined to the blunt ends so that they may become cohesive.
Linkers are short, chemically synthesized, self-complementary, double-stranded

oligonucleotides which contain within them one or more restriction sites. These
linkers can be fused to the blunt-ended DNA, thereby supplying the latter with a site
for staggered cut. That is, the ends can now be cut with restriction endonuclease.
The joining is catalyzed by DNA ligase. DNA ligase comes from different sources
but T4 ligase is the preferred one. T4 DNA ligase uses ATP as the cofactor, which
serves to adenylate the amino group of a lysine residue in the enzyme. 5' phosphoryl
terminus of DNA is also adenylated in the same manner. The DNAs are then joined
liberating AMP in the process. See Fig. 5.4.
Site of cleavage
Blunt DNA segment
Ligase
Linkers
Blunt DNA segment joined to linkers

Restriction endonuclease
DNA with cohesive ends
Fig. 5.4 Joining of linkers to the blunt-ended DNA

27
5.4.3.2 Cloning vectors
A cloning vector is a genetic element into which a foreign gene can be inserted,
recombined, and replicated. They are also called vehicles. Cloning vectors are generally
designed to allow recombination of a foreign DNA at a special site known as
restriction site. Since the foreign DNA is joined to the self-replicating vector, the
replication of the latter automatically ensures the replication of the foreign DNA
inserted into it. Cloning vectors also contain selectable and scorable marker genes and
an attachment site. In complex vectors like shuttle vectors the ori sites come from two
different organisms. This makes the hybrid (recombinant) gene compatible to both
types of organisms. When expression is desired, it also contains promoters (for
detailed understanding, go through microbial genetics, Chapter 8). The restriction
site should not fall within the ori site, as this will destroy the replication ability.
Some of the important vectors (all of them genetically engineered) are plasmids (e.g.,
pBR322, pUC, etc.),  vectors, cosmids, phagemids, YAC (Yeast Artificial
Chromosome), etc. See Fig. 5.5 for the example of plasmid vector. Refer to
microbial genetics (page 48) for discussion on plasmids.
Desirable properties of cloning vectors
The fundamental requirements of a cloning vector can be summarized as:
 It should replicate autonomously: must be under relaxed control

 It should be easy to isolate and prepare
 It should be easy to deal with in transformation reactions
 It should have suitable markers for easy detection
 In gene transfer, it should integrate either itself or the foreign DNA into
chromosome
 It should have as many target sites as possible, with only one site for one
enzyme
 The expression vector should have suitable control elements, e.g.,
promoters, operators, binding sites for ribosomes, etc
Pst I Eco RI
Ampicillin resistance
(Ampr) Tetracycline resistance
(Tetr)
Hind III
pBR 322
Ori site
Bam HI
Sal I
Fig. 5.5 An example of plasmid vector
28
Charon and  phages
Wild  phage has one cos site and 5 Eco RI sites. A sizeable portion of DNA of this
virus is unessential. The phage genome has att and ori sites. Modified  phages have
been prepared by snipping out unessential regions of DNA. Charon 16 is one such
modified vector which has one Eco RI site. Since the foreign DNA has to be inserted
into Eco RI site of the phage vector, the vector can be called an insertion vector.
Charon 4A is another modified  phage vector that has 3 Eco RI sites. During
enzymatic digestion some portion of the vector therefore gets removed for
accommodating the foreign DNA. Charon 4A is therefore a replacement vector. While
including the restriction sites in vectors, some markers are used to ascertain whether
chimerization has occurred or not. The restriction sites generally fall within such
markers. Markers that are responsible for resistance to antibiotics are called selectable
markers. If the resistance to antibiotics due to the vector gene is lost it can be
concluded that chimerization has occurred because the insertion of foreign gene into
the resistance gene inactivates the latter. On the other hand, if resistance persists,
chimerization has not taken place. In other words, the resistance gene is still intact
and no insertional inactivation has taken place.
Another type of marker, called scorable marker, is also used in cloning vectors. One
such marker gene is lac Z gene. It is responsible for β-galactosidase activity (see later).
 phage contains double stranded DNA. The DNA is linear inside the particle but
becomes circular as it enters the host. The recircularization is due to the presence of
cohesive ends (termed cos site) of about 12 nucleotides at each end. The cos site may
also lead to linear linking between the λ DNAs to form concatemers. The cos sequence
is also involved in the packaging of viral DNA into the protein head. The packaging
is promoted by viral proteins Nu 1 and A. The phage has the uniqueness of being
able to package DNA of sizes between 38-52 kilobase pairs (kbp). Since about 40%
of the phage DNA can be snipped out (because it is unessential) an equivalent size
(about 25 kbp) of foreign DNA can be inserted or replaced into the phage DNA.
Plasmids can incorporate only about 15 kbp, which means  phages can allow
packaging of much bigger sizes of foreign DNA.
Cosmid vectors
These are essentially plasmids that contain a minimum of 250 bp of  DNA. The
vector has an ori site, restriction site, cos site and selectable markers. Cosmids can
take up to 45 kbp long DNA inserts. The packaged cosmids infect the host cells like
 particles, but once inside the host they replicate and propagate like plasmids.
5.4.4 JOINING PASSENGER DNA (FOREIGN DNA)
Usually, the double stranded foreign DNA as well as the chosen vector is cut with
the same restriction enzyme. Both should have the restriction site for the enzyme
used. Thus, similar cuts are obtained in both of them. The two can therefore form
complementary strands for automatic pairing. The sealing is achieved with DNA
ligase either from E. coli or T4. T4 DNA ligase is more active and it uses ATP.
29
During mixing, a large number of foreign DNA is used to increase the probability of
base-pairing to make chimeric DNA. All three types of molecules may react to form
any one or more of the following products: circularized foreign DNA, circularized vector,
and circularized recombinant. Circularized foreign DNA does not pose problem as it
lacks origin of replication. Recircularization of vector is undesirable in that
chimerization efficiency is decreased. To prevent recircularization of the vector, its
DNA is treated with alkaline phosphatase. The enzyme digests 5' phosphoryl group of
the vector DNA thereby preventing reclosure. The digestion leaves a nick even after
reclosure or chimerization. The nick can later be repaired by the host’s system.
When foreign DNA with blunt ends is to be used, it is first joined at both the ends
with appropriate linkers with the help of T4 DNA ligase. Adaptors can also be used to
make chimeric DNAs. Adaptors differ from linkers in that they have restriction sites
for two (or more) different enzymes. With the use of two different enzymes, two
different sticky ends in the same DNA can be produced. This comes handy when the
restriction site of foreign DNA is different from that of the cloning vector. The
adaptor serves here as a joining factor. See Fig. 5.5, 5.6 and 5.7.
Site 1 Site 2
Restriction enzyme 1 Adaptor Restriction enzyme 2
Foreign DNA Ligase Cloning vector
Adaptor
Foreign DNA Cloning vector
RECOMBINED DNA
Fig. 5.6 Linking with adaptor molecules
Blunt foreign DNA
Linker with Eco RI site Linker with Eco RI site

T4 DNA ligase Plasmid
vector
Eco RI digestion
Eco RI digestion
T4 DNA ligase
Digested
plasmid
vector
Recombinant
DNA
Fig. 5.7 In vitro production of recombinant DNA
30
5.4.5 INTRODUCTION OF RECOMBINANT VECTOR INTO HOST
A good host must have following properties:
 Is easy to transform
 Supports replication of rDNA.
 Lacks restriction enzymes, e.g., strains of E. coli K12.
 Is deficient in normal recombination function so that the DNA insert is not
altered by natural recombination events.
The most widely used hosts are the special strains of E. coli and Bacillus subtilis.
Bacillus subtilis has certain demerits: it cannot store the DNA insert in a stable state
for a long time. The organism is nevertheless used as a good host for study related to
expression of extracellular cell products such as enzymes.
5.4.5.1 Plasmid introduction
E. coli cells are generally poorly accessible to DNA molecules, but treatment with
CaCl2 makes them permeable to DNA. The process involved is poorly understood.
Growing E. coli cells are suspended in 50 mM CaCl2 at a concentration of 108-1010
cells/ml. The cells may be incubated for 12-24 hrs for increasing the frequency of
transformation. The recombinant vector is then added; efficient transformation
requires only a few minutes. The frequency of transformed cells is 106-107 per g of
plasmid DNA (1 transformation out of 10,000 plasmids). Molecular weight of
chimeric DNA in the range 3105 to 8106 can result in successful transformation.
Another method being used is electroporation technique. In this technique, electric

pulse is used to facilitate entry of foreign DNA into the host cell.
5.4.5.2 Introduction via  phage vectors, cosmids, etc.
 phage vectors have cos site needed for the packaging of recombinant vector into
phage heads. When recombination is carried out in vitro, DNA inserts of varying
lengths can recombine (see Fig. 5.8).
The phage has the ability to pack a total DNA size of between 38-52kbp only.
Depending on the actual size of the  phage vector, the foreign DNA recombined
and packed is of about 25 kbp. Cosmids, being smaller, can recombine and pack
up to 45 kbp of foreign DNA. When the foreign DNA is mixed with specially
prepared head and tail proteins in cell extracts, packaging of recombinant DNA
and assembly of the particle occur under the influence of viral proteins Nu 1 and
A.
It must be noted again, only the right size of DNA gets packed. The frequency of
recombination is much higher than in CaCl2 method. Special techniques and mutants
are required for the preparation of empty heads, tails, and DNA-free terminase. The
packed particles are then allowed to infect special strains of E. coli. When  vector is
31
used, the recombinant DNA integrates to the host DNA. When cosmids are used,
they replicate like plasmids.
Recognition site Foreign DNA
15kbp 25kbp 10kbp

Eco RI digestion
15kbp 10kbp
25kbp lac z Eco R I
Ligase Cos site
Charon vector
15kbp 10kbp
25kbp
X X
Empty
phage
head
25kbp Chimeric DNA
Fig. 5.8 In vitro recombination using phage vector
5.4.6 DETECTION OF RECOMBINANT CELLS (CLONES)
Because of markers present in vectors (spanning the restriction sites) and the special host
used, identification of the transformants is quite easy. During recombination, two things
can happen: (i) vector will recircularize without taking the foreign DNA, or (ii) vector becomes
chimeric by taking up the foreign DNA. During transformation, three things can happen: (i)
the host receives only the vectors, (ii) the host receives recombinant vectors, or (iii) the host does not
receive anything (or only the foreign DNA). The sorting out of all these is quite interesting.
An example of selection of transformants is provided in the following paragraphs.
5.4.6.1 Examples of recombinant identification
When plasmid is used
 Vector used: pBR 322

 Host used: selected strain of E. coli (sensitive to Ampicillin and Tetracycline)
 Restriction enzyme used: Pst I.
 Selectable markers used: Ampicillin resistance (Ampr) and Tetracycline
resistance (Tetr)
After reaction, the host cells are grown on a normal medium. The growth is then
replicated (replica-plated) on a medium amended with ampicillin and tetracycline. The
growths on all the plates are compared. The normal plate is called master plate. The
master plate supports, ideally, the growth of all host cells. The original host cell
cannot grow on either of the replica plates because the organism is sensitive to
32
antibiotics. Following observations (Table 5.4) and conclusions can be drawn after
comparing the growth on different plates. See Fig. 5.9 for clarity.
Since the restriction enzyme we have used is Pst I, recombination at Tetr is out of
question. Therefore, only 1, 2 and 4 (Table 5.4) are possible.
Table 5.4 Observation and conclusion of test
Observation Conclusion
Growth in Ampicillin+ and Entry of intact plasmid
Tetracycline+ media
Growth in Tetcycline+ media but Recombination in Ampr gene
not in Ampicillin+ media
Growth Ampicillin+ media but Recombination in Tetr gene (but not possible
not in Tetracycline+ media with pBR 322 in the above case because we
have used Pst I as the restriction enzyme)
No growth in both Ampicillin+ Host cell did not receive anything or received
and Tetracycline+ media only foreign DNA
No growth
Growth
d d'
a c a' c'
b b'
Master plate Replica plate
(Ampicillin-amended)
a and c: transformed with only cloning vector Note:

b and d: transformed with recombinant vector Tetracycline-amended media is superfluous
(i.e., successful transformation) here because restriction enzyme specific to
that site has not been used
Fig. 5.9 Detection of recombinant cells with the help of markers
When charon-16 is used
Charon-16 has a scorable marker, lac Z. Lactase-negative (lac¯) strain of E. coli is

chosen as the host. The host cells are first spread over a special medium (called X-gal
medium) to form a lawn (rich growth). The medium contains a compound, 5-bromo-4-
chloro-3-indoyl-β-D-galactopyranoside. Over this lawn of E. coli, the reconstituted phage
particles are spread and the color of lysis (plaques) observed. Recombinant E. coli
cells yield colorless plaques. This is because of the insertional inactivation of lac Z
gene spanning the restriction site. Cells that have received intact charon vector
(i.e., only the phage DNA packed) produce plaques of blue color. This is because
the intact lac Z has β-galactosidase activity. The organism bearing this gene (passed
from phage) can break down X-gal (lactose analog) to produce color. Thus, only
the colorless transformants are the true transformants.
33
5.4.7 SELECTION OF SPECIFIC CLONES
Selection of transformants is relatively easy. However, selection of the desired

transformants is complex. Their selection is very important because we are
interested in only those transformants that have picked up the right gene. There are
two basic approaches used for this: (i) looking for the gene products, and (ii) looking for the
gene itself.
5.4.7.1 Looking for the gene product
One basic requirement of this technique is that the host cell itself should not
produce the desired gene product. There are several methods for testing this gene
product. One common method is to use antigen-antibody reaction. The process is also
known as immunological test. See Fig. 5.10 for the outline of the protocol.
Cells on replica plate
Partial lysis
Lysed cells with
exposed proteins
(antigens) Filter paper with
Antibody 1
Unmatched pair
Spontaneous matching of
antigen and Antibody 1
Antibody 1 - Antigen
Antibody 1*
complex
(radioactively labeled)
Antibody 1 - Antigen -
- Antibody 1* complex
X-Ray filming
1 1
2 6 2 6
Transformed
7 7
clolonies
3 5 3 5 (2, 3. 6, 7)
4 4
Master plate X-Ray image
Fig. 5.10 Antigen-antibody reaction for detecting clones
First of all, replica-plates of the colonies are prepared from the master plate. The
cells in the replica plates are then lysed by exposure to chloroform or high
temperature. Then a cellulose filter (or polyvinyl filter) onto which specific antibody
has been fixed is pressed over the lysed cells. If the antibody finds its corresponding
antigen (the gene product) in the lysed cells, it will bind to it. The filter is taken out,
along with the bound antigen, and incubated with yet another radioactively labeled
antibody. The gene product (i.e., antigen) gets sandwiched between two antibodies.
After reaction, the excess antibody is washed away. The filter is now X-ray imaged to
determine the position (and presence) of gene product in the lysed colonies.
34
Comparison of the image with the master plate will reveal the presence and position
of cells (clones) containing the desired gene. The desired colonies can now be picked
up from the master plate for subculturing.
5.4.7.2 Searching for the gene itself
This is commonly known as colony hybridization. Replica plates are prepared on a

nitrocellulose filter disc. The disc is placed on a gelled medium (nutrient) and incubated
to develop colonies. Cells growing on the disc are nourished through the gelled
medium. The disc is then placed on a blotting paper soaked in 0.5 N NaOH
solution. The alkali diffuses into the nitrocellulose, lyses the cells, and denatures their
DNA. Thereafter, the disc is neutralized with tris (hydroxymethyl) aminomethane-
HCl buffer. This results in binding of the DNA in the same position as the colony.
To fix the DNA properly, the disc is baked at 80°C. It is then incubated with a
radioactively labeled probe (20-40 nucleotide, labeled with P32). The unbound probes
are washed away and the discs are X-ray imaged to compare with the master plate
for determining the colony and position of cells containing the desired gene.
5.4.7.3 Hybrid Arrested Translation (HART)
HART is a fusion of two techniques, viz., immunological and colony hybridization. HART
can identify a given DNA in a clone even if neither DNA nor RNA probe is
available, so long as one knows which protein one is looking for!
In this method, each clone’s DNA is extracted and purified and the two strands are
separated from one another. Raw RNA is also extracted from the donor cell (the cell
from which the foreign DNA was taken). The extracted DNA and RNA are then
mixed in a cell-free protein synthesis system where everything needed for protein synthesis
is present. If the foreign DNA is present in the extract it will hybridize with the
complementary RNA extracted from the donor cell. This particular RNA being
hybridized will not be free for taking part in protein synthesis. In other words, all the
other mRNAs in the mixture begin busily directing protein synthesis except for those
bound to DNA. So, if the protein one is looking for is expressed in the mixture, the
gene one wanted is not present in the clone. To find protein, one can use antigen-
antibody reaction as described in immunological method.
35
CHAPTER 6
PRESERVATION AND MAINTENANCE OF INDUSTRIAL CULTURES
6.1 INTRODUCTION
Microorganisms for the production of industrially important products are useful

only if they can be maintained indefinitely in healthy, pure, and genetically stable
form.
Industrial culture collection consists of stock cultures. A stock culture may be simply
defined as a culture which serves as a source of inoculum. Stock cultures are of two
types:
1. Working stock culture

2. Primary stock culture
Working stock cultures are maintained in a vigorous and uncontaminated condition

and are used frequently. They must be checked frequently for characteristic features
and contamination. In contrast, primary stock cultures are kept for long-term storage
for later use: they are used to produce new working stock cultures as per need.
6.2 PRIMARY STOCK CULTURE
As this culture is meant for reserve, it is stored in an uncontaminated state for

prolonged periods. The cultures are maintained in a state of low physiological activity
(suspended animation). There are several primary stock culture methods available,
ranging from low risk to high risk. High-risk methods, as evident, use risky albeit
simple methods, often requiring frequent subculturing. Should this be the case,
selection must be made of the methods that specify a minimum number of transfers,
as with each transfer the chances of contamination and mutation increase
significantly. Some of the general methods of preservation and maintenance of
primary stocks are:
 Periodic transfer
 Oil overlay
 Preservation in soil
 Desiccation
 Lyophilization (freeze-drying)
 Use of low temperatures
6.2.1 PERIODIC TRANSFER
This method is also called active transfer, subculturing, or serial transfer. The temperature
of storage and the type of medium chosen should support a slow, rather than rapid,
growth. This reduces the frequency of transfers. Slants (agar slopes) and broth
cultures are the most widely used methods. The culture, after maturation, can be
preserved (and maintained) in the refrigerator for a period of 2-6 months. Although
simple, these methods are the least desirable. Mutation cannot be prevented and the
frequent transfers increase the chances of contamination and genetic changes.
6.2.2 OIL OVERLAY METHOD.
Many bacteria and other organisms can be preserved by this method for 1 month to
2 years. In this method, an agar slant growth is overlayed with sterile and inert
mineral oil (½" above the tip of the slant surface: see Fig. 6.1) and stored cold. The
unique advantage of this method is that some of the growth can be removed to
inoculate a fresh medium without contaminating the stock. In other words, the stock
can be used for a number of times. This is not possible in subculture method (= serial
transfer method). Another advantage of this method is the control of water and gas
exchanges in the medium. There are some limitations also. For example, this method
is not suitable for the preservation of sporulating molds because it sometimes leads
to loss of sporulation property (and sometimes, biochemical activity also). The most
common mineral oil used is the liquid paraffin. It is sterilized at 180°C in hot air
oven for an hour before using for overlay.
Mineral oil 1/2 inch
Slant surface
Medium
Fig. 6.1 Mineral oil overlay
6.2.3 LYOPHILIZATION
Lyophilization is probably the most satisfactory method for long-term preservation

of those microorganisms which can withstand the rigor of the process. In this
process, a dense cell suspension (in stationary phase) is placed in small vials (ampules
or ampoules), frozen under vacuum (200 μm Hg) at -60 to -70°C, and vials sealed
using oxygen-natural gas flame (Fig 6.2). The suspension is prepared in various
protective media such as bovine serum, media containing sugars, or skim milk. It is
also advantageous to include inositol (5%). The whole is stored under refrigeration
in dark. The organism remains viable for as long as 30 years.
37
Vacuum pump Seal
Condenser
(by flaming open end)
Ground-glass Dry ice,
joint alcohol Asbestos packing
(provides insulation
during sealing)
Vials containing
Outer tube
frozen bacterial
suspension Inner vial
(cotton plugged)
Glass flask for 10 Dewar flask containing lyophilized
to 100 small vials specimen
Fig. 6.2 Lyophilization process for the preservation of cultures
The advantages of lyophilization are as follows:
 Requires no subculturing
 Ease of transportation
 Low cost
 Less storage space
The only disadvantage of lyophilization is that not all microorganisms can be

subjected to this method.
6.2.4 PRESERVATION IN SOIL
Soil can be used either as a carrier or a growth medium. If used as a carrier,

abundant spores of bacterial or fungal species must be prepared in advance. The
spores are then placed in sterile soil and the resulting preparation dried in air or
under vacuum.
For many fungi, maintenance by use of soil is very useful. Moist soil may be
inoculated with the fungus and may be allowed to grow until sporulation has
completed (see Fig. 6.3). This type of culture may be used both as primary and as
working stock culture. Preparation of murcha (an amylolytic starter culture indigenous
to Nepal) is very much similar to this, differing only in the use of starchy material in
place of soil. An example of preservation in soil is given in Fig. 6.3.
6.2.5 USE OF LOW TEMPERATURE
Cells are prepared as a dense suspension in a medium containing a cryoprotectant

such glycerol or dimethylsulfoxide. Ampules are prepared and frozen at a controlled
rate to -150°C. Initially, the temperature is allowed to go down approximately at the
rate of 1°C/min up to -20°C. Thereafter the temperature is brought down as rapidly
as is possible. Electrolytes should be kept at as low a concentration as possible.
Finally, the ampules are stored in liquid nitrogen refrigerator either by immersing
(-196°C) or in a gas phase (-150°C). This method has been successful with many
species, including human cells, which cannot be preserved by lyophilization. Most
species remain viable for 20-30 years. The method, however, is very expensive. The
38
expense arises from the amount of nitrogen needed for replenishment. A common
laboratory method for preservation by low temperature is given in Fig. 6.4.
20% soil + 78% sand + CaCO3
Distribute in tubes
Sterilize at 130oC for 8-15 hrs
Cool Mold spores
Sterile soil medium
Grow for 10 days
Keep in desiccator
Store cold
Fig. 6.3 Preservation of mold in soil
2 ml glycerol + Growth from agar slope
Emulsify
Methanol Mix CO2
Put into vials
Freeze rapidly to -70oC

Store at -40oC
Fig. 6.4 Simple laboratory method for low-temperature preservation
6.2.6 DESICCATION OR DRYING
This method is useful for preserving yeast cells. Cells are kept directly in contact
with silica gel desiccant. Silica gel is kept in a screw-capped container (to a depth of
about 1 cm, sterilized in hot air oven). The culture is grown to a stationary phase in a
suitable medium and then suspended in 5% skim milk medium. The latter is finally
transferred to gel without saturating it (the gel). The heat developed should be
dissipated quickly. The gel is dried at room temperature for 2 weeks and finally put
in an airtight container for storage at 4°C. The culture is stable for several years.
Alternatively, cells may also be preserved by desiccating them on a filter paper kept
over silica gel in a desiccator.
39
6.3 MAINTENANCE OF PURITY OF CULTURE
A variety of chemicals and antibiotics are used for maintaining purity in culture
collection. Some common and easily available compounds and their application are
given in Table 6.1.
Table 6.1 Some common and easily available compounds and their application
Compound Concentration (mg/L) Use

Rose Bengal 35 Inhibits colony growth
Neomycin sulfate 20 Inhibits actinomycetes
Cycloheximide (actidione) 20-200 Inhibits yeasts and some fungi
Penicillin 40 Inhibits bacteria
Griseofulvin 2-20 Inhibits fungi
Invasion of fungal culture by mites is a very common problem. For this, low risk
methods such as mineral oil overlay, lyophilization, or liquid nitrogen storage can be
used. Rooms for storing cultures as well as those for subculturing should be easily
cleanable, have low microbial load, and provided with filtered air. The surfaces and
walls should withstand the harshness of cleaning agents. Periodic disinfection should
be carried out with UV radiation and other disinfectants such as formaldehyde,
ethanol, isopropanol, hypochlorite, etc. In case the contamination is very high, entire
room must be fumigated. Fumigation can be achieved by boiling formalin to release
formaldehyde. Approximately 0.5 ml formalin should be used for each cubic feet of
air space.
6.5 SOME CULTURE COLLECTION CENTERS
 American Type Culture Collection Center (ATCC)

 Commonwealth Mycological Institute (CMI)
 Northern Region Research Laboratory (NRRL)
40
CHAPTER 7
CONCEPT OF BIOTECHNOLOGY
7.1 INTRODUCTION
The term biotechnology came into being in the 1970’s when scientists learned to alter
precisely the genetic constitution of living organisms by processes without traditional
breeding practices. Biotechnology is defined by different persons and organizations
in different ways: for example, those of US National Foundation, European
Federation of Biotechnology, the IUPAC, etc. This is natural because, by its nature,
the area covered under biotechnology is very vast and the techniques used are highly
divergent. This has often made a precise definition of the subject rather difficult.
Although different definitions of biotechnology differ in their approach, content and
emphasis, the two main features common to them all are: (i) utilization of biological
entities (microorganisms, cells of higher organisms – either living or dead), their components or
constituents (e.g., enzymes), in such a way that (ii) some products or service is generated. This
product or service should, obviously, enhance human welfare. In essence, therefore,
biotechnology concerns with the exploitation of biological agent for generating
useful products/services. Production technologies pertaining to agriculture,
horticulture, and animal husbandry also utilize biological entities to generate useful
products. But these activities are not regarded as biotechnology since they are long
recognized and established disciplines in their own right. However, the exploitation
of animal and plant cells cultured in vitro as well as their constituents for the
generation of products/services is an integral part of biotechnology.
Although the term biotechnology is of recent origin the discipline itself is very old.
Man has long been in association with microorganisms and has gainfully employed
them since time immemorial. Historically, biotechnology was an art rather than a
science where techniques of manufacture were well worked out and reproducible, as
exemplified in by the production of wines, cheeses, beers, etc., but the molecular
mechanisms were not understood. For the sake of clarity, the chronology of some
important developments in biotechnology is given in Table 7.1
It is ironical that the major advances in biotechnology were the aftermath of post-
wars. During the First World War, Germans were forced to develop the technology
for glycerol (needed for manufacturing explosives) production when their supply of
vegetable oil was disrupted due to the British naval blockade. Similarly, British
resorted to acetone-butanol fermentation using Clostridium acetobutylicum due to the
German interferences with the normal supply of these chemicals. The First World
War also left citrus orchards of countries like Italy in ruins; this resulted in a great
jump in the prices of citric acid which was extracted from citrus juice. As a result,
the technology for citric acid production using Aspergillus niger was developed. The
production of the antibiotic penicillin by Penicillium notatum was discovered in 1930
by Alexander Fleming, but its commercial production began, again, only during
Second World War. But subsequent developments in chemical pharmaceutical
production using microorganisms have been very rapid and have covered a very
wide range indeed.
Man has continued his quest for improving the natural capabilities of
microorganisms, making them capable of novel processes, and discovering
microorganisms with new capabilities. This has led to the development of recombinant
DNA technology, which allows man to modify microorganisms and other organisms to
create in them highly valuable, novel and naturally non-existent capabilities.
With the major advances in our understanding of microbiology and biochemistry,

genetics, chemistry, chemical- and process-engineering, several molecular
innovations have now become possible. Unprecedented changes can now be
brought about in living system. Transgenic plants and animals are heralding a new
age in agriculture, and gene therapy in humans may eradicate previously
incapacitating diseases. In the environment, biotechnology is allowing major
improvements in water and land management and also remediating the pollution
generated by over-industrialization.
Table 7.1 Chronology of important developments in biotechnology
Activity Year
Yeasts used to make wine and beer ~ 6000 BC
Yeasts used for making leavened bread ~ 4000 BC
Sewage treatment systems using microbes developed/established 1910 AD
Large-scale microbial production of acetone, butanol and glycerol 1912-14
Large-scale production of penicillin 1944
Mining of uranium with the aid of bacteria 1962
First successful genetic engineering experiments 1973
Marketing of human food of fungal origin (United Kingdom) 1980
The use of monoclonal antibodies for diagnosis approved (USA) 1981
Approval of the use of insulin produced by genetically engineered 1983
microbes (USA and UK)
Animal interferon produced from genetically engineered microbes 1984
(GEMS) approved for the protection against diseases
Animal experiments by British scientists
Thus, in its simplest form, biotechnology employs microorganisms to convert simple

organic molecules (e.g., sugar) into more useful products (e.g., alcohol) and to
produce some unique biochemicals (e.g., antibiotics). On the other extreme of the
spectrum are ranged the sophisticated techniques of recombinant DNA technology,
hybridoma technology, enzyme technology and enzyme engineering, etc.
42
7.2 BIOTECHNOLOGY AS AN INTERDISCIPLINARY ACTIVITY
Biotechnology is truly interdisciplinary in nature, exhibiting a bewildering array of

sub-disciplines: microbiology, chemistry, biochemistry, genetics, molecular biology,
immunology, cell- and tissue culture, physiology, chemical-, biochemical-, and process
engineering. A distinction needs to be made between interdisciplinary and
multidisciplinary natures. The term multidisciplinary describes a quantitative extension of
approaches to problems that commonly occur within a given area. It involves the
marshalling of concepts and methodologies from a number of separate disciplines
and applying them to a specific problem in another area. In contrast, interdisciplinary
application occurs when the blending of idea that occurs during multidisciplinary
cooperation leads to the crystallization of a new disciplinary area with its own
concepts and methodologies. In practice, multidisciplinary enterprises are almost
invariably mission-oriented. However, when true interdisciplinary synthesis occurs the
new area will open up a novel spectrum of investigations. Fig. 7.1 shows the
interdisciplinary nature of biotechnology.
Electronics
Fig. 7.1 The interdisciplinary nature of biotechnology
7.3 SCOPE AND IMPORTANCE OF BIOTECHNOLOGY
Biotechnology has rapidly emerged as an area of activity having a marked realized as

well as potential impact on virtually all domains of human welfare, ranging from
food processing, protecting the environment, to human health. As a result, it now
plays a very important role in employment, production and productivity, trade,
economics and economy. This is clearly reflected in the emergence of numerous
biotechnology companies throughout the world, including India, and the movement
of noted scientists, including Nobel Laureates, to some of these companies. The
total volume of trade in biotechnology products is increasing sharply every year, and
it is realistically accepted that by the early 21st century it will be contributing trillions
of dollars to the world market. Unfortunately, very few people realize that
biotechnology affects over 30% of global economic turnover by way of health care,
food and energy, agriculture and forestry and this economic impact will grow as
biotechnology provides new ways of influencing raw material processing. Many
commentators are confident that the 21st century will be the century of
43
biotechnology, just as the 20th century was the era of electronics. A summary of the
main areas of applications of biotechnology is given in the following paragraphs:
7.3.1 Bioprocess technology
Historically, the most important area of biotechnology, namely brewing, antibiotics,

mammalian cell culture, etc.; extensive development in progress with new products
envisaged, namely polysaccharides, medically important drugs, solvents, protein-
enhanced foods; novel fermenter designs to optimize productivity.
7.3.2 Enzyme technology
Use for the catalysis of extremely specific chemical reactions; immobilization of

enzymes; to create specific molecular converters (bioreactors). Products formed
include L-amino acids, high fructose syrup, semi-synthetic penicillins, starch and
cellulose hydrolysis, etc. Enzyme probes for bioassay.
7.3.3 Waste technology
Long historical importance but more emphasis now being made to couple these
processes with the conservation and recycling of resources; foods and fertilizers,
biological fuels.
7.3.4 Environmental technology
Great scope exists for the application of biotechnological concepts for solving many
environmental problems – pollution control, removing toxic wastes; recovery of
metals from mining wastes and low-grade ores.
7.3.5 Renewable resources technology
The use of renewable energy sources, in particular, lignocellulose to generate new

sources of chemical raw materials and energy ethanol, methane and hydrogen. Total
utilization of plant and animal material.
7.3.6 Plant and animal agriculture
Genetically engineered plants to improve nutrition, disease resistance, keeping

quality, improved yields and stress tolerance will become increasingly commercially
available. Improved productivity, etc., for animal farming. Improved food quality,
flavor, taste and microbial safety.
It is being argued that, although the global production of food may be sufficient to
feed the world’s population, problems of poverty and distribution of food resources
mean that hunger and malnutrition are still endemic in the developing world. In the
face of these issues, biotechnology should be used to develop food crops which
offer greater nutritional benefits so that the poorest people can obtain adequate
nourishment from smaller quantities of food.
44
7.3.7 Health care
New drugs and better treatment for delivering medicines to diseased parts.
Improved disease diagnosis, understanding of human genome.
Biotechnology, thus, has unlimited potential in view of its capability to generate an

unlimited range of valuable and useful products/services concerned with virtually
every aspect of human existence. For example, in the mid-1991, over 130
biotechnologically derived pharmaceuticals, aimed at everything from hemophilus to
AIDS, from anemia to leukemia, were under regulatory review in the US.
The new biotechnology, however, demands very high expertise and skill, and
continued heavy funding coupled with dedicated effort. Therefore, highly
industrialized countries have a dramatic edge over the less industrialized ones, more
so, over the developing countries both with regard to the research and development
activities, and the exploitation of market potential.
7.4 POTENTIAL HAZARDS OF BIOTECHNOLOGY
The early studies on gene manipulation provoked wide discussion and concern at the
possible risks that could arise with certain types of experiments. Thus, it was
believed by some people that the construction of recombinant DNA molecules and
their insertion into microorganisms could create novel organisms that might
inadvertently be released from the laboratory and become a biohazard to human
beings or the environment. In contrast, others considered that the newly designed
organisms, with their additional genetic material, would not be able to compete with
the normal strains present in nature. The present views of gene manipulation are
becoming more moderate as the experiments have shown that this work can proceed
within a strict safety code. The standards of containment enforced in the early years
for recombinant DNA studies were unnecessarily restrictive and there has been a
steady relaxation of the regulation governing much of the routine genetic
engineering activities. However, for many types of study, particularly with
pathogenic organisms, the standards will remain stringent. The physical containment
in rDNA studies includes the use of sterile techniques, containment hoods, and
specially designed laboratory to prevent vector containing the rDNA form being
transferred or escaping from the containment to natural ecosystems. Biological
containment involves the use of organisms with specially constructed, weakened
genotypes as vectors in cloning experiments. Ideally, these organisms should be
unable to survive under conditions existing in natural ecosystems.
One of the greatest risks of genetic engineering is the possible abuse of the
technology for biological warfare. The recent anthrax episode after the 11th
December (2001) bombing of the World Trade Center in America is an example of
how dangerous genetically engineered microbes (GEMS) could be. Biological
weapons are prohibited by International Treaty, but such treaty may of course be
disregarded by independent groups (guerilla movements, terrorists, organized crime).
The governments that watch over the Biological Weapons Treaty should therefore
closely monitor the consequences of genetic engineering. With all these risks in view,
National Institutes of Health (NIH) have established guidelines for researches
involving rDNA molecules. Under these guidelines the NIHs serve an overseeing
45
role by sponsoring risk-assessment program, certifying new host systems, serving as
information clearing house, and coordinating Federal and local activities.
There is also concern regarding the use of GEMS in vaccines. The poliovirus can be
taken as an example. The oral polio vaccine (OPV) consists of weakened form of
poliovirus. Salk vaccine, an alternative form, consists of killed virus. Because of low
cost and ease of administration, OPV is the most widely used polio vaccine. OPV
viruses differ from the virulent virus by only a few mutations and the condition in
the gut can favor their reversion to pathogenic form. Besides, the OPV can persist
for years in people with impaired immune systems, where they mutate faster than in
normal populations. HIV-infected people worldwide in particular can serve as a
reservoir. Another danger from OPV is the vaccine plant itself. Since vaccines are
produced from virulent forms, an escape from the plant can wreak havoc. So
factories would have to use high levels of containment, making the vaccine
expensive.
There has been growing concern in the domain of transgenic plants also. The main
concern relates to the possibility (i) of transgenic plants becoming persistent weeds, (ii) of gene
transfers from them to other plants making the latter more persistent and invasive, (iii) of their
being detrimental to environment, and (iv) leading to resistance build-up overtime in pathogens.
The impact on plant ecosystem is of considerable concern. The use of transgenic
plant in mass scale not only interferes with the diversity that has its own natural
management system but also may adversely affect the environment by bringing
about genetic changes in wild types and even wiping out some of the species. The
safety of produce is also of considerable debate. Since the assessment of risk is a
time-consuming process, it would be nothing if not hazardous to use a wait-and-watch
approach after introducing the transgenic plants.
As of now, there isn’t any perceivable hazard from animal biotechnology. Problems
of course exist. The social opinion on transgenic animal research is almost divided in
the middle. The main opposition stems from ridiculous assumptions like empathy
between humans and animals while at the same time trying to maintain a divide by
considering transgression for inter-animal transfers of genes.
46
CHAPTER 8
MICROBIAL GENETICS
8.1 INTRODUCTION
Genetics is the science of inheritance and variability. It attempts to explain the

differences and similarities between organisms and the way in which characters are
passed from parents to offsprings.
8.2 GENETIC MATERIAL
These are elements responsible for the transfer of genetic traits (characteristics/
properties/information) from parent(s) to the progeny (offspring/daughter or sister
cell).
8.2.1 TYPES OF GENETIC MATERIALS
Nucleic acids are the basic units of genetic materials. Deoxyribonucleic acid (DNA,
see Fig. 8.1) are the universal units but RNA can also play the same role when RNA
is the sole genome: this is especially true in the case of RNA viruses such as Tobacco
Mosaic Virus (TMV), tumor viruses, etc. RNAs in all other organisms are non-
genetic materials (because they are not transferred to the progeny).
o
34A
o
10A
A = Adenine
T = Thymine
C = Cytosine
G = Guanine
Major groove Minor groove
Fig. 8.1 Simplified diagram of normal DNA
The most conclusive proof of DNA as the genetic element came from the elegant
experiment performed by Fredrick Griffith in 1928 (transformation phenomenon in
pneumococci). In 1953, with Watson and Crick’s work, DNA was universally
accepted as the genetic element. The evidence that RNA could also serve as genetic
material came from A. Gierer and G. Schram’s work (1956) with TMV, an RNA-
virus.
8.2.1.1 Chromosomes
Chromosome refers to the bulk of DNA present in an organism. Prokaryotic and

eukaryotic chromosomes differ from each other by several counts. Prokaryotic
chromosomes consist of a single, circular, double-stranded DNA existing in a highly
condensed (folded, negatively super-coiled) form. The size of the chromosome (and
also the size of gene or any DNA segment) is usually measured in terms of base pair
(bp), or kilobase pair (kbp) for much larger segments. An idea about the chromosome
size can be gathered from examples given in Table 8.1.
8.2.1.2 Mini-chromosomes
These refer to extra-chromosomal genetic elements more widely known as plasmids

and episomes. Episomes are special types of plasmids which can reversibly integrate
(fuse) with the main chromosome. Plasmids (and also episomes) are extra-
chromosomal self-replicating, double-stranded, closed, circular DNA present in the
cytoplasm. The number in a single cell may range from zero to several. A number of
host properties are specified by them. A single plasmid may contain 3 to several
hundred genes.
Table 8.1 Chromosome sizes of different organisms
Organism Chromosome size (bp) No. of genes

Escherichia coli 4.7106 4000
Mycoplasma genitalis -- 470
Human 3109 80,000
The autonomous replication ability of plasmid is due to a unique DNA sequence

(gene) from where the replication starts. The site of this origin of replication is
denoted by ori. Similarly, some plasmids integrate with the main chromosome due to
yet another DNA sequence (the site being denoted by att for attachment). It is
therefore important to note that all plasmids must have an ori site. Such and other
sites in plasmids are commonly referred to as modules. Modules, then, are DNA
segments performing a specific function. Each module may contain one or more
genes. A simplified diagram of plasmid with modules is shown in Fig. 8.2.
Att
Ori: site of origin of replication
Tr: site of tetracycline resistance
Att: site for attachment
The concentric circle represents double-stranded DNA
Ori Tr
Fig. 8.2 Simplified diagram of plasmid
A number of different types of plasmids have been shown to occur in organisms.

Some of the more important ones are:
F-plasmid or F-factor (F for fertility)
This is possessed by microorganisms such as strains of E. coli. The module in the

plasmid is responsible for sexual reproduction by conjugation. The strain having this
48
plasmid produces a hollow conjugation tube (pili) to attach to F-negative strain (the
female) and passes varying proportions of the plasmid through this tube into female.
Col-plasmid or Col-factor
This plasmid carries genes responsible for producing colicin, which is a bacterial toxin
that kills colicin-negative strains. Col-factor also provides immunity to colicin-
positive strains against their own colicin.
R-plasmid
This plasmid is responsible for the resistance to antibiotics.
Ti-plasmid
This induces tumor in plants. The plasmid integrates (through wound in plant) stably
with the plant DNA. The plasmid codes for enzymes responsible for the synthesis of
plant growth hormones. As a result, callus, galls and tumors are formed.
Agrobacterium tumefaciens, a soil bacterium is responsible for this plasmid.
Ri-plasmid
It is present in Agrobacterium rhizogenes, another soil bacterium. The plasmid is

responsible for root induction.
Yeast plasmid
This plasmid may be of integrating, episomal, or replicating type. It may be circular

or linear. 2 μm-plasmids (pronounced two micrometer plasmids) are the most important
plasmids present in many strains of Saccharomyces cerevisiae and Saccharomyces uvarum.
These plasmids are covalently closed, and exist in 50-100 copies.
8.2.1.3 Transposable genetic elements
They are also called jumping genes, cassettes, transposons, mobile genes, or insertion sequence.
They are small mobile DNA sequences that move around chromosomes with no
regard to homology. The random insertion and ejection of these genes in the host
chromosome may lead to mutation. This mutation produces a range of effects, from
benign to lethal.
8.2.1.4 Viral DNA
The viral DNA can be single- or double stranded. Further, they can be either linear
or circular. Single-stranded DNAs are usually circular. Some examples are given in
Table 8.2.
49
Table 8.2 Some examples of viral DNA
DNA type Representative virus

Single-stranded, circular Coliphages M13, fd, Ф X174
Double-stranded Coliphages T1 to T7. , μ
8.2.1.5 Mitochondrial DNA
Eukaryotic microorganisms (such as yeasts) have mitochondria. Mitochondria have

their own machinery. During cell division mitochondria must also be passed to the
progeny, which means that mitochondrial DNA is also passed. This is how the
progeny faithfully inherits mitochondrial DNA.
8.2.1.6 DNA in endosymbionts
Certain intracellular parasites such as bacteria and virus maintain symbiotic

relationship with host cells. The symbiotic forms are self-reproducing and look like
cytoplasmic inclusions. Sometimes, they exhibit an infection-like transmission with a
hereditary continuity of their own. Such endosymbionts are usually denoted by the
letter σ, κ, μ, etc. A good example of κ particles is the endosymbionts called Lyticum
flagellatum. The organism consists of a protozoan, Paramecium tetraurelia and κ
particles. κ particles in turn consist of symbiotic bacterium Caedobacter taenospiralis and
an infectious virus. The virus controls the toxic viral protein that makes the
endosymbiont protozoa a killer strain. The protozoan in turn synthesizes folic cid
required for the κ particle. Conservation of this killer property from one generation
to another implies that DNA in these particles also serves as a genetic element. It
must be noted that the killer strain attacks only the sensitive strains.
8.2.1.7. RNA genetic material
mRNA, tRNA, and rRNA are non-genetic RNAs in eukaryotes and prokaryotes. In
RNA viruses, however, RNA constitutes their sole genome. They therefore use
RNA as the genetic element. Examples of RNA genetic elements are given in Table
8.3.
Table 8.3 Examples of RNA genetic elements
RNA virus RNA type

Coliphages MS2, F2, R17, QB Messenger, single stranded
Raus sarcoma virus Messenger, single stranded
Polio virus Messenger, single stranded
Rabies virus Non-messenger, single stranded
50
8.3 THE GENETIC CODE
DNA, and in some cases RNA, are the genetic materials. DNA in particular, is the
master carrier of hereditary characters. It carries the genetic information in coded
language. The four common bases, viz., Adenine, Guanine, Cytosine, and Thymine
can be likened to alphabet and the sequential triplet arrangement out of these bases
can be likened to a code word. Assortment of these code words leads to code language.
Although the code is primarily preserved in DNA it is customarily represented in
terms of mRNA language. The code words then become codons. Combination of
these four bases taken three at a time yields 64 theoretical codons. When the code
words are translated into decipherable words, they mean amino acids. Out of the 64
codons, however, only 61 codons imply amino acids. They are thus called sense
codons. The rest 3 are nonsense codons, as they do not code for any amino acid. They
are also called stop codons.
The genetic code is therefore the system of mRNA sequences that designate particular amino
acid during the process of translation.
The triplet AUG (and sometimes GUG) is called initiator codon, as it is the first
codon to be encountered during translation (see later). UAA (also termed ochre),
UAG (amber) and UGA (umber) are the three nonsense codons. The initiator and the
nonsense (or stop) codons are collectively called punctuation codons.
Codons are grouped into 16 families, each with four codons characterized by its first
two bases only. The codon families differ from each other with respect to their third
(i.e., 3′) base only. There are 8 unmixed codon families, viz., UC, CU, CC, CG, AC,
GU, GC, and GG. See Table 8.4 and 8.5 (the shaded boxes represent unmixed
codon family) for detail.
Table 8.4 The unmixed codon family
Codon family Amino acid specified Codon family Amino acid specified
UC Serine AC Threonine
CU Leucine GU Valine
CC Proline GC Alanine
CG Arginine GG Glycine
In the unmixed family (Table 8.5), the third base can be any one of A, U, G, and C.
The third base does not alter the specification for amino acid. There are 8 mixed
codon families, each specifying more than one amino acid, depending on the third
base of the codon. In the UU family, for example, UUU specifies for phenylalanine
while UUC specifies for leucine.
51
Table 8.5 The universal codon
1st base 2nd base 3rd

(5' base) U C A G base
UUU UCU UAU UGU U
Phe Tyr Cys
UUC UCC UAC UGC C
U Ser
UUA UCA UAA UGA Stop A
Leu Stop
UUG UCG UAG UGG Trp G
CUU CCU CAU CGU U
His
C CUC CCC CAC CGC C
Leu Pro Arg
CUA CCA CAA CGA A
Gly
CUG CCG CAG CGG G
AUU ACU AAU Asn AGU U
Ile Ser
AUC ACC AAC AGC C
A Thr
AUA ACA AAA AGA A
Met Lys Arg
AUG ACG AAG AGG G
GUU GCU GAU GGU U
Asp
GUC GCC GAC GAC C
G Val Ala Gly
GUA GCA GAA GAA A
Glu
GUG GCG GAG GAG G
8.3.1 CRACKING THE GENETIC CODE
In 1961, Nirenberg and Matthaei carried out a decisive experiment to crack the
much-hypothesized genetic code. They used artificially synthesized poly U mRNA in
a cell-free extract. The extract contained the entire requirement for protein synthesis.
With poly U mRNA, they obtained a polypeptide polyphenylalanine as the end product.
This showed that the triplet of U codes for phenylalanine. Similar experiments were
carried out with poly A, poly C, and poly G to crack the codes for lysine, proline,
and glycine respectively. Nirenberg also showed that a codon consisted of base
triplet and that the reading of mRNA during translation was sequential and non-
overlapping. A simple illustration of the principle of his experiment is:
Nirenberg used polyribonucleotides of alternate nucleotide of the sequence

CACACACACACACACACACA. Since only two amino acids (histidine and
threonine) were obtained in an alternating sequence during translation experiment in
cell-free system, it was clear that the triplets CAC and ACA were used. Note above
that the combination of triplet can be either ACA or CAC irrespective of where the
reading started. If a codon of duplet is used, only one combination (either CA or
AC) is possible, which means that the synthesis of two different amino acids would
be impossible. A codon of quartet would also have the same result. This study thus
showed that a codon consists of a base triplet and the reading is sequential and without a
break.
He also used mRNA of the sequence AAGAAGAAGAAGAAGAAGAAG. Note

that the triplet combination can be either AAG or AGA or GAA. In other words,
the mRNA consisted of three possible codons. When experimented in a cell-free
system a homopolypeptide of lysine was obtained. It was clear that the reading was
sequential and without a break. Note again in the above discussion, lysine was
coded by AAA. Since lysine was obtained from the triplet AAG as well, it is clear
that a single amino acid can be coded by more than one codon.
52
By 1966, all the 64 codons were cracked. That is, codons for all the 20 amino acids
were deciphered. The universal codon, which appears in Table 8.5, is a standard
form for most microorganisms for their chromosomal DNA. The codons are
slightly different for DNA present in organelles like mitochondria, chloroplast, etc.
8.3.2 CHARACTERISTICS OF GENETIC CODE
There are certain properties of codons that have helped us generalize the genetic
code and the codons. Some of these properties (called characteristics hereafter) are:
 The code is triplet

 The codon is non-overlapping: the same base cannot serve in common for two
juxtaposed codons during translation. There are some exceptions though,
for example, ØX174 (a virus) codes for more proteins than would be
predicted by their nucleotide content, which obviously means overlapping.
 The codon has polarity: it is always read in the direction 5′3′.
 The genetic code has punctuation codons
 The genetic code is universal: there are exceptions though, for example, UGA
refers to stop in mammals but tryptophan in yeast mitochondria.
 The code is unambiguous: generally, the same codon will not code for another
amino acid. There is exception here also. For example, GUG codes for
methionine as well as valine.
 The code is degenerate: there are 61 sense codons whereas only 20 amino acids.
It is obvious that the same amino acid is coded by more than one codon
(often called synonyms or synonymous codons). In fact, except methionine and
tryptophan all amino acids have synonyms. Such a code is then said to be
degenerate. Leucine, serine, and arginine have as many as 6 synonyms (see
Table 8.5).
 The code is commaless: the code is read continuously, without a spacer. In other
words, the enzymes translate the code without skipping certain sections of
codons.
8.3.3 DEGENERACY AND THE BIOLOGICAL ADVANTAGE
Degeneracy can be of two types, (i) complete, and (ii) partial. In complete degeneracy,
the synonyms are not of the same codon family, e.g., CGG and AGA for arginine.
Partial degeneracy occurs in unmixed codon families, e.g., CGG and CGA for
arginine.
Degeneracy of genetic code has certain biological advantages. For example, it

permits the synthesis by organisms of essentially the same complement enzymes and
other proteins varying widely in amino acid composition. Degeneracy also provides
the organism with a mechanism for surviving mutagenic effects. For example,
transversion of ACC to AGA has no lethal effect on the organism as both the
triplets code for the same amino acid, threonine.
53
8.3.4 WOBBLE HYPOTHESIS
Since there are 61 codons but only 20 amino acids, some amino acids must be coded
by more than one codon. Such codons are called synonyms and the code is called
degenerate. Also, since the number of tRNA (that works as an adaptor molecule during
translation) is more than 20 but less than 61, some amino acids can be carried by
more than one tRNA. Such tRNAs are called isoacceptors. The tRNA forms a
complementary base pairing with codons using its corresponding anticodon present in
the molecule. However, since the number of tRNAs is smaller than that of codons,
some of the tRNAs must form base-pairing with more than one codon. Such a base
pairing leads to abnormal bonding.
According to Crick (1966), the ability of the same anticodon of the tRNA to read
different codons (synonyms) is due to flexibility or wobble in the pairing among bases
of the codon and anticodon. The hypothesis is known as wobble hypothesis and the
exceptional pairing, wobble pairing (Fig. 8.3 and 8.4). Wobble pairing does not follow
standard base pairing rule. The hypothesis states that the base at the 5′ end of the
anticodon is not spatially confined because the other two bases help it to form
hydrogen bond with any of several bases located at the 3′ end of the codon.
However, in most cases, it is essential that the first two bases of the codon should
form standard base pair. This state is called two out of three condition and is
particularly true of unmixed codon families (Table 8.5).
Table 8.6 Allowed base-pairing combination according to wobble hypothesis
5' base of the anticodon 3' base of the codon

C G
A U
U A G
G C U
I U, C, A
Normal Abnormal
Amino acid
3'
5'
tRNA
Anticodon
3' 5'
Normal base Anticodon
A
5' UUC 3' mRNA codon CGI
3' UUU 5' 5' GCG 3'
Abnormal base Codon
Fig. 8.3 Normal and wobble pairing Fig. 8.4: Wobble pairing
The first two bases are of capital importance in the base pairing. They imply factor
of genetic stability because a point mutation at the third position of the base will have
54
no influence in its coding ability. Nevertheless, the codon has altered, and this
particular mutation is called silent mutation. However, the wobble hypothesis fails to
explain degeneracy of genetic code in some cases. For example, it cannot explain
some of the observed pairing such as in Fig. 8.8.
8.4 CENTRAL DOGMA AND TEMINISM
Forwarded by Crick (1958), the central dogma of protein synthesis states that genetic
information flows from nucleic acids to proteins and not vice-versa. The first step of
the central dogma is known as transcription. In this step, the message flows from
DNA to mRNA and does not involve a change of code since DNA and RNA are
complementary. The second step involves a change of code from nucleotide
sequence to decipherable amino acid sequence. This step is called translation. The
scheme can be illustrated as:
DNA RNA Proteins

Transcription Translation
Replication
8.5 TEMINISM (CENTRAL DOGMA REVERSE)
In 1970, Temin and Baltimore discovered an enzyme that could use mRNA as the
template for the synthesis of DNA. The enzyme is now called RNA-dependent DNA
polymerase or reverse transcriptase. He showed that RNA virus could replicate RNA with
the enzyme RNA-dependent-RNA polymerase. This finding showed that the
information does not necessarily flow DNA  RNA in one direction but can also
be reversed. The improved version of central dogma can therefore be represented
as:
Transcription Translation
DNA RNA Proteins
Reverse
transcription
Replication
8.6 TRANSCRIPTION
8.6.1. THE RNA
The RNAs are formed using DNA as the template, by the normal polymerization
mechanism. The complementary RNA produced initially is called a primary transcript.
There are 4 broad classes of RNAs, viz., (i) transfer RNA (tRNA), (ii) messenger RNA
(mRNA), (iii) ribosomal RNA (rRNA), and (iv) heteronuclear RNA. The last one is found
in eukaryotes only.
Transfer RNA
This is also called soluble RNA and accounts for 15% of total RNAs in a cell. The
tRNA molecules serve a number of functions, the most important of which is to act
as a specific carrier of activated amino acids (see later). Thus, there are at least 20
55
tRNAs in every cell to account for the 20 amino acids. The number of tRNAs is
variable between 20-64 (both not inclusive), depending on the organism.
All the tRNAs have a common design and consist of 3 folds giving each a cloverleaf
structure (Fig. 8.5). The base sequence at the 3′ end of all tRNAs is cytosine-
cytosine-adenine. During translation, amino acids bind to their terminal adenosine
via 3′ OH group of its ribose. Each tRNA also has a recognition site on which the
anticodon is present. The anticodon forms hydrogen bonding with respective codon
on the mRNA. In general, tRNAs contain 70-90 nucleotides only. They may
sometimes contain rare bases such as inosine and methylated bases. All tRNAs have
secondary structures which are responsible for the characteristic function they carry
out.
A 3'
C
C
P
5'
5' 3'
Anticodon
Codon
GC-rich region 3' 5'
Anticodon
Fig. 8.5 Schematic diagram of tRNA
Messenger RNA
It is also called template RNA. Messenger RNA is the most heterogeneous of all the
RNA types with respect to size and stability. It accounts for 5% of total RNA in a
cell. It is synthesized on the surface of DNA template. Thus, it has base sequence
complementary to DNA and carries genetic information or message (hence its name)
for the assembly of amino acids. mRNAs are very unstable in bacterial systems: they
have a half-life of about 2 min. Mammalian mRNAs have half-lives ranging from few
hours to one day. The short half-life of prokaryotic mRNA is related to the need for
these organisms to adapt to quickly changing surrounding environment.
Ribosomal RNA
It is the most stable form of RNA and is found in association with ribosome. It is
also the most abundant and accounts for 80% of total cellular RNA. rRNA
represents about 40-60% of ribosome by weight. It combines with some 55 different
proteins to form complex structures called ribosome whose molecular weight is about
3 million dalton. Ribosomes are sites of protein synthesis. Unlike mRNA, rRNA is
not an informational element (does not carry genetic information).
The ribosomes, as also the rRNAs, are classified according to their sedimentation
coefficient expressed in Svedberg unit, S. Svedberg unit (also called sedimentation
coefficient) is defined as the sedimentation velocity per unit field of centrifugal force. It
56
has a unit of cm/sec given by: S = v/rω2 where S is the Svedberg unit, v is the
sedimentation velocity (i.e., rate of migration, cm/sec) of the solute particles in the
direction of centrifugal force, r is the distance in cm of the solution from the
rotational center, and ω is the angular velocity of rotation. A sedimentation
coefficient of 110-13 cm/sec is expressed as one Svedberg unit.
The characteristics of ribosomes vary somewhat in different organisms. The

ribosomes from animals and eukaryotes consist of 40S and 60S subunits, which
together form an intact 80S particle. Bacterial ribosomes consist of 30S and 50S
subunits that can be dissociated into ribosomal and rRNA particles. Combination of
30S and 50S particles gives an intact 70S particle (not the arithmetic sum). See Fig. 8.6(a)
and 8.6(b) for the summary. It has been shown that prokaryotes have 21 small
proteins and 33 large proteins.
70S Ribosome
30S
50S
16S RNA
23S RNA
5S RNA
21small, specific proteins
called S1, S2, S3,....S21
33 large, specific proteins

called L1, L2, L3,....L34
Fig. 8.6(a) The make up of bacterial ribosome
80S Ribosome
40S
60S
18S RNA
23S RNA
5S RNA
23 proteins
49 proteins
Fig. 8.6(b) The make up of mammalian ribosomes
57
8.6.2 MECHANISM OF TRANSCRIPTION
The synthesis of RNA using DNA as the template is called transcription. Here, the
coded message preserved in the DNA is transcribed into mRNA language. The
transcription process is similar to replication of DNA in following aspects:
 Transcription as well as replication has polarity. The polymerization takes

place in the 5’3’ direction
 Replication as well as transcription cannot occur without a template DNA
 The basic event in replication as well as transcription is the polymerization
of respective nucleotides
There are fundamental differences, though. The differences mentioned in the

following paragraph must be carefully borne in mind by the students. See Fig. 8.7 for
an idea about the basic mechanism of polymerization of RNA during transcription.
 Replication cannot start without the presence of a primer but transcription

does not require a primer
 Replication is a symmetrical process. Both the strands are duplicated
simultaneously. In transcription, the process is almost always asymmetrical.
Only one of the strands of the double stranded DNA is used as the
template. The other strand is primarily for stabilization. The strand that is
involved in coding is called sense strand. It is used for transcribing different
species of RNAs. The corresponding regions of the complementary DNA
are non-coding regions or antisense as they do not serve as template for any
RNA transcription. But other regions of the second strand can function as
the coding or sense strand for other tRNA species. Such transcription of
two different sets of RNA species on the templates of two different DNA
strands of a DNA duplex is called asymmetric transcription.
 The enzymes used in replication and transcription are quite different.
Prokaryotic replication uses DNA polymerases (Pol I, Pol II, Pol III, etc.) but
the transcription requires a single RNA polymerase
 Replication has high fidelity because of the presence of proofreading enzyme
(Pol I). Transcription has no mechanism for proofreading. Proofreading
mechanism in replication is related to genetic stability, which is essential for
the survival of the organism. On the other hand, proofreading in
transcription need not be of high fidelity. The errors that occur in
transcription are not inherited by the offsprings because RNA is not a
genetic material
58
P
5' 5'
3' P
P P
A A U
P P P
T T A
P G P P
G P G C
P P P P
C P C G
P P P
T A T A
3' P P P P
C G C G
P P P P
C G C G
P P P P
T A T A
P P P P
A U A U
P P P P
A U A U
P P
3' 3'
Template P Template P
DNA P DNA P
5' 5'
mRNA mRNA
3'
5' 5'
(a) (b)
Fig. 8.7 Polymerization of mRNA
8.6.3 BASIC STRUCTURAL FEATURES OF PRIMARY RNA TRANSCRIPT
The following are true of any primary RNA transcript in prokaryotes:
 Presence of pppA or pppG at the 5′ end

 Presence of 5′ UTR (untranslated region, also called 5′ leader region)
 Presence of 3′ UTR (also called 3′ leader region)
 Polycistronic nature of mRNA
 Presence of spacers in the transcript
Prokaryotic mRNAs are polycistronic. That is, they carry the information for the
production of multiple polypeptides. Each region of mRNA responsible for the
coding of polypeptide is called cistron. In polycistronic mRNA, the cistrons are
separated by non-coding sequences called spacers. See Fig. 8.8 for the schematic
representation of prokaryotic primary RNA transcript and Fig. 8.9 for the eukaryotic
primary RNA transcript.
Coding 1
Coding 3 3' UTR
Coding 2
5' UTR
Spacer
pppA
or Spacer
pppG
Fig. 8.8 Prokaryotic primary RNA transcript
59
The primary RNA transcript of eukaryotes is different from that of prokaryotes. The
main features in eukaryotic primary RNA transcript are: (i) Presence of cap at the 5′
UTR (ii) Monocistronic nature, and (iii) Poly A tail.
5' UTR 3' UTR

5' cap A A A A( A)200-300
Coding region Poly A tail
Fig. 8.9 Eukaryotic primary RNA transcript
The 5′ ends of eukaryotic mRNAs have a 7-methyl guanylate attached by 5′ to 5′

triphosphate linkage. This structure is called cap. The functions of cap are (i) to make
the translation efficient, and (ii) protect the RNA from exonucleases. See Fig. 8.10
for the structure of cap.
O CH3
+
HN N
7
H2N N Cap 1
OCH2 N
O
Cap 2
HO-P=O
O
HO-P=O
O Cap 3
HO-P=O
OCH2
O Base 1
O OCH3
HO-P=O
OCH2
O Base 2
O OCH3
Fig. 8.10 The cap of the eukaryotic mRNA
8.6.4 PROKARYOTIC RIBOSOMAL RNA
There are three kinds of prokaryotic rRNA, viz., (i) 23S rRNA (2904 nucleotides)
that is a component of 50S ribosome, (ii) 16S rRNA (1541 nucleotides) that is a
component of 30S ribosome, and (iii) 5S rRNA (120 nucleotides) that is a
component of 50S ribosome.
60
8.6.5 ORIGIN AND PROCESSING OF rRNA TRANSCRIPT
Prokaryotic rRNAs arise from the processing of a large, 30S precursor rRNA. Seven
genes produce rRNA. Each gene produces 30S precursor rRNA that is processed to
discrete, functional rRNA. All seven genes contain the sequences that become 23S,
16S, and 5S rRNA. Within the transcribed portion of these genes are some of the
tRNA genes. Different rRNA genes contain different tRNA genes. Upon formation
of the 30S precursor, the non-functional spacer sequences are removed by a series of
specific endonucleolytic cleavages by the enzyme ribonuclease P and ribonuclease III. After
removal from the spacer sequences, some of the bases in the final rRNA undergo
base modification: they are methylated. This modification is needed for the rRNAs to
be fully functional.
Many tRNAs are formed from the processing of precursor rRNA. Those that are
not formed in this manner arise from large precursor transcripts. The tRNA genes
are clustered, and each of the transcripts that form functional tRNAs is removed by
the enzymes ribonuclease P and ribonuclease D. All tRNAs have CCA at their 3′ end.
Some tRNA transcripts do not contain CCA. This sequence is added to those
lacking by the enzyme tRNA nucleotidyl transferase. Many of the bases of tRNA are
modified. These modifications consist of various methylations and other more
extensive modifications of some of the bases. The modifications are needed for the
tRNAs to adopt their unique, functional conformations. See Fig. 8.11 for the
mechanism of tRNA and rRNA formation.
3'
Spacer sequence
30S precursor rRNA
RNAse P
+
RNAse II
Fig. 8.11 The processing of prokaryotic RNA from the primary transcript
8.6.6 THE TRANSCRIPTION PROCESS IN PROKARYOTES
The process of RNA synthesis directed by DNA template is termed transcription. It

occurs in 3 steps, viz., (i) Initiation, (ii) Elongation, and (iii) Termination.
8.6.6.1 Initiation
This step implies the recognition of a region of the helicoidal double-stranded DNA
molecule (the region is called promoter), the binding of RNA polymerase
(holoenzyme) to the DNA, localized separation of the two strands producing a short
single-stranded region (about 17 base pairs at a time), one of which will serve as a
template for the pairing of ribonucleotides, and the selection of the first
ribonucleotide of the RNA chain.
61
The bacterial polymerase
Unlike DNA polymerases, this enzyme does not have a proofreading activity.
Bacteria have a single polymerase (called DNA-dependent RNA polymerase or simply,
RNA polymerase) responsible for the synthesis of all cellular RNAs (tRNA, rRNA,
mRNA). It contains six subunits, viz., α, α, β, β′, ω, and σ. The complete enzyme
(holoenzyme) can be represented by α2ββ′ωσ. The σ factor has a loose attachment. The
core enzyme therefore consists of only α2ββ′ω.
There are several different σ factors in E. coli, each being specific for binding to the
promoter site at 3′ end of a specific coding part of the template DNA. The core
enzyme is primarily responsible for elongation. Fig. 8.12 is an oversimplified
illustration of the core and holoenzyme.
' '
 
+ 
 
 Sigma factor 
Core enzyme Holo-enzyme
Fig. 8.12 Model of prokaryotic RNA polymerase
Unlike replication, transcriptional initiation does not require a primer. Promoter

sequences are responsible for directing RNA polymerase to initiate transcription at a
particular point of the template DNA. In bacteria, a promoter consists of a sequence
of about 40 bp on the upstream site of the transcription initiation site. There are two
common patterns (consensus) consisting of 6 bp each, called (respectively) Sequence -10
(or Pribnow- or TATA box) and Sequence -35. There are about 200 promoters in E. coli,
the model organism. See Fig. 8.13 for the schematic representation of a typical
prokaryotic promoter sequence.
-35 -10
-2 -1 0 +1+2+3
DNA 5' TTGACA TATAAT 3'
-35 sequence TATA box
Fig. 8.13 Promoter sequence in prokaryotic DNA
By convention, the starting point of the transcription is numbered +1. The bases
situated downstream (right hand side from the starting point) are numbered +2, +3,
+4, etc. The sequence (consensus) shown above is not the actual sequence of the sense
strand. They are from the partner DNA and are therefore representative of RNA
transcript.
The core enzyme has the ability to bind to any section of the DNA. It is the σ factor
that causes the holoenzyme to bind preferentially and more tightly to the promoter
sequence. The σ factor therefore initiates transcription by enabling the holoenzyme
to recognize the proper binding site. The binding of σ (as a part of the holoenzyme)
to the promoter site facilitates partial opening or melting of DNA double helix. The
62
part that melts is generally rich in A:T bases so that the opening is easier compared
to the G:C counterpart. The binding of holoenzyme immediately opens the double-
stranded DNA over an 11 base-pair region immediately downstream the initiation
site. This gives rise to a transcription bubble (Fig. 8.14) around the initiation site and
provides the holopolymerase an easy access to the nucleotides in the unwound stretch
of the DNA sense strand.
Enzyme movement
Rewinding
point Partner DNA
DNA double helix

3'
5' Unwinding point
RNA Template DNA
RNA polymerase
Fig. 8.14 The initiation of transcription
After the transcription initiation site has been exposed by the formation of the
transcription bubble, an appropriate ribonucleoside 5′ triphosphate from the
surrounding medium gets its base hydrogen-bonded to the corresponding base of
the coding strand of the template DNA. The first nucleotide is usually pppA, or
sometimes, pppG, but always a purine nucleotide. Thereafter the next complementary
ribonucleoside triphosphate comes for the polymerization. The β and γ phosphates
are eliminated from the second ribonucleotide in the process. During the
polymerization, α2 binds to the promoter, β to the ribonucleotide, and β′ to the
template DNA.
If rifampicin is administered to the prokaryotes, the compound binds to β subunit and

inhibits initiation. Once initiated, elongation (polymerization) cannot be inhibited.
8.6.6.2 Elongation
During or immediately following the formation of the first phosphodiester bond, the
polymerase releases its σ factor and changes into core enzyme. Because of the
lowered affinity of the core enzyme to the promoter (because σ factor has been
released) the former is now no longer restricted. It is free to move along the coding
strands towards 5′ direction.
The released σ factor may join another core polymerase to form a holopolymerase
for fresh chain initiation. As the polymerase is translocated progressively along the
coding strand in the 3′5′ direction, the transcription bubble also moves in the same
direction while RNA elongation proceeds in 5′3′ direction. The holoenzyme covers
about 60 bp, and the bubble comprises about 17 bp. The hybrid DNA-RNA covers
about 12 bp. The passage of the enzyme is followed by the displacement of the
preformed RNA chain from the hybrid state and the previously-open DNA helix is
reconstituted.
63
In E. coli, at 37°C, the mRNA is synthesized at a rate of 40-50 nucleotides/sec. That
of T3 and T7 is 200/sec at 37°C.
As each RNA polymerase molecule moves downside along the gene to transcribe a
progressively elongating RNA transcript, other RNA polymerase molecules follow it
in close succession in binding to the promoter, in moving downstream along the
same gene and transcribing successive RNA transcripts. The onward translocation of
successive polymerase molecules lends an arrowhead appearance to the electron
micrograph of gene undergoing transcription (Fig. 8.15).
5' Template DNA

RNA polymerase
3' Termination site
RNA transcript
3'
Initiation site
5'
Fig. 8.15 Transcription in prokaryotes
8.6.6.3 Termination
In prokaryotes, e.g., E. coli, there are two basic methods of termination, viz., (i) factor-
independent termination, and (ii) factor-dependent termination.
Factor-independent termination
A number of E. coli genes carry a transcription termination signal in the form of

specific consensus base-pair sequences in the DNA duplex. The signal consists of a
palindromic sequence rich in G:C followed by a region rich in A:T. The RNA
transcript of this signal region consequently has a poly U 3′ tail preceded by a hairpin
loop. The loop causes the RNA polymerase to slow down. The RNA-DNA hybrid
beyond the loop is unstable because of weak A:U bonds and consequently the RNA
falls off the template. The DNA reconstitutes and the enzyme is liberated. See Fig.
8.16 for the diagrammatic representation.
Factor-dependent termination
Particular sequences act as the termination sequence in the presence of a factor

called rho (ρ), a hexameric protein with an ATPase activity. A short hairpin loop is
formed in ρ-dependent termination also. The RNA polymerase pauses on the DNA
on reaching its termination site, which is not well defined yet for such genes. The ρ
protein meanwhile binds to a recognition sequence on the RNA transcript in 5′3′
direction. On reaching the RNA polymerase halted at the termination site, the ρ
factor unwinds the RNA-DNA hybrid double helix with the energy of the hydrolysis
of ATP. This helps release the polymerase from the DNA.
64
Terminator sequence of DNA
3' GCGCTGC AAAA GCAGCGC AAAAA
5' CGCGACG TTTT CGTCGCG TTTTT
Inverted repeat
Template DNA
3' GCGCTGC AAAA GCAGCGC AAAAA
5'
5' RNA
transcription
being terminated
Partner DNA being opened C G

G C
C G
G C
A T
hairpin loop of C G
tRNA G C
Fig. 8.16 Factor-independent termination of transcription
8.7 TRANSLATION
Translation is the synthesis of proteins under the direction of specific genetic codes
(represented as codons in mRNA). Ribosomes and tRNA participate in translating
the message of the genetic code into amino acid sequence of the nascent peptide.
Amino acids are polymerized sequentially into a peptide, starting from its N-terminal
end following the 5′3′ sequence of codons in the mRNA.
Prokaryotes translate the cytoplasmic proteins in the cytosol and the exportable- and
membrane proteins on the cytoplasmic surface of the plasma membrane.
Protein synthesis consists of 3 distinct steps, viz., (i) Initiation, (ii) Elongation, and (iii)
Termination. But before these steps are followed sequentially, the amino acids have to
be activated: in other words, the amino acids do not spontaneously link together. The
activation involves formation of aminoacyl tRNA, a “charged” amino acid.
8.7.1 FORMATION OF AMINOACYL tRNA (CHARGED tRNA)
tRNA serves as an adaptor molecule by bringing the specific amino acid coded by the
mRNA. The joining of the tRNA with the specified amino acid is illustrated in Fig. 8.17.
The respective synthetases ensure that only the correct amino acids are bound to the
corresponding tRNA. This specificity apart, the enzymes also have proofreading
activity. Most synthetases have a synthetic site that does not accept amino acids larger
than the specified size and a hydrolytic site for degrading amino acids smaller than the
specified size.
65
-amino acid + ATP Aminoacyl-AMP-Enzyme complex
Aminoacyl tRNA
synthetase/ligase tRNA
AMP + Enzyme
Aminoacyl tRNA
tRNA
NH2
5'
3' N N
C*
C* N
O N
C-terminal O
C=O C* refers to cytosine
Aminoacyl
CH-R
moiety
NH2 The joining takes place between 3' end
N-terminal of tRNA and C-terminal of amino acid
Fig. 8.17 Activation of amino acid before actual translation
In translation, the first codon to be read is AUG, which is for methionine. The
methionine brought in the first step of the translation (as against within the peptide
chain) is of a different type, viz., N-formyl methionine. The specific initiating tRNA used
here is called N-formyl methionine tRNA or tRNAfmet, which is formed according to Fig.
8.18.
Methionine
Aminoacyl tRNA
tRNA fmet synthetase
met tRNA fmet

Formate (from N10-formyl Transformylase
tetrahydrofolate)
Tetrahydrofolate
fmet tRNA fmet
Fig. 8.18 Formation of formylated methionine tRNA
8.7.2 INITIATION
The main features of the initiation step are: (i) binding of mRNA to ribosomes, (ii)
selection of initiation site, (iii) formation of charged tRNA, (iv) binding of the tRNA (to
mRNA) bearing the first amino acid.
The first event is the formation of a 30S initiation complex. Since prokaryotic ribosome
exists as 70S unit, it must first be dissociated into 30S and 50S subunits. This
66
dissociation requires the action of initiation factors IF-1 and IF-3. See Fig. 8.19 for
illustrated representation.
IF-1, IF-2 IF-1

50S +
30S IF-2
70S ribosome 50S ribosome
Fig. 8.19 Formation of 30S initiation complex
The initiation factor remains bound to 30S. IF-3 now helps 30S complex to bind to
the initiation site in mRNA. A purine-rich sequence called Shine-Dalgarno sequence (also
referred to as SD sequence, leader sequence, etc.) within the mRNA (within 10 nucleotides
upstream the initiation codon AUG) base-pairs with 16S rRNA. The mRNA base
sequence is AGGAGGU or similar to it. The binding of 30S also needs S1, S18, and
S21. See Fig. 8.20 for the illustration of mechanism of binding.
SD sequence
5' 3'
-AGGAGGU- -AUG----
mRNA
3'-UCCUCCA-5'
(16S rRNA) IF-2
IF-1 30S ribosome
Fig. 8.20 Transient base-pairing between mRNA and rRNA
IF-2 (another initiation factor) in combination with GTP, brings the fmettRNAfmet to
the initiator codon. IF-3 dissociates upon binding of fmettRNAfmet. See details in Fig.
8.21.
5' SD seq AUG 3' mRNA

IF-2
IF-1
IF-3
IF-1
IF-2
GTP IF-3
fmet tRNAfmet
Fig. 8.21 The binding of first amino acid
The release of IF-3 allows 50S to combine with 30S to form 70S initiation complex.
This new formation causes release of IF-1 and IF-2. GTP is removed by hydrolysis
into GDP + Pi. See Fig. 8.22 for detailed illustration.
67
The ribosome covers a region of 35-40 nucleotides. This can be shown by a method
called foot-printing, which is analogous to DNA finger-printing. The region covered by
the ribosome is protected against enzymatic degradation.
The 70S complex is now thought to consist of 2 sites, viz., P site and A site. The P site
refers to peptidyl site, where peptide chain grows, and A site refers to aminoacyl site,
where the new incoming charged amino acid is accepted. The first activated tRNA,
however, is bound to the P site rather than the A site as just described. See Fig. 8.23
for detail.
IF-2
IF-1
Initiation complex
70S ribosome
GDP + Pi
IF-1
IF-2
50S ribosome
Fig. 8.22 Formation of 70S initiation complex
8.7.3 ELONGATION
Elongation entails: (i) attachment of a new Aminoacyl tRNA to site A, (ii) joining together of
two amino acids (polymerization) by peptide bond formation – the process called
transpeptidation, and (iii) moving of one codon towards 3′ end of mRNA – the process called
translocation.
Three elongation proteins are required for the process, viz., EF-Tu, EF-Ts, and EF-G,
where EF refers to elongation factor, Ts to thermostable nature of the factor, and Tu
to the thermounstable nature of the factor. EF-G is a factor responsible for catalyzing
translocation. Elongation factors account for 5-10% of all proteins. The involvement
of the elongation factors is quite interesting. The delivery of the new charged tRNA
to the site A is affected by EF-Tu-GTP complex. After being bound, EF-Tu-GTP
breaks into EF-Tu-GDP + Pi. Another factor, EF-Ts now replaces GDP form EF-
Tu-GDP to form a new complex EF-Tu-EF-Ts. Intervention by GTP again
dissociates the new complex to EF-Tu-GTP and EF-Ts. EF-Tu-GTP is free to
perform another cycle again. See Fig. 8.23 for detail.
The activated amino acid attached to the tRNA in the P site, initially fmettRNAfmet, is
transferred to the amino group of the Aminoacyl tRNA in the site A. This is
catalyzed by peptidyl transferase, which is an integral part of 50S subunit (i.e., L2, and
L16). The P site becomes empty of amino acid. The reaction is also called
transpeptidation. See Fig. 8.24 for detail.
68
Peptidyl site Aminoacyl site
P A 3' mRNA P A 3' mRNA
70S ribosome
Pi
EF-Tu-GDP
Charged, EF-Tu-GTP EF-Tu

new tRNA EF-Ts
GDP
EF-Ts GTP
Fig. 8.23 Involvement of elongation factors in translation
After transpeptidation follows translocation. The ribosome moves one codon in the
5′3′ direction along the mRNA. This movement releases the uncharged tRNA from
ribosome. Very soon, this tRNA falls off the mRNA and enters the tRNA pool. The
relative position of the site A is translocated to that of site P, leaving again a new site
for the Aminoacyl tRNA. The translocation is catalyzed by EF-G.
Another ribosome assembly may occur immediately the previous assembly moves
some 80 nucleotides ahead. Beads of such ribosomes seen attached to an mRNA are
called polyribosome or polysome. The rate of translation in E. coli is about 15 amino acids
per sec.
Peptidyl site Aminoacyl site Peptidyl site Aminoacyl site

P A P A 3' mRNA
3' mRNA
Peptidyl transferase A site with polymerized
amino acids
P A P A
O O O
CO CO CO
CH R1 CH R2 CH R2 + H2O
NH NH2 NH
O
CO
CH R1
NH
Fig. 8.24 Mechanism of elongation of the peptide chain
8.7.4 TERMINATION
No tRNA pairs with stop codons. Instead, the stop codons are recognized by release
factors RF-1 and RF-2. The former recognizes UAA and UAG, the latter, UAA and
UGA. A third factor in association with GTP promotes termination. The binding of
release factors induces peptidyl transferase to release the polypeptide in the tRNA in
69
the P site by hydrolysis. The ribosomal unit then separates in a GTP-dependent
manner. The 30S ribosome may move along the mRNA until another Shine-
Dalgarno sequence is encountered and the translation resumes, or it may completely
dissociate from the mRNA. The dissociation of 70S unit needs IF-3. The natural
termination of protein synthesis uses nonsense codons but it appears that the signal is
not limited to a single nonsense triplet: the serious disadvantage in dependence on a
single nonsense codon is that it can at any time mutate to sense codon thereby
upsetting the process.
During termination, the release factors bind to site A. The same peptidyl transferase
now serves as the hydrolase. With the usual mechanism, the empty tRNA comes out
of the assembly and eventually falls off the mRNA. See Fig. 8.25 for an idea about
movement of ribosome and termination of protein synthesis. See Fig. 8.26 for the
recapitulative diagram of transcription and translation.
Movement of ribosome 30S

Nonsense codon ribosome
UAG
3' mRNA UAG
3' mRNA
RF-1 + RF-2 + RF-3 IF-3, IF-1
GTP GDP+Pi 50S

ribosome
Fig. 8.25 Termination of translation
DNA
Transcription
ATP, Enzymes Degradation
rRNA mRNA tRNA
Activated tRNA
30S
50S
30S
50S Ribosomal proteins
Amino acids
Fig. 8.26 Recapitulative diagram of transcription and translation
70
Note:
 Streptomycin affects S12 and causes misreading of mRNA

 Tetracycline inhibits binding of charged tRNA to the codon by binding itself
to 30S ribosome
 Chloramphenicol binds to 50S and blocks transpeptidation
 Erythromycin inhibits translocation
 The above drugs affect only prokaryotes
 The fidelity in E. coli is 1 error in 2000
8.8 METABOLIC REGULATION
A living cell is in a dynamic state. For proper growth and maintenance, the cell
requires a highly integrated coordination between anabolic and catabolic processes.
Since the genomic DNA is the key to every expression in the cell, a highly efficient
mechanism must be available to achieve the delicate balance of many processes. The
mechanism is collectively called gene regulation. Although cells contain the genetic
capacity to synthesize a large number of proteins, not all of these are present (or
synthesized) at any given time. Many of these are synthesized only as and when
required. The regulation may range from a modest repair of DNA to complex
reactions. Besides, gene regulation is also responsible for orchestration and
maintaining functional difference that exist in cells during development.
The control of metabolism in a cell depends on: (i) Enzyme concentration, (ii) Substrate (co-
substrate) concentration, (iii) Action of activators and inhibitors, and (iv) Modification of enzymes.
The concentration of enzyme is controlled at two levels, viz., transcription and

translation. The transcription is operated through protein factors which interact with
particular nucleotide sequence (generally located upstream of genes) and the RNA
polymerase itself. Modulation of transcription of a gene is performed by the joint
action of RNA polymerase and a series of regulatory proteins (activators, repressors,
terminators, anti-terminators), which in the presence of co-factors, interact with the
DNA (or mRNA) at the level of particular nucleotide sequences (targets).
In the following paragraphs, regulation of protein synthesis (also enzyme synthesis)

will be discussed. There are 6 points at which the amount of protein can be
regulated, viz., (i) synthesis of primary transcript, (ii) post transcriptional processing, (iii) mRNA
degradation, (iv) translation, (v) post-translational modification of proteins, and (vi) protein
degradation. See Fig. 8.27 for the summary.
71
DNA (gene)
Transcription
mRNA
Post-transcriptional processing
mRNA degradation Nucleotides
Translation
(Inactive proteins)
Post-translational processing
Modified proteins/enzymes
(functional)
Protein degradation Amino acids
Fig. 8.27 Summary of regulation of protein synthesis
8.8.1 REGULATION OF GENE EXPRESSION
The synthesis of particular gene product is controlled by mechanisms collectively

called gene regulation. Evidently, although cells have an enormous genetic capacity not
all the proteins are synthesized all the time. Many of them are produced only on
special occasions and in response to some environmental stimuli. For example,
antibodies are produced in response to antigens. Such controlled and regulated
synthesis of gene products leads not only to cellular economy but also to homeostasis. It is
obvious that genes have myriad activities. Consequently, genes have been variously
named:
 Structural genes: they code for enzymes, tRNA, rRNA, mRNA, proteins,
indeed anything that has a structure
 Regulatory genes: they code for products that regulate expression by structural
genes. They may or may not be located near the structural gene. Regulatory
genes are usually not considered as part of operon
 Architectural genes: they are responsible for integration of proteins into the
structure of the cell
 Temporal genes: they control the time and place of action of other genes and
largely control the differentiation of the cells and tissues of the body
The regulation of metabolic process can be possible by regulation of enzyme

concentration, coenzyme concentration, concentration of activator and inhibitor,
and by varying the specific molecular activity of the enzymes by covalent
modification of the proteins.
Exhaustive investigations have established that regulation of gene expression in all

life forms may occur at three levels: (i) transcription, (ii) translation, and (iii) post-
translation.
72
8.8.1.1 Regulation at the level of transcription
In bacteria, there occur several mechanisms of gene regulation at the level of

transcription. A notable method depends on whether the enzyme being regulated act
in catabolic or anabolic pathway. For example, in a multi-step catabolic system the
availability of the molecule to be degraded commonly determines whether the
enzymes of the pathway will be synthesized or not. In contrast, in biosynthetic
pathway, the final product is often the regulatory molecule.
The molecular mechanisms for each of the regulatory patterns differ greatly and are
of one of the two types: (i) negative regulation, or (ii) positive regulation.
In negative regulation, transcription is inhibited through the binding of an inhibitor

molecule to the DNA. To remove the effect of this inhibitor molecule, an antagonistic
molecule (called inducer) is required. Thus, the resumption of transcription requires
neutralization of the effect of inhibitor.
In positive regulation, an affector molecule activates a promoter by binding to the

DNA. See Fig. 8.28 for the illustrated explanation of positive- and negative
regulations.
Inhibitor
DNA
Gene not expressed
Inducer
DNA
mRNA
ribosome
Inducer-inhibitor complex
(Inactive, removed) polypeptide
Gene expressed
(Negative regulation)
Promoter
DNA
Gene not expressed
Affector
DNA
mRNA
ribosome
polypeptide
Gene expressed
(Positive regulation)
Fig. 8.28 Summary of Negative-and Positive regulation
73
The lac operon
lac operon is an example of negative regulation. Before we proceed, let us become

familiar with some terminologies of operon.
 Operon: an operon is a group of coordinately regulated genes (i.e.,

polycistronic), the products of which typically catalyze a multienzymatic
pathway and its controlling elements. An operon is operated through a
common promoter.
 Inducible genes: they are genes used for the production of inducible enzymes
when a specific inducer is present. For example, production of the enzyme
β-galactosidase is induced by the presence of its substrate, lactose, in the
medium. Ordinarily, when lactose is not supplied, the organism does not
synthesize the enzyme: it would be a waste!
 Constitutive genes: these refer to prokaryotic genes whose expression is not
regulated. The products of the genes are produced at a constant, often low,
rate.
The purpose of lac operon
The purpose of lac operon in E. coli is to make enzymes needed to metabolize

lactose. In E. coli, lac operon consists of two classes of genes, viz., structural- and
regulatory genes. The schematic representation of lac operon in E. coli is given in Fig.
8.29.
i p o z y a
DNA
CAP site Structural genes
Fig. 8.29 Schematic representation of lac operon genes in E. coli
Notations:
i = lac repressor (regulator), which has its own promoter in DNA and SD sequence
in mRNA
p = promoter
CAP site = site within the promoter needed for binding affector molecule for
positive regulation (promotes transcription; see catabolite repression, page 76)
o = operator, the site where the repressor due to i gene binds for inhibiting
transcription
z = structural gene that codes for β-galactosidase
y = structural gene that codes for galactoside permease
a = structural gene that codes for thiogalactoside transacetylase (uncertain function)
z, y, and a all have separate SD sequences with stop codons in between.
74
Regulation by lac operon
In the absence of lactose in the medium, lac operon does not function. This is
because lac repressor synthesized by gene i tightly binds to the operator thereby
blocking the transcription. Lac repressor is a constitutive molecule that is
continuously expressed in low amounts. It is a diffusible tetrameric protein that
diffuses along the DNA to reach the operator. It has one binding site per unit. The
site is specific for inducer.
The presence of inducers such as allolactose (true inducer: see Fig. 8.31) or isopropyl
thiogalactoside (IPTG, also referred to as gratuitous inducer: see Fig. 8.31) the negative
regulation by lac repressor is relieved. Upon binding of the repressor and inducer to
form inducer-repressor complex, the structure not only prevents the binding of other
repressors to o site but also causes the ones already bound to fall off. The RNA
polymerase is now free to move ahead for transcription. Lactose serves as an inducer
indirectly, in the form of allolactose. A small amount of lactose is converted to
allolactose form. With the depletion of lactose, allolactose will also deplete. The
repressor again binds to the operator thereby quickly stopping the transcription. The
unstable mRNA is now rapidly degraded. See Fig. 8.30 for detailed illustration.
Repressor
molecule
P P O
Lac I Lac Z Lac Y Lac A
Initiation Initiation
CAP site
RNA polymerase
Operator +
Promoter Repressor
Allolactose
molecules
(inducer)
Repressor
P P O
Lac I Lac Z Lac Y Lac A
Initiation Initiation
Fig. 8.30 Schematic representation of lac operon in E. coli
75
Catabolite repression (positive regulation)
When lactose is supplied to E. coli, lactose-degrading enzymes are rapidly synthesized

(5000 per cell in several minutes). However, if glucose is supplied, the preference
switches back to glucose. This switching to the preferred fuel is known as catabolite
repression, as it is due to the catabolite glucose that repression has occurred. It is
important to note, however, the presence of glucose does not so much repress the expression of the
lac operon as does the absence of glucose enhance the expression.
Functioning of lac operon not only requires an inducer but also a positive regulatory
system. The system consists of cAMP (cyclic AMP), a site within the promoter called
CAP site, a CAP (Catabolite Activator Protein: dimeric in nature) factor, and CAP gene
remote to the operon. CAP is a regulatory factor synthesized separately. cAMP is
inversely related to glucose concentration. Depletion of glucose increases the level of
cAMP, which then complexes with the constitutively produced regulator CAP to
form cAMP-CAP complex. Upon binding of this complex to the CAP site, it
stimulates β-galactosidase production by 50 fold.
CH2OH CH2OH
O O
O
Lactose
-D-galactosidase
CH2OH
O OCH2
O
Allolactose
CH2OH CH2OH CH2OH

O O O SH(CH3)2
+
Galactose Glucose Isopropyl thiogalactoside (IPTG)
Fig. 8.31 Substrates of β-galactosidase
Tryptophan operon
The tryptophan operon in E. coli is responsible for the synthesis of the amino acid
tryptophan. Regulation of this operon occurs in such a way that when tryptophan is
present in the growth medium, trp operon is not active. That is, when adequate
tryptophan is present, transcription of the operon is inhibited; however, when its
supply is insufficient, transcription occurs. The trp operon is quite different from lac
operon in that tryptophan acts directly in the repressor system rather than as an
inducer.
Tryptophan synthesis is controlled by two mechanisms, viz., (i) Negative repression by

corepression, and (ii) Control by attenuation. Brief descriptions of both the control
mechanisms are given in the paragraphs to follow.
76
Negative regulation by corepression
The regulatory protein of the repressor system of the trp operon is the trp R gene
product. This protein is called trp aporepressor and does not bind to the operator
unless tryptophan is present. The trp R gene has its own promoter and is remote
from the operon. When tryptophan concentration is high the same tryptophan
functions as corepressor: it binds with the aporepressor (i.e., trp R gene product) to
form an active trp repressor. The latter binds to the operator of the trp operon and
blocks transcription by blocking the movement of RNA polymerase. This type of
regulation by the cell is used for rough control of tryptophan synthesis. Fine control
is possible with yet another mechanism called attenuation.
P O Leader Trp E Trp D Trp C Trp B Trp A
Arrangement of genes in trp operon
Active (tryptophan deficient condition)
Aporepressor
RNA
Not able to bind to O polymerase

Transcription
Inactive (tryptophan present)
Aporepressor Tryptophan
Activated repressor

No transcription
Fig. 8.32 Negative regulation by corepression
The structure of trp operon
Tryptophan is synthesized in five steps from chorismic acid, each requiring a particular
enzyme. In E. coli chromosome the genes encoding these enzymes are located
adjacent to one another in the same order as they are in the biosynthetic pathway;
they are translated from a single polycistronic mRNA. The genes are called trp E, trp
D, trp C, trp B, and trp A. They are structural genes. Just upstream the first structural
gene (i.e., trp E), are attenuator (trp a), leader (trp L), operator (O), and promoter (P)
genes. See Fig. 8.32 for detail.
77
Attenuation
This method of regulation can occur only in prokaryotes, because of the coupling of
transcription and translation. The fundamental feature of attenuation is that, at low
tryptophan concentrations, the operon makes full-length mRNA; but at high
tryptophan concentrations, transcription is prematurely terminated.
The transcript of trp L has four complementary segments (1, 2, 3, and 4). Segment 2
is complementary both to 1 and 3 (see Fig. 8.33). Segment 3 is complementary to
both 2 and 4. On this transcript, 3 types of hairpin loops (1:2, 2:3, 3:4) are thus
possible. In vivo, however, one of the last two (2:3 or 3:4) occurs. This is because the
ribosomes closely follow the RNA polymerase and segment 1 is not free from
hairpin loop. The loop 3:4 acts as the transcription terminator because RNA
polymerase cannot cross this loop. The transcription prematurely terminated in this
way is called attenuation. Such a transcript has (in trp attenuation) an incomplete
140-nucleotide RNA transcript of L gene, which is eventually released.
Hairpin loop
1 2 3 4
mRNA transcript
trp trp
Leader sequence
Fig. 8.33(a) Transcript of trp L
Non-termination loop
1 2 3 4
Leader trp
peptide trp trp Ribosome RNA
trp proceeding polymerase
slowly advancing
Fig. 8.33(b) Attenuation regulation at low tryptophan concentration
1 2 3 4
Leader trp
peptide trp trp
trp RNA
polymerase
Ribosome proceeding stopped
rapidly to segment 2
Fig. 8.33(c) Attenuation regulation at high tryptophan concentration
78
8.8.1.2 Translational control
In prokaryotic gene regulation at the translational level, the lifetime of mRNA

molecule may be genetically determined. Enzymatic degradation of mRNA is from
the 5′3′. The average lifetime of many mRNAs of E. coli is only two minutes at
37°C. The specific nucleotide sequence at the 5′ end may influence its susceptibility
to enzymatic digestion. Further, catabolic enzymes are denied access to mRNA when
the ribosome coats them. Hence, the lifetime of mRNAs may also be correlated with
the number of free ribosomes available at any given moment to translate mRNA
molecules. Bacteria vary their rates of protein synthesis by varying their ribosomal
content rather than by varying the translational rate.
79
CHAPTER 9
CONCEPT OF FERMENTATION TECHNOLOGY
9.1 INTRODUCTION
The term fermentation came from the Latin verb ‘fervere’, which means to boil. It is
clear now that the boiling impression was due to carbon dioxide bubbles quickly
moving up to the surface of the liquid medium.
Fermentation has many definitions. In a biochemical sense, fermentation is an

anaerobic process in which substrate is utilized for energy production without the
involvement of molecular oxygen. In industrial microbiology, fermentation is simply
a large-scale microbial process. This implies that the process can be both aerobic and
anaerobic. Indeed, almost all of the industrial fermentations are highly aerobic
processes Thus, production of beer, sake, jand, wine, penicillin, etc., all are examples
of fermentation.
Fermentation process requires the presence of both the substrate and the conversion
agent (microorganism and/or enzymes). For large scale production, the process also
requires a large vessel for accommodating the organism and the substrate. For
improved yield, a carefully controlled environmental- and cultural condition is
required. Since the yield and productivity is a function of microbial property,
selection and improvement of microbial strains has become a norm in industrial
fermentations. The overall fermentation event can be presented as:
Microorganisms or Enzymes
Substrate 
Products or Services
9.2 FERMENTER AND FERMENTATION PROCESS
The tank or vessel in which industrial fermentation is carried out is called fermenter
(fermentor) or bioreactor. It is a vessel/system which provides the organism with
favorable place for multiplication and product formation. There are several types of
fermenters, some of which you will come across in the paragraphs to follow. The
spellings fermenter and fermentor are interchangeably used, although the term fermenter
refers to (lexically) the causative agent of fermentation, viz., the microorganism. In
industrial microbiology, many terms have contextual implications. For instance,
when talking about microbial production of alcoholic beverages, the term alcohol
fermentation does not mean the microbial conversion of alcohol into other products.
Rather, it refers to microbial conversion of substrates into alcohol.
The terms fermenter and bioreactor are often used interchangeably. In a very strict
sense, all fermenters are bioreactors but not all bioreactors are fermenters. Although
biochemical conversion occurs in both of them, the term fermenter has been
conventionally associated with product generation for human, animal, or plant use.
Bioreactor, on the other hand encompasses all the biochemical conversion
processes, including those that generate service, e.g., waste treatment.
9.2.1 THE COMPONENT PARTS OF A FERMENTATION PROCESS
Regardless of the type of fermentation (with possible exception of some

transformation processes) an established process may be divided into six basic
component parts (see Fig. 9.4 also), namely:
1. Formulation of the medium for culture propagation (inoculum build up)

and the main fermentation
2. Sterilization of the medium, fermenter, and ancillary equipment
3. Production of active pure culture in sufficient quantity to inoculate the
production vessel
4. Growth of the microorganism in the production fermenter under optimum
condition for the product formation
5. Extraction of product and its purification
6. Disposal of effluents produced by the process
9.2.1.1 Medium formulation
Formulation of medium is a very important part of the fermentation process. The

composition of media for pre-fermentation is quite different from that for main
fermentation. The pre-fermentation stage implies propagation of the pure culture
from the stock culture (which may be in slants, vials, flasks, plates, etc.). This stage is
also known as inoculum build-up (see later). The inoculum build-up phase is
concerned with rapid propagation of the cells and so the medium is generally richer
in nutrient level.
The main fermentation is concerned with accumulation of the desired

metabolite/product. In most cases, the organism does not accumulate the metabolite
in very rich growth medium. Sometimes they have to be starved and sometimes
some key compounds must be added or removed from the medium to force the
organisms to synthesize the metabolite. This is where the importance of medium
formulation comes in. For this reason, industries spend a good amount of resources
on medium formulation. The findings from the researches have been very useful for
fermentation industries. Several excellent examples of medium formulation (e.g., for
glutamic acid, citric acid, tetracycline, etc.) have been given elsewhere in this book
(pages 328, 319, and 289).
9.2.1.2 Sterilization and asepsis
Microbiological processes cannot run successfully if contaminations occur.

Contaminants may come from various sources and due to various reasons.
Therefore all the potential sources of contamination must be sterilized prior to
fermentation.
Sterilization refers to freedom from all life forms. This can be achieved by physical
means (heat, radiation, filtration, etc.) or with chemical agents (e.g., sodium
81
hypochlorite). In almost all cases, the medium used for the fermentation should be
either pasteurized or sterilized. Very often, the fermenter itself and the ancillary parts
must be sterilized. The air that is to be supplied during the fermentation should also
be sterile. Some “protective” fermentations (e.g., alcohol and wine production) may
not have stringent requirements for the main fermentation but there are many
fermentations which are very sensitive to contamination.
Medium is usually sterilized in batch or continuous process. The continuous process

normally utilizes plate-heat exchangers or flash sterilizers, using hot water or steam
as the heating agent. Air sterilization can be done by passing through filter beds of
glass wool, fiber, etc., of 5-15 m pore size. The packing material gets contaminated
in due course and this is rendered sterile by injecting steam (Fig. 9.1).
Asepsis refers to measures adopted to create a germ-free condition. In microbiology,

this relates to prevention of contamination of the sterilized material. In fermentation
industries, aseptic techniques are used to avoid contamination during subculturing,
inoculum build up, fermentation, and sampling. A few examples of aseptic
techniques are described in the following paragraphs.
steam
packing
material
to fermenter
air
stainless
steel casing
condensate
Fig. 9.1 Sterilization of air
Aseptic sampling
During fermentation, samples are regularly taken out of the fermenter to monitor
the progress of the process. This interruption can result in contamination of the
fermentation medium if aseptic techniques are not used. The pipelines and valves for
handling sample should be rendered sterile both before and after sampling. Fig. 9.2
gives an example of the arrangement for aseptic sampling.
B
A steam
fermenter C steam
condensate
D
sample
Fig. 9.2 Protocol for aseptic sampling
82
During the normal fermentation process, valves A, B, C, and D remain closed.
When sampling is to be done, valve B is opened to let in steam through valves C and
D. After passing steam for a specified period, the valves are again closed. Valve A is
now opened and the sample allowed to fill up the space between the valves A, B, C
and D. Valve A is now closed and then valve D opened to receive the sample. After
receiving the sample, the pipeline is sterilized once again as described before.
Aseptic inoculation
Inoculum for the final fermentation is prepared separately and aseptically. Once
prepared, it is transferred to the main fermenter aseptically so that the final
fermentation remains as contamination-free as possible.
Usually, the final inoculum tank is directly linked to the main fermenter and the
transfer takes place under pressure created by air supplied to the inoculum tank.
There are provisions also for steam-sterilizing the pipelines. See Fig. 9.3 for an idea.
steam
filter
seed tank
sterile air main fermenter

sterile air
condensate
Fig. 9.3 Protocol for aseptic inoculation
9.2.1.3 Inoculum build-up
The preparation of a population of microorganisms from a dormant stock culture to

an active state of growth that is suitable in the final production stage is called
inoculum development or inoculum build up. Inoculum build up generally starts in flask
cultures (called shaker flask or shake flask, Fig. 9.4).
The culture is grown in a rich medium for 2 to several days under aerobic condition.
Thereafter the culture is transferred aseptically to a bigger vessel for propagation.
Once again growth takes place for 2 to several days. The transfers are continued for
a few more times until the desired amount of inoculum is produced. At each step,
the volume is increased by 20-200 times. The culture should be in log phase.
Everything must be carried out aseptically. As mentioned earlier, the medium for
83
inoculum build up is quite different from the one used for the final fermentation.
You will find several examples of inoculum build up and medium formulation in the
later chapters (pages 151, 217, 279, etc.)
Stock culture
Main fermenter
(Production vessel)
Shaker flask
Propagator
Propagator
Sterile air
supply
Inoculum build-up
Culture fluid
Cell separation
Biomass Cell-free supernatant
Product extraction
Effluent treatment Product purification
Product packaging
Fig. 9.4 The component parts of a fermentation process
9.2.2 TYPES OF FERMENTERS / FERMENTATION PROCESSES
Various schemes have been used for classifying fermentation process. Some of the
commonly used schemes and the classifications are:
 Aerobic and Anaerobic

 Batch, Continuous, and Fed-batch
 Solid state culture (= Surface culture) and Submerged culture
84
You will see considerable overlapping in the above schemes. For example, solid state
(also called solid substrate) and surface culture are batch processes while submerged
fermentation can be either batch or continuous. It must also be noted that there are
several variations in each category. For example, a continuous fermenter can be of
tower type, cascade type, air-lift type, etc. A brief description of basic types of
fermentation processes is given in the following paragraphs.
9.2.2.1 Solid-state fermentation
It is a fermentation in which the microbial growth occurs on moist, non-soluble

substrate in the absence or near-absence of free-flowing water. The solid substrate
acts as a source of carbon, nitrogen, minerals and a growth surface which absorbs
water necessary for microbial growth. In addition, the solid substrate provides
anchor points for the growth and propagation of microorganisms. Solid state
fermentation (also called koji process, see later) is a highly aerobic process. The
microorganisms get their nutrients by hydrolyzing (breaking down) the substrates. In
other words the microorganisms are good producers of extracellular (i.e., produced
and sent out of cell) hydrolytic enzymes. Most indigenous fermented foods and a
few enzymes are produced by this method. Some of the important examples of
solid-state fermentation are mushroom cultivation, jand, kinema, natto, sauerkraut,
tempeh, sake, and miso production. All solid-state fermentations are batch processes.
The process cannot handle more than about 1000 kg per batch. This is because the
medium is not liquid and the control of temperature, pH, nutrient distribution, and
aeration is very not easy.
The solid substrate chosen are usually wheat, maize, soybean, rice, and wheat bran.
The substrate is initially rendered sterile either by autoclaving or cooking and cooled
before inoculating with the organism. The inoculum is prepared aseptically
separately. When the inoculum is a mold, it is called koji. The substrate, which may
or may not need to be supplemented, serves as a rich source of nutrients. Such
substrates support the growth of mycelial organisms which can grow at high nutrient
concentrations and produce a variety of extracellular enzymes.
The fermentation may take place in trays (rotary or stationary), compartments, or

even in a room. When large-scale production is used, temperature and humidity is
controlled by passing conditioned air. Some examples of solid-state fermentation are
given in later sections.
The main advantages of solid substrate fermentation are:
1. Low cost (due to simple device and less manpower)

2. Readily carried out on home scale
3. Reduction of the fermentation- and liquid effluent volumes
4. Reduced risk of bacterial contamination because of low moisture level
The limitations of solid substrate fermentation are:
1. Difficult to scale up
85
2. Difficult to control and monitor different factors, e.g., pH, temperature,
nutrient distribution, etc.
3. Gas exchange is difficult (O2 supply and CO2 removal, that is)
4. Problem of heat exchange
More recently, solid substrate fermentations have been used to produce extracellular
enzymes, fungal toxins, and fungal spores.
9.2.2.2 Submerged fermentation
It is a process in which organisms are forced to grow in a submerged state (that is,
state in which cells are dispersed in liquid medium). Submersion is carried out using
suitable mixing device or technique. The oxygen required by the microorganism is
supplied by passing forced air through the medium. Thus, although the
microorganisms are aerobic, they do not experience lack of oxygen. The advantages
of submerged fermentation are:
1. Can be operated in large volumes and even in a continuous mode

2. Temperature, pH, oxygen concentration, etc., can be closely controlled
Some of the examples of submerged fermentation are: production of beer, vinegar,

ethanol, baker’s yeast, etc. For beer and ethanol, only the initial phase is truly
submerged. During the latter part, fermentative metabolism is maintained simply by
not supplying oxygen. The organism quickly consumes the available oxygen and then
shifts the metabolism toward ethanol production. The intimate mixing is possible
even without an agitator. The CO2 evolved during alcohol generation rises to the
surface and sets the whole broth in continuous motion.
9.2.2.3 Batch process
This is the simplest type of culture in which microorganisms grow in a vessel with
limited amounts of nutrients under optimum environmental condition. Unlike
continuous culture, the microorganisms pass through all stages of microbial growth
cycle, viz., lag phase, log phase (exponential phase), stationary phase, and decline
phase, exhibiting a sigmoid growth curve (Fig. 9.5). Completion of each cycle
constitutes a batch. The downstream processing (Fig. 9.4) is done at the end of each
cycle. The vessel is then prepared for receiving the next batch. This preparation
requires certain time period, called down time, which is equivalent to unproductive
period. This is what makes batch process less efficient than the continuous process.
9.2.2.4 Continuous fermentation
In this, the microorganisms are maintained in exponential phase (log phase) so that
the growth remains constant throughout the fermentation period (which may even
run for years). This is possible by continuous feeding (of fresh medium) and
withdrawal (of finished product). Both these activities need very delicate balance.
You will find several examples of continuous fermentation (See microbial
production of ethanol, page 219; and semisynthetic penicillins, page 133).
86
Continuous process is far more efficient and economical than any other processes
because:
1. There is no down time as in batch process

2. Since the culture is in log phase there are no lag-, stationary-, or decline
phases
3. The process can be automated, which means less manpower is needed
There are certain limitations, however. For example, since the process runs for very
long periods of time contamination can be a problem. Besides, the source of raw
material must not dry up.
10
0
0 2 4 6 8 10 14 16
Time (hrs)
Fig. 9.5 The bacterial growth curve
9.2.2.5 Fed-batch fermentation
Basically, it is a batch culture that is fed continuously with fresh medium without
simultaneous removal of the original culture medium from the fermenter. This
results in continuous increase in volume of the medium in the fermenter. The
feeding and growth rate is adjusted such that the fermentation is complete by the
time volume reaches the predetermined level. This method is used when high
concentrations of nutrients turn out to be counter-productive due to shift in the
pattern of metabolism, catabolite repression, etc. Bakers yeast is produced by this
method (see page 153).
9.3 FERMENTER DESIGN
The basic function of a fermenter is to provide a controlled environment for the

growth of a microorganism or a defined mixture of organisms to obtain the desired
product. This is where development and design of fermenter comes in. Let us now
look at some important aspects fermenter design.
87
The problem
1. Selection of the best type of reactor for the particular reaction

2. Determination of the best operating conditions
The objective of design
The objective of design is to be able to describe the effects of operating conditions

on the performance of a bioreactor and to compare alternative designs with
economic criteria. For this, an in-depth knowledge of (and experience in)
microbiology, biochemistry, thermodynamics, microbial and biochemical kinetics,
fluid mechanics, mass and heat transfer, and economics is essential. Even with such
knowledge and experience an ideal fermenter can rarely be made. Fermenter
designing, ultimately, is a matter of compromise.
The designer’s job
The designer’s primary job is to construct, at the lowest possible cost, a fermenter,
and design in features such that control will be possible over reasonable ranges of
the important process variables (e.g., oxygen concentration, temperature, pH, etc.)
and that the operation will be reliable and contamination-free. To achieve these
ends, he must, at the minimum provide for:
1. Adequate heat and oxygen transfer

2. Aseptic and sterilization procedures
3. Reliable foam control
4. Good spatial definition of environmental conditions
5. Simple, rapid, and thorough cleaning systems
6. Responsive, reliable, and appropriate monitoring and control systems
7. Appropriate materials for construction and reliable fabrication methods
8. Possibility that the fermenter will be used for more than one product
(flexibility)
9.3.1 FERMENTER CONFIGURATIONS
There are different types of fermenters. The structure and design of fermenter is
called ‘fermenter configuration’. Fermenters vary both with respect to configuration
and capacity. In the former case, the primary variations are in geometric ratios and
the types and number of impellers used. There are many fermenters which cannot be
covered by general rule (e.g., tower fermenter, air-lift fermenter, tubular fermenter,
etc.). Generally, the design decisions are made on the basis of conventional
‘wisdom’, professional advice from equipment manufacturers, personal experience,
and hearsay. Stated differently, it is very difficult to design a fermenter. Its designing
needs the involvement of microbiologists, engineers, and technologists. A very
common fermenter type is described next.
88
9.3.1.1 The stirred tank fermenter
The stirred tank fermenter (STF) is used almost universally in fermentation

industries. The capacity of the fermenter ranges from 100,000-500,000 gallons (1 US
gallon = 3.79 liters, 1 imperial gallon = 4.55 liters). A typical stirred tank fermenter
(batch) is shown in Fig. 9.6.
Description of parts
Sight glass: This is used to view what is inside the fermenter. A powerful lamp placed
on the opposite side of the fermenter provides light for viewing.
Agitator/impeller: This is a motor-driven mixing device that agitates the liquid
medium. Agitation fulfills several functions. It helps breakdown the air
bubbles, distributes the nutrients uniformly, and helps remove the heat
developed during fermentation.
Cooling/heating coil: This is used for removing or adding heat to the fermenter. To
remove heat, cold water is passed from the lower end. To add heat, steam is
passed from the upper end.
Baffle: This is placed against the wall of the fermenter. During agitation, the liquid
tends to form vortex if there is no baffle. Baffle works by foiling formation
of vortex. It also helps in better mixing of liquid. Usually, a small gap is left
between the wall and the baffle so that the liquid can pass through it. This
gap helps in automatic cleaning of the wall of the fermenter by an action
referred to as “scouring”. A fermenter has at least four baffles.
Steady bearing: This is used to hold the shaft that carries the impeller.
Air duct/pipe: This is used for supplying air in the medium.
Inoculum port: This is used for the introduction of culture
Feed port: This is used for introducing the fermentation medium (= feed).
Pressure gauge: This is use for measuring the air pressure inside the fermenter.
Temperature probe: This is used for continuous measurement and monitoring of
temperature of the medium.
pH probe: This is used for the continuous measurement and monitoring of pH of the
medium
Dissolved oxygen probe: This is used for the continuous measurement and monitoring
of dissolved oxygen concentration of the medium
9.3.1.2 Fabrication of stirred tanks fermenters
The material for construction should withstand sterilization. Mild steel fermenter is
often used. For 300,000-400,000 liter capacity, 7 mm plates may be used for the
sides of the vessel and 10 mm plate for the top and the bottom. The top (and
sometimes bottom) of the vessel should be hemispherical to withstand internal
pressure. The shape of the vessel is almost always cylindrical. Quite often, the
bottom section is made conical to facilitate product removal. In certain
fermentations, wooden, concrete or plastic fermenters are also used.
89
Gear box Motor
Aseptic
seal
Feed Pressure
Inoculum gauge
Sight glass
Shaft
Working level
Steam in Dissolved O2 probe
Hot water Temp probe

out
Agitator/Impeller
pH probe
Cooling and
heating coil
Baffle
Cold water in
Sampling
point
Steady
bearing Condensate
out
Air duct
Product out
Fig. 9.6 A typical batch, stirred tank fermenter
9.3.1.3 Geometric ratios of stirred tanks fermenters
Geometric ratios are very important in the construction of a fermenter. For a stirred
tank fermenter, the commonly used ratios are Liquid height/Tank diameter, Impeller
diameter/Tank diameter, Baffle width/Tank diameter, and Impeller height/Tank
diameter, etc. Typical data of the ratios and other related details of a stirred tank
reactor are given below. See Fig. 9.7 for the notations used in the data.
Operating volume = 170 dm3 P/V = 0.74

Liquid height, (L) = 150 cm P/W = 0.77
Liquid height/Tank diameter, (L/D) = 1.7 P/Y = 0.77
Impeller diameter/Tank diameter, (P/D) = 0.33 P/Z = 0.91
Baffle width/Tank diameter = 0.0.098 H/D = 2.95
Impeller height/Tank diameter = 0.37
90
Aseptic
inoculation pipe Stirrer shaft
seal
Working
level
Impeller V
Baffle W H
L
Sampling Y
P
point
Sterile Z
air line
Air
sparger Drain
point
D
Fig. 9.7 Typical dimension for a stirred tank fermenter
9.4 AERATION AND AGITATION
The purpose of aeration in a submerged fermentation is to supply adequate oxygen

to the microorganisms. Oxygen is supplied to the submerged fermentation in the
form of sterile air. The aeration rate is in the range 0.25-2.0 vol/vol/min.
Agitation serves a double purpose, namely:
1. Diminishes the size of air bubbles to give a bigger interfacial area for oxygen
transfer and to decrease the diffusion path
2. Maintains a uniform environment throughout the vessel content
Based on the method of aeration and agitation, fermenters can be classified as

(i) mechanically agitated, and (ii) non-mechanically agitated.
Baker’s yeast production is a very good example of non-mechanically agitated

fermentation (see page 154). In it, the air passing into the fermenter itself agitates the
medium. In such fermenters, the ratio of height to diameter is usually not greater
than 5:1. The type of aeration-agitation system used in a particular fermentation
depends on the characteristics of fermentation process under consideration. The
mechanically agitated aeration system is usually required in fungal and
actinomycetous fermentations. Such fermentations develop very complex rheology
as the fermentation progresses (due to microbial growth) and may require specially
designed impellers.
91
9.4.1 COMPONENTS OF MECHANICALLY AGITATED SYSTEMS
9.4.1.1 The impeller
There are four basic designs of impellers, viz., (i) Disc turbine, (ii) Marine propeller,
(iii) Open turbine, and (iv) Vaned disc (see Fig. 9.8). The most widely used impeller
is the open-turbine (= flat-bladed turbine). It can break up a fast air stream without
itself becoming flooded with air bubbles. Ideally, the impeller should be 1/3 to
1/2 Dt (tank diameter) above the base.
Disc turbine Vaned disc Open turbine Marine propeller
Fig. 9.8 Different types of impellers
9.4.1.2 Stirrer glands and bearings
These are required for the satisfactory sealing of stirrer shaft assembly. Most
industrial fermenters have mechanical seal (see Fig. 9.9 for an over-simplified
schematic). This seal is composed of two parts, one part is stationary in the bearing
house and the other one rotates along with the shaft. The two components are
pressed together with expanded bellows. The meeting surface should be precision-
machined. Steam condensate is used for the lubrication of mechanical seal.
Shaft
Housing
Stationary counter-face
Exit port for steam condensate
Moving counter-face
Shaft muff
Entry port for steam condensate
Bellows
Fig. 9.9 Schematic drawing of mechanical seal
9.4.1.3 Baffles
Baffles improve aeration and prevent vortex formation. Four baffles are normally
needed. A small gap is needed between the baffle and the vessel wall for scouring
action (which enables self-cleaning of the walls of the vessel). See Fig. 9.5 and 9.7 for
an idea about baffles.
92
9.4.1.4 The air sparger
It is a device for introducing air into the medium in the fermenter. The two most
widely used types of spargers are: (i) Nozzle sparger and (ii) Orifice sparger.
Nozzle sparger consists of a single open or partially closed pipe for providing stream
of air bubbles. Ideally, the pipe should be positioned centrally below the impeller
and as far away as possible to ensure that the impeller is not flooded with air
bubbles. See Fig. 9.10 for the schematic drawing.
Orifice sparger system is widely used in yeast production where mechanical agitation
is not done. The air sparged in the medium does the mixing. For example, baker’s
yeast production in a 200 m3 vessel uses orifice assembly with 24 side tubes
containing in all 30000 holes of 1.5 mm diameter each. See Bakers yeast production
for the schematic drawing (page 154).
Paddle
Air bubbles
Air supply Nozzle
Fig. 9.10 Schematic drawing of nozzle
9.4.1.5 Cooling
Since fermentation is a metabolic process, large amount of heat is generated. This is

undesirable for obvious reasons. Internal cooling coil is most satisfactory for faster
cooling. Agitator has an important role here too. When cooling coils are used, 50-70 m2
may be taken as an average contact area for a 55000 dm3 fermenter.
9.4.1.6 Foam control
Foam is an undesirable aspect. Foams are generated primarily due to denatured

microbial- and other proteins. Foaming reduces the vessel capacity and thus the
productivity. Foams may also enter the various entry ports of the fermenter if left
uncontrolled. This entry in due course leads to contamination. Besides, the product
also oxidizes due to increased exposure to air. Foam can be controlled by two main
methods, namely, (i) mechanical defoaming, and (ii) chemical defoaming.
Mechanical defoamers rely on discs, propellers, brushes, etc., for defoaming (see
vinegar production, page 264 also). In the chemical defoaming method, antifoams
such as alcohols, fatty acids, esters, silicones, sulfonates, etc., are added in controlled
amounts. They all work by reducing the surface tension and thus suppressing the
foam. In automatic control systems, sensing probes are inserted at a suitable distance
93
above the working level. As soon as the foam touches this probe the electrical circuit
closes and this activates systems for the introduction of chemical antifoams.
9.5 BASIC VARIABLES FOR MONITORING FERMENTATION
The most important variables that need control during fermentation are:
1. Temperature
2. Agitation rate
3. Aeration rate
4. Dissolved O2 activity
5. pH
6. O2 and CO2 partial pressure in the exhaust gas
9.5.1 MEASURING / SENSING DEVICES
Temperature
Thermocouples are the cheapest but do not have resolution. Resistance Temperature
Detector (RTD, made of platinum) is probably the best for critical applications but it
is very expensive. The principle involved in the latter instrument is that electrical
resistance changes with change in temperature. The accuracy is ± 0.25%.
Aeration
Rotameters and thermal mass flow meters are used for monitoring the supply of air.
Rotameter is a very simple air flow measuring device. It consists of a vertically
mounted glass tube with an increasing bore and enclosing a free-moving float (Fig.
9.11 gives an idea about the principle on which the rotameter works). The position
of the float in the graduated glass tube is indicative of the flow rate.
Float
Graduated,
tapered glass tube
Air flow
Fig. 9.11 Working principle of rotameter
94
Agitation rate
The rate of rotation is sensed by tachometers
Dissolved O2
Electrodes are used for the continuous measure of dissolved O2. Oxygen selectively
diffuses through the membrane and produces signal. The current is directly
proportional to the activity of O2 in the broth but not to the concentration.
Pressure
Normally, pressure is measured by Bourdon tube gauges, which in turn are of

different makes.
pH
Electrodes of various types can be used for the continuous measurement of pH.
9.6 FERMENTER SCALE UP
Determination of optimum condition for fermentation, e.g., medium requirements,

aeration, agitation, temperature, pH, duration, etc., using a microbe producing a
metabolite is called process development. Consideration of cost, labor, time, and
space make it mandatory to carry out the process in 3 distinct stages: (i) laboratory
process using flasks, (ii) scaling up using small to medium fermenters and, finally,
(iii) production scale fermentation experiments.
The information gained from small fermenter is used to predict/determine the

proper fermentation conditions for the large fermenters; this is called scale-up of the
process. The use of small fermenters saves cost (media, etc.), labor, time, etc., allows
replicated studies and keeps the production fermenters free for operations.
However, the information generated from small fermenters is not entirely applicable
to production fermenters. As a result, some production-scale experiments are always
required at the time of process development and later during production. In such
cases, a pilot fermenter may be included.
Scale up is extremely important yet one of the most complicated aspects of industrial
microbiology. An understanding of the problems of scale up is extremely important
because rarely does a microbial process behave the same way in large-scale
fermentation as in small scale laboratory equipment. In industrial fermentation,
mixing and aeration becomes complex. As the size of the fermenter is increased, the
surface-to-volume ratio also changes. Scale up of an industrial process is, therefore,
the task of the biochemical engineer, who is familiar with gas transfer, fluid
dynamics, mixing, and thermodynamics.
95
CHAPTER 10
BASICS OF ENZYME TECHNOLOGY
10.1 INTRODUCTION
Enzymes are biocatalysts that catalyze (govern, initiate and control) biochemical
reactions with specificity and at a rate compatible with cellular reactions. They are
produced by living cells but can act independently of cell if appropriate
environmental conditions are created. Almost all enzymes are proteins. As of now,
however, partly due to the discoveries of enzyme-like compounds that defy the
conventional concept of enzymes in properties and makeup, other newer terms have
evolved. To this end, several non-protein molecules can also carry out reactions
similar to enzyme-catalyzed reactions. Some of these molecules are ribozymes,
synzymes, etc. There are also quite a few non-traditional enzymes, e.g., extremozymes,
abzymes, etc. A brief treatment of the abovementioned new enzymes will shortly
follow but it must be noted here that some enzymes are active only when coenzymes
(cofactors) are present. Such incomplete enzymes are called apoenzymes. The fully
functional form (after combining with coenzyme) is then termed holoenzyme. Almost
all vitamins of the B group function as coenzymes. The illustration of coenzyme-
requiring enzyme is given in Fig. 10.1.
Apoenzyme + Coenzyme = Holoenzyme

Non-functional Non-functional Functional
Fig. 10.1 Combination of apoenzyme and coenzyme to make holoenzyme
It must be clear from the very beginning that an enzyme cannot initiate a reaction that
cannot occur spontaneously. It also does not alter the overall free energy change of the
reaction.
10.1.1 ABZYMES
Abzymes are antibodies that function as enzymes. They are also called catmab
(referring to catalytic monoclonal antibodies). Antibodies, by definition, have
evolved to recognize and bind to the ground states of the molecules they are specific
to. In contrast, enzymes have binding sites that preferentially bind to the transition
state of their substrate molecules. A catalytic antibody is produced in response to
molecules that have a structure similar to the proposed/expected transition state of
the reaction to catalyze which the antibody is sought.
Abzymes are usually artificial constructs but naturally occurring abzymes have also
been observed in normal individuals (e.g., anti-vasoactive intestinal autoantibodies)
and individuals with autoimmune problems.
10.1.2 RIBOZYMES
These are RNA molecules that have catalytic power. Ribozymes are so far known to
catalyze only two reactions: (i) cleavage of RNA, and (ii) cleavage of DNA. The catalytic
power of ribozymes is due to their 3-D structures, which are able to generate in
them substrate-specific binding sites.
10.1.3 EXTREMOZYMES
These are enzymes that function optimally only under extreme conditions of
temperature, pH, etc., e.g., DNA polymerase from Pyrococcus furiosus that has half-life
of 20 hrs at 95°C and functions optimally at 90°C.
10.1.3 SYNZYMES
These are generally synthetic polymers, sometimes proteins, which have enzymatic
activities. Synzymes must possess two functional sites: one for substrate binding and
the other for catalysis. Cyclodextrin is a non-protein molecule in which 6, 7, 8, 9, or 10
α-1,4-linked D-glucose residues are joined head to tail in a ring (called α-, β-, γ-, δ-, and
ε-cyclodextrins respectively). When pyridoxal coenzyme is attached to C6 hydroxyl
group of β-cyclodextrin, it acts as a natural transaminase.
10.2 CLASSIFICATION AND NOMENCLATURE OF ENZYMES
Enzymes can be classified according to various schemes, such as:
1. Substrate acted upon
Table 10.1 Examples of enzymes whose names are derived from the substrate acted on
Substrate Enzyme
Protein Protease (proteinase)
Carbohydrate Carbohydrase
Lipid Lipase
Penicillin Penicillinase
Sucrose Sucrase
Polyphenol Polyphenolase
97
2. Type of reaction catalyzed
Table 10.2 Examples of enzymes whose names are derived from the reaction they
catalyze
Reaction type Enzyme

Isomerization Isomerase
Oxidation Oxidase
Dehydrogenation Dehydrogenase
Hydrolysis Hydrolase
Aldolization Aldolase
Transamination Transaminase
3. The IUB system
This system of classification takes into account the overall chemical reaction. Although
complicated, the IUB (International Union of Biochemistry, 1961) system is precise,
descriptive, and informative. Enzymes are classified and named by the Commission on
Biochemical Nomenclature. All the enzymes are classified into 6 groups; subclasses
also occur. Each enzyme is given a code number, the interpretation of which gives
many details regarding the enzyme. An enzyme may be denoted in one of the
following three accepted ways: (i) a four-number code following the letters EC (for
enzyme commission), e.g., EC 3.2.1.26 (this is for invertase: the first number refers to
enzyme class, the second to subclass, the third to sub-subclass, and the fourth to serial
number of the enzyme within a subclass), (ii) its systematic name based on the above
classification, e.g., β-D-fructofuranoside fructohydrolase for what is commonly called invertase
or even sucrase, or (iii) its recommended name, e.g., β-D-fructosidase (for invertase, that is).
The classes and their examples appear in Table 10.3.
Table 10.3 Classes and examples of enzymes (IUB system)
S.N. Class Common example

1 Oxidoreductase Dehydrogenase, oxidase, oxygenase
2 Transferase Transaminase, kinase
3 Hydrolase Lipase, peptidase, phosphatase
4 Lyase Decarboxylase, hydratase
5 Isomerase Phosphohexoisomerase, mutase
6 Synthetase/Ligase Acetyl-ScoA synthetse, glutamine synthetase
Hydrolases are the most commonly used enzymes, accounting for nearly 80% of all
commercially produced enzymes. A major share of it constitutes proteases (used in
detergent, dairy, meat and leather industry) followed by carbohydrases (28%), and
98
lipases (5%). The usage of enzymes in various industries is as follows: detergents
(34%), dairy-related uses (14%), starch processing (12%), textile applications (11%),
beverages and brewing (7%), animal feed (7%), bakery (5%), and others (9%).
10.3 CATALYTIC POWER OF AN ENZYME
The catalytic power of an enzyme is measured in terms of activity. Some of the terms
used for this are turnover number, enzyme unit, and specific activity.
1. The turnover number is defined as the number of substrate molecules

converted into product per unit time when the enzyme is fully saturated
with substrate. The values of turnover vary widely with different enzymes
and depend on conditions in which the reaction is taking place. However,
for most enzymes, the turnover numbers fall between 1-104/s. The turnover
number of 6105 per sec for carbonic anhydrase is one of the largest
known.
2. The enzyme unit is the amount of enzyme which will catalyze the
transformation of one mole of substrate per minute under defined
conditions.
3. Specific activity is expressed as units of enzyme per milligram of protein.
10.4 FACTORS AFFECTING ENZYME ACTIVITY
The main factors that affect enzyme-catalyzed reaction are (i) temperature, (ii) pH,
(iii) enzyme concentration, (iv) substrate concentration, (v) presence of inhibitors, (vi) presence of
allosteric affectors, (vii) covalent modification, (viii) metal activators, and (ix) redox potential.
Although a full discussion on the above factors is beyond the scope of this book, a
brief treatment is given in the following paragraphs.
10.4.1 EFFECT OF TEMPERATURE
Enzymes are sensitive to temperature changes. They have their own optimum
temperature at which the catalytic activity is maximum. The optimum temperature for
most enzymes, with certain exceptions, is around 40°C. At low temperatures the
reaction is slow; and at higher temperatures, the enzymes, being protein, get denatured
and cease to function. The effect of temperature (hypothetical) is shown in Fig. 10.2.
Optimum
temperature
Optimum pH
Temperature pH
Fig. 10.2 The effects of temperature and pH on enzyme activity
99
10.4.2 EFFECT OF pH
The pH changes profoundly affect the ionic character of the amino- and carboxylic-
groups on the enzyme molecule and therefore markedly affect the catalytic site and
conformation of an enzyme. Without an appropriate conformation, an enzyme
cannot function as desired (see later). Besides, low or high pH values can cause
considerable denaturation and hence inactivation of the enzyme. Like temperature,
the pH should also be in optimum range for maximum activity (Fig. 10.2).
10.4.3 EFFECT OF ENZYME CONCENTRATION
As is true of any catalyst, the rate of an enzyme-catalyzed reaction depends directly on

the concentration of the enzyme. Provided that there is sufficient amount of substrate,
increase in concentration of the enzyme will yield a first order kinetics (Fig. 10.3)
10.4.4 SUBSTRATE CONCENTRATION
With a fixed enzyme concentration, an increase in substrate concentration will at

first result in a very rapid rise in the velocity of reaction rate. As the substrate
concentration continues to increase, however, the increase in the rate of reaction
begins to slow down until, with a large substrate concentration, no further change in
velocity is observed. This is because at high substrate concentrations the active sites
of the enzymes (see later) are completely filled (saturated) and can catalyze no more
reaction than their full capacity.
3x
2x
1x
Time of reaction
Fig. 10.3 Effect of enzyme concentration on reaction rate
The mathematical equation that defines the quantitative relationship between the
rate of enzyme-catalyzed reaction and the substrate concentration for simple system
and thus fulfils the requirement of a hyperbolic curve is the Michaelis-Menten
equation:
Vmax  S 
v
Km  S 
100
Where, v = observed velocity (reaction rate) at a given substrate concentration,
[S] = substrate concentration at any given instant (expressed in moles/L), Km =
Michaelis-Menten constant (expressed in moles/L), and Vmax = maximum velocity
of reaction (at saturating concentration of the substrate). The hyperbolic curve due
to Michaelis-Menten equation appears in Fig. 10.4.
10.4.5 EFFECT OF INHIBITORS
A number of compounds have the ability to combine with certain enzymes, but do
not serve as substrates. These compounds therefore block catalysis by the enzyme.
Such compounds are called inhibitors. Inhibition in enzyme-catalyzed reactions can be
grouped into three broad types: (i) competitive inhibition, (ii) non-competitive inhibition, and
(iii) uncompetitive inhibition, a brief treatment of which shortly follows.
Maximum velocity, Vmax
Zero order kinetics

(Phase II)
V/2 Mixed order kinetics
1st order kinetics

(Phase I)
Km Substrate concentration [S]
Fig. 10.4 Effect of substrate concentration on reaction rate
10.4.5.1 Competitive inhibition
In this category, the inhibitor has structural analogy with substrate of the enzyme
and thus competes with it in order to bind to the active site (see later) of the enzyme.
This inhibition is also called substrate analog inhibition. The enzyme can therefore
combine with either the substrate or with the inhibitor and following equilibrium
may exist:
ES E+P
EI
Where, I = inhibitor, and S = substrate. The enzyme involved in the formation of EI

complex cannot function as a catalyst; only ES (enzyme-substrate complex) will
allow the formation of the reaction product.
The phenomenon can also be related to the Michaelis-Menten constant, Km.

Consider a typical enzyme-catalyzed reaction:
101
K1 K

E  S  ES  E  P
3
K2
Where P = product, and other notations carry usual meanings. The ratio of
dissociation of ES to its formation is given by:
Dissociation of ES K 2  K3
Km  
Formation of ES K1
We can thus see from the initial equilibrium that Km value increases in the presence
of inhibitor as some of the enzymes are being simultaneously utilized for the
formation of EI complex. This EI reversibly breaks down to E and I producing an
effect equivalent to the dissociation of ES.
The maximum velocity, on the other hand, will remain the same. Thus, succinic acid,
which is readily oxidized to fumaric acid by succinic dehydrogenase, is competitively
inhibited by malonic acid because the structure of malonic acid closely resembles
that of succinic acid. See below for the structural analogy of succinic and malonic
acid.
COOH
CH2 COOH
CH2 CH2
COOH COOH
Succinic acid Malonic acid
The inhibition, however, can be reversed by increasing the concentration of the

substrate, succinic acid. The proportion of enzyme molecules combining with the
inhibitor (and therefore competitive inhibition) depends on: (i) substrate concentration,
(ii) inhibitor concentration, and (iii) affinity of the enzyme for the substrate and the inhibitor.
Competitive inhibition exists for all enzymes. A non-metabolic structural analog is
generally a competitive inhibitor.
Numerous practical applications are based on competitive inhibition, particularly in

chemotherapeutics: fight against bacteria, control of weeds, parasites, etc. The basic
idea is to inhibit an enzymatic reaction which is of capital importance in
microorganisms being fought. A classic example is that of sulfamides, analogs of para-
aminobenzoic acid (a compound indispensable to many bacteria - but not to man -
for the synthesis of folic acid).
NH2 NH2
COOH SO2NH2
p-amino benzoic acid Sulfanilamide
102
The sulfamides compete with para-aminobenzoic acid for the active site of the
bacterial enzyme catalyzing the transformation of this derivative into folic acid. This
explains the bacteriostatic effect of sulfamides.
Another beautiful example of competitive inhibition is that of methanol on alcohol

dehydrogenase. Oxidation of methanol produces formaldehyde, which is toxic to all
biological tissues. This accounts for the toxic effect of methanol. As an antidote,
ethanol is administered to the methanol-poisoned patient. Ethanol competes with
methanol for the active site of alcohol dehydrogenase. Methanol will be slowly
excreted away.
10.4.5.2 Non-competitive inhibition
In this, the inhibitor compounds bind either to the enzyme (but to the site other
than the active site), or to the ES complex (to form ESI complex), or to both. But
the inhibitors are not displaced by increasing the substrate concentration; the
inhibition is irreversible. Evidently, enzyme from the inhibitor-enzyme complex
cannot be available any more so as to form the final product. This leads to decrease
in Vmax and can even become zero on total inhibition. The Km value, however, is
unaltered.
A good example is the reaction of iodoacetamide on triose phosphate dehydrogenase, a

sulfhydryl enzyme.
Enzyme ~ SH + ICH2CONH2 Enzyme ~ SH + CH2CONH2 + HI

(Iodoacetamide) (Enzyme-acetamide complex)
Many drugs are based on this principle. Thus, penicillin blocks the cell wall synthesis
of bacteria by preventing bridge formation between N-acetylmuramic acids.
A highly dangerous nerve poison diisopropyl fluorophosphates works by inhibition of

acetyl cholinesterase, the enzyme immediately associated with nerve function.
One potential use of non-competitive feature of inhibitors in enzymatic reactions is

that they can be added to a reaction mixture to rapidly reduce or arrest the reaction
when it has proceeded to the desired stage.
10.4.5.3 Uncompetitive inhibition
Some inhibitors can combine reversibly with ES complex only. They are therefore
called uncompetitive inhibitors. These inhibitors have no affinity for substrate alone.
They also bear no resemblance to substrate and therefore yield no products(s). The
reaction can be shown by:
I ESI
 ES 
E  S 
Where, the notations carry usual meanings.
103
This type of inhibition is found in multisubstrate reactions. Both Km and Vmax are
altered but the slope is the same as that of an enzymatic reaction free of inhibitors.
Experimentally, the value of Km is generally obtained from a graph known as
Lineweaver-Burk plot. It is a reciprocal plot and uses rearranged form of Michaelis-
Menten equation given below:
1  Km  1 1
  
v  Vmax   S  Vmax
The nature of graphs for various inhibitions can be shown as in Fig. 10.5.
Non-competitive inhibition
Competitive inhibition
No inhibition
1
Vmax1 1
Vmax2
Uncompetitive inhibition
1
Vmax3
1
[S]
Fig. 10.5 Reciprocal plots of v and [S] in the presence of different types of inhibitors
10.4.6 EFFECT OF ALLOSTERIC AFFECTORS
All allosteric enzymes have quaternary structures. In addition to catalytic sites where the
substrates bind, these enzymes have one or several allosteric sites, which can be
located on a different polypeptide chain. The allosteric affectors (activators or
inhibitors) need not have structural analogy with the substrate. A distinct feature of
this enzyme is that it does not exactly exhibit Michaelis-Menten kinetics. This is
principally because the enzyme is not a simple one that is subject to inhibitions as
described before. The kinetics of allosteric enzymes in the presence of allosteric
affectors is shown in Fig. 10.6.
Allosteric activator or inhibitor does not change the Vmax. Only the Km values are
different. When an allosteric activator binds to the allosteric site, there results a slight
modification of the conformation of the enzyme – called allosteric transition
(reversible) – which changes the conformation of the catalytic site. The site, in
general, acquires a conformation more favorable to the binding of substrate; the
affinity of the affected enzyme for the substrate increases (Km’' < Km’). Even the
shape of the curve can change from sigmoid (a characteristic of allosteric enzyme) to
the hyperbolic form. The binding of allosteric affectors that activate or increase
substrate-binding capacity of enzymes is called cooperative binding. If the activator is
the substrate itself, for example, as in the case of hemoglobin-oxygen reaction, it is
called a positive homotropic response.
104
Vmax
Vmax
2
2 1 3
1 In absence of allosteric affector
2 In presence of allosteric activator
3 In presence of allosteric inhibitor
Km' Km'' Km'''
Fig. 10.6 Kinetics of reaction catalyzed by an allosteric enzyme
When the activator is a compound other than the substrate itself, the response is
called heterotropic (positive) response. Hemoglobin is therefore a homotropic enzyme.
Phosphofructokinase, which uses AMP as an activator, is a heterotropic enzyme. See
Fig. 10.7 for a simplified explanation of allosteric transition caused by allosteric
activator.
Active site with a

conformation not Active site adapted to
favorable for the Substrate
the substrate and
binding of substrate, S (not bound)
having bound it
A Activator S
Allosteric activator
Vacant allosteric site bound to the
allosteric site
Fig. 10.7 Allosteric transition caused by allosteric activator
When an allosteric inhibitor binds to allosteric site, an allosteric transition (also

reversible) takes place causing a change in the active site, which takes a conformation less
favorable to the binding of substrate. The affinity of the enzyme for the substrate
decreases (Km''' > Km', Fig. 10.7). The curve in this case is a true sigmoid. The Vmax,
however, remains unchanged. Although most allosteric inhibitors are competitive by
nature (Km changed, Vmax unchanged, and reversible), non-competitive inhibitions also
occur. In this case, two or more affectors may compete for the same allosteric site(s). See
Fig. 10.8.
Allosteric enzymes play a very important role in metabolic regulation, which occurs
through allosteric controls. The sigmoidal curve denotes response which acts, in a sense, as
an off-on switch and so provides a much more sensitive control than the hyperbolic
response.
105
Substrate, which
Active site having can no longer be
bound substrate bound to active site
S
Inhibitor
S Active site with
conformation no longer
favorable to the binding
Vacant allosteric site of substrate, S
Allosteric inhibitor having
bound to allosteric site
Fig. 10.8 Allosteric transition caused by allosteric inhibitor
10.4.7 EFFECT OF COVALENT MODIFICATION
The activity of numerous enzymes is controlled, not by formation of complexes between

enzymes and regulatory molecules, but by covalent modification of the enzyme.
Some monomeric enzymes, particularly those responsible for protein digestion (pepsin,
trypsin, chymotrypsin, etc.), are synthesized in catalytically inactive forms (called
proenzymes or zymogens). It would be extremely damaging to cells if they were
synthesized in active form. Only after they reach the intestine that they are converted
into active forms. This conversion consists of cleavage of one or several peptide
bonds. The cleavage initiates a reorganization of the spatial structure of the enzyme
and the specific hydrolytic reaction occurs.
10.4.8 EFFECT OF METAL ACTIVATORS
The rates of enzyme-catalyzed reactions are altered by certain ions which behave as
activators. A large number of enzymes requiring nucleoside di- and triphosphates
invariably need divalent metal ions like Mg+2 or Mn+2, which are occasionally
replaceable. Many enzymes require monovalentpage cations, usually Na+, K+, or
NH4+ for maximum catalytic activity. Amylases require Cl¯ ions for activity while
carbonic anhydrase, chymotrypsin and alkaline phosphatase require Zn+2. Usually,
these ions interact with the substrate so that the latter’s binding with the active site
will be most favored. See Fig. 10.9 for example of involvement of metal ions. If
metals form only loose and easily dissociable complexes and can easily release the
metal without denaturation, the enzymes are called metal-activated enzymes. Those
enzymes that do not release metals easily are called metalloenzymes. Some of the
important and familiar enzymes that need metals for catalytic action are enolase,
carboxypeptidase, hexokinase, glutamine synthase, etc.
O O O
Ribose Adenine ~ O P O P O P O
O O O
Mg+2
[ATP Mg]-2 complex
Fig. 10.9 The involvement of Mg+2 in favoring substrate binding

106
10.4.9 EFFECT OF REDOX POTENTIAL
Many enzymes are sensitive to oxidizing or reducing agents. The oxidizing or

reducing ability of an enzyme is measured in terms of redox potential. The redox
potential is the electromotive force, measurable in millivolts developed by the
solution when in physical contact with the platinum electrode as compared to
normal hydrogen electrode at zero potential. The redox potential of an enzyme is
either negative or positive owing to its relative reducing or oxidizing ability in
comparison to hydrogen.
10.5 MECHANISM OF CATALYSIS
The mechanism of enzyme-catalyzed reaction is a composite description of all the

events that take place at a molecular and atomic level, from the initial binding of the
substrate to the release of the product.
The essential steps involved in an enzyme-catalyzed reaction are: (i) binding of substrate
to the enzyme, (ii) conformational changes of enzyme or substrate or both as a result of binding, (iii)
changes in chemical bonding by way of transition states and intermediates, (iv) further
conformational changes on formation of products, and (v) release of product to the solvent.
Enzymes reduce the overall activation energy, denoted by ∆G. Even the modest
reduction in the value of ∆G leads to a very large increase in the reaction rates. For
example, ∆G of an uncatalyzed breakdown of H2O2 into water and oxygen is 76
kJ/mole. The enzyme catalase requires only 30 kJ/mol for the same reaction. This
lowering of energy is enough to yield 9108-fold increase in the reaction rate, enough
to reduce the time from years to seconds.
An oversimplified summary of the mechanism of an enzyme-catalyzed reaction is

given in the following paragraphs:
Binding of substrate is probably the most important step in enzyme catalysis. An

enzyme is a unique polypeptide which folds in the solution in a defined 3-D structure.
Usually the polar amino acids orient outwards and come in contact with the aqueous
medium. Non-polar amino acids are found in the interior side. All enzymes have
unique 3-D site(s) for binding substrate(s) in specific orientation. The site is called active
site (catalytic site). To this end, some enzymes have been categorized as serine enzymes,
lysine enzymes, sulfhydryl enzymes, according to the essentiality of specific amino acid
residues like serine, lysine, or cysteine-SH, respectively, at their active sites.
Substrate molecules are comparatively much smaller than the enzyme molecule. For
example, consider invertase of molecular weight 127,000 against its substrate sucrose
of molecular weight mere 342!
The side chain groups of the amino acids, e.g., -NH2, -COOH, -CH2OH, etc., serve
as the catalytic group. Not all the amino acids involved function alike. One category
constitutes the specificity site and enables recognition of the substrate to enable
reaction. The second category of amino acids participates in the chemical
transformation of the substrate. These two categories form the catalytic part of the
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active center. Other participating amino acids are necessary for maintaining the
adequate conformation of the allosteric sites (which control functioning of active
site) and adequate positioning of the enzyme within the cells (for example,
association of the enzyme with another enzyme to form a multienzyme complex).
The configuration of the active center, according to modern hypotheses and

evidences, is not rigid. It has great flexibility. The association of the substrate with
the active center induces very regularly a change in conformation which results in
the induced-fit or induced-adjustment of the structure of the active center. The side
chains, which participate in the functioning of catalytic part of active center, change
conformation during attachment of the substrate to form the familiar enzyme-substrate
complex, but also very often during catalytic transformation of the complex to form
reaction products. It is quite obvious that any event leading to the change in natural
conformation of the active center will lead to inactivation of the enzyme. The
enzyme-substrate complex is formed mainly by non-covalent bonds, such as hydrogen
bond, electrostatic bond, Van der Waal force, and hydrophobic interaction. Enzymes such as
transaldolase use covalent bondings also.
Chemically, the enzymatic catalysis involves interactions between functional groups

of substrate and enzyme molecules. This is in fact the central stage of reaction. It is
difficult to generalize the events but in essence, amino acid side chains may act as
acids or bases and thereby catalyze reactions. Electrons may be transferred during
the course of reaction. Covalent and other bonds are formed for the reaction to
occur but are later broken to form reaction products. Once the product is formed,
the enzyme again changes its conformation to allow release of the product. Although
the description is rather mechanical and straightforward, the chemical explanation,
again, is quite complicated. Refer to allosteric regulation of enzymes (Fig. 10.7 and
10.8) also.
10.6 ENZYME KINETICS
Enzyme kinetics is the study of reaction rates of enzymes. It is an indispensable part

of enzymology. Enzyme kinetics has dual purpose, viz., to understand the normal and
abonormal metabolism of organism as a whole, and to elucidate the nature of enzyme process itself.
Because of the complexity of the nature, property and function of enzymes,
however, no single kinetics can characterize all enzymes. In the following paragraphs
is described a classical treatment of enzyme kinetics (for steady state) due to
Michaelis and Menten (and therefore called Michaelis-Menten equation). The equation
is derived employing certain assumptions and using Briggs and Haldane derivation
for steady-state kinetics. The equation yields a saturation hyperbolic curve typical of the
simplest, single-substrate-single displacement enzyme reaction.
The basic assumptions used in the derivation are: (i) only a single substrate and a single
product are involved, (ii) the process proceeds essentially to completion, (iii) the concentration of the
substrate is much greater than that of the enzyme in the system, (iv) an intermediate enzyme-substrate
complex is formed, and (v) the rate of decomposition of substrate is proportional to the concentration of
the enzyme-substrate complex.
108
The steps for the derivation are as follows:
A typical enzyme-catalyzed reaction involves the reversible formation of an enzyme-

substrate complex, ES, which eventually breaks down to form enzyme, E, again and
the product, P.
K1 K

E  S  
 ES 
3

EP
K 2 K 4
Where, S is the substrate, K1, K2, K3, and K4 are the respective reaction rate
constants.
A few milliseconds after the enzyme and the substrate have been mixed, a
concentration of ES builds up and does not change as long as S is in large excess and
K1 >>K3. This condition is called the steady state of reaction, since the rate of
decomposition just balances the rate of its formation. We can therefore safely write,
for steady state:
Rate of formation of ES = Rate of decomposition of ES or,
K1[E][S] + K4[E][P] = K2[ES] + K3[ES]

 ES  K1 S  K 4  P        (i)
[E] K 2  K3
However, at an early stage of reaction, the rate of formation of the product, P is very
small; the rate of formation of ES from E + P is even smaller. This would enable us
to safely rewrite the initial equation:
K1 K

E  S  ES  E  P
3
K 2
Therefore, using K4 [P] ~ 0 in equation (i), we have,
 ES  K1 S
[E] K 2  K3
Using Km = (K2 + K3)/ K1, and rearranging,
 E   K m        (ii)
 ES S
But [E] and [ES] are not measurable values. We can resolve equation (ii) if we
consider that the total enzyme concentration [E]t in the reaction consists of free
109
enzyme [E], and the enzyme-substrate complex, [ES]. The free enzyme
concentration is, therefore,
 E   Et   ES        (iii)

Using (iii) in equation (ii),
Et  ES Km

ES S
 E t K m
  1        (iv)
 ES S
Since the terms cannot still be readily measured, we can take the help of following
relation: the maximum velocity, Vmax, is attained when the total enzyme, [E]t, is
completely complexed with saturating amounts of S, or
Vmax   Et
Moreover, the initial velocity, v, is proportional to the enzyme present as ES

complex at a given concentration of S, or,
v   ES
Vmax  E t
         (v)
v  ES
Using relation (v) in equation (iv),
Vmax Km
  1
v S
Vmax S
v        (vi)
K m   S
Equation (vi) is the required Michaelis-Menten equation. The graphical

representation is given in Fig. 10.4.
110
10.6.1 UNIT AND SIGNIFICANCE OF Km
If we choose v = half of Vmax,
Vmax Vmax S


2 K m  S
Km  S
This shows that the unit of Km is the same as that of [S], i.e., moles/L. It can also be
seen that Km represents substrate concentration, [S], at v = ½ Vmax
Km can be defined in many ways. It can be considered as substrate concentration

when the reaction rate is half of the maximum rate. That is, Km = [S], when v = ½
Vmax. Also, since Km = (K2 + K3)/K1, it is a ratio of complex dissociation to complex
formation rate. Evidently, at Km values greater than unity the dissociation of ES
complex dominates.
Km signifies many important meanings, some of which are:
1. If Km is known, the fraction of sites filled, fES, at any substrate

concentration can be calculated from the following equation:
fES 
v

S
Vmax K m  S
2. High Km values indicate weak binding between E and S to form ES. Low Km
values indicate strong binding of E and S. This is particularly true if K2 >> K3,
which means that the product formation is negligible. Equilibrium exists
between E+S and ES and Km will equal dissociation constant.
3. Km values can be used to identify whether a given enzymatic reaction is free
from inhibitions, as also the type of inhibition.
4. Km values can be used to predict Vmax of a reaction at a given substrate
concentration. Under practical condition, the observed velocity, v, becomes
Vmax at [S] ≥ 100Km. The reaction is then independent of [S] and exhibits
zero order reaction. The basic assumption is that at [S] >>Km:
Vmax S
v becomes
K m   S
Vmax S
v
 S
 v  Vmax
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5. Km value can be used to evaluate dependence of v on [S]. Under practical
conditions, this relation can be obtained at [S] ≤ 0.01Km. The basic
assumption is that at Km >> [S],
Vmax S
v becomes
K m   S
Vmax S
v
Km
 v  S 
The reaction is then of 1st order.
The Km values of enzymes differ greatly. However, for most enzymes, the general
range is between 10-1 and 10-6 mole/L. Km values are not absolute constants; they
depend on the source of enzyme, environmental conditions such as temperature,
ionic concentration, and particular substrate.
10.6.2 DETERMINATION OF Km Value
Km of an enzyme-catalyzed reaction is determined usually by graphical methods such

as Woolftees plot, Lineweaver-Burk plot, etc. For reasons of simplicity and sufficient
accuracy, the Lineweaver-Burk plot is more extensively used. In this method, only a
small number of experimental points are required. Furthermore, Vmax can be readily
evaluated by extrapolation. The plot is also called Double reciprocal plot. The plot is
actually a rearranged form of Michaelis-Menten equation:
Vmax S
v
K m   S
1 K m  S
 
v Vmax S
1 K  1 1
  m  
v  Vmax  S Vmax
The last equation is in the form of y = mx + c; 1/v and 1/[S] represent variables y
and x respectively; Km/Vmax and 1/Vmax represent constants m and c respectively (see
Fig. 10.10).
112
1 Km
Slope, m =
v Vmax
1 1
Km Vmax
1
[S]
Fig. 10.10 A typical Lineweaver-Burk plot
The experimental results are plotted as follows: a double plot of 1/v values on the
ordinate and 1/[S] values on the abscissa is made. A straight line is obtained from
which the value of Km is calculated. While plotting, to obtain greater accuracy, the
line is extended to a point where 1/v = 0. It may be noted, mathematically 1/v = 0 is
impossible. The line is further extended to a point where 1/[S] = 0. These
manipulations are required because, under practical condition, the value of 1/[S]
never touches the ordinate. For, this would mean that 1/[S] = 0, which is
mathematically impossible.
Note that at 1/v = 0, 1/[S] = - 1/Km, from which the value of Km can be calculated.
Similarly, for Vmax, we can use 1/[S] = 0 from the graph, in which case Vmax = v.
This is equivalent to saying that at infinitely high substrate concentration, v = Vmax,
which has already been stated at the very outset.
10.7 GENERAL PROPERTIES OF PROTEIN ENZYMES
In brief, to suit the context, properties of protein enzymes can be stated as follows:
 They are proteins

 They exhibit specificity in reaction
 They are biocatalysts
 Their activities can be controlled
The very proof for their being protein is the presence of amino acid residues. With
some exceptions, all protein enzymes are globular in structure. Like proteins, they all
have primary-, secondary-, tertiary-, and sometimes, quaternary structures. They get
denatured by agents that denature proteins. A very remarkable feature of enzyme
makeup is the diversity they exhibit with respect to organization/arrangement. Thus,
based on the degree of association and function, protein enzymes are grouped into
(i) monomeric enzymes, (ii) oligomeric enzymes, and (iii) multienzyme complex.
Monomeric enzymes are the simplest in makeup. They consist of a single

polypeptide. Some of the familiar examples of monomeric enzymes are pepsin, trypsin,
etc.
113
Oligomeric enzymes contain at least two and as many as 60 or more polypeptide
subunits. Oligomeric enzymes have a wide range and diversity of functions. Some of
the important categories of oligomeric enzymes are: isozymes (e.g., lactate
dehydrogenase), allosteric enzymes (e.g., aspartate transcarbamylase), bifunctional enzymes
(e.g., tryptophan synthetase in E. coli), etc.
A distinction needs to be made between oligomeric enzymes and multienzyme

complex. Multienzyme complexes are actually aggregates of a number of enzymes.
They are all engaged in a sequential series of reactions in the transformation of
substrates into products(s). The enzymes are tightly associated and all attempts to
dissociate them lead to complete inactivation of the enzyme. In essence,
multienzyme complex consists of an organized mosaic of enzymes in which each of
the components is so located as to allow effective coupling of the individual reaction
catalyzed by these enzymes. An excellent example of multienzyme complex is the
one involved in oxidation of pyruvic acid to acetyl-SCoA and CO2. The enzyme has
a molecular weight of about 4 million and consists of three separate catalytic
activities, viz., that of pyruvic dehydrogenase, dihydrolipoyl transacetylase, and a dihydrolipoyl
dehydrogenase. An illustration appears in Fig. 10.11.
NAD+
[FADH2] NADH+H+
CO2
CH3CHOH-TPP [LA] Dihydrolipoyl
Lipoic acid dehydrogenase
Pyruvic Dihydrolipoyl
dehydrogenase transacetylase [FADH]
CH3CO-LAH
Thiamine
CH3 COCOOH pyrophosphate CH3CO-SCoA
(Pyruvic acid) CoA-SH (Acetyl-SCoA)
Fig. 10.11 Oxidation of pyruvate to acetyl-SCoA
One of the most important distinctions between a chemical catalyst and the enzyme
is the specificity. Unlike chemical catalysts, enzymes are very specific in catalyzing
reactions. They can unequivocally select substrates or reactions, which is not
possible in chemical catalysis. Specificity may be observed on the one hand in the
type of reaction catalyzed and on the other hand in the substrate for reaction.
Enzymes also have the ability to distinguish between isomers.
10.8 MECHANISM OF ENZYME BIOSYNTHESIS
Metabolism is principally regulated by a change in the rate of enzyme reaction, which

in turn is controlled at the levels of transcription, translation, and post-translation. Enzyme
concentration is varied by two mechanisms, viz., controlled synthesis, and controlled
degradation.
Enzyme synthesis is controlled by induction or repression. Many of the enzymes

produced/used commercially are of inducible type. The biosynthesis of inducible
enzymes is triggered only when the corresponding substrate (the inducer) is
114
presented to the organism. Often, inducers are analogs or derivatives of substrates,
e.g., IPTG (isopropyl thiogalactoside) for β-galactosidase. Induction occurs at the
level of transcription. The gene is rapidly transcribed and the resulting mRNA
translates for enzyme production.
Repression can be of two types, viz., feedback repression and catabolite repression.
Feedback repression results from the accumulation of end products in concentration
more than needed by the cell. This is called allosteric regulation. In this, the end
product or an intermediate binds to the allosteric site of the enzyme resulting in a
conformational change in the enzyme. This is unfavorable for the enzymatic
reaction. When the end product diminishes, the system works again (the binding is
relieved).
Catabolite repression occurs when an organism is grown in a readily metabolizable

carbon source, glucose as against lactose for E. coli, for example. The repression is at
the level of transcription but the mechanism does not represent an off-on system. It is
meant only for fine-tuning of control. Feedback control, on the other hand is used for
rapid control.
10.9 THE KINETICS OF ENZYME BIOSYNTHESIS
Biochemical processes are extremely complex and sensitive to a number of factors

and rendering their mathematic modeling is most difficult. This is why commercial
fermentation processes have not been significantly optimized in an engineering
sense.
Excellent contributions to the development of kinetic models of enzyme formation

stem from Terui and associates (1967). The represented models refer to hydrolases
produced commercially. They are based on the assumptions that the rate limiting
ability of enzyme forming system corresponds to mRNA and that the specific rate of
enzyme production is proportional to the quantity per cell of mRNA. Hence, for
growth-associated enzyme production the following hypothetical relation was
proposed:
dE d
 a  b k
dt dt
Where, μ = specific growth rate (h-1), k = monomolecular decay rate constant of the
specific mRNA (h-1), a and b = system constants, with b representing rate of growth-
associated repression exerted at the level of transcription, E = growth-associated
enzyme production.
The equation becomes very complex when the enzyme production occurs at
stationary phase.
E  Em e


 k  t  tm 
    e  
K1 e
 t  tm

 k t  tm
E due to the mRNA carried E due to the turnover synthesis

over from the growth phase and degradation of mRNA
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10.9.1 MANIPULATION OF BIOSYNTHESIS
For an industrial production of microbial enzymes, of first and foremost importance

is the selection of suitable strains. They are then progressively improved in
laboratory. A number of methods are available for the improvement, most of then
aimed at overcoming the inherent control mechanisms of the microorganisms over
enzyme synthesis. The techniques are of two main categories: (i) manipulation of
genetic function, and (ii) manipulation of cultural- and process conditions.
10.9.1.1 Manipulation of genetic function
This entails mutation programs for (a) desensitizing controlling enzymes, (b)
producing auxotrophic mutants, (c) enzyme engineering, (d) producing leaky cells, (e)
inserting multiple genes, and (f) mutation to constitutivity.
10.9.1.2 Manipulation of cultural- and process conditions
This entails following approaches: (a) introduction of inducer, (b) mixed culture to
aid removal of end products, (c) use of slowly metabolizable substrates, (d) control
on feed rate, and (e) selective removal of corepressor.
10.10 ENZYMES IN VARIOUS INDUSTRIES
Enzymes are obtained from animal tissues, plants, bacteria and fungi (including
yeast). The bulk of enzymes, both in terms of quantity and variety, are derived from
microorganisms, higher plants being the distant second and animals being the least
important. The only animal enzyme to be produced in quantities greater than
2MT/year is rennin or chymosin obtained from calf stomach. See Table 10.4 for
production data as of 1996.
Table 10.4 World production of enzymes
Enzyme Source Annual production (MT)

Protease Bacillus (bacteria) 550
α-amylase Bacillus (bacteria) 350
Amylase (α and β) Barley malt > 200
α-amylase Aspergillus (mold) 20
Glucoamylase Aspergillus (mold) > 100
Rennet Mucor miehei (mold) 25
Pectinase Aspergillus (mold) 20
Papain Papaya latex > 10
10.11 PRODUCTION OF MICROBIAL ENZYMES
At present, over 2,000 enzymes have been isolated and characterized, about 1000 of
them are recommended for various applications, and about 50 microbial enzymes
116
have industrial applications. Based on the area of use, microbial enzymes can be
classified as (i) fine chemicals, and (ii) industrial enzymes. Fine chemicals are confined to
laboratory use for research purposes and hence require a very high degree of
purification. This is also true if the enzymes are to be used in diagnostic kits, e.g., in
biosensors. Industrial enzymes, on the other hand do not warrant such high degree
of purification, particularly if the enzymes are to be used in an industrial scale.
10.12 PRODUCTION ASPECTS
Microbial enzymes are relatively high-value, low-volume products. In most cases the
fermentation process is largely secondary to downstream processing. This is
particularly true of intracellular enzymes. If a ready source of enzyme is available,
e.g., Aspergillus niger mycelia after gluconic acid fermentation (the mycelia are a rich
source of glucose oxidase), the fermentation process can be totally circumvented.
However, in the case of extracellular enzymes fermentation is obligatory. It must be
emphasized here that adequate attention must be given to the design of the
fermentor. In general, it is advisable to use small, batch-type, stirred-tank, aerated
fermenters designed to allow considerable flexibility in use.
A large-scale enzyme production entails, in the first stage, choice of strains that
produce large amounts of the concerned enzyme. It is also important to note here
that extracellular enzymes are preferred. This is basically because the downstream
processing of intracellular enzymes is very costly. The cells have to be ruptured to
release the enzymes, which again is not easy. The enzymes need extensive
purification because they come contaminated with, among other things, nucleic
acids. Added to these aspects, the enzymes cannot be produced in large amounts
under cellular environment: this is because of the repression and feedback inhibition
encountered by the synthesizer cell. Thus, for the overproduction of enzymes, some
mechanisms must be available for counteracting these inherent feedback inhibition
and repression systems.
The criteria for the choice of strains are:
 Extracellular enzymes are preferred

 High yield of enzyme
 Ability to grow in cheap substrate
 Minimal by-product formation
 Ease of recovery
 Freedom from antibiotic activity
 Non-pathogenic
10.13 ADVANTAGES OF MICROBIAL ENZYMES
The preference of microbial enzymes to enzymes from other sources stems from:
 Normally high specific activity per unit dry weight

 Independent of seasonal variability
117
 Enzymes of a wide range of properties and stability produced
 Microorganisms are easier to manipulate genetically for the improvement
 Due to short generation time, the productivity is comparatively high
 The downstream processing is comparatively simple
 Even whole cells can be used as an enzyme source, which is impossible in
other cases
 Enzyme engineering in microorganisms is both possible and pragmatic
 Some special enzymes, e.g., reverse transcriptase, heat-stable DNA
polymerase, DNA ligase, etc., can be obtained from microorganisms only.
10.14 GENERAL PRODUCTION AND PURIFICATION METHODS
The raw materials for industrial enzyme fermentations have normally been limited to
substances that are readily available in large quantities at low cost, and are
nutritionally safe. Some of the most commonly used substrates are starch
hydrolysates, molasses, corn steep liquor, whey, and many cereals. Microbial
enzymes are produced by two main methods, viz., (i) solid substrate cultivation, and (ii)
submerged cultivation.
10.14.1 SOLID SUBSTRATE FERMENTATION
Solid substrate methods of producing fungal enzymes have long-standing historical

applications, particularly in Japan and Far East countries.
In this method, the microbial growth and product formation occur on the surface of
the solid substrate. The system is suitable for the production of extracellular
enzymes, certain valuable chemicals, toxins, and fungal spores. Some of the enzymes
produced commercially by solid substrate fermentation are given in Table 10.5.
Table 10.5 Enzymes produced by solid-state fermentation
Enzyme Organism (examples) Substrate Function

Cellulase Trichoderma viride Wheat bran Cellulolytic (in detergents, etc.)
Amylase Aspergillus oryzae Rice Saccharification
Protease Aspergillus oryzae Wheat bran Proteolysis
The media in solid-substrate fermentations for enzyme production are mainly based
on wheat bran. This material is particularly suitable because of its high content of
nutrients and large surface area. Other materials such as rice, soybeans, etc., can also
be used but they must first be cracked to suitable size to allow profuse superficial
growth of the relevant organism. The type of enzyme desired also dictates the choice
of the material. The amount of water needed for moistening the substrates is in the
range 30-70%. The process is used largely for molds or other mycelial bacteria, e.g.,
Streptomyces species. It must be noted here, the organisms used in solid substrate
fermentations are highly aerobic and are good producers of extracellular enzymes.
This has to be so because they have to simplify the complex substrate presented to
them before the nutrients can be taken inside the cell for intracellular metabolism. In
118
industrial fermentation, effort is expended to accentuate this natural capability of the
organism. Thus, the medium is often supplemented with some salts or other suitable
components. The pH is adjusted with an acid. The medium is sterilized normally by
autoclaving (with stirring). Fungal spores (or other cultures) are inoculated on the
surface. Aeration is achieved by circulating conditioned air over the surface. The
conditioned air helps maintain the humidity, reduces the temperature, supplies
oxygen, and removes the CO2 generated in the metabolic process. The temperature
should be controlled within narrow limits. This calls for use of appropriate cooling
systems in large fermenters.
The main advantage of solid-substrate process is the low investment required. Also,
being low in moisture compared to submerged process, the enzyme concentration in
the fermented medium is much higher. Of the limitations, the following three may
be mentioned: (i) labor intensive, (ii) requires more space, (iii) greater risk factor.
As such, solid-substrate fermentations are difficult to scale up. Consequently,

fermentation is carried out in small batches. The distribution of heat, air, and
nutrient is also very difficult. Nevertheless, industries have come a long way using
solid-substrate fermentation, which are mainly of two types, viz., (i) Thin Layer
Technique (tray process), and (ii) Deep Bed Process.
10.14.1.1 The thin layer process (cabinet method)
The process works with substrate layer (2-4 cm thick) spread on wooden or metallic
trays. After inoculation with spores, the trays are incubated in air-conditioned rooms
or cabinets (hence cabinet method). The heat produced by the growing culture is
removed by passing moistened, cool air over the surface of the culture. Cooling
systems can also be provided. A typical flow sheet of tray method of enzyme
fermentation is given in Fig. 10.12.
Dilute acid
Nutrients
Mixing Steam cooker/cooler
Bran
Spores Grinder
Conditioned air Extractor

Substrate
Crude
Tray enzyme
solution
Cultivation cabinet Drying tunnel
Fig. 10.12 Thin layer solid state fermentation
119
10.14.1.2 The deep bed process
The deep bed process is a modification of the traditional solid-substrate (= solid-

state) fermentation. The process solves most of the problems associated with
traditional processes. A typical deep bed process uses substrate layers of 2-6 feet.
Deep bed plants are fully automated. The substrate is sterilized by first moistening
with acidified water and then injecting steam to give 95°C for 15 min.
Decontamination of the substrate, e.g., bran, can be carried out using bactericides
such as formaldehyde. Inoculation of the sterilized medium is carried out with
spores in a dry or suspended form. Cooling system coupled with passage of
conditioned air is universal in solid-state fermentations.
10.14.2 SUBMERGED CULTURE
Submerged fermentations were not as widespread until recently. The fermentation

on commercial scale is carried out in continuous stirred tank bioreactor (CSTBR; this is
the same as STF mentioned elsewhere, page 93) of stainless steel with capacities
ranging from 10-15 m3. With enzyme fermentations, the formulation of the
production medium and to a lesser extent, control of fermentation conditions, play
major roles in the success of the process.
The production medium should basically contain an energy source, nitrogen source,
and any special growth requirements (amino acids, vitamins, etc.). However, good
growth is not enough to obtain a high enzyme yield. Inducers may have to be used.
As inducers are rather expensive, it is preferable to use constitutive mutants, which
do not require the inducer. Where catabolite repression is observed, slow feeding in
fed-batch mode (= extended culture method) is desirable. Incremental feeding of slowly
metabolizable sugars is also possible. The presence of certain surfactants in the
production medium increases the yield of certain enzymes. Non-ionic detergents,
e.g., Tween-80, are frequently used. Most enzyme fermentations are carried out at
neutral pH. The change in pH is controlled by adding buffers. An alternative is to
add certain compounds, which upon metabolism, bring about change in pH in the
desired direction. See Fig. 10.13 for the generalized scheme of production of liquid
microbial enzyme and Fig. 10.14 for generalized scheme for extraction and
purification of enzymes.
10.15 GENERAL PROCESS OF ENZYME RECOVERY
The downstream processing of enzyme fermentation can be as simple as drying of

substrate to as involved as chromatographic purification. The complication depends
on: (i) whether crude or high-grade enzyme is to be produced, and (ii) whether the
enzyme is intracellular or extracellular. Whatever the concentration/purification
technique, the fermentation broth is usually cooled to about 4°C immediately after
completion of the fermentation to arrest contamination and degradation of the
enzyme.
The recovery of fungal enzymes is normally straightforward and usually involves

centrifugation or filtration. Bacterial enzymes are more difficult to
concentrate/purify. Typically, bacterial broths are treated with coagulating or
120
flocculating agents such as calcium phosphate to separate the bacterial cell and the
colloids. The cells can later be filtered using 2-3% diatomaceous earth as the body
feed.
Reverse
Agar medium osmosis
Slant
culture
Inoculation Main Drum filter

tank fermentor
Mycelium
waste
Air supply NaCl
Bacterial
filter
Standardization Cool
Filter storage
Packaging
Fig. 10.13 Schematics of stages in the production of a liquid enzyme preparation
When the fermentation is of solid-substrate type (e.g., mold bran fermentation) and
the enzymes are extracellular in nature, the extraction is basically a washing process.
Countercurrent techniques of percolation are the most frequently used unit
operation. In many cases, the mold bran is dried prior to extraction. The actual
extraction may be done on demand. The solvent used for the extraction is
universally water. Certain components, e.g., buffer, salt, etc., may be added to
facilitate extraction or improve stability in solution.
The extent of purification is dictated by the intended use of the enzyme, viz.,
industrial use, food use, and laboratory use. In the industrial category, for economic
reasons of enzyme application, a concentration up to 10-fold is usually satisfactory.
For example, enzyme products employed in detergents contain about 5-10%
protease while amylase preparations for use in flour treatment contain only about
0.1% pure amylase. However, in applications where high purity enzymes are
required, e.g., in enzymic hydrolysis, 1000-fold purification is quite common. In
some applications, such as baking and dextrose manufacture, the presence of
contaminating enzymes must be very low or rigidly controlled.
Crude preparations, although much easier to produce, suffer from the decreased
stability. Since the trend in enzyme applications is towards use of liquid preparations,
stabilization is a very important aspect.
10.15.1 RECOVERY OF EXTRACELLULAR ENZYMES
The general steps in sequence are centrifugation (to remove cells) at low temperature
(alternatively, vacuum filtration with filter aid) and purification. Purification may
involve any one of the following general techniques: Membrane filtration, Gel filtration,
121
Adsorption, Ion-exchange or Precipitation. A brief treatment of the different
concentration/purification process is described in the following sub-sections.
Solid
substrate Submerged
culture culture
Extraction Cell mass
Extract Solid-liquid separation
Clear culture liquid, extract
Precipitation Solvent removal Partition separation
Drying Liquid concentrate
Solid concentrates
Stabilization
Standardization
Finished product
Fig. 10.14 Extraction and purification of an enzyme
10.15.1.1 Ultrafiltration
It is used for concentration and demineralization of solutions of proteins, sugars,

and organic solutes. The method is typically a membrane separation technique. From
normal filters it differs by the size range of particles to be separated (molecular
cutoffs between 500 and 300,000).
10.15.1.2 Reverse osmosis
It is also a membrane separation technique. It is used for the recovery of dissolved

proteins, ionic salts and small organic molecules. Unlike ultrafiltration, it can
separate molecules of similar sizes. In particular, reverse osmosis allows only water
to pass through.
10.15.1.3 Gel permeation or exclusion chromatography
This separates molecules on the basis of size. The separation is unique in that the
molecules that happen to enter the channels in the gel body will be delayed in
passing out while those that do not enter the same pass out fast. Consequently, large
molecules come out faster than the small molecules.
122
10.15.1.4 Chromatography
This separation technique is based on ion exchange, hydrophily/lipophily, etc., and

is carried out in columns. The partially purified solution is passed through the
column that contains immobilized ion-exchange components. As the liquid
percolates down, the relevant enzymes are selectively retained in the column due to
interaction with the immobilized counterparts while the rest pass through the
column unhindered. In the second phase, the bound enzymes are eluted from the
complex with a suitable solvent. Once again the column becomes ready for receiving
a new charge.
10.15.1.5 Precipitation
Separation by salting out (in electrolytes) is one of the oldest and yet the most
important methods of enzyme concentration/purification. An additional treatment
of the topic is therefore appropriate here.
The logarithm of decrease in protein solubility in concentrated electrolyte solutions

is a linear function of increasing salt concentration (ionic strength), as described by
the equation:
τ
log s  B  K s
2
Where s = the solubility of protein (g/L);  = ionic strength (mol/L); K' = salting
out constant (dependent on protein and salt type); B' = intercept constant
(dependent on pH, temperature, and the nature of protein in solution).
The above equation implies that electrolyte concentration required for protein
precipitation varies with protein concentration.
The influence of the most important precipitation parameters can be outlined as

follows: Higher valency salts produce higher ionic strength than lower valency salts.
At constant ion strength, protein solubility increases with increasing distance (in
both directions) from its isoelectric point. As a result, lower ionic strength is
required for precipitating when carried out at the isoelectric point of the protein.
The most commonly used salt for protein precipitation is ammonium sulfate. The
reasons can be found in the high solubility of the salt, low price, non-toxic nature (to
most enzymes), and enzyme-stabilizing property. Enzymes prepared by precipitation
with ammonium sulfate are often stable for years when stored at low temperatures.
In very simple terms, the precipitation of enzymes/proteins with salt can be

described as follows: Protein solubility tends to increase when salt is added to the
solution. This phenomenon is called salting in. With further addition, however, the
salt will begin to compete with the protein for water and at some point, force the
protein to precipitate out. This phenomenon is called salting out. When ammonium
sulfate is used for the precipitation, terms like 25% saturation, 50% saturation, 75%
saturation, etc., are frequently encountered. These refer to the percentage by volume
123
of saturated ammonium sulfate used for the precipitation. For instance, 25%
saturation means mixing of 1 volume of saturated ammonium sulfate with 3 volumes
of enzyme/protein solution.
Precipitation of enzymes with solvents is less common for large-scale purification.

The main reasons behind it are high cost of solvent, equipment and processing, risk
of explosion, and tendency of enzymes to denature at processing temperatures above
4°C.
Solvent precipitation is based on the fact that the solubility of enzymes decreases
with the decrease in dielectric constant () of the solvent. The concentration required
is lower the less hydrophilic the solvent is. Thus, an increasing precipitation effect
can be achieved in the series methanol (25 = 33), ethanol (25 = 24), and isopropanol
(25 = 18). Besides aliphatic alcohols, acetone (25 = 20) is often used as a precipitant.
In the solvent category, polyethylene glycol (mol wt: 6000) appears to be the best in
that it does not bring about enzyme denaturation and is relatively independent of
temperature and ionic strength. However, there is a strong dependence on hydrogen
ion concentration. The best results are obtained at the isoelectric point of the
enzyme to be precipitated. The hydrogen ion concentration can be easily adjusted
with acid or alkali. As the solubility of a protein molecule is lowest at its isoelectric
point, successive precipitation of different enzyme species can be affected from the
same solution by altering the pH. The precipitated enzyme can now be easily
separated by centrifugation.
10.15.2 RECOVERY FOR INTRACELLULAR ENZYMES
The separated cells are washed free from impurities and subjected to any one of
the following cell disruption techniques: (i) Chemical/Biochemical, viz., autolysis, or
(ii) Physical disruption, viz., homogenization (e.g., in Manton Gaulin homogenizer) or
bead milling (e.g., Dynomill). The subsequent purification technique is the same as
for extracellular enzymes. An additional step must be included for the removal or
reduction of the contaminating nucleic acids and cellular debris.
10.16 CONVERSION TO STORAGE FORM
Enzymes are usually very unstable (due to denaturation, microbial degradation,

photochemical effect, charge destabilization, etc.) in aqueous solutions. This calls for
appropriate treatment of enzyme solution for storage. Some of the practical methods
of treatment are:
 Use of highly concentrated solutions of salt and sugar (to repress microbial
growth)
 Conformation- or charge stabilization and/or protection from dilution-
dissociation by using buffers, glycerol, substrates, or inhibitors
 Protection of active site thiol via disulfide exchange by thiols, redox dyes,
oxygen-binding agents, or chelating agents
 Inhibition or removal of proteolytic enzymes
124
Following the above treatment, it is imperative that the preparation be stored at low
temperature and at suitable pH in appropriate packaging material.
10.17 AMYLASE PRODUCTION
Of the various enzymes, amylases play probably the most important part in food
technology. The use ranges from production of alcoholic grain beverages (whiskey,
beer, sake, etc.), non-alcoholic beverages (soft drinks, coffee, etc.), confection, corn
syrup, to pharmaceutical products (digestive enzymes).
Amylase is the collective name given to a group of enzymes characterized by their

ability to hydrolyze 1,4-glucosidic linkage in polysaccharides (e.g., starch and
glycogen). There are two main subgroups of amylases, viz., α-amylase and β-amylase. α-
amylases are also known as endoamylases, keeping with their random hydrolytic action
within the starch molecule. β-amylases (called exoamylases by analogy) liberate maltose
units by hydrolyzing the starch molecule sequentially from the non-reducing end. Both
the enzymes, however, are unable to hydrolyze glucose polymers of starch linked by
an α-1,6-glucosidic bond (see Fig. 10.15).
Microbial amylases are extracellular enzymes. Depending on the source organism,

the amylases exhibit differing properties especially with respect to mode of action,
products of hydrolysis, pH and temperature optima, etc.
CH2OH CH2OH CH2OH

O O O
O O O
-1,6-linkage
CH2OH CH2OH CH2O CH2OH (branching point)
6CH2OH
non-reducing   O O 5 O
end 4 1
O O O O O 3 2 reducing
end
CH2OH CH2OH CH2OH 6CH2OH 6CH2O CH2OH
O O O 5 O 5 O O
4 1 4 1
O O O O 3 2 O 3 2 O
-1,4-linkage
Fig. 10.15 Simplified structure of starch molecule
10.17.1 MICROBIAL PRODUCTION OF α-AMYLASE
The enzyme (α-1,4-glucan-glucanhydrolase, EC.3.2.1.1) is produced industrially from

bacteria as well as fungi (mold).
10.17.1.1 Fungal α-amylase
Fungal α-amylase is produced industrially from Aspergillus oryzae and Aspergillus niger
by either solid-state or submerged fermentation process.
125
Solid-state process
In the solid-state method, wheat bran serves as the basic component of the medium.
The treatment of the medium, inoculation, and fermentation is not much different
from a typical solid-state fermentation described elsewhere for molds. Fermentation
is carried out at 30°C for 1-4 days. The recovery consists of either extraction in
water or drying of the mold bran to produce crude enzyme. For reasons of
consistency, the preparation must be carefully standardized for activity according to
the intended use.
Submerged fermentation
Submerged fermentation exhibits marked rheological complexity (because of gradual

mycelial growth) and consequent aeration problem. A typical fermentation medium
is given in Table 10.6.
It is to be noted that glucose has not been included in the above medium. The basic
reason for this is that glucose exerts catabolite repression thereby interfering with
the enzyme yield. The pH is monitored with organic acids (citrate, gluconate, etc.) or
alkalizing nitrogenous compounds (nitrates, urea, proteinaceous matter, etc.) but the
shift in pH should be gradually towards alkalinity as the fermentation progresses.
This is true of bacterial process as well. The basic reason for this is the tendency of
α-amylase to denature at pHs below 6. When buffering is needed, CaCO3 may be
added.
Table 10.6 Medium composition for submerged fermentation of α-amylase
Component Amount (g/L)

Corn starch 24
Na2HPO4 47
Cornsteep liquor 36
KCl 0.2
CaCl2 1
MgCl2.6H2O 0.2
10.17.1.2 Bacterial α-amylase
Bacterial α-amylase (mol wt: ~ 50,000) is produced industrially from Bacillus

amyloliquefaciens and Bacillus licheniformis. Bacillus subtilis is also a good candidate. The
production is done in submerged mode. A typical composition of the main
fermentation medium is given in Table 10.7.
A temperature in the range of 30-40°C is satisfactory. The pH should be near

neutrality but not below 6. The production of amylase begins when the bacterial
count reaches 109-1010 cells per ml after about 10-20 hrs, and continues for another
100-150 hrs.
126
Bacterial amylase is commonly produced with minimum purification. The enzyme is
preserved in 20% NaCl. The most active liquid preparations contain 2% active
amylase. The most active solid preparations contain 5% active amylase. Highly active
and purified preparations are obtained by precipitation and/or adsorption
techniques.
Table 10.7 Medium composition for bacterial α-amylase production
Component Amount (%)

Ground soybean meal 1.85
Autolyzed brewer’s yeast fraction 1.5
Distiller’s dried solubles 0.76
Enzymic casein hydrolysate 0.65
Lactose 4.75
Antifoam 0.05
MgSO4.7H2O 0.04
Water 90.40
10.18 PROTEASES
Protease is a generic term for proteolytic enzymes that use proteins and peptides as
the substrate. The microbial proteases which are of interest for application in the
food industry are all of the endopeptidase type and are all extracellular enzymes.
There are many different types of proteases produced by an extraordinarily large
number of microorganisms, but in actual practice the enzymes prepared
commercially are of very limited number and types and they are derived from very
few organisms.
Proteases can be divided into two main groups, viz., (i) acid proteases, and (ii) alkaline
proteases. The proteases are sometimes classified in a manner meaningful to each
specific purpose, for example, serine proteases, metalloproteases, thiol proteinases, etc.
The industrial production of microbial protease is carried out on a large scale by a

number of companies in Europe, Japan, and the United States. The microorganisms
involved are species of Bacillus (Bacillus subtilis, Bacillus licheniformis) and some genera of
molds (Aspergillus oryzae, Aspergillus niger, Mucor miehei, Mucor pusillus, etc.). The bulk of
bacterial proteases go to the detergent industries, followed by leather tanning- and
food industries while fungal proteases go to food- and pharmaceutical industries.
10.18.1 ALKALINE SERINE PROTEASE
Alkaline serine protease, called Subtilisin Carlsberg, is the most widely used alkaline
serine protease. It is obtained from Bacillus licheniformis by submerged fermentation.
Subtilisin Novo is another alkaline serine protease of commercial interest. Although
it is distinct from Subtilisin Carlsberg, it possesses many similar properties.
A typical medium for the production of Subtilisin Carlsberg by submerged

fermentation is given in Table 10.8.
127
Other media are also available for this purpose. The temperature of fermentation in
the range of 30-40°C has been found to be satisfactory. The pH of the production
medium is kept at 7.0, as low pHs markedly lower the yield. The production of
enzyme begins when maximum cell growth is achieved after 10-20 hrs and this
continues at an almost constant rate till the completion of fermentation.
Table 10.8 Medium composition for the submerged fermentation of Subtilisin

Carlsberg

Starch hydrolysate 50
Soybean meal 20
Casein 20
Na2HPO4 3.3
At the end of the productive fermentation, protease is the only protein present in the
production medium. The reason for this is the occurrence of hydrolysis of all
proteins present in the medium by protease. The yield may be 10% of the initial
protein content of the medium. The enzyme is marketed primarily in the form of
dust-free granules (see later). The granules contain 1-5% enzyme. The enzyme
remains stable in liquid preparations, which contain about 2% of the enzyme.
10.18.2 FUNGAL ALKALINE PROTEASE
Fungal alkaline protease is mainly produced from Aspergillus species, in both solid-
substrate and deep tank fermentations. Solid substrate cultures, extensively used in
Japan, are carried out with wheat or rice bran or whole grains as the basic substrate.
The production is inhibited be NH4+ but promoted by nitrate- and sodium salts of
aspartate and glutamate.
10.18.3 ACID PROTEASE
Acid proteases constitute the most interesting group of proteases with respect to use
in food industry. They are characterized by maximum activity and stability at pH 2.0-
5.0. The molecular weight is around 35,000. Acid proteases are low in basic amino acid
content and have low isoelectric pH. They are sensitive to SH-reagents, metal
chelators, and heavy metals, and are generally stable in the acid range (pH 2-6), but are
rapidly inactivated at higher pH values.
Acid proteases of commercial importance are exclusively produced from fungal

sources and are tentatively divided into two subgroups by their physiological
characteristics: pepsin-like protease (from Aspergillus niger var macropus and some
species of Penicillium and Rhizopus) and rennin-like protease (from Mucor miehei, Mucor
pusillus, etc.).
10.18.3.1 Production method for acid protease
The enzyme can be produced by either semi-solid culture or submerged culture,

depending on the fungal species employed. For example, Mucor pusillus is cultivated
128
on a semi-solid medium. The medium consists of 60% wheat bran with water. The
optimum temperature of fermentation is 30°C. The fermentation lasts for 3 days.
The yield is 3,200 Soxhlet units per gram of wheat bran. 1 Soxhlet uint is the amount
of enzyme activity which can coagulate 1 ml of milk solution in 40 min. The yield of
enzyme can be increased by addition of ammonium salts. Finally, the enzyme is
extracted with water. See Table 10.9 for medium composition of the fermentation.
Table 10.9 Medium composition for the submerged fermentation of acid protease
Component Amount (%)

Starch 4
Soybean meal 3
Ground barley 10
CaCO3 0.5
The fermentation is carried out at 30°C for 7 days. The yield is about 3,500 Soxhlet
units per ml of broth.
The enzyme preparations, which contain 0.2-0.3% active enzyme, are marketed at
concentration of 10,000-1,50,000 Soxhlet units per ml.
10.8.4 RENNET PRODUCTION BY Mucor miehei
Many microorganisms are capable of producing rennet, the milk clotting enzyme
used in cheese production. Microorganisms like Rhizomucor miehei, Rhizomucor pusillus,
Endothia parasitica, Aspergillus oryzae, and Irpex lactis are extensively used for rennet
production. The aspartyl protease from Mucor miehei is commonly used as a chymosin
substitute in cheese making. This enzyme has a high ratio of MCA/PA (milk clotting
activity/proteolytic activity).
Rennet production using Mucor miehei can be carried out in solid-state as well as
submerged fermentation. The strain (e.g., Mucor miehei NRRL 3420) is maintained on
Sabouraud agar slants at 15°C. To recover the spores, the culture is grown on
Sabouraud agar plate or Raux bottles at 35°C for 72 hrs. Thereafter the spore
suspension (about 106 spores/ml) is grown in sterile broth. Growth as well as
fermentation occurs in a medium maintained at pH 6. For submerged fermentation,
inclusion of cornsteep liquor (2.2 g/liter), casein (2-4 g/liter), and glucose (18 g/liter)
in the medium appears to be optimum. Molasses and sucrose are not considered
good carbon sources. The highest enzyme activity occurs after about 48 hrs of
cultivation. The aeration rate is maintained at around 2 vol/vol/min (with agitation).
For solid-state fermentation, wheat bran is moistened with 0.3N HCl and sterilized
in autoclave. Inclusion of casein (0.1-0.2%) and skim milk powder (5%) in the bran
gives better result. The enzyme can be recovered by extraction with water and
subsequent centrifugation.
129
10.19 IMMOBILIZED ENZYMES
The use of enzymes in a soluble or free form must be considered as very wasteful
because the enzyme generally cannot be recovered at the end of the reaction. This is
where immobilization technique comes in. Present applications of immobilized
enzymes are confined mainly to industrial processes, e.g., production of L-amino
acids, organic acids, and fructose syrup. The future potential for immobilized
enzymes lies in novel applications and the development of new products rather than
as an alternative to existing processes using free enzymes.
Enzyme immobilization may be defined as confining the enzyme molecules to a

distinct phase from one in which the substrates and the products are present; this
may be achieved by fixing the enzyme molecules to or within some suitable material.
It is critical that the substrates and the products move freely in and out of the phase
to which the enzyme molecules are bound (confined). Immobilization of enzymes
does not necessarily render them immobile; in some methods of immobilization,
e.g., entrapment and membrane confinement, the enzymes are freely mobile within their
phase, while in cases of adsorption and covalent bonding they are, in fact, immobile.
10.19.1 GENERAL TYPES OF IMMOBILIZATION
In practice, both physical and chemical methods are routinely used for enzyme
immobilization. Physically, enzymes may be adsorbed onto an insoluble matrix,
entrapped within a gel, or encapsulated within a microcapsule or behind a semi-
permeable membrane. Chemically, enzymes may be covalently attached to solid
supports or cross-linked. Before a brief treatment on the available methods of
immobilization, see Fig. 10.16 for the summary of techniques used for the same.
ENZYMES OR CELLS
Bonding Physical entrapment
Physical bonding Covalent Gelation Encapsulation

chemical bonding (mostly cells) (enzymes)
Flocculation Adsorption Surface Cross-linking

to surface carriers precipitate
carriers
Carrier-free Carrier-free method
method (mostly for enzymes)
(cells only)
Fig. 10.16 Summary of cell and enzyme immobilization principles
10.19.1.1 Adsorption
The enzymes are adhered to the surface of carrier matrix (support) due to the
combination of hydrophobic effect and formation of several salt links per enzyme
molecule. The most widely used supports are carboxymethyl cellulose (CMC) and
Diethylaminoethyl cellulose (DEAE cellulose). See Fig. 10.17 for the principle.
130
10.19.1.2 Covalent bonding
A large number of chemical reactions have been used for the covalent binding of
enzymes by way of their non-essential functional groups to inorganic carriers such as
ceramics, glass, iron, zirconium and titanium; to natural polymers such as sepharose
and cellulose; and to synthetic polymers such as nylon, polyacrylamide and other
vinyl polymers and copolymers possessing reactive chemical groups. In many of
these procedures the covalent binding of enzymes to the carriers is non-specific, i.e.,
the binding of the enzyme to the carrier is by way of the enzyme’s chemically active
groups distributed at random. More recent studies have attempted to develop
techniques of enzyme immobilization in which the enzyme binds to a carrier with
high activity without affecting its catalytic activity.
Because of the covalent bond formed between the enzyme and the carrier molecule,
the binding is very strong. The most commonly used matrices are agarose, cellulose,
and polyacrylamide. The most useful amino acid residue (of the enzyme) in covalent
bonding is that of lysine. Glutaraldehyde is another important support. An additional
feature of glutaraldehyde is that it not only binds enzyme but also cross-links them (see
Fig. 10.17).
10.19.1.3 Entrapment
The entrapment of enzymes in gel matrices is achieved by carrying out the

polymerization or precipitation/coagulation reactions in the presence of the enzyme.
Polyacrylamides, collagen, silica gel, etc., have all proved to be suitable matrices but
the entrapment process is relatively difficult and results in low enzyme activity.
During the polymerization, the enzyme molecules get entrapped within the gel or
fiber of the carrier and it may or may not involve formation of covalent bonding.
Cellulose triacetate, agar-agar, gelatin, alginate, etc., are some of the widely used
support (see Fig. 10.17).
Enzyme E E E
E E
molecule E
E E E E
E E E E E
E
E E
Adsorption Entrapment Encapsulation
E
E
E E E CHO CHO HC=N-Enzyme
E E Matrix
E Immobilization using glutaraldehyde
E E E
Covalent bonding
Fig. 10.17 Different immobilization methods
131
A very straightforward entrapment procedure usually carried out in laboratories is
the entrapment of cells or enzymes in sodium alginate gel. A 3-4% aqueous solution
of sodium alginate is prepared and the required amount of cell suspension is added
(and mixed). With the help of a pipette or a syringe, the mixture is transferred drop-
wise to a 2% aqueous solution of calcium chloride. Calcium chloride is a denaturant
that causes hardening of the gel surface by exchanging sodium ions for calcium ions.
The gel droplets change into porous, plastic beads. The beads can be taken out,
washed with water or buffer, and used in place of enzyme. They can also be stored
for future use.
10.19.1.4 Membrane confinement
In this method, the enzyme molecules are confined within a semi-permeable membrane.
Encapsulation is one of the most widely used methods of membrane confinement. Nylon,
liposomes, polyacetic acid, etc., may be used for the encapsulation (see Fig. 10.17).
It must be noted that not all immobilization techniques yield equally efficient
enzymes. Some of the differences, especially with respect to advantages and
limitations, among the immobilization techniques appear in Table 10.10.
Table 10.10 Comparison of different types of immobilization techniques
Method Advantages Disadvantages

Covalent Not affected by pH, ionic Active site may be modified; costly
attachment strength of the medium process
or substrate
concentration
Covalent Enzyme strongly bound, Loss of enzyme activity during
cross-linking thus unlikely to be lost preparation; not effective for
macromolecular substrates; regeneration
of carrier not possible
Adsorption Simple with no Changes in ionic strength may cause
modification of enzyme; desorption; enzyme subject to microbial
regeneration of carrier or proteolytic enzyme attack
possible; cheap technique
Entrapment No chemical modification Diffusion of substrate to, and product
of enzyme; enzyme not from the active site; preparation difficult
subject to microbial or and often results in enzyme inactivation;
proteolytic action continuous loss of enzyme due to
distribution of pore size; not effective for
macromolecular substrates
Immobilized whole cells are becoming increasingly utilized and tend to eliminate the
tedious, time-consuming and expensive enzyme purification steps. Immobilization
of whole cells is normally achieved by the same methods as for cell-free enzymes.
The greatest potential for immobilized cell system lies in replacing complex
fermentations such as secondary product formation (i.e., semi-synthetic antibiotics),
132
in the continuous monitoring of chemical processes (via enzyme electrodes), water
analysis and waste treatment, continuous malting processes, nitrogen fixation, and
synthesis of steroids and other valuable medical products.
The advantages of immobilized system over normal biocatalysts (cells or enzymes) are:
 Permits the reuse of the component enzyme(s)

 Ideal for continuous operation
 Product is enzyme-free
 Permits more accurate control of catalytic process
 Improves stability of enzymes
 Allows development of a multi-enzyme reaction system
 Offers considerable potential in industrial and medical use
 Reduces effluent disposal problems
 Allows savings in downstream processing
 The reaction is specific and faster
 Certain stereochemical reaction are impractical with chemical methods
Immobilized systems also have their associated limitations, the important ones of
which are:
 The cost of enzyme is high

 The activity is sometimes inferior to free enzymes because of undesirable
conformational changes resulting from immobilization
 Not all enzymes can be immobilized by general methods. Suitable protocols
must be developed in such cases.
It is also possible to make a comparison between immobilized cells and immobilized

enzymes. Immobilized enzymes offer the advantage of minimum byproduct
formation and a high product yield (because the substrate is not converted to
biomass). On the other hand, enzymes can be less stable. Besides, it is difficult to
run an enzymatic process that requires multienzyme complex. At this point,
immobilized cells have tremendous advantage over purely enzymatic process. A
whole cell can in fact be considered a bag of enzymes and therefore has a far greater
potential.
10.19.2 USE OF IMMOBILIZED ENZYMES IN INDUSTRIAL PROCESSES
In industrial practice, immobilized enzymes or immobilized whole cells are

generally utilized within the confines of bioreactors. Bioreactor systems can have
many forms depending on the type of reaction and stability of the enzyme. Some
of the important types of enzyme reactors are (i) stirred tank reactor, (ii) membrane
reactor, and (iii) continuous flow reactor (e.g., packed bed reactor, continuous flow stirred tank
reactor, and fluidized bed reactor). A schematic sketch of each of the above reactors is
given in Fig. 10.18.
133
Product
Product
Enzyme
  (bed): 15cm
Stirred tank Substrate Gas

reactor (STR) Membrane reactor Substrate
Filter bed: 200 Fluidized bed
Thickness: 50 Packed bed reactor reactor (FBR)
Fig. 10.18 Different types of enzyme reactors
10.19.2.1 Production of high fructose corn syrup (HFCS)
D-glucose is only 70% as sweet as sucrose and is comparatively less soluble in water.
Fructose is 30% as sweet as sucrose and is twice soluble in water. Therefore glucose
syrup is treated with glucose isomerase to produce high fructose syrup (42-55%
fructose). The enzyme is obtained from Actinoplanes missouriensis, Bacillus coagulans, and
Streptomyces species. The enzyme is immobilized by cross-linking with glutaraldehyde
and the process of isomerization carried out in continuous packed bed reactor
(PBR). The enzyme is used continuously for about 150 days, which is equal to 3 half-
lives of the enzyme. 1 kg of enzyme yields 10-11 MT of 42% fructose syrup. See Fig.
10.19 for the flow diagram of production of high-fructose syrup.
Glucose syrup
Purification
Mg++ as cofactor
Concentration
(35-40% dry matter)
Heating
(55 -60oC, pH 7.5-8)
Passage through PBR

(60 oC)
pH lowered to 4.5
Purification
Evaporation
(70% dry matter)
Fig. 10.19 Production of high fructose syrup by enzymatic method
10.19.2.2 Production of semi-synthetic antibiotics
134
Semi-synthetic penicillins are produced by a combination of microbial and enzymatic
or chemical process. 6-aminopenicillanic acid (6-APA) is the principal intermediary
in the manufacture of semi-synthetic penicillins. This must first be obtained in
sufficient amounts. Under suitable conditions, Penicillium chrysogenum produces large
amounts of 6-APA by interrupted biosynthesis. Interrupted biosynthesis is now no
longer economical. Once the 6-APA is obtained, it can be joined by chemical means
with any other desired side chain. The trade production of 6-APA starts from either
penicillin G or V. The side chains in these natural penicillins are first removed and
the nucleus recovered for the chemical fusion with the desired side chain. The
hydrolysis of penicillin G and V can be carried out with the help of an enzyme called
penicillin acylase or amidase. Acylases are produced by yeasts, bacteria as well as molds
but the commercially used penicillin acylase probably comes from molds and
bacteria (E. coli). Microbial hydrolysis of natural penicillins is favored at high
temperature and alkaline pH. Bacterial acylase is more specific for the hydrolysis of
penicillin G. Mold acylase is specific for penicillin V. The reaction can be reversed
when the pH is reversed.
Bacterial acylase
pH 8. 35-40 C
 Side chain  6-APA
Penicillin G 
pH 5
Semi-synthetic penicillins have been developed to overcome the shortcomings in the

natural penicillins. Today, thousands of such semi-synthetic penicillins have been
prepared and a number of them have been found to be superior to natural penicillins
in oral absorption, acid stability, and resistance to penicillin-inactivating enzymes.
See Fig. 10.20 for some examples of some semisynthetic penicillins.
S CH3
CH CONH CH3
NH2
N COOH CH3
O C C CONH S
CH3
Ampicillin
N C
OCH3 O N COOH
O
S CH3
CONH Propicillin
CH3
OCH3
N COOH
O CH3
CH CONH S
Methicillin CH3
CH2
N COOH
CH3 CH3 O
CH CONH S
CH3 Oxycillin
CH3
N COOH
O
Phenethicillin
Fig. 10.20 Some examples of semisynthetic penicillins
The enzymatic hydrolysis of penicillin V or G can be carried out using immobilized

acylase. There are several patented- and industrial methods of acylase
immobilization. A typical method used by Hindustan antibiotics involve covalent
135
linking of the enzyme in cyanogen bromide-activated sephadex 200 (see Fig. 10.21).
Either batch or semi-continuous method can be used. The reactors can range from
STR to PBR. In a packed bed reactor (Fig. 10.18 for an idea about PBR), the enzyme
has been used for 100 days yielding 2 MT of 6-APA per kg of immobilized enzyme.
In essence, the process of 6-APA production is as follows:
Penicillin substrate is dissolved in methanol and passed through the reactor for the
hydrolysis to take place at about 36°C. A pH of about 8 is maintained with 4N
NaOH during the reaction. When the enzymatic hydrolysis is over, the released side
chains are extracted with organic solvent and the 6-APA isolated by precipitation at
pH 4.2 (adjusted with 6N HCl). The precipitate is filtered, washed with methanol,
and dried under vacuum. In general, the purity of the product is more than 90% and
the yield is about 86%.
At least 3500 MT of 6-APA are produced each year, requiring about 30 MT of the
enzyme.
NH
OH O – C – NH – Enzyme
+ CNBr + Enzyme
OH OH
+ O
O – C – NH – Enzyme
OH
Fig. 10.21 Immobilization of penicillin acylase
Before bringing the 6-APA and the side chains together for condensation, the side
chains must be activated. These days, the activation of 6-APA is done by silylation
(replacement of an acidic hydrogen on the compound with an alkylsilyl group, for
example, N-trimethylsilylacetamide and other silicon hydride derivatives). The
process entails mixing of 1 equivalent of 6-APA with 1-2 equivalents of silylating
agent in an inert solvent, allowing the reaction, bringing the mixture to isoelectric
pH for precipitation, and recovery by filtration.
A method described in a patent for the production of ampicillin trihydrate is as

follows:
52.5 g of N-trimethylsilylacetamide was added to a suspension of 6-APA in 350 ml of

methylene chloride under agitation. The reaction was carried out at 40°C for 2 hrs
and then cooled to -25°C. Abundant precipitation occurred during the cooling stage.
43.3 g of -phenylglycyl-chloride-HCl (an acyl halide) was added at -25°C and the
temperature was brought to -5°C. After holding for 1.5 hrs, 450 ml of water was
added to the mixture. pH was adjusted to 4.5 with dilute NH4OH. The mixture was
then agitated for 1 hr at 10°C. The precipitate was filtered, washed 2 times with 75 ml
portions of water, 3 times with 125 ml portions of acetone, and dried at 35°C. The
yield was about 81%.
136
10.19.3 USE OF IMMOBILIZED ENZYMES IN BIOSENSORS
Biosensors are group of sensors that possess a biological sensing layer, comprising a
receptor and an antibody or enzyme intimately associated with a transducer. These
produce a signal (electrochemical, optical or thermal) which is suitably calibrated.
Glucose biosensor is an example. An Indian version of this biosensor uses glucose
oxidase that is immobilized on an electrode surface. It can assay more than 100
samples per run, is stable for several weeks and is sensitive to 0-15 millimoles. The
enzyme is trapped in a liposomal bag. Biosensors hold an immense potential in fields
as diverse as diagnosis, agriculture, environmental monitoring and defense. See Fig.
10.22 for component parts and working principle of a biosensor.
Analyte
Amplifier Processor Display
Transducer
Biological component
Physical component
Fig. 10.22 Component parts of a biosensor
10.20 ENZYME ENGINEERING
Improvement in the activity and usefulness of an existing enzyme or creation of a

new enzyme by making suitable changes in its amino acid sequence is called enzyme
engineering. Enzyme engineering is a part of the larger activity of protein engineering.
Enzyme engineering utilizes recombinant technology to introduce the desired
changes in the amino acid sequences of enzymes. It must be understood that genetic
engineering in which whole genes are transferred from one organism to another is
not enzyme engineering. Enzyme engineering rests on modification of amino acid
sequence of the concerned enzyme.
The chief objective of enzyme engineering is to produce an enzyme that is more

useful for industrial and/or other applications. The various properties of an enzyme
that may be modified to achieve this objective are as follows:
 Improved kinetic properties

 Elimination of allosteric regulation
 Enhanced substrate reaction specificity
 Increased thermostability
 Alteration in optimal pH
 Suitability for use in organic solvents, and
 Increased/decreased optimal temperature, etc.
137
10.20.1 PRINCIPLES OF ENZYME ENGINEERING
The structure and function of an enzyme molecule, or for that matter any protein
molecule, are chiefly determined by its amino acid sequence, i.e., its primary structure.
Therefore any change in the properties of an enzyme is always reflected in its
primary structure. Conversely, a change in the amino acid sequence should alter the
properties of the enzymes. But this is not always the case because the enzymatic
properties, etc., are changed only when amino acid changes are introduced in certain
critical regions of the proteins. Therefore it is of great importance to know the critical
regions for the various functions of an enzyme, and to be able to predict the effect
of specific amino acid changes in these areas on the various functions. However, the
knowledge of the relationships between amino acid sequence and three-dimensional
structure and properties of enzymes, obtained from a larger database, is only partially
operative. It allows an explanation of the changes in structure and function on the
basis of the changes in amino acid sequence, but it does not allow a dependable
prediction of the influence of genetic amino acid changes on the structure and
function of an enzyme.
It may, however, be reasonable to anticipate that as more elaborate databases and

improved softwares become available, it should become possible to predict with far
greater confidence the structural and functional changes in enzymes produced by the
specified changes in their amino acid sequences. The effectiveness of enzyme
engineering will be greatly enhanced then, and this activity may have a tremendous
influence on enzyme technology.
138
CHAPTER 11
YEAST TECHNOLOGY
11.1 INTRODUCTION
Yeasts are fungi, which in a stage in their life cycle, occur as single cells, reproducing
commonly by budding or less frequently by fission. They lack chlorophyll. Hence
they must live a saprophytic or in some cases parasitic lifestyle. They have rather
rigid, thick cell walls, have a well-organized nucleus with a nuclear membrane, and
have no motile stages. With some exceptions, they do not form mycelial structures.
They are typically 4-5 μm in size.
11.2 TAXONOMIC CONSIDERATION
In the currently accepted classification, yeasts are arranged hierarchically into

subdivisions, families, subfamilies, genera and species. At the highest level,
identification is based on fundamental aspects of yeast sexuality (Ascomycotina or
Basidiomycotina), or the lack of it (Deuteromycotina). Ascomycotina is a subdivision
that includes ascosporogenous yeasts. By analogy, Basidiomycotina includes
basidiomycetous yeasts. Deuteromycotina includes those that do not produce sexual
spore.
The above classification scheme, however, does not necessarily imply sporulation as
the dominant mode of reproduction. Out of the four main subdivisions of the
fungal kingdom Mycetae, the abovementioned three subdivisions contain yeasts but
the main domain is Ascomycotina. This subdivision contains the more familiar
yeasts such as Saccharomyces, Schizosaccharomyces, Hansenula, Debaryomyces, Endomycopsis,
Kluyveromyces, etc. The Deuteromycetes or “fungi imperfect” includes Brettanomyces,
Bullera, Candida, Cryptococcus, Kloeckera, Rhodotorula, Torulopsis and a number of
important molds such as Aspergillus and Penicillium. Basidiomycotina is lesser known
to contain yeast. Leucosporidium, Filobasidium, Rhodosporidium, etc., are present in this
subdivision. Molds such as Rhizopus and Mucor are included in the fourth subdivision
Zygomycotina. See Fig. 11.1 for a recapitulative diagram.
As of now, there are 86 genera of yeasts consisting of 597 species. Genera are
primarily distinguished on the basis of morphological characteristics and sporing
details. Speciation, on the other hand, relies heavily on nutritional requirements.
11.3 REPRODUCTION IN YEASTS
In general, yeast can reproduce sexually as well as asexually. Asexual mode consists
of (i) budding, (ii) pseudomycelia formation, (iii) fission, (iv) bud-fission, (v) budding
and fission, and (vi) clamp connection.
Mycetae
(Fungal kingdom)
Amastigomycota Mastigomycota Gymnomycota
Ascomycotina Basidiomycotina Deuteromycotina Zygomycotina

Domain of yeasts
Main domain
of yeasts Domain of molds
Examples: Examples: Examples: Examples:

Saccharomyces Filobasidium Yeast Mucor
Hansenula Leucosporidium Candida Rhizopus
Schizosaccharomyces Rhodosporidium Kloeckera
Debaryomyces Rhodotorula
Kluyveromyces "Fungi imperfect"
Torulopsis
Endomycopsis Brettanomyces
Mold
Aspergillus
Penicillium
Fig. 11.1 Recapitulative diagram for classification of yeast
The sexual mode of reproduction in yeasts consists of sporulation in which yeast

sexuality can be homo- or heterothallic. Parasexuality, in which yeast alternates
between haploid and diploid phases without the formation of sexual spores, is also
observed. Of the asexual modes, budding and fission are by far the most important
ones.
11.3.1 BUDDING
The development of a new bud begins with a localized weakening of the mother cell
wall (because of weakening of sulfhydryl bridges in the wall protein). The bud
continues to grow and after the nucleus copy has migrated to the budding cell,
septum wall is formed. The pattern of budding is variable. In S. cerevisiae and some
other ascomycetes it is multilateral and can occur at any point except at a previous
budding site. Other yeasts have bipolar or even tripolar budding pattern in which
budding occurs repeatedly at the same point. Such variations in budding often give
characteristic morphology to yeasts. See Fig. 11.2a for some typical budding patterns
and the resulting cell morphologies.
Laboratory Saccharomyces yeast is produced by cross-breeding. The yeast has a sexual

life cycle. Haploid cells have one set of chromosomes (n = 16) and are of either
mating Type a or mating Type . Such haploid cells cannot sporulate. When mixed
together, a-cells can mate with -cells, forming diploids (2n = 32) zygotes containing
a double set of chromosomes. Both haploid and diploid cells can multiply asexually
by budding. Under certain starvation conditions, the diploid cells can undergo
sporulation, resulting in the formation of asci containing 4 spores. These spores
contain the haploid (n = 16) number of chromosomes and can germinate to give rise
to two a-, and two -cell cultures. Lager brewing yeast strains are genetically more
complicated, being species hybrids carrying the tetraploid (4n = 64) number of
140
chromosomes. Furthermore, they are heterozygous (carrying more than one type of
certain gene). Sporulation and subsequent intercrossing of the spore clones may
form new combinations of genes, resulting in yeast strains with altered
characteristics, some of which may be attractive to the brewer. See Fig. 11.2b for an
idea about the mechanism of cross-breeding.
S. cerevisiae (ellipsoidal to spheroid): Trigonopsis (triangular): Pityrosporum (flask-shaped):

multilateral budding tripolar budding budding at one pole
Candida (pseudomycelia): Hanseniaspora (apiculate shape):

budding at shoulders bipolar budding
Fig. 11.2a Budding pattern and resulting morphology of yeast cells
PAIRING
HAPLOPHASE DIPLOPHASE HAPLOPHASE
a a/ 
SPORULATION
GERMINATION GERMINATION
Fig. 11.2b Cross-breeding mechanism in yeast
11.4 INDUSTRIAL CATEGORIZATION OF YEASTS
Yeasts can be categorized in several ways. In industries, yeasts are generally

categorized as:
1. Culture yeast versus Wild yeast
The yeast used in an industry for a particular process is called culture yeast. The rest
becomes wild yeast for that particular process. As an example, brewer’s yeast from
San Miguel can be a wild yeast for Tuborg beer.
141
2. Top yeast versus Bottom yeast
Top yeasts rise to the top of the fermentation broth after the fermentation has
completed. By analogy, bottom yeasts settle down after the fermentation has
completed. S. cerevisiae var cerevisiae is the representative yeast for top yeast. S.
cerevisiae var uvarum (carlsbergensis) is an example of bottom yeast. There is one very
important biochemical difference between top- and the bottom yeast. Top yeasts
cannot utilize melibiose, (a disaccharide) while bottom yeasts can. This is because
bottom yeast possesses a gene called MEL, which is responsible for the production
of an enzyme called melibiase (α-galactosidase) needed for the breakdown of
melibiose. Bottom yeasts can also utilize raffinose, a trisaccharide. Top yeast does
not have MEL genes. It yeast can utilize only 1/3 of raffinose (see Fig. 11.3 also).
Another fundamental difference between top yeast and bottom yeast is the ability to
produce alcohol. Top yeasts generally produce more alcohol (up to or above 18%
abv) than the bottom yeast (8% abv). The notation abv is used here for “alcohol by
volume”.
CH2OH CH2OH CH2OH CH2OH

O O O O
O
O
-D-galactose -D-glucose Lactose
Melibiose CH2OH CH2OH

O O
Melibiose
Melibiase
O
O
CH2OH
CH2OH CH2OH O
O O
+ CH2OH
-D-galactose -D-glucose Sucrose
Raffinose
Fig. 11.3 Melibiase activity in bottom yeast
3. Flocculent versus Powdery yeast (non-flocculent)
Flocculent yeasts are those that flocculate after maturation or completion of

fermentation, e.g., top and bottom yeast. Powdery yeast derives its name from its non-
flocculent nature. The cells remain suspended in the broth to give a turbid
impression (like powder). Flocculation refers to reversible aggregation of cells to
form a fluffy mass (floc). Flocculation has great importance in fermentation in that it
helps in the removal of cells from the fermentation broth.
11.4.1 SOME IMPORTANT USES OF YEASTS
Yeasts are probably the oldest of microorganisms used (and cultivated) by man. Man
has used them for bread-making and alcohol fermentation since pre-historic times.
142
Today, yeasts have much more diverse uses. Some of the more important uses of
yeasts are:
 Production of Single Cell Protein (food and fodder)

 Production of enzymes (lactase, invertase, lipase)
 Microbiological assay
 Production of leavened breads
 Production of alcoholic beverages (beer, wine, etc.)
 Production of vitamins (riboflavin)
 Production of flavors
 Genetic engineering studies
11.5 FOOD YEAST
Food yeasts come in three important forms: (i) active cells, (ii) killed cells, and
(iii) processed products.
11.5.1 DRIED YEAST
Dried Yeasts are nutritional concentrates employed as a source of protein and vitamin
B-complex to enrich food and feed. Dried Yeasts are heat-killed yeasts obtained
either by primary fermentation or separated and recovered as cell concentrates from
brewing and distilling residues. Baker’s yeast, Candida, Rhodotorula, and Cryptococcus
(torula) can also be used for the same. Details of production of the source yeast are
similar to those of bakers yeast production.
To qualify as food yeast, Dried Yeast must be of acceptable type in terms of flavor,
color, microbial purity, chemical composition and vitamin content. Additionally, the
yeast must conform to the specifications covered by three recognized definitions of
Dried Yeast: (i) The International Union for Pure and Applied Chemistry (IUPAC),
(ii) National Formulary (NF-XII) of American Pharmaceutical Association, and
(iii) FDA. See Table 11.1 for the IUPAC Standard.
Table 11.1 IUPAC Standard for Dried Yeast (food)
Parameter Value
Moisture % (max) 10
Ash% (max) 10
Live bacteria count (max) 7500/g
Mold count (max) 50/g
Nitrogen % (min) 7.2 (≈ 45% protein)
Thiamine μg/g (min) 10
Riboflavin μg/g (min) 30
Niacin μg/g (min) 300
Lead and arsenic μg/g (max) 5
Starch and bacteria of the genus Salmonella Nil
143
11.5.1.1 Dietary use of Dried Yeast
 In fabricated foods such as baked goods, baby foods, geriatric foods, and as
extenders. The concentration varies between a few tenths to about 2%.
 In health food industries as solid tablets or dry powders. The preparation is
usually fortified with water-soluble vitamins.
11.5.2 YEAST AUTOLYSATES
Yeast autolysates (self digests) are produced by the induced action of intracellular
enzymes (principally proteases) on polymeric proteins of yeast cells. Autolysis is not
limited to proteins: carbohydrates and nucleoproteins are also degraded by their
respective hydrolytic enzymes.
The production of yeast autolysates is carried out at temperatures which kill the yeast
cells but do not inactivate the hydrolases (that is, at a temperature between 40 and
55°C). It is advantageous to initiate autolysis by the addition of plasmolyzing agents,
which may also serve as antiseptics to suppress the growth of thermophilic bacteria.
Some of the more common antiseptics are chloroform, toluene, thymol, phenol,
ether, ethyl acetate, and formaldehyde.
Autolysis is carried out at a slightly acidic pH. It may proceed for 12-36 hrs
depending on the degree of hydrolysis required. Customarily, proteolysis is carried
out to a point where 25-50% of the nitrogen of the cell is present as α-amino
nitrogen. The whole is then pasteurized at 80-90°C, cooled, and filtered with
diatomaceous earth. The filtrate can now be concentrated under vacuum to a paste of
80% solids. Frequently, salt is added to the autolysate before spray-drying since the
material is generally used as a condiment. A typical composition of the paste is given
in Table 11.2.
The starting material can be brewers yeast (either bitter or debittered) or bakers
yeast. The composition of commercially available autolysates varies greatly with the
source, the processing conditions, and the addition of MSG, salt, and 5'-
ribonucleoside. Most commercial products are indeed extracts since autolysis has
been followed by filtration to remove the insoluble cell wall debris. The extracts are
completely water-soluble and form clear solutions whose colors vary between amber
and brown.
Table 11.2 Typical composition of yeast autolysates paste
Parameters Percentage
Total solids 80
Salt 15
Ash (other than added salt) 6
Total nitrogen 6.5-7.5
α-amino nitrogen 2-4
pH 5-6
144
11.5.2.1 Uses of yeast autolysate
Due to pleasant meat-like flavor and aroma, it finds use in soups, gravies, meat
dishes, pet foods, and generally as condiments. In Australia and New Zealand, and
to some extent in Great Britain, autolysate in paste form is used as bread-spread.
Yeast autolysate finds use in food fermentation industries as fermentation nutrient.
It is also an indispensable component of routine media used in microbiology
laboratory
11.5.2 FEED YEAST
In the West, use of yeast as an ingredient in feed and feed concentrate is well
established. Indeed, a major fraction of the brewers yeast and Candida utilis is used in
feed. A special application is the use of active dry yeast or compressed yeast (see
later) in the preparation of ‘yeast culture’. This feed supplement is prepared by
seeding a cereal grain mash with yeast cells, incubating, and then drying the seeded
mash under controlled condition so that live yeast cells, enzymes, and other heat-
sensitive nutritional factors are preserved.
For the most part, however, feed yeast is produced as Dried Yeast which can be
categorized as Primary Dried Yeast, Brewers Dried Yeast, Distillers Dried Yeast, Torula
Dried Yeast, etc. To qualify as feed supplement, Dried Yeast must be non-
fermentative, and contain not less than 40% protein. A typical composition of
Brewers Dried Yeast is given in Table 11.3.
Recently, Candida utilis (also known as feed- or fodder yeast) has received
considerable attention. Unlike bakers yeast, Candida utilis does not have stringent
cultural and environmental requirements. The respiratory capacity of Candida utilis is
higher than that of bakers yeast. It is immune to “glucose effect” (see later, page
154) and can utilize a wide range of cheap substrates. Additionally, since the
organism is versatile, very little alcohol is generated, which means much of the
carbon source is diverted to storage polysaccharides. See Fig. 11.4 for the metabolic
route in Candida utilis.
Table 11.3 Typical composition of Brewers Dried Yeast
Parameter Value
Protein (%) > 45
Fat (%) ≈2
Fiber (%) ≈2
Ash (%) 7-8
Thiamine Adequate amounts
Riboflavin Adequate amounts
Niacin Adequate amounts
Pantothenic acid Adequate amounts
145
Sugar Direct storage
Storage polysaccharides
Energy efficient
Glycolysis
Pyruvate Ethanol
Active respiration
CO2 + H2O
Fig. 11.4: Metabolic route in Candida utilis
Yeasts are commercially grown for feed yeast production only if the process is
economically justifiable, e.g., as in the case of Candida utilis. In other cases, yeast is
obtained from other sources such as brewery, bakers yeast industry, distillery, etc.
When yeast is grown primarily for feed yeast production, it is commonly referred to
as Primary Grown Yeast. See Fig. 11.5 for generalization of the description made
above.
Baker's yeast
Saccharomyces cerevisiae Distiller's yeast Dried yeast
Brewer's yeast (secondary)
Industrial yeast
Candida utilis
Rhodotorula Primary grown yeast
Fig. 11.5 Use of yeast for feed production
11.5.2.1 Production outline of fodder (feed) yeast
The method of fodder yeast production from sugar sources is similar to that of
bakers yeast production, except for the utilization of continuous culture techniques.
The functional property of bakers yeast is not a requirement here because the cells
are used as a protein source only.
The fermentation vessel is sized according to the desired production level. The
specific growth rate, μ, of microorganisms is generally limited to a defined region for
optimum cellular protein level and biomass-to-substrate yield. Fermenters of 1500 to
2000 m3 are not uncommon. The aeration systems used for bakers yeast production
do not work in fermenters of sizes above 200 m3 and so the aeration system
becomes more sophisticated. Two types of aeration systems seem appropriate here.
They are airlift design and the Rumanian plant aeration system. In particular, ICI
(Imperial Chemical Industries)-airlift fermenters have no mechanical parts for
agitation. The forced air passing through the fermenter is responsible for the
required degree of mixing. The operating region most suitable (industrially) is a
dilution rate of 0.1 to 0.2 h-1. Under this arrangement, a combination of high
biomass yield, high cell protein content, and economically practicable oxygen supply
is possible.
146
11.6 BAKERS YEAST
Bakers yeast belongs to Saccharomyces cerevisiae. There are many different strains in this
group, all of them having somewhat different qualities. The yeast manufacturer
therefore takes great pain to find out strain most suited to various local conditions.
Improved strains are chosen not only on the basis of yield, growth rate, genetic
stability, etc., but also for the following properties:
 Gas generation rate

 Stability on storage
 Ability to withstand drying
 Osmotolerance
 Low alcohol generation
 Ability to disperse readily in water
 Mild flavor
 Ability to propagate readily
11.6.1 NUTRITION FOR BAKERS YEAST
As with all organisms, yeast needs energy source, carbon source, electron source,
nitrogen, minerals, growth factors, water, and oxygen for growth and reproduction.
The description made in the following paragraphs relates to nutritional requirements
of bakers yeast.
11.6.1.1 Carbon source
In bakers yeast production, the most important carbon source is the sugar present in
molasses. Molasses can be either from beet or sugarcane. Usually, the two molasses
are mixed in a suitable ratio for the mutual supplementation of nutrients and growth
factors. Some of the important physicochemical properties of cane and beet
molasses are given in Table 11.4.
Table 11.4 Physico-chemical properties of cane and beet molasses
Description Beet Cane

Dry matter (%) 74-78 75
Fermentable sugar (%) 45-47 46-52
Invert sugar (%) 0.2-1.2 15-20
Nitrogen compounds (%) 6-8 2-3
Betaine* (%) 3-4 --
Glutamic acid (%) 2-3 --
* Betaine, (CH3)3N+CH2COO–
Ash (%) 10-12 10-15 (= trimethyl amino acetate) is a
Potassium (%) 2-7 1.5-5 nitrogenous compound found
Vitamins only in beet molasses. It is not
Biotin (ppm) 0.04-0.13 1.2-3.2 utilized by yeast. Betaine
pH 7-9 5-6 increases BOD of the effluent.
147
Molasses treatment
Molasses as it comes in tank cars is of about 80° brix with a fermentable sugar of
about 45-50%. It first undergoes an initial treatment, the purpose of which is to
remove colloids, firm particles, and to kill unwanted microorganisms. In the normal
procedure, molasses is diluted to 35-45° brix and pH adjusted to 5 by adding H2SO4.
Clarification is done by centrifugation or filtration. The clarified molasses is heated
to boiling point and kept at this temperature for a couple of hours after which the
liquid is drawn out from the settled sediments. The molasses is now cooled to either
75°C or 20°C and stored until required. See Fig. 11.6 for the protocol for molasses
treatment.
Molasses, 80o brix
Dilution to 45o brix

H2SO4
Filtration
pH adjustment
(pH = 5)
Boiling
Leaving for 2 hrs

Sediments
Cooling
(20oC or 75oC)
Storage
Fig. 11.6 Treatment of molasses before using as an the medium
Alternatively, HTST treatment (140°C for 4 sec) can be done using live steam. The
use of extreme storage temperatures ensures that the contaminants that might gain
entry during the course of storage do not multiply.
11.6.1.2 Nitrogen source
Amino acids in beet molasses comprise about one-third of total nitrogenous

compounds present in it. All of these amino acids (present mainly as glutamic acid)
are quickly utilized by the yeasts. Rest of the requirement must be met by external
addition of nitrogen source, usually ammonia, ammonium salts (ammonium sulfate,
ammonium phosphates, ammonium nitrate, ammonium chloride), and urea. If cane
molasses is used, the entire requirement of nitrogen must be met by external supply.
11.6.1.3 Minerals
Yeast needs a wide range of minerals. The more important ones are phosphorus,
sulfur, potassium, magnesium, calcium, sodium, barium, zinc, iron, and manganese.
In bakers yeast production, the only mineral supplied is phosphorus. Other minerals
are automatically inclusive in the medium because of the complex nature of
molasses. For trade fermentation, phosphorus is supplied in the form of H3PO4,
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KH2PO4, Na2HPO4, NH4H2PO4, etc., because it is the inorganic phosphate (and not
the elemental phosphorus) that is assimilated by yeasts. Sulfur, if required, may be
added as K2SO4 or Na2S2O3, although the yeast can also utilize sulfur-containing
amino acids.
11.6.1.4 Growth factors
Growth factor requirement varies amongst species and strain, as does the actual
concentration. A typical data for the most common strain of S. cerevisiae is given in
Table 11.5.
Table 11.5 Growth factor requirement for S. cerevisiae
Growth factor Requirement (mg/L)

Inositol 125
Ca-pantothenate 6.25
Pryridoxine-HCl 6.25
Thiamine-HCl 5.0
Nicotinic acid 5.0
Biotin 1.0
Folic acid, p-aminobenzoic acid, and riboflavin are occasionally added to the medium
although normally the yeast can synthesize them all. Biotin requirement in particular
is very critical in bakers yeast production using molasses. Beet molasses, being low in
biotin, must be blended with cane molasses (10-20% cane molasses) to achieve the
balance. The mixing of cane and beet molasses is desirable for other reasons also,
namely, for balancing nitrogen and reducing sugar levels.
11.6.1.5 Anaerobic growth factors
Yeast requires ergosterol and unsaturated fatty acids for the synthesis of its cell membrane
components. This requirement vanishes when the yeast is supplied with oxygen:
oxygen is utilized by the yeast, among other things, for the synthesis of ergosterol
and unsaturated fatty acids. This fact implies that ergosterol and unsaturated fatty
acids are anaerobic growth factors for yeasts.
11.6.1.6 Water
Commercial production of bakers yeast requires water to be potable. Contextually,

the term ‘potable’ implies ‘fit for drinking purpose’. Stated differently, water should
be acceptable in terms of all physicochemical qualities. See Table 11.7 for other
requirements.
149
Table 11.6 Water standard for bakers yeast production
Parameter Requirements
Oxygen demand (ppm) 10
Total hardness (ppm CaCO3) 150 (max)
Iron (pm) 0.1 (max)
Manganese (ppm) 0.05 (max)
Total count
At 21°C 100/ml (max)
At 37°C 10/ml (max)
Coliforms and Clostridium. perfringens 0/100 ml
11.6.1.7 Oxygen requirement
Oxygen is required for the synthesis of cell components as well as respiratory

metabolism. A quantitative reflection of the oxygen requirement can be shown by
the following stoichiometry:
200 g sucrose  100 g yeast solids

+   +
 
10.32 g NH3    140.14 g CO 2
   
 
100.44 g O2   78.12 g H 2 O
Thus, theoretically, 1 g of O2 is required for every gram of dried yeast produced.

Under practical condition, however, the supply of oxygen should exceed the
theoretical value by several fold. This is because oxygen is supplied in the form of air
and partly because the supplied air is vented off to facilitate agitation. The cells thus
consume only about 20% of the oxygen supplied, the rest being vented away.
11.6.2 CULTURAL ENVIRONMENT FOR BAKERS YEAST
Besides supplying a well-balanced medium, attention must also be paid on various

requirements such as pH, aeration rate, and temperature of fermentation. Yeasts
exhibit a wide range of pH and temperature optima for growth. The pH may range
from 2.8-6.5 and temperature 0-47°C. However, the majority of the commercially
important yeasts grow best at pHs between 3.5 and 5 and temperatures between 10
and 33°C. It must be noted, the pH and temperature optima for growth may not be
the same as those for metabolite- or product formation. For bakers yeast, the
optimum pH is 4.5-5 and the optimum temperature is around 30°C.
11.6.3 OUTLINE OF BAKERS YEAST PRODUCTION
The principal sources of carbon and energy for bakers yeast production are the
fermentable sugars from cane- and beet molasses. Whenever possible, a judicious
mixture of these two is used as the substrate. The fermentation medium is
150
supplemented with ammonia, ammonium salts, or urea for nitrogen. For
phosphorus, orthophosphates are used. The medium must also contain other
minerals and growth factors in adequate amounts.
Customarily, the production is carried out in multiple stages. The inoculum is built up
separately in a series of propagators before the final pitching. The final trade
fermentation is carried out under highly aerobic condition, and with incremental
feeding (fed-batch mode) of the sterilized molasses wort of 45 brix. The feeding
and the aeration rates are controlled such that the reducing sugar content of the
medium at any instant is about 0.2 g/lit, the specific growth rate () about 0.2, and
the respiratory quotient (RQ) about 1. The controlled feeding is required to shift the
process from fermentative mode to respiratory mode. The optimum pH and
temperature are 4-6 and 30C respectively. Normally, the duration of fermentation is
about 8-20 hrs. The yeast increases by five to tenfold (~ 3 generations) and the
concentration of yeast solids reaches 4-6% at the end of the fermentation period.
The fermentation is terminated with half an hour of maturation in which the feeding
is stopped but aeration is continued. The nominal capacity of the main fermenter is
200 m3. About 20 MT of sales yeast (yeast mass with 27% dry matter) can be obtained
per batch.
After maturation, the yeast cells are recovered using a battery of centrifuges. The
step produces yeast cream of about 15-18% dry matter. The cream is stored under
refrigeration for future use or can be passed through filter press for further
concentration (27-28% yeast solids). Alternatively, concentration may also be
achieved by a rotary vacuum filter (Fig. 17.10a and 17.10b). The resulting compressed-
yeast is blended with suitable emulsifiers and plasticizers, and extruded in the form of
semi-plastic rectangular blocks. Packaging is done in wax paper. Storage of packaged
yeast in cold room marks the final stage of processing. The compressed yeast has a
storage life of 10 days at 5-8C.
The productivity is ~ 3 g/lit/hr and under optimum condition, the practical yield is
0.50 kg yeast of solids per kg of fermentable sugars utilized. A recapitulative diagram
of bakers yeast production (multistage type) is shown in Fig. 11.7.
11.6.4 PRODUCTION DETAIL OF BAKERS YEAST
11.6.4.1 Preservation and maintenance of culture
Although “low risk” methods are highly desirable, simple and rapid methods such as
“active transfer” are more widely used. Selected strains of S. cerevisiae are prepared
conventionally on agar stabs or as slant cultures and stored under refrigeration at
4°C. Transfer to fresh slant (subculturing) is done every 3-6 months. Malt Yeast
extract Glucose Peptone Agar (MYGP) or other commercial media may be used for
the subculturing.
11.6.4.2 Inoculum build-up
In practice, bakers yeast is not produced by direct pitching in the main fermenter.
Rather, it is preceded by a sequence of smaller fermentations (and therefore
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multistage) in which pitching yeast is grown. Typically, yeast is first grown in
laboratory in a medium containing 5-7.5% sugar. This is then taken to the first
propagating plant. Under light aeration and positive pressure, it is left for 20 hrs. The
whole is transferred aseptically to an even bigger second stage propagator. Again,
light aeration takes place and after 16 hrs, the content is again transferred in toto to yet
another large propagator. The propagating vessels are kept in a separate room so
that the entire work can be carried out aseptically. The final propagator needs about
26 hrs. This vessel can be of closed or open type but the propagators preceding it are
necessarily closed vessels.
Filter
Cane Beet
molasses molasses Pre-
treatment
Storage tank
Main fermentor
Propa-
Propa- gator
gator II
I
Slant Shaker
culture flask
Air Extruder
Centrifuge
Cold room
Compressed yeast
Fig. 11.7 Recapitulative flow diagram of bakers yeast production (multistage)
Aeration maintains the positive pressure, which is later used also for the aseptic
pressure-transfer of inoculum. A protocol for inoculum- and seed yeast build-up for
a 200 m3 fermenter is shown in Table 11.7.
From Table 11.7 it is clear that alcohol is generated only in steps A and B. Here, the
sugar concentration of the medium is relatively high (because of batch nature). The
fermentation is midway between fermentative and respiratory metabolism. Step C is
carried out in a manner similar to the main fermentation to be carried out later on.
The vessel capacity of step C (and also the configuration) need not be different from
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that of the final fermenter. Step C, like the main fermentation, proceeds in a fed-
batch mode. The medium is fed in incremental manner, closely corresponding to the
actual carbon requirement for the respiratory growth of participating yeast cells. The
vigorously growing yeast cells quickly assimilate alcohol formed in steps A and B.
The final propagator (vessel of step C) has a capacity of about 200 m3, which yields
about 20 MT (27% solids) of yeast. The yeast cells are collected by centrifugation.
The resulting biomass, called ‘yeast cream’ can be now used for pitching 6 main
fermenters each of 200 m3 capacity. Each main fermenter will in turn yield 20 MT of
sales yeast. If the seed yeast is not to be used immediately, it can be stored for a
week under refrigeration.
Table 11.7 Scheme for inoculum build-up for a 200 m3 fermenter
Stage Molasses consumed *Yeast produced Alcohol generated Duration

(kg) (kg) (L) (hr)
Lab. 1 0.1-0.2 -- --
Step A 40 9 9 20
Step B 1000 200 250 16
Step C 22000 20000 0 26
* Yeast produced in terms of 27% solids (also called sales yeast)
11.6.4.3 Pitching
The addition of inoculum to the main fermenter is called pitching. A basic difference
between bakers yeast and other fermentations is that the former uses yeast cream
(solid, called seed yeast) instead of liquid inoculum.
Into vats that have been sterilized with steam and cooled, are pumped adequate
amounts of potable water, various chemicals (phosphates, ammonium salts) and a
small amount of prepared medium. Seed yeast is then added at a rate of 3 MT per 200
m3 fermenter. The yeast cream from the propagator is usually stored in cooled tanks
(0-4°C) for 1-2 weeks before actual use in the final fermenter. Sometimes, a short acid
treatment is also given (pH ≈ 2) to seed yeast before use. This will reduce the level of
microbial contaminants without affecting the seed yeast. The air supply is
immediately opened and the incremental feeding of nutrient (prepared molasses)
started.
11.6.4.4 Main fermentation
The main fermentation is carried out in fed-batch mode. Bakers yeast fermentations
are not carried out under conditions of exponential growth. Since the fermentation is
carried out in fed-batch mode, the constant feeding (without simultaneous removal
facilities) does not permit exponential growth but can only provide a constantly
diminishing rate. The fermentation must be complete by the time the medium
reaches the predetermined level. The duration of fermentation for a typical fed-batch
production for bakers yeast is 8-20 hrs.
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Successful bakers yeast production requires monitoring of several process variables,
such as aeration, temperature, feed rate, etc. Brief descriptions of some these
important factors are given in the following paragraphs.
Feed rate control
The requirement of the feed rate control arises from the fact that bakers yeast
exhibits glucose effect (Crabtree effect) at hexose concentrations above 5% in broth.
Glucose effect refers to the shifting of metabolism from respiratory mode to
fermentative mode. This occurs because the organism tends to attain cell economy.
That is, when adequate amounts of readily assimilable substrates are available, the
organism does not use the Tricarboxylic Acid Cycle and Electron Transport Chain which
are energy intensive: the organisms meet their energy requirement from fermentation
alone. Since fermentation by S. cerevisiae leads to ethanol production, this is an
undesirable aspect: it leads to substrate loss. Bakers yeast production therefore
requires that feed rate be controlled so that the nutrients are instantly utilized for the
respiratory metabolism only. The shift from fermentative to respiratory metabolism
is called Pasteur Effect. It may be noted, the fundamental difference between bakers
yeast and ethanol production is that the former entails Pasteur effect while the latter,
glucose effect. In bakers yeast production, the feed rate is maintained such that
glucose concentration of the medium at any instant is below 0.2 g/L.
Aeration
Bakers yeast production is a highly aerobic process, requiring (theoretically) 1 g O2

per gram of bakers yeast (dry) produced. Aeration systems used in bakers yeast
production vary considerably (for instance, agitated-, stationary-, and proprietary
systems) but the most common is the stationary aeration system. In the common
type, the bottom is covered by a horizontal, diametrically placed main pipe
containing 24 side tubes provided with a total of 30,000 holes each of 1.5 mm
diameter. The air is sparged as fine bubbles, the movement of which also aids in
agitating the medium (see Fig. 11.8).
Air pipe
Side tubes
with holes
Fig. 11.8 Stationary aeration system
Customarily, filtered air containing 21-30% O2 is used for aeration. Since only 20%
of the supplied O2 is consumed before finally being vented out, the supply of air far
exceeds the theoretical requirement. The aeration is typically controlled at 0.8-1.4
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vol/vol/min. Concentrated O2 gas mixture is not preferred as this leads to
disturbance in yeast metabolism: yeast’s ability to respire and ferment becomes
impaired.
Cooling
Cooling can be carried out with internal cooling coils. Bakers yeast fermentation is
carried out at 28-30°C. High temperature is undesirable because this leads to
contamination problem. The performance of the yeast is also affected. Cooling
requirement is great in bakers yeast production because the heat liberated during
aerobic growth is very high: it is about 3.5 kcal/g of yeast solids produced.
Defoaming
Considerable foaming occurs in bakers yeast production. This is why about 1/3
space of the fermenter is left as headspace. Foam can be controlled by adding
antifoams like silicone oil, edible oil, surface-active agents, etc.
Growth rate, generation time, and generation
In bakers yeast production, extreme growth rates are not desirable. Feed rate and
aeration are adjusted such that the growth rate always remains very near to 0.2.
Control of aeration serves double purpose: it maintains Respiratory Quotient (RQ =
mole of CO2 generated per mole of O2 consumed) at an optimum of 1.0 and growth
rate at around 0.2. High level of aeration increases growth rate, liberates more CO2,
disturbs RQ balance, and eventually leads to “respiratory” fermentation.
The growth is expressed as an average. It does not remain constant throughout the
fermentation period. The growth rate decreases with the corresponding
generation time: 3 hr  5 hr  7 hr. Therefore, for a 15-hr fermentation, 3
generations and an 8-fold multiplication can be expected.
11.6.4.5 Final stage of fermentation
Maturation of yeast cells marks the final stage of the fermentation. Maturation is
required to impart yeast cells the ability to withstand future adverse conditions (e.g.,
extruding, drying, etc.). The maturity is expressed as percentage of budding. A low
percentage of budding reflects maturation and the resultant improved stability on
storage.
Maturation is achieved by sharply reducing the feed rate towards the end of the
fermentation. Even after terminating the feeding, aeration is continued for another
half an hour.
11.6.4.6 Yield and productivity
During fermentation, growth of bakers yeast decreases rapidly at concentrations

exceeding 3-4% yeast solids in the broth. For practical reasons, commercial bakers
yeast fermentation is terminated at solids concentration of 4-6%.
155
The average productivity of a typical fermentation is 3 g/lit/hr. In practice, ethanol
formation is minimized and kept below 0.1%. Using an ordinary beet molasses with
addition of necessary growth factors and nutrients, approximately 0.5 kg yeast solids
can be obtained per kg of fermentable sugars. This compares well with a number of
theoretical views.
11.6.4.7 Harvesting
The final broth is separated for yeast cells in a battery of vertical, nozzle-type,
continuous centrifuges which can develop a *G of 4000 to 5000 (Fig. 11.9). This is
sufficient to affect the separation of cells which have a water content of 62 g/100 g
cells and a density of 1.133 g/cm3. The first pass (along with washings) through such
a centrifuge triples the yeast concentration. Additional passes produce yeast cream
with 18-20% solids. The cream can be stored at 1-4°C for several days.
* G refers to velocity of settling of particles due to gravity. It depends on viscosity

() of the liquid, diameter (D) of the particle, acceleration (g) due to gravity, and
density () of the particle as follows:
D2  g
Velocity of settling of particles due to gravity =
18
Velocity of settling due to centrifugation depends on radius (r) and angular

velocity () of the centrifuge in place of acceleration due to gravity (g). The
relation is as follows:
D 2  2 r
Velocity of settling of particles due to centrifugation =
18
Feed
Lighter liquid
Heavier liquid
Solid Solid
Fig. 11.9 Nozzle-type vertical centrifuge
156
11.6.4.8 Filtration/Compression
Yeast cream is further concentrated by filtration (by pressing, which accounts for the
term compressed or pressed yeast). Filter aids must not be used as they contaminate the
yeast. Plate and frame filter press is normally used but rotary continuous vacuum
filters are now getting increasingly popular. In the latter type, the drums are
precoated with edible starch and a small amount of salt before filtration. Salt is
added to reduce the moisture content of the yeast mass. After filtration the salt can
be removed by spraying water. The press cake, called sales yeast, contains 27-28%
yeast solids. Bakers yeast obtained by vacuum filtration has slightly higher solids
content (30-33%). Salt works as a dehydrating agent. Theoretically, it is roughly 11
times as effective as sucrose. The equation showing the dehydration effect is: πV =
nRT, where π is the osmotic pressure and n is the number of solute particles. In the
case of Na+Cl¯, there are two particles per mole (58.5 g). On the other hand, sucrose
(342 g) contains only one particle.
11.6.4.9 Extrusion and packaging
The yeast cake is mixed in a blender with small amounts of emulsifier and cutting
oil (soybean or cottonseed oil). These additives, which may be added at the rate of
0.1-0.2%, facilitate subsequent extrusion and provide better, lighter appearance.
Mono- or diglycerides, sorbitan esters, and lecithin are the commonly used
emulsifiers.
The blended cake is extruded through nozzles in the form of continuous, thick
ribbons with a rectangular cross-section (open-throated nozzles). This is cut into
appropriate lengths to form the well-known shape of packaged bakers yeast (~ 500 g).
The packs are immediately cooled in refrigeration chamber with a vigorous air
circulation. Generally, a cooling period of 24-48 hrs is needed.
11.6.4.10 Storage stability
The storage stability of compressed yeast at 5-8°C is quite good (about 10 days). A
loss of 3.5% gassing activity may occur. To some extent, the stability also depends
on processing conditions, maturity, etc. Cells with nitrogen contents between 6-7%
show excellent stability. Temperatures above 10°C are detrimental to yeast quality.
The storage stability of compressed yeast can be checked by determining the effect
of storage on viability (by plate count method).
11.6.4.11 Contaminants
The most numerous are lactic acid bacteria of the genera Lactobacillus and Leuconostoc.
The counts are normally between 104 and 109/g.
11.6.4.12 Wastewater
Typically effluent from bakers yeast industry contains 0.1-0.3 kg BOD/kg yeast
produced. Beet molasses contribute to more BOD than cane molasses because of
betaine. The general method of reducing BOD (by ~ 80%) is anaerobic purification.
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The organic compounds are decomposed to CO2 and methane. Methane may be
used in factory boilers.
11.6.4.13 Quality control
The quality of bakers yeast is judged by three important categories of tests, namely,
(i) Chemical, (ii) Microbiological, and (iii) Physical (see Table 11.8).
Table 11.8 Test of bakers yeast
Test category Test Description

Chemical Dry matter
Nitrogen
Phosphorus
Trehalose Disaccharide of glucose with no
reducing property
Microbiological Total count of living cells
E. coli count
Streptococcus fecalis
Wild yeasts and molds
Physiological Raising power
Keeping quality Leavening power after storage at
30°C for 2 and 4 days, 20°C for 7
days, and 5°C for 4 weeks.
Autolysis No. of days at 35°C before the yeast
becomes soft.
11.6.5 ACTIVE DRY YEAST
The production of Active Dry Yeast (ADY) begins with the selection of bakers yeast
which will yield the desired characteristics on drying. In order to produce an ADY
with acceptable leavening activity, viability and storage stability, following factors
should be taken into account during drying:
 Drying temperature
 Drying rate
 Final moisture content
At present, the only method used on commercial basis starts from press cake. The
cake is subdivided into thin strands or fine particles. Drying is carried out with
currents of air at temperatures which keep the temperature of the yeast itself below
40°C. The yeast chosen for drying should be mature enough to withstand the
harshness of drying. The common methods of drying are:
 Continuous belt tunnel dryers
158
 Rotolouver dryer
 Through circulation dryers
 Air-lift (fluidized-bed) dryer
 Spray dryer
Air-lift dryer is probably the most popular of dryers. It can be used either as a batch-
or a continuous process. For batch operation, the extruded yeast strands are fed into
a drying chamber with a metal screen or perforated plate at the bottom. Heated air is
blown from the bottom through the yeast particles in a fluidized bed. Emulsifiers
and swelling agents are often added to the yeast preparation (suspension) prior to
drying. The drying time may vary between 10 min and 4 hrs. For rapid drying
(between 10 and 30 min) an inlet air temperature of 100-150°C can be used at the
beginning, while keeping the temperature of the yeast itself at 24-40°C. Temperature
of the yeast particles up to 50°C is not detrimental at the end of the drying period. A
multichamber continuous air-lift dryer patented by Pressindustria (1977) uses the
conditions given in Table 11.9.
Table 11.9 Yeast drying protocol used by Pressindustria (1977)
Description Measurement
Air velocity 4000m/h
Air flow volume 4000m3/h
Air temperature
1st chamber 46°C
2nd chamber 36°C
3rd chamber 32°C
4th chamber 30°C
Retention time 3h
Yeast productivity 160-350kg/h
Using the protocol given in Table 11.9, the final moisture content of the ADY can
be brought down to 7%. In general, the survival rate of yeast in fluidized-bed dryer
is about 85%, which is not possible using other drying methods.
In commercial practice, sorbitan monostearate is used in ADY at concentrations

between 0.5 and 2 % based on dry weight of yeast cells. The addition improves
dehydration characteristics of ADY. Butylated Hydroxy Anisole (BHA) may be used at
the rate of 0.1% for the protection of low-moisture ADY against oxidative rancidity.
Such ADYs are available under the trade name protected ADY.
Generally, ADY is dried to moisture content of 7.5-8%. This level represents a

compromise between the demand for good quality ADY (good bake activity), which
is higher at higher moisture levels, and good stability, which is better at lower
moisture level.
159
11.6.5.1 Packaging
The ADY is normally given a protective packaging in an atmosphere with less than
2% O2, or under vacuum. Yeast for consumer trade is usually packed in aluminum
foil-laminated, heat sealable plastic pouches. The inert gas for smaller packages is
usually N2.
11.6.5.2 Stability
ADY loses about 7% of its bake activity per month at ambient temperatures and has
a useful life of 1-2 months. If packaged in an inert atmosphere or vacuum, it loses
only 1% of activity per month, with an annual loss not exceeding 10%. Under good
packaging, a good quality ADY will have a shelf-life of 1 to 2 years. The stability of
ADY is also related to its chemical composition (Table 11.10). Nitrogen content of
about 7% is thought to be optimum.
Table 11.10 Composition of ADY
Parameter Range
Moisture, % 7.5-8.5
Nitrogen, % 7 (based on yeast solids)
Phosphorus (as P2O5), % 1/3  nitrogen content
11.6.5.3 Rehydration
Rehydration of ADY is done in warm water (40°C), and it takes about 5 min. If cold
water is used, a loss of 20-25% yeast solids can occur by leaching. Among the
materials that may leach during rehydration, glutathione (a tripeptide of γ-glutamyl-
L-cysteinylglycine) has an important bearing on the bake quality of bakers yeast. This
compound activates proteolytic enzymes in the flour, which in turn affect gluten
membrane in the dough. The slackened protein membranes can no longer hold back
CO2 and the loaf becomes flat.
11.6.6 INSTANT ACTIVE DRY YEAST (IADY)
This preparation can be directly used with the flour (without rehydration). The
fundamental difference between ADY and IADY is the processing. IADY uses
threads as small as 0.5 mm in diameter for drying. Emulsifiers such as sorbitan
monostearate, citric acid esters are used. The rest is similar to ADY production.
160
CHAPTER 12
BREWING TECHNOLOGY
12.1 INTRODUCTION
Beer is one of the most important alcoholic beverages. The term is generic and
implies undistilled alcoholic drinks made from malted barley and adjuncts, and are
flavored and preserved with hops. The art and science of brewing is called zymurgy.
12.1.1 TYPES OF BEER
There are two major types of beers, which are distinguished mainly on the basis of
fermentation differences. They are (i) Lagers and (ii) Ales.
12.1.1.1 Lagers
Most bottom-fermented beers are called lager beers, or simply, lagers. This term was
derived from the German verb lagern, which means to lay down, or store. Historically,
storing for long periods was an integral part of lager beer production: it served to
mature and clarify beer. This significance no longer exists in modern lager beers.
Lager beers are produced employing the yeast Saccharomyces carlsbergensis (synonym:
Saccharomyces uvarum), a bottom-fermenting yeast.
12.1.1.2 Ales
Most top-fermented beers are called ales. The yeast employed is Saccharomyces
cerevisiae, a top-fermenting yeast. Usually, ale fermentation occurs at a temperature
higher than that for lager. The duration of fermentation, however, is shorter.
Another fundamental difference between lager and ale is in the method of mashing.
Lagers are produced by decoction mashing (see later, page 183) while the ales are
produced by infusion mashing.
Both lagers and ales can be further classified on the basis of color and body, into light
and dark. It has to be remembered that there are several other beers, which do not
neatly fall in the aforementioned categories. Lambic beers, for example, are produced
by spontaneous fermentation.
The fundamental differences between light and dark beers are shown in Table 12.1.
Some examples of beer and recapitulative representation of classification of beers are
shown in Fig. 12.1.
Table 12.1 Fundamental differences between Light and Dark beers
Beer type
Characteristics
Light Dark
Color Light Dark
Body (gravity) Weak Heavy
Palatability Dry Sweeter *Attenuation refers to weakening of beer in
terms of nutrient content. A well-fermented
Attenuation* Full Not full beer is supposed to be attenuated because the
Hops level Medium to high Low yeast has utilized almost all of the substrate.
BEER
Lager (bottom-fermented) Ale (top-fermented)
Light-colored Dark-colored Light-colored Dark-colored

Examples: Examples: Example: Examples:
------------- ------------- ----------- -------------
Pilsner Munchner Pale ale Mild ale
Dortmunder Bock beer Stout
Porter
Fig. 12.1 Classification of beer
12.1.2 COMPOSITION OF BEER
Beer contains water, alcohol, carbohydrates, CO2, organic acids, resins and essential
oils, proteins and amino acids, higher alcohols, minerals, and congeners. These
constituents come from the raw materials, yeast, and fermentation. A typical
composition of beer is given in Table 12.2.
Table 12.2 Normal composition of beer
Components Amount
Water, % 88-92
Alcohol, % 4-5
Acidity, % 0.15-0.5 (as lactic acid)
CO2, % 0.4-0.5
Nitrogenous substances, % 0.58-0.74
Sugar, % 0.9-2.6 (as maltose)
There are several exceptions, though. For instance, Switzerland produces one of the
world’s strongest beers (14% abv). Switzerland is also the first country to market
alcohol-free beer.
162
12.1.3 ALCOHOL-FREE BEER
These beers were developed in view of concern over the effects of alcohol
consumption on health and partly in an attempt to provide an acceptable social drink
for motorists.
The most common means of production involves post-brewing removal of alcohol

from beer. Thermal evaporation under vacuum is currently used. Such equipment is
typically capable of producing an end product with an alcohol content of about
0.03%. The beer retains the brewed taste. There has been considerable interest in the
development of yeast strain also, which produces the brewed flavor but is unable to
complete the alcoholic fermentation.
12.1.4 THE STORY OF CARLSBERG BEER
The best known of all Scandinavian (i.e., Denmark, Norway, Sweden, and Iceland)
breweries is the Carlsberg of Denmark, which dates from 1847. The story runs thus:
Christien Jacobson of Denmark visited Munich several times. He studied the
bottom-fermenting technique and brought back the yeast from there in 1846 to start
his own brewery the following year. He named the brewery after his son Carl and the
fact that the brewery was on a hill, berg. In 1883, Carlsberg brewers identified and
isolated a single cell yeast culture which has left the name in the annals of lager
brewing as carlsbergensis. Tuborg was a rival of Carlsberg but these companies merged
in 1970.
12.2 MATERIALS REQUIRED FOR BEER MAKING
The principal materials required for beer making are: (i) Yeast, (ii) Malt, (iii) Hops,
(iv) Adjuncts, and (v) Water. A brief discussion on each of these materials is given in
the following paragraphs.
12.2.1 BREWERS YEAST AND ITS MANAGEMENT
Depending on the type of beer, either S. cerevisiae or S. uvarum is used. Whichever the
yeast type, it must possess certain properties in order to make a good beer. Some of
the most important properties required of these yeasts are:
1. Rapid growth rate (but without excessive yeast growth)

2. Efficient utilization of maltose and maltotriose with good conversion to
ethanol
3. Ability to withstand stress imposed by alcohol concentrations and osmotic
pressure encountered in breweries
4. Reproducible production of the correct levels of flavor and aroma
compounds
5. Ideal flocculation characteristic for the process employed.
6. Good handling characteristics (e.g., retention of viability during storage,
genetic stability, DOG resistance, killer strain resistance, etc.).
163
The selection of mutants resistant to glucose analog, 2-deoxyglucose (= DOG), has
proved extremely useful. These mutants have the ability to utilize glucose and
maltose simultaneously: they are insensitive to catabolite repression.
The most widely used method for stock-culturing in breweries is active transfer,
which may be either broth culturing or slant/slope culturing.
12.2.1.1 Broth culture
The yeast is inoculated and incubated at 28°C for 2-4 days in MYGP broth.
Thereafter it is stored at 2-4°C. Subculturing is done every 2-3 months.
12.2.1.2 Slant/slope culture
Either MYGP agar or Universal Beer Agar (UBA) slopes are inoculated and incubated
at 28°C for 2-4 days and stored at 4°C. Subculturing is done every 6 months. If
stored under paraffin oil, the culture is stable for a year.
12.2.1.3 Quality control
Several tests are available for the quality control of yeasts. To ensure that the
contamination and mutant levels are low (whether stock-culture or recycled yeast)
following general tests may be carried out.
 Microscopic examination: Screening can be done for unusual size, shape, and
morphology for the evidence of wild yeasts. The examiner must be familiar
with the diagnostic morphological characteristics of the culture yeast,
though.
 Cycloheximide in UBA: Cycloheximide added at the rate of 2-4 μg/ml in the
medium selectively inhibits the culture yeast. All yeasts capable growing in
such a medium are therefore wild.
 Lysine/Crystal violet/Fuchsin sulfite media: These three media are used to detect
wild yeasts in the presence of culture yeasts. Culture yeasts cannot utilize
lysine as the sole source of carbon and are inhibited by crystal violet and
fuchsin sulfite.
 Test for heat resistance: The yeast culture is heated at 53°C for 10 min. Wild
yeasts survive this treatment but the culture yeasts are rapidly destroyed.
 TTC overlay method: Upon prolonged use, some culture yeasts mutate to
Respiratory Deficient Mutants (RDMs). These mutants cannot oxidize
glucose and their number exceeding 10% of total yeast cells is considered
unsatisfactory. Triphenyl tetrazolium chloride (TTC, a colorless salt) is used
to detect the presence of (and also enumerate) RDMs. In essence, the
method involves aerobic growth of yeast culture in general growth medium
at 20-30°C and then overlaying the colonies with 20 ml of molten TTC
overlay agar. After about 3 hrs at room temperature, colonies begin to
differentiate. Pink to red colonies are respiratory sufficient while the
colorless colonies are RDMs.
164
12.2.1.4 Yeast purity
Pure yeast strains are prerequisites for good brewing performance and product
uniformity. The purity of brewers yeast is most precisely analyzed by DNA
fingerprints. Using this technique, the yeast chromosomes are first separated
according to size by gel electrophoresis. Chromosomal DNA bands fluoresce in UV
light when stained with ethidium bromide. Each of the analyzed strains exhibits a
unique chromosomal DNA pattern (Fig. 12.2).
1 2 3 4 5
2000 Kbp
Kbp = kilobase pairs
1, 2, 3 and 4 refer to
brewer's yeast type
200 Kbp
Fig. 12.2 Electrophoretic analysis of yeast chromosome
Wild yeasts in beer
Wild yeasts in brewing are undesirable for a host of reasons, the more important of
which are:
1. Poor flocculation characteristic in wild yeasts produce haze and turbidity in

beer
2. Wild yeasts may form film or pellicle on the surface of the wort/beer, which
is objectionable
3. Super-attenuation may result due to utilization of maltodextrins by wild
yeasts. This affects the body, taste, and alcohol content of the beer.
4. Off-flavors may be produced
12.2.2 MALT
Malt is cereal, usually barley, which has been allowed to germinate for a limited
period of time and the growth terminated by drying. Malt is used for following
reasons: (i) as an enzyme source, (ii) as a substrate, and (iii) for flavor and color.
European beers use two-row variety barley for malting. The US beers use six-row
varieties for the same. The latter has lower malting quality but has higher level of
enzyme activity and hence suitable when adjuncts (see later) are used.
Before a new barley variety can be accepted for the production of malt, it is tested in
micro-, pilot-, and production brewing trials. Some of the varieties accepted in many
165
European countries are Alexis, Barke, Prestige, and Scarlet, all of which are 2-row
variety.
Recently, newer varieties of barley, collectively called proanthocyanidin-free barley (e.g.,

Chamant, Prominant, etc.) are being used for preparing beer with better haze (turbidity
that appears when beer is cooled) stability and greater brilliancy.
12.2.2.1 Safe storage of barley
Barley should be stored at a low temperature and moisture content below 12% to
avoid fungal problems and reduction in seed vigor (see Table 12.3). The molds most
implicated are species of Aspergillus, Penicillium, and Fusarium. These molds, Fusarium
in particular, produce metabolites (not yet characterized) that have been implicated
for gushing in beer, where the beer spontaneously gushes from the bottle on opening.
Gushing in beer is a very severe quality defect.
Table 12.3 The influence of moisture and storage temperature on seed vigor
Seed moisture
Temp. °C 10% 12% 14% 16% 18%
0 16 years 6 years 2 years 1 year 190 days
6 9 years 3 years 1.3 years 210 days 105 days
12 5 years 1.6 years 240 days 110 days 55 days
18 2.3 years 290 days 115 days 50 days 25 days
24 1 year 130 days 55 days 25 days 12 days
30 210 days 55 days 22 days 10 days 5 days
12.2.2.2 Testing for barley vigor
Fast and even germination during malting is an essential quality parameter of malting
barley. Prior to malting, all barley lots are analyzed for both germination percentage
and germination rate, measured as germination index.
Just after harvest, especially after a cold summer, the barley may exhibit dormancy,
preventing germination. Dormant barley has to be stored for a certain span of time
until it has developed full germination capacity. In order to analyze the germination
capacity of a barley lot just after harvest, the dormancy has to be broken. Carlsberg
Research Laboratory has developed a method for breaking this dormancy, in which
the barley is treated with H2O2.
12.2.2.3 Selection of barley grain for malting
Barley selected for use in the malting industry must meet special quality
specifications. Accepted malting barleys have to modify (see later) evenly and produce
a finished malt whose properties lie within the brewer’s specifications. The malt
166
quality of a given barley variety is determined by its genetic background and the
physical conditions during growth, harvest, and storage.
Malting quality has to be tested in micro-, pilot- and industrial malting trials.
Thereafter, brewing trials are done in pilot and production scale.
The physical condition of the barley must meet specifications concerning:
 Germination: min. 97% after 3 days

 Germination index: min. of 6.0
 Moisture content: 12%, max.13%
 Protein: > 9% and < 11.5%
 Grading: 90% > 2.5mm
 -glucan: max. 4%
 Variety purity: min. 99%
12.2.2.4 Malting
The key physiological events of malting, which determine the quality of the final
malt, include rapid and uniform germination, the synthesis of hydrolytic enzymes in
the scutellum and aleurone tissues surrounding the endosperm (Fig. 12.3) and finally
the degradation of endosperm cell walls, described as modification. Gibberellin, which
is a plant hormone that regulates the physiological events, is produced by the
germinating embryo.
During germination, enzymatic hydrolysis of endosperm mobilizes the nutrients and

energy reserves of the grain for the growing barley plant. Growth of germ is an
unwanted incidental to the making of malt, because it leads to respiration and
growth of new parts. After germination to a desired extent, the growth of the
embryo is therefore terminated by drying. It is customary to further dry the malt in
kilns for long-time storage. In summary, malting process involves:
 Collection of suitable stocks of barley

 Storage of the cereal until required
 Steeping in water
 Germination of the grain
 Drying and curing in the kiln
The selected malt grains are steeped in water at 10-16°C to raise the moisture
content of the grains to 42-46% whereupon germination begins. Steeping is done to
provide correct environment for the synthesis of hydrolytic enzymes and their
controlled action on the cell wall of the reserve protein, hordein. The distinction
between steeping and germination, however, is artificial. A steeping time of 70-72
hrs is not uncommon, and usually, steep water is changed every 4-10 hrs. Water
enters the grain via the embryo, and after 24 hrs, the first visible sign of germination
is the appearance of the root as a white “chit”. See Fig. 12.3 for the cross-section of
barley grain.
167
Aleurone
Endosperm
Scutellum
Embryo (germ)
Husk/hull
Fig. 12.3 The barley grain
Germination can be carried out by various methods, e.g., on concrete floor, in drum
germinators, compartments, and pneumatic germinators.
Modern malting employs combined steeping-germinating technique. The equipment

consists of a cylindroconical tank of a short height. The grain is transferred to make
a shallow grain bed. Every 4-6 hrs, water is changed and the barley is intensively
aerated from the bottom following each drainage. Air can also be introduced in the
steep-water. Aeration is essential because germination cannot occur in absence of
O2. CO2 that accumulates during germination is drawn out for 10-15 min every hour
or so. Germination can be accelerated and regulated by post-steeping application of
gibberellic acid (a plant growth hormone) to initiate enzyme production by the
aleurone layer. It is possible to enhance the passage of gibberellic acid into the
aleurone layer by mechanically abrading the husk and underlying layer. This process
is used commercially to accelerate malting or to assist the malting of poor quality
barley. The application of gibberellic should be as early as possible. Typically, 0.03-
0.08 g of this hormone is needed per 1000 kg of barley.
In modern practice, condition during germination is controlled by passing

humidified, temperature-controlled air through the germinating grain. Continued
growth of embryo, with the appearance of rootlets and acrospires, can lead to root
entangling. The grain bed is regularly turned with a rotating screw to prevent grain
matting together. It is desirable to reduce malting loss by limiting growth and
168
respiration and this may be achieved by resteeping. A final steep at 40°C may also be
employed to achieve root killing. The whole process may take 8-9 days. Tropical
grains may require temperatures above 24°C for malting.
Barley is preferred over other grains for the preparation of malt. This is because of
following reasons:
 The grain retains husk, which affords protection during transportation and
storage
 Coarse husk particles aid in wort filtration
 Comparatively low germination temperature of the barley
 Comparatively high β-amylase content of the malt.
Assessment of the completion of malting is carried out organoleptically, by chewing

and rubbing. The loss due to respiration and growth is about 6-10% on dry basis.
Modification
Proteolysis of aleurone protein is the first hydrolytic reaction to occur. The enzymes
then move on to the endosperm. Endospermic starch and storage proteins are
enclosed in the cell walls composed mainly of β-glucans. The endospermic
hydrolysis is therefore preceded by hydrolysis of β-glucans by glucanases. Once the
cell walls are removed, starch and proteins are hydrolyzed to varying degrees by their
corresponding enzymes. α- and β-amylases are the most important carbohydrases
from brewery point of view. The sugars and amino acids thus produced are
mobilized for the nutrition of the growing barley plantlet.
 amylase is already present in the grain but increases during the initial phase of
germination.  amylase is undetectable in the ungerminated barley but is formed
during the latter phase of germination.
A term frequently used by malsters is modification. The term refers to alteration of

structure of three main categories of polymers in the barley endosperm, namely:
 Cell wall polysaccharides

 Reserve protein, hordein
 Starch granules
A well-modified malt is one in which all the cell walls have been eliminated by
hydrolysis. Overmodification means extensive damage (corrosion) of starch granules.
The cell walls of barley endosperm primarily contain β-glucan and pentosans. β-
glucans must be degraded as completely as possible because they are highly viscous.
They complicate recovery of malt extract, affect filtration, and impede wort run off.
The development of the acrospires and the progress of modification of the kernel
run approximately parallel to one another.
169
12.2.2.5 Drying and curing in kilns
The degree of modification required depends on the type of beer to be brewed and
when this has been achieved, malting is stopped by drying.
These days, several types of highly efficient kilns are available for drying and curing
of germinated barley. Modern kiln types include single floor circular kilns, two floor
circular kilns, high performance kiln with loader and unloader, and vertical kilns. The
duration of kilning is 18-20 hrs. After drying and curing, the rootlet fraction (called
“culms”, and it constitutes 3-4% of malt) is removed in a malt deculming screw.
Before dispatch, the grain is subjected to mechanically operated brushes to give the
kernels an attractive, polished appearance.
Drying can be carried out in 2-3 stages. A typical three-stage method involves:
 Initial stage: 50-60°C until 25% moisture content is achieved

 Second stage: Temperature slowly increased to 71°C. Moisture content of malt
is lowered to 12%
 Final stage: This stage is also called curing or kilning. The malt is dried at 71-
92°C (sometimes even higher temperatures are used) until the moisture
content of barley drops to 4-5%
There are, of course, considerable variations in the time-temperature combination.

For instance, a final temperature of 95-105°C is used for the production of dark
malt. Such malts are not primarily used as enzyme sources. They only impart malt
favor and color.
The grains are immediately cooled to 38°C or lower. The final stage eliminates the
green, grainy taste and supplies the characteristic malt flavor. Higher temperatures
increase acidity. The color will also darken due to Maillard reaction.
Purpose of kilning
 Removal of water to obtain keeping quality of the highly perishable unkilned

green malt
 Interruption of germination, i.e., inhibition of metabolism by drying
 Formation of aroma, taste, and color by curing
Effects of curing on endogenous enzymes
 α- and β-amylases are only slightly decreased during drying. During curing,
β-amylase is inhibited to a greater extent compared to α-amylase.
 Proteolytic enzymes are destroyed noticeably only at curing temperatures
above 100°C
 Catalase activity is strongly diminished during drying, and curing
temperatures completely inactivate it
 Polyphenolase activity is hardly affected
 Lipase activity is partially inactivated
170
The kilned-and-cooled malt is dropped into collecting hoppers, sprouts severed over
sprout cleaner (deculming device), and then stored for 3-6 weeks to several months
before blending and shipping. Before shipping, malts from different lots are blended
to meet the specification. This is essential because it is seldom possible to produce
malt of the same quality (despite utmost care in malting). Malt with moisture content
higher than the specification is called slack.
12.2.2.6 Malt milling
Malt milling is carried out in the brewery. There are two types of malt milling
processes, viz., (i) dry milling, and (ii) wet milling. However, there are several
variations within each milling type.
Dry milling
Dry milling can be carried out in roller mills or hammer mills. There are several
types of roller mills and may contain 2 to 6 rollers. The six-roller mill (see Fig. 12.4
for the principle) is the best and most frequently used type of grist mill. Its three
pairs of rollers are called (a) primary crushing roller pair, (b) husk roller pair, and (c)
grits roller pair.
There is a vibrating screen unit with two different mesh sizes suspended between
each pair of rollers. These screens sort the milled material from the preceding pair of
rollers into three fractions, viz., (i) coarse components (husks with attached grits),
(ii) middle components (grits), and (iii) fine components (fine grits and flour). The
flour portion is led directly into the grist case since it is not milled any more. The
husks are crushed, with as little damage to the husks as possible, by the second pair
of rollers. The grits are ground by the third pair of rollers to the extent desired.
Preliminary
Feed crushing pair
Distributor roller Husk with attached grits
Grits
Flour Husk crushing
roller pair
Upper vibrating
screen
Lower vibrating
screen
Grits roller pair
Fig. 12.4: Principle of six-roller malt milling
The bulk density of the malt thus milled is about 20.8 lb/ft3. It is important to note
that husks are not discarded. A typical composition of malt that goes into mashing
is: husk 15%, coarse grits 23%, fine grits 34%, and flour 28%. The main objective of
adding husk is to facilitate filtration. Husks also provide some flavor components to
beer. Dry milling produces good extract but the wort run-off time is longer.
171
In modern type mash filters, filtration is performed by means of small pore
polypropylene filter cloths with a spent-grain layer thickness of only 4 cm. In such
cases husks are not needed as filter material and the malt can be ground finely. Fine
milling is done using a hammer- or pin mill. In a hammer mill, swinging
hammerheads are attached to a rotor that rotates at high speed inside a hardened
casing. The principle is illustrated in Fig. 12.5. The material is crushed and pulverized
between the hammers and the casing and remains in the mill until it is fine enough
to pass through a screen which forms the bottom of the casing.
Feed
Hammer
Stator Rotor
Fine product
Fig. 12.5 Principle of hammer milling
Wet milling
Wet milling of malt can be carried out by several methods. Today, the conventional
wet mills are no longer manufactured. A variant of wet milling process called “steep
conditioning” is widely used. A steep conditioning operates as follows (Fig. 12.6):
CIP
Grist hopper
Water supply
Cleaning nozzles
Feed roller
Cleaning nozzles
Crushing rollers
Spray nozzles
Mashing pump
Fig. 12.6 Schematic of steep conditioning
172
In the grist hopper the weighed amount of grist is stored and is supplied with hot
water in the conditioning stage for about 60 s. The water temperature is kept at
around 60-70°C. The rapid uptake of water by the kernel makes the husk rubbery.
The grain contents are then comminuted by a pair of rollers. The milled grist is
intimately mixed with water (by means of spray nozzles) at mashing-in temperature
and transported by means of a pump from below into the mash vessel. Some oxygen
uptake occurs during milling because of the longer grist mashing-in time and this is
undesirable. These days, there are systems that inject CO2 during milling to minimize
the entry of oxygen.
12.2.3 ADJUNCTS
Adjuncts are defined as non-malted carbohydrate materials of suitable composition

and properties that beneficially complement (go well with, often contrasting
characteristics) and supplement (add to make complete) barley malt. Adjuncts are
used primarily as a cost-saving initiative but other advantages can also be achieved,
for example, it produces beer:
 With lighter color

 That is less satiating and has thinner taste
 With greater brilliancy
 With enhanced stability
 With superior chillproofing quality (see page 209)
Adjuncts can be classified into two groups, viz.:
1. Starchy materials: cassava, maize, corn, rice, etc.

2. Sugar and sugar syrup: Today’s liquid adjuncts are clear, colorless, non-
crystallizing liquids consisting of carefully controlled mixtures of glucose,
maltose, maltotriose, and maltodextrins. Liquid adjuncts have following
advantages:
 Better wort run off
 Clearer wort
 Better control over kettle operation
 Increased productivity (more hectoliters per brew)
12.2.4 HOPS
Hops as used in brewing are the dried blossoms of the female hop plant (Humulus
lupulus, Fig. 12.7) or their preparations (extracts, powders, pellets, etc). Hops are
primarily employed in the brewing process for imparting a bitter flavor to beer. Since
hops also contain many unique components, the flavor they impart to beer is of
unique hop character. Hops have other beneficial functions also, viz.:
 Contribute to palatefulness, colloidal stability, and head retention (page 208)

 Have antiseptic properties
 Tannins present in hops aid in precipitation of undesirable nitrogenous
substances of the wort by forming protein-phlobaphene complex (page 188)
173
Fig. 12.7 A feemale hop conne
112.2.4.1 Hop vaarieties
TThere are threee species of hops,

h viz., (i) Humulus lupullus (common hhop) (ii) Hummulus
jajaponicus (Asian hop), and (iii) Humulus yunnanensis (Y Yunnan hop)). From brew wing
ppoint of view,, hops are off two main tyypes: (i) bitter hops, and (ii) aroma hoops.
BBitter hops aree used universally. Some exxamples of bitter hop varieeties are: Brewwers
ggold, Northernn brewer, Galeena, Target, ettc.
112.2.4.2 Hop coomponents and chemistry

c
A good beer needs

n a good aroma,
a an acceeptable palate,, and a great ddeal of flavor tthat
iss provided byy hops. It has been said: “M Malt is the souul of beer and yeast gives it life
bbut the kiss off the hops is thhe consummaation of that liffe”.
HHop constitueents of imporrtance in brew wing are hop resins (14-21% %), essential oils
((0.5-1.5%), poolyphenols (2--5%, about 800% of which are anthocyaanidins), proteeins
((20%), and minerals (8%). Essential oilss are responsible for hop aaroma. The m main
ccomponents (80-90% conntribution) off essential oills are terpenne hydrocarbbons
((myrcene, hum mulene, and caaryophyllene).. There are abbout 200 comppounds in thee oil
ffraction of thee hops. Resinss are responsiible for the plleasant bitternness in beer. TThe
ttotal hop resinn can be classiffied as shownn in Fig. 12.8.
Hopp resins
Sofft resins Harrd resins

(Solublee in hexane) (Inssoluble in hexanne,
no brewing
b value)
-resins
 
-resins
(FForm lead salt, which is
innsoluble in methhanol) C
Contain 5 -acidds
C
Contain 5 -acidds LLupulone
C
Colupulone
Humulone
H A
Adlupulone
C
Cohumulone P
Prelupulone
A
Adhumulone P
Postlupulone
P
Prehumulone
P
Posthumulone
Fig. 12.8 Hoop componentts

1174
Alpha-acids (-acids) are of prime importance in brewery (Fig. 12.9). Breweries
therefore use α-acid content as a criterion in purchasing hops. Normally, hops
contain 6-8% -acids and 3-4% beta acids (-acids). In the improved varieties,
however, the α-acid content can reach as high as 19%, or even more.
O O O O
R R
Where,
HO OH HO OH R = isobutyl, isopropyl,
OH sec butyl, etc.
-acid -acid
Fig. 12.9 General structure of α- and β-acid
12.2.4.3 Hop processing
Only female hop cones are used (Fig. 12.7). The picked hops contain 75-80% water.
Drying is performed on belt driers or in small firms, in batches in kilns. The hops
are carefully dried at a maximum temperature of 60°C to a water content of 8-12%.
The dried hops are then cooled and packed into rectangular bales of 508040 cm3,
typically weighing about 90 kg.
Because hop components are very unstable, stabilization of the dried hops is
essential. For this, the sacks of loosely packed hops are emptied into sulfur treatment
chamber at the packaging plant. Sulfur is burnt beneath this chamber and the sulfur
dioxide produced acts as a disinfectant and antioxidant. The sulfur dioxide is then
driven off by fresh air.
The hops thus prepared can be used as such or processed further into different
forms. A brief description of processed hops is given in the sub-sections to follow.
12.2.4.4 Hop storage
Hops are unstable at higher temperatures and in environment having access to air.
They are generally stored at 2-4°C. However, this does not fully arrest the changes:
they still lose their bittering potential and the α-acid content (at a slower rate,
though). The decrease in α-acid content is greater than the bittering potential
because the oxidation products of α-acids also have some residual bitterness. -acids
also contribute to bitterness but it is about 9 times less bitter than -acids. Bitterness
value is therefore expressed as:
β-fraction
Bitterness value = α-acids 
9
175
12.2.4.5 Some commercial hop products
Of the commercial hop products, hop pellets (and their variants), hop extracts and iso-α-
acid extract are the most important.
Hop pellets
A very effective way of preserving the contents of hops is pelletization. In this

process the dried hops are milled to a powder and then compressed into pellets.
Three types hop pellets are available commercially, viz., (i) pellets type 90, (ii) enriched
pellets, and (iii) isomerized pellets.
In the production of type 90 pellets, 90 kg of powder, containing all the important

contents of the original hops, are produced from 100 kg of intact hop cones. Hop
pellets are prepared by hammer-milling of dried hops to powder followed by
pressure-pelletizing in dies. The dense pellets, which are roughly 4.5 mm  4-6 mm
in size, are vacuum-packed and stored under refrigeration. Instead of vacuum
packaging, CO2 or nitrogen can also be used as a protectant.
Manufacture of enriched pellets is much more complicated. All the resins and the
oils are present in the lupulin gland of the hops. Since the glands have a natural
particle size of ~ 0.15 mm the task of removing them from the cone is not easy. For
this, gentle milling and sieving at low temperature (-35°C) is done in a special
machine. The finely milled material contains lupulin gland and about 50% of the
dried cone mass. For the production of enriched pellets, the lupulin glands must be
intact and not crushed.
Enriched pellets give about 10% more bittering yield compared to cone hops. The
packaging of enriched pellets is similar to that of pellet 90.
Isomerized pellets are produced by adding magnesium oxide (as a catalyst for
isomerization of -acid to iso--acid) to the milled hops before pelletizing. The
pellets are produced as usual, packed, and then placed in a warm chamber at 50°C
until isomerization is complete.
Hop extracts
Hop extracts are concentrations of α-acids. Hop extracts have several advantages
over pellets, powders, and cones. Improved hop utilization is the most important
factor contributing to cost saving. In conventional brewing in which hops are boiled,
bitter principles are lost to the extent of 75% during boiling. The loss figure when
hop extract is used is 5-20%. Depending on the type of hop extract, it is also
possible to add the extract at various stages of wort boiling.
Some of the important advantages of hop extracts are:
 Improved hop utilization

 More consistent bitterness between successive brews
176
 Improved stability on long-term storage
 Reduced transportation, storage and handling costs
Hop extracts are prepared by extraction with liquid CO2 or ethanol, the former
being superior. In order to be able to use a gas such as CO2 as an extractant it must
have a density similar to that of a liquid. This high density in the region 0.9-1.0
kg/liter is obtained by compressing the CO2 gas.
In principle, CO2 exists in two states which can be used for extraction. The critical
pressure of CO2 is 73 bar and the critical temperature 31°C. Above this pressure but
below this temperature CO2 is liquid but its solvent properties are very limited.
Above the critical points the terms used are supercritical CO2 or fluid CO2.
For hop extraction purposes, useful solubility properties are obtained with
supercritical CO2 pressures of 120 bar and above. Worldwide, hop extraction is
nowadays performed with supercritical CO2 at pressures of 150-300 bar and
temperatures of 32-100°C.
The hops to be extracted are put, as pellets, into the extraction vessel and the latter
are brought to extraction pressure (Fig. 12.10). Liquid CO2 at 60-70 bar is drawn
from a working tank and compressed to extraction pressure. The heat exchanger is
set to produce the extraction temperature and the CO2 is pumped through the
extraction vessel. The bittering and aroma substances are thereby dissolved in the
CO2. The enriched CO2 passes into the separation tank. Before this, the pressure is
reduced to 60-70 bar in the expansion valve and the CO2 evaporated in the heat
exchanger. As a result the CO2 loses its ability to act as a solvent and the extract is
separated in a container. The gaseous CO2 is liquefied in the condenser and returned
again to the extraction circuit.
Expansion
valve
Heat
Extraction
exchanger
vessel
Condenser
Separated
extract strainer
Heat
exchanger
CO2 tank
Compressor
Fig. 12.10 Schematic of liquid CO2 extraction
Extraction with liquid CO2 is the best method available. This solvent is highly
selective: essential oils and α-acids are extracted sequentially. The final product is
free from solvent and is stable at room temperature
177
Iso-α-acid extract
α-acids themselves are not bitter. It is the iso-α-acid (Fig. 12.11), produced during
wort boiling, which is responsible for the bitterness. Thus, boiling of hop extract is
obligatory. Preisomerized α-acids are now available. They can be directly metered into
the final beer. The preparation begins with the extraction process. The hop extract is
then isomerized by various means. One method is to treat the extract with aqueous
sodium- or potassium carbonate to obtain respective salts of iso-α-acids.
CO2 extract is used to produce isomerized extract. The extract is heated and
emulsified with degassed water. This emulsion is heated further and isomerized by
the addition of a basic catalyst (Mg++). This isomerization must be controlled. After
the isomerization, hop waxes and uncharacterized soft resins, which are of no use in
the further processing, are removed. The solution is then cooled and brought to a
pH of 7-8. This results in precipitation of the -acids and they are removed. As a
result of a further pH adjustment to 5-6 the unisomerized -acids are precipitated
and they are then removed by a separator.
Free iso -acid is now precipitated by the pH reduction to 2 and stored in this form
until final packing. Only shortly before delivery to brewery is the iso--acid
converted to the potassium salt and then brought to the sales concentration and
filled into containers.
OH O O O O O
5 5
4 4
HO HO
HO O OH + OH
OH O O
-acid iso--acids
Fig. 12.11 Conversion of -acid into iso--acid
Reduced, isomerized α-acid extract
This special type of isomerized hop extract is obtained by simultaneously reducing

and isomerizing α-acids by treating with borohydride. This extract permits the use of
clear bottles for packaging without the development of sunstruck flavor (page 202).
Hop extract powder
It is prepared by spraying hop extracts onto silica gel. In order to make the hop
extract powder pour easily, at least 30-40% silica gel is required. Instead of silica gel,
hop powder or hop pellets can also be used.
12.2.5 BREWING WATER
Brewing water must meet not only the general requirements for potable water but
also other specific requirements in order to:
178
 Ensure proper mash pH
 Ensure efficient hop extraction
 Produce good kettle break
 Allow sound fermentation
 Produce acceptable color and flavor in the finished beer
In almost all breweries, the incoming water is passed through activated carbon and
in some cases, also through ion-exchange resin. Since the mineral composition of
natural water is very much subject to variation, most brewers consider it
advantageous to control the composition by external manipulation. The water may
be rendered soft and salt mixtures such as gypsum (CaSO4) added in controlled
amounts to give known hardness. Addition of CaSO4 to make it similar to the
natural water of Burton Upon Trent (of United Kingdom) for brewing of beer is called
burtonization. For lager beers, soft water is desirable at 200-300 ppm hardness.
Hardness, particularly due to calcium, has many important functions, especially
during mashing. For a good brew, it is normal to maintain 60-70 ppm Ca++ content.
Alkalinity is also important, and is maintained at 25 ppm.
12.2.5.1 Functions of Ca++
 Protects α-amylase from heat destruction

 Stimulates activity of proteases and amylases
 Increases the yield of fermentable extracts
 Controls the mash pH. Ca++ precipitates out calcium phosphate and
decreases pH to 5.4 from an initial pH of 6.
 Aids in flocculation of proteinaceous materials and yeast cells
 Aids in oxalate removal. Oxalate is poisonous.
12.3 OUTLINE OF BEER PRODUCTION
The brewing process proper starts with the mashing of barley malt and adjuncts.
Mashing entails cooking of properly ground malt and malt adjuncts (for example, in
the ratio 97:3) at a combination of time-temperature so that substrate constituents
are degraded to forms readily assimilable by yeasts. Mashing is followed by
separation of the solids, which can be carried out either in mash filters or lauter tuns.
The liquor, called wort, is then boiled. Hops are added at a rate of about 0.4% and
boiling carried out for a total period of about 1.5-2.5 hrs. Hop residues are then
strained off, trub (the precipitate) removed in a whirlpool separator, and the hopped
wort cooled to a pitching temperature of about 10C. The main fermentation consists
of adding vigorously growing yeast inoculum (~ 10 million cells per ml of wort) in
the cooled, pre-oxygenated wort at pH 5 and fermentable sugar concentration
around 12%, and allowing anaerobic fermentation at: 8-12C (about 14 days) for
lagers, and 12-18C (about 8 days) for ales. Fermentation is carried out in
cylindroconical vessels, allowing 25% headspace. Considerable amounts of foam and
heat may develop. Foaming can be controlled using antifoams like sorbitan esters,
alcohols, etc. The heat (and therefore the fermentation rate) can be controlled by
internal cooling coils. The CO2 evolved is collected and purified for future use.
During the course of fermentation, the pH drops to 4, the yeasts utilize 90% of the
179
fermentable carbohydrates and nitrogen, and the cell population increases by 4-6
folds. The settled (or floating) yeast cells can be removed after cooling to 0C by
sedimentation (or skimming) respectively. The green beer (also called ruh beer) is
pumped to a storage tank for fassing while the yeast is recycled. Fassing may take
place for several weeks to several months. Depending on the process followed,
fassing may be called lagering, aging, or maturing. The process may or may not involve
secondary fermentation, called kraüsening. A sizeable fraction of yeast biomass and haze
settle down during the fassing period. The process can be hastened by immediate
centrifugation of the green beer. The beer is then passed through kieselguhr filter
and a polishing filter, CO2 is amended to 0.5%, and packed in containers.
Pasteurization can be done before or after packing by chemical- or physical (heat,
filtration) means. See Fig. 12.12 for the flowsheet of beer production.
MALTED BARLEY
Milling Adjuncts (cereals)
Ground malt Cereal cooker Brewing water
Mash tub
Pure culture
Lauter tub Hops
Propagation
Brew kettle
Spent grain BEER
Hop strainer
Yeast Pasteurization
Whirlpool separator Yeast recycle
Hop residue Bright Beer Tank (BBT)
Yeast
inoculum Separated yeast
Wort cooler Carbonation
Hot trub
Wort aerator
Fermenter Clarifier Ageing Filter

CO 2 Purification
Fig. 12.12 Outline of beer production
The production steps include:
1. Mashing
2. Wort boiling
3. Wort cooling
4. Wort oxygenation and pitching
5. Fermentation
6. Yeast and particle removal
7. Aging and final processing
8. Carbonation, pasteurization, and packaging
180
12.4 PRODUCTION DETAIL
12.4.1 MASHING
Mashing is a process whereby a combination of time and temperature is used to

cook malt and/or adjuncts so that malt enzymes are activated to hydrolyze the
substrates and produce as much of valuable soluble portions of malt and/or adjuncts
as possible. These soluble fractions are needed for yeast nutrition and metabolism.
The objectives of mashing can be summarized as:
 To dissolve as much of readily soluble fractions of the ingredients as

possible. This fraction constitutes 10-15%
 To render soluble (through enzymatic actions) substances which are
insoluble in their natural state
 To change the chemical structures through simultaneous enzymatic action,
of some constituent substances in a planned and predictable manner.
From unit-operation point of view, mashing entails (i) mixing, (ii) enzymatic
reactions, (iii) liquid-solid separation, and (iv) elution. The major enzymes and their
reactions are:
 -and  -amylases
Starch 
 Dextrins  maltose
 -glucanases
 -glucans  Low molecular weight carbohydrates
Proteins  Peptides and amino acids
proteases
Other enzymes of importance are limit dextrinase, xylanase, and -glucosidase.
The malt starch is degraded to the extent of 90-95%. The undegraded part consists
of dextrins. Malt protein is degraded to the extent of about 40%. Phosphatase
enzymes also help in releasing phosphate, which in turn is utilized by the yeasts.
Mashing begins with doughing in, which is simply mixing of ground malt and/or
adjuncts with brewing water. This is then heated in vessels called mashing vessels
(also called mash converter, mash tun, mash tub, and mash cooker: see Fig. 12.13).
The amount of water to be used is variable but in general is 1.6-3.2 times the weight
of grist for infusion mashing and 3.2-5.4 times for decoction mashing. If the adjuncts
(see later) consist of cereals, they are given an additional preliminary mashing in cereal
cooker (vessel similar to mash converter but smaller in size) before adding them to the
main mash. Some amount of malt (~10%, but this may be omitted if the adjunct is
less than 20% of the grist) is also added in cereal cooker. Of the cereal adjuncts, rice
is the most difficult to mash because the starch granules are very small and firmly
embedded. The granules swell only slowly in water.
The temperature of water to be added for the doughing in (called striking heat) is also
very critical and its calculation is quite involved.
181
The mashing vessel is similar to the brew kettle. The schematic of a modern mash
converter with provisions for agitation, heating arrangement and CIP is shown in
Fig. 12.13. The mash heating coils consist of semicircular welded-on pipes. The
mixing is normally achieved in a premasher (grist hydrator) built into the mashing-in
pipe (Fig. 12.14) before the actual mashing begins. Premasher is a mechanical unit
that prepares the grist in the slurry form for mashing.
top cover
vapor chimney
interior light
viewing and
access opening
CIP cleaning
heating pipe
(semi-circular)
insulation
stirrer
mash inlet
drive motor and outlet
Fig. 12.13 Schematic of a modern mash converter
water supply
hydrator
pre-mashing chamber
Fig. 12.14 Premashing equipment
During mashing, the stirrer (present at the bottom of the vessel) must be run at a
controlled, slow speed for good contact and reaction with malt enzymes.
Basically, there are two main methods of mashing, viz., (i) infusion mashing, and
(ii) decoction mashing. But there also exist a number of variations. Since the
efficiency of mashing is dependent on pH, the mash pH is maintained at around 5
with food-grade acids like lactic acid.
The time-temperature combination during mashing is very important. For instance,

prolonged mashing may liberate too much nutrients, which will consequently
182
encourage the growth of contaminating organisms, or even overgrowth of culture
yeasts. This will significantly affect the organoleptic quality of the final beer.
Whatever the mashing technique, mashing entails following four common,

obligatory events:
1. Mashing in: Mixing of malt and water

2. Protein pause (rest): Release of peptides and amino acids
3. Sugar pause: Release of maltose and maltodextrins
4. Mashing off: Degradation of residual starch, and inactivation of enzymes
12.4.1.1 Infusion mashing
There are two variations in this technique, viz., (a) temperature programmed infusion, and
(b) single temperature infusion, the former being the industry standard. Infusion
techniques are used for the production of ales. The temperature-programmed
infusion technique uses two sets of temperatures, viz., (i) 38-50°C for about 1 hr, and
(ii) 65-70°C for a few minutes. Both the sets are obligatory.
The lower temperature favors the activity of proteolytic enzymes and the mashing
period is called protein-rest period. The higher temperature favors starch
saccharification and dextrinization. The heat is provided by internal heating coils or
welded-on pipes (Fig. 12.13). The mashing may follow in either direction. When the
mashing is carried out in the direction (i)(ii) it is called upward infusion. By analogy,
the variation (ii)(i) is called downward infusion. Whatever the method followed,
mashing is terminated by raising the temperature to 75°C, or a little above. This
increase in temperature is sufficient to destroy enzymes. The mash is filtered at this
temperature. See Fig. 12.15 for the different enzymatic reactions taking place during
mashing.
12.4.1.2 Decoction mashing
This is used for the production of lager beers. In general, slightly less modified malts
are used here. Water is added at the rate of 3.2-5.4 HL/100 kg grist and the
temperature maintained at 40°C after mixing. The temperature is raised in steps until
about 75°C. The temperature increment is achieved in an interesting manner. A
portion of mash (about 1/3rd) is withdrawn and boiled for a short period of time
and returned to the main mash. Upon mixing, the heated mash raises the
temperature of the entire mash bulk. The mixture is left as such for 20-30 min and
the next portion is taken out again for the boiling. The enzymes in the boiled
portions have been destroyed, but the cell walls of the grain are softened and starch
is liquefied. Diastatic action is facilitated in this manner. The process may be called
single-, two-, three-, etc.-, mash depending on the number of times portions are
removed for heating. The single-mash method is essentially an infusion mashing
technique. Usually, the decoction mashing employs up to three-mash level.
During the rest period, the mash separates into two fractions, viz., (i) the “thin
mash” that occupies the upper portion, and (ii) the “thick mash” that occupies the
183
bottom portion. Thick mash consists of undissolved mash components. During
decoction mashing, it is the thick mash that is taken for incremental boiling.
80 4
3 77oC
70
68oC
60
2
50 52oC
saccharification
40 1
38oC starch
30 gelatinization
20 protein degradation
10 cell wall degradation
0 20 40 60 80 100 120 140

Time (min)
Fig. 12.15 Relation of enzyme action with mashing time-temperature regime
During mashing, the rest period is controlled carefully. Prolonged rest period leads
to excess break down of the mash components, which is not desirable. Excessively
hydrolyzed mash promotes growth of contaminating microorganisms and also leads
to poor foam properties of the beer.
12.4.2 SEPARATION AND WASHING OF MASH
At the end of the mashing process the mash consists of a watery mixture of
dissolved and undissolved substances. The aqueous solution of the extract is called
the wort; the insoluble part is referred to as the spent grains. The process of separation
of the soluble portion from the spent grains is called lautering (or mash separation).
Several types of mashing- and mash separation systems are available today. Great
innovations have taken place in the mash filter designs. There are two basic types of
mash separators, viz. (i) lauter tun (= lauter tub), and (ii) mash filters.
12.4.2.1 Mash filter
Mash filters are basically variants of membrane-assisted plate and frame filter press.
They have provisions for compression and CIP also. The device can handle 12-14
brews per day. The amount of sparging water needed is about 2.5 HL per 100 kg
malt. A typical mash filter assembly is given in Fig. 12.16. See Fig. 12.17 for the
structural units of plate and frame.
Fig. 12.18 shows the principle of mash filtration in a modern mash filter designed by
Meura S. A., Belgium. The system has filter cloth as well as membrane. During
filtration, the wort passes through the filter cloth while the grains accumulate in the
frame. After the mash supply finishes, the filter is blown with compressed air to
recover the entrained wort. Next, sparging water is passed through another opening
to elute the residual extract. Finally, the filter is once again blown with air to recover
as much of the extract as possible. The filter needs to be cleaned and disinfected
184
after the frames become full with the spent grain cake. The cake can be removed by
dismantling the assembly from the movable end. The filter assembly must be cleaned
by CIP (cleaning-in-place) technique every one week or so.
main mash supply
mash supply to
movable end
mash supply to
control valve
fixed end
manometer
movable shut off valve
handle end plate
pump plate and frame thermometer
mixing unit
cold water
supply pipe
hot water
supply pipe
run-off tap water sparge
wort grant valves
carrier fixed end
beam plate
Fig. 12.16 Mash filter
frame
water supply
mash supply at the top
water supply
at the bottom
filter cloth
plate
tap
Fig. 12.17 Arrangement of plate, frame and filter cloth in mash filter
185
Wort
Mash
Mash
Wort
(A) Filter is ready in closed position (B) Introduction of mash
Compressed air
Wort Wort
Water
Water
Wort Wort
(C) Compression with air after (D) Sparging with water to
mash filtration is over recover the residual wort
Compressed air
Wort
Spent
grain cake
Wort
(E) Compression with air to (F) Dismantling of plates and frames
conclude the filtration for removing spent grain
Fig. 12.18 Mash filtration mechanism
186
12.4.2.2 Lauter tub
Lautering machines are becoming increasingly popular these days. The system,
however, is more complex than the mash filters. Lauter tub (also called lauter tun)
consists of a cylindrical vessel with false bottom, a raking unit, and several ancillary
parts (Fig. 12.19). The tank is first filled with recycled liquor (from the previous lot)
to cover the bottom. The mashed grain is introduced on the false bottom to form a
bed. The grain bed is agitated with the help of raking unit to facilitate extraction of
the wort. The “sweet wort” that is leached out finds its way through the false
bottom, ending up in a collecting vessel called “grant”. The first run of the wort is
recycled until the required degree of clarity is obtained. The residual soluble fractions
are eluted by sparging with hot water (75°C) through a spray manifold at the top
until the level of soluble fractions in the wash drops down to less than 1%. The run-
off time is 2-3 hrs and 98-99% recovery is possible. The spent grains can be
removed and dried for animal feed. After the extraction has finished, the vessel and
the pipelines are cleaned by CIP process.
There are several variations of the lauter tub. A design by GEA-Huppmann is given
in Fig. 12.19. See Fig. 12.20 for the schematic of the same design.
Sparger manifold
S-shaped spent
grain removal blade
Double shoe
raking knife
Geometrically optimized
wort run-off system
Central run-off
system
Fig. 12.19 Cut-away view of a modern lauter tub
Sparger manifold
Grain bed
Collecting pipe
Fig. 12.20 Principle of lautering in lauter tub
187
12.4.3 WORT BOILING
The sweet wort is taken to a vessel called brew kettle or copper (as it is usually made of
copper, see Fig. 12.22) and boiling started. The heating is done using internal heating
coils to achieve vigorous ebullition. Requisite amounts of hops are added at this stage
and boiling carried out for 1.5-2 hrs. The boiling fulfills various objectives:
 Isomerizes α-acids and extracts hop components

 Precipitates unwanted nitrogenous materials (hot trub)
 Removes undesirable volatile compounds
 Sterilizes the wort
 Concentrates the wort (about 15% volume is lost by evaporation)
Many complex reactions occur during wort boiling. All enzymes are destroyed. Some
proteins are coagulated and some, together with simple nitrogenous compounds,
interact with carbohydrates and/or tannins to give visible precipitates called hot break
or hot trub. The hopping rate is adjusted to roughly 8 g α-acid/HL of wort (or to
some defined final bitterness, e.g., 25 bitterness unit).
During kettle operation, the wort becomes slightly more acidic since the melanoidins
formed on boiling are acidic and the hops also contribute some acid. The color of
wort also becomes darker for the same reason.
There are many variations of the wort kettle. A modern wort kettle with internal
boiling is shown schematically in Fig. 12.21.
vapor condenser
wort kettle
internal
boiler
condensate
steam
Fig. 12.21 Schematic of wort kettle (brew kettle) with internal heating
188
Fig. 12.22 The exterior of brew kettle
The boiling protocol for the kettle design shown in Fig 12.21 is as follows:
1. Heating up to 100°C in about 15 min

2. Initial boiling at 100°C for about 10 min
3. Heating up to 102°C in about 10-15 min
4. Boiling under pressure at 104°C for about 15 min
5. Final boiling at 100°C for about 10 min
This design reduces the boiling time by 50% in comparison to conventional boiling.
At the end of the prescribed boil period, which may be based on time, evaporation
rate, or a combination of two, the wort is pumped through strainers (hop strainers) to
remove hop residues. The hot trub and any hop particles remaining in the copper
must be removed for subsequent processing of the wort. Whirlpool separation is the
most elegant method for break removal and is the least costly alternative of all trub
removal methods. In modern breweries, centrifuges are also used for the same.
A whirlpool is a vertical cylindrical vessel with no internal fittings, into which wort is
pumped tangentially. This produces a sustained rotational flow in the vessel, which
causes the hot break to settle in the shape of a cone in the middle of the vessel. The
clear wort can be drawn off at the side of the bottom. A vessel with a conical recess
in the middle has been shown to retain the trub better. See Fig. 12.23 for a typical
design of the whirlpool.
189
circular
motion
feed wort
hot trub
clear wort
Fig. 12.23 Schematic of whirlpool separation
The way in which the wort is pumped into the whirlpool is particularly important.
The wort inflow velocity should not exceed 5 m/s. The clear wort is removed from
the whirlpool after a rest period of 20-40 min. In some variations, however, the wort
is drawn even whilst the sedimentation is taking place in the lower part.
12.4.4 WORT COOLING
After trub separation the wort is cooled in tubular or plate-heat exchanger.

Additional trub, called cold trub, can be removed in this stage. Cooling fulfils several
objectives:
 Reduces the wort temperature to pitching temperature

 Aids in the removal of cold trub
 Reduces temperature to microbiologically safe zone
12.4.5 WORT OXYGENATION/AERATION
Brewery fermentation differs from most fermentations in that oxygen is supplied only
once. The purpose of wort aeration is not the promotion of yeast growth as such, but
the promotion of biosynthesis of lipids required for yeast growth. The oxygen
supplied is used by the yeast for the synthesis of ergosterol and unsaturated fatty acids,
which are integral components of cell membrane. Supply of these preformed
components obviates the need of aeration. Wort oxygenation is achieved, usually
with the incoming wort, by passing sterile air to a level of 8-10 ppm.
12.4.6 YEAST HANDLING AND PITCHING
Under normal conditions yeast cells grown during one fermentation cycle are used as
inoculum in subsequent cycles. It is necessary to maintain yeast in a satisfactory
physiological condition before reuse and, in this context, glycogen level within the
cell is of prime importance. Yeast is usually stored in beer or water. Handling
procedures should avoid the inclusion of air and the yeast should be rapidly cooled
to 4-6°C. A larger inoculum size should be used if the yeast has low glycogen
content.
190
Glycogen is an energy reserve for yeasts. During the first 6 hrs after pitching, the
yeast depends solely in the intracellular glycogen for the synthesis of lipids and
sterols needed for growth.
Two tests, the acidification power, and specific O2 uptake rate have been developed, both
of which correlate well with fermentation performance. Either test may be used to
determine optimum pitching rate or to reject yeast of unsatisfactory performance.
Pitching yeast is now recognized as the major source of bacterial contamination,

especially Lactobacillus, Pediococcus, and Obesumbacterium. These organisms are involved
in beer spoilage. Therefore, if recycling is desired, the yeast is purified before
pitching using any one of the following methods:
1. Repeated washing with sterile water

2. Treatment with dilute acids (pH 2.2-2.4) for 1-1.5 hrs. Acids such as tartaric
H3PO4, or H2SO4 can be used
3. Treatment with 0.75% acidified Na- or ammonium persulfate. The
temperature should be maintained below 5°C. Additional treatment with
antibiotics such as nisin, polymixin, penicillin and neomycin has also been
proposed.
It is advisable to acid-wash yeast only after 7-8 repitching (not at every repitching).
Reculturing from stock culture (pure culture) is done after every 20-30 repitching.
Typically, the pitching rate (final) is about 2 g pressed yeast per liter. This works out
to be a quarter crop from previous fermentation or 1 million cells/ml/°Plato. The
term °Plato (degree Plato, °P for short) is a hydrometric measure, where 1% sucrose
is equivalent to 1°Plato.
When top yeast is to be recycled, the first skimming, being most contaminated, is
discarded. Only the middle skimming is used. In the bottom yeast also, the middle
portion is desirable. The upper portion of crop tends to be of non-flocculating type
while the bottom portion of the crop tends to be early flocculating type, both of
which are undesirable in brewing.
12.4.6.1 Determination of yeast cell concentration
The correct number of yeasts cells in each batch is very important. Yeast number
representing 1 million cells/ml of wort/°Plato is considered the correct pitching
rate. Yeast cells are counted using hemocytometer and adjustment made
accordingly. The counting may be done on the crop or on wort after pitching. A
brief description of the cell counting process is given in the following paragraphs.
Using a clean, dry flask, take a small sample from the fermenter. Swirl the sample
around to help break up any clumps of yeast and to make sure it is properly mixed.
The swirling also helps remove any gas in solution. The hemocytometer and the
cover slip should be clean and dry. Place the cover slip over the counting areas.
Next, affix the pipette into the pipette pump. Pull out ~ 2 ml of beer into the
pipette, and then purge out 2-3 drops to clear the tip of any differentiation that may
191
occur. Immediately place the tip of the pipette on the V-shaped groove (see Fig.
12.24) and gently fill the counting area under the cover slip without disturbing the
cover slip; only a drop or so is needed. The counting chamber must be completely
filled, but not overfilled. The sample should not run out into the canals or bulge at
the edges.
Raised Area
FILL HERE
FILL HERE
Canal Area Lower counting chamber
Hemocytometer
Coverslip
Fig. 12.24 Hemocytometer showing counting chambers and fill points
The sample on the hemocytometer is now ready to be viewed under the microscope.
Without spilling any of the sample, carefully place the hemocytometer on the
microscope stage. Using 10× objective lens, frame up one of the counting chambers.
You should see a grid with 25 large squares, each of which contains 16 smaller
squares. You can count all of the cells within the 25 squares, or, when there is a large
number of cells, you can count the cells in the four corner squares and in the center
square (Fig. 12.25) and multiply the total by 5. To count cells within an area, switch
to 10× eyepiece with 40× objective lens, frame up the counting area of one of the 25
large squares, and take a count.
To eliminate the chance of counting a square twice or a cell twice, it is important to

use a standardized counting procedure. Always count in one direction (left to right,
top to bottom, for example). Cells touching the top or right-hand boundaries are not
counted. Cells touching the bottom or left-hand boundaries are counted (Fig. 12.26).
Cells that are budding are counted as one cell, unless the daughter cell is equal to or
greater than one-half the size of the mother cell, in which case the daughter cell is
counted as a separate cell (Fig. 12.27).
If there are more than ~ 50 cells per large square (that is, per square that contains 16
smaller squares) dilute the sample to be counted. The dilution factor must be used to
back-calculate the final cell count. It is important to dilute and count samples of
yeast slurry as soon as possible after sampling to prevent inaccurate counts due to
192
cell multiplication. Because there can be a high degree of inaccuracy inherent in this
procedure due to human error, perform a second count to confirm the first count.
1 2
4 5
Fig. 12.25 Counting area grid on a hemocytometer counting chamber
Do not count
this cell
Count this cell
Fig. 12.26 Close up view of a counting square showing counting protocol
daughter cell
1/2 mother cell

mother cell count as two cells
daughter cell
1/2 mother cell

mother cell count as one cell
Fig. 12.27 Decision for counting yeast buds based on size
The total number of cells per ml of sample is calculated as follows:

Cell count per ml = N × 5 × F × 104
Where, N = number of cells counted in 5 large squares; F = fold of dilution made.
For example, if you counted a total of 300 cells in your 5 large squares (without
dilution of sample), the total cell count per ml of sample is given by:
300 × 5 × 104 = 1.5 × 107 cells per ml
193
12.4.7 YEAST PROPAGATION
When pure culture is to be used, a stepwise propagation is used. There are three
distinct stages in pure yeast propagation, viz., (i) isolation of suitable yeast cells,
(ii) growth in the laboratory, and (iii) growth in the plant.
For industrial use, authentic yeast cultures are used. Thus, it should be less general to
frequently isolate yeast for the industrial production of beer. Nevertheless, if desired,
yeasts can be isolated by micromanipulator technique from the actively fermenting
beer. The following discussion relates, typically, to the propagation of commercially
available brewers yeast.
12.4.7.1 Yeast propagation in the laboratory
The primary stock culture (which may be in vials, slants, or other storage forms) is
initially cultured in 5 ml of sterile wort. After incubation for a couple of days, the
culture is transferred to a flask with 50 ml of sterile wort and incubated again for a
couple of days. Next, the contents are transferred to 500 ml of sterile wort. Further
multiplication of the yeast is carried out until 20 liters of green beer in the vigorously
fermenting stage (the high foam head stage) is obtained.
Vessels larger than 10 liters are made of chrome-nickel steel and are called Carlsberg
flasks (Fig. 12.28). A small Carlsberg flask has 8-10 liters capacity while a large
Carlsberg flask may have 20-25 liters capacity. The yeast needs to grow aerobically
and for this, the Carlsberg flask is equipped with sterile air filter. It also has all the
provisions for aseptic propagation of the yeast cells.
Sample tap
Inoculation connection
with rubber membrane Sterile air filter
Thread seal
Fig. 12.28 The Carlsberg flask
12.4.7.2 Yeast propagation in plants
Further multiplication of the yeast occurs in the brewery in yeast propagation plants
(closed type) or open growth vessels. There are several methods of yeast propagation
but all of them use sterile wort. Small breweries carry out propagation by “milk
churn process” in which 40-liter milk cans can be used for the vessel. Large
breweries use cylindroconical vessels (CCV) for the propagation. The vessels have
194
facilities for aeration, agitation, CIP, and temperature monitoring. The propagation
occurs in a batch process at about 22-25°C. Several variations are used with the CCV
system, notable among which are Conti-Prop system, Prof Back system, and
Wackerbauer single vessel system.
In the Conti-Prop system, the culture is grown in CCVs to a vigorous growth phase
(called “high krausen” in brewery) for 24-36 hrs and the total contents of the vessel
are pumped aseptically into the next largest vessel and topped up with sterile aerated
wort. This process continues until the required amount of yeast is obtained.
In the Wackerbauer single vessel procedure (Fig. 12.29), the contents (10 liters) of
the Carlsberg flask (at high krausen) are pitched aseptically into 25 HL of sterile wort
medium previously placed in a CCV of about 50 HL capacity. The CCV is equipped
with heating/cooling jacket, aeration lance, and CIP system. The culture is
propagated for 36-48 hrs at 20°C with aeration at intervals (1 min aeration every 15
min for the first 24 hrs, and 1 min aeration every 5 min for the second 24 hrs). This
“high krausen” culture is pitched into 500 HL of wort for fermentation.
12.4.8 FERMENTATION
Brewery fermentations have traditionally been considered to be of two distinct types:

(i) top fermentation and (ii) bottom fermentation. Traditionally, ales were fermented
in relatively shallow, circular or rectangular vessels while the lager fermentations
were carried out in similar but deeper vessels. As of now almost all breweries use
cylindroconical vessels (see Fig. 12.30).
Air supply through an air filter

CIP
Aeration lance Propagator
Heating/Cooling mantle
Wort inlet Pure culture to CCV
CIP
Fig. 12.29 Wackerbauer propagator
At least 25% of the vessel capacity is used as headspace to allow for foams that
generate during the high krausen. The charging and discharging of wort, beer, and
yeast are done from the bottom.
195
CCustomarily, lager beers aree fermented at
a 6-12°C for 8-14 days andd ales at 12-200°C
ffor 4-7 days. Fermentation
F proceeds
p mosst rapidly at hiigher temperaatures but the risk
oof flavor defeect is greater and the temmperature is a compromise between thhese
ffactors.
Vacuuum
releaase Veent
valve
3.2m
11.43m
Yeast in * Wort in
*Yeast ouut
*Beer outt
Fiig. 12.30 Beer fermenting vessel
TThe most serrious situationn faced by thhe brewer is a stuck fermenntation. The m main
mmedium contaains roughly 12% sugar (measured( as °Plato). Fivee to 6 hrs aafter
ppitching, nutriient uptake coommences. Tw wenty four too 48 hrs after pitching, clummps
oof foam calledd krausen appear on the surfface due to raapid evolutionn of CO2: a deepth
oof up to 1 m may be form med. This perriod of vigoroous fermentatition is called high
kkrausen and coorresponds to logarithmic phase
p of yeastt growth. Yeaast growth durring
tthe fermentatiion amounts to 3-5 times the original number. As tthe fermentattion
pproceeds, the specific gravitty decreases from
fr an initial of about 1.0448 down to 1.01-
11.016. The proocess is referrred to as attenuuation. Fully attenuated
a beeers have very low
leevels of carbbohydrates annd amino acidds. Fig. 12.31 is represenntative of typpical
pphysicochemiccal changes occcurring durinng fermentation.
TThe temperatuure is maintainned by internaal cooling coills. Temperaturre control is vvery

immportant for controlling thhe fermentatioon rate. Excesssive foaming ccan be controolled
uusing antifoam ms such as neuutral fats and oils, sorbitann esters, alcohool, polyetherss, or
ssilicone oils. In
I brewing industry, there are a numberr of advantagges to controllling
ffoam, some off which are:
 The feermenter capaacity can be inncreased by upp to 20%

 Lesserr protein denaaturation
1196
 Improved foam stability
 Improved hop utilization (less bitter principles lost)
pH
100% 5.5 yeast 100%
ethanol
5.0
4.5
sugar
4.0 pH
3.5 nitrogen
0
2 4 6
Time (days)
Glucose, fructose, sucrose
Maltose
Maltotriose
Period of utilization of sugar
Fig. 12.31 Changes during beer fermentation
CO2 generated during the fermentation is recovered, purified, and stored (page 212)
for adjusting CO2 level during finishing. The fermentation also lowers the pH to 4.0
and 3-5% alcohol by volume is produced. The dissolved CO2 content in the final
broth amounts to 0.3%. The main fermentation, called primary fermentation, produces
a young- or green beer (also termed ruh beer), which has varying fates.
If the broth is not fully attenuated, it is subjected to secondary fermentation. Secondary

fermentation is simply an extension of primary fermentation but at a very much
reduced rate because of lower temperature and lesser concentration of both yeast
and fermentable sugars. In order to invigorate the yeast and accelerate the secondary
fermentation, priming sugars or syrups may be added. The fermentation occurs in
closed tanks so that the beer becomes fully attenuated during the process.
Fully attenuated beer can also be subjected to secondary fermentation. In this case,
about 10-20% of the vigorously growing yeast from the fresh batch is added to the
attenuated beer. This addition of young beer to initiate a secondary fermentation is
called krausening.
The most important objective of the secondary fermentation is flavor maturation of

green beer. The unique quality the beer attains in the process is simply unmatchable.
The flavor maturation is due to:
197
 Reduction in the concentration of H2S.
 Reduction in the concentration of acetaldehyde
 Reduction in the concentration of diacetyl (= vicinyl diketone or VDK)
 Formation of esters that contribute to flavor
Other advantages of secondary fermentation are as follows:
 Clarifies beer
 Produces self-carbonated beer
 Chillproofs (page 209) and stabilizes beer
Krausening produces high quality beer but the primary fermentation yeast should be
removed as completely as possible otherwise it spoils the beer.
The secondary fermentation proceeds at 5-6°C for a week or until it reaches the
attenuation limit. Yeast is removed every 2-4 days, the last removal naturally being
before filtration. An important indication to maturation is the reduction in diacetyl
level. The maturation period is therefore also called diacetyl rest period during which
the diacetyl level drops to about 0.1 ppm. Only after diacetyl removal is the beer
cooled to the lagering temperature of -1°C and the cold lagering phase is continued
for a week.
The removal of diacetyl during maturation is considered to be a decisive criterion.

Consequently, most breweries begin cold lagering only after the diacetyl content has
been reduced to less than 0.1 ppm.
Diacety is the most important immature beer aroma. Above the threshold value it
gives beer undesirable slipperiness in the mouth and an unclean, sweetish to
revolting taste, which in higher concentration is responsible for the aroma of butter.
Because pentanedione also acts in a similar way, although with a substantially higher
taste threshold, these substances are considered together. They are referred to as
vicinal diketone (VDK) because both compounds are diketones. The breakdown of
these vicinal diketones occurs parallel to other maturation reactions during the beer
conditioning process and is therefore nowadays regarded as the essential criterion
for the state of maturation of a beer.
The formation and removal of vicinal diketones occurs in three stages. The starting
point for vicinal diketone synthesis is the pyruvate formed as an intermediate during
respiration and fermentation. Next, the yeast converts pyruvic acid to acetohydroxy
acids, which in turn are excreted out of the cell. The acetohydroxy acids give rise to
vicinal diketones by oxidative decarboxylation outside, independent of yeast cell.
The diacetyl formed can only be removed again by the yeast cells and the removal
occurs by reduction reactions. The yeast’s ability to remove diacetyl is about ten
times as great as its rate of formation during fermentation. Diacetyl removal is very
temperature dependent and increases greatly with increasing temperature.
Consequently, storage of beer at 18°C for about 15 days after the secondary
fermentation is not uncommon.
198
Spontaneous clarification during krausen storage is due to the positive pressure
developed by CO2. At least 10-15 fold reduction in turbidity is possible. Clarification
of beer during maturation is hastened by adding fining agents such as bentonite,
isinglass, Irish moss (carrageenan), and silica gel at the onset of aging.
12.4.9 YEAST AND PARTICLE REMOVAL
A major portion of yeast is removed by sedimentation or skimming before

maturation. Yeast collection in CCV system is different from conventional system.
In the CCV system, for the bottom-fermented beers, the yeast is removed from the
cone before the beer is transferred (for top-fermented beer, the yeast removal occurs
after the beer has been drawn). Yeast is collected several times at short intervals.
Basically, the yeast should be used again for repitching as soon as possible. To
activate its metabolic processes it should be aerated for 2-3 hrs before pitching.
Washing and sieving weaken the yeast and also introduce the risk of microbiological
infection. Therefore these should be avoided if possible. If the yeast is stored for
only 2-3 hrs, cooling is not necessary, but the longer it is stored the more important
cooling becomes. In pauses between brews, yeast should be stored at 0°C under beer
containing residual extract or under water. For longer storage, however, it must be
pressed and stored cold.
The young beer is further clarified either by secondary fermentation or by

mechanical means such as centrifuges and filters. Centrifuges (Fig. 11.9) are primarily
used (but is not obligatory) for removing gross impurities before carrying out
kieselguhr filtration (discussed shortly). Today, a large number of filter types are
available, e.g., diatomaceous earth filter, sheet filter, pulp filter, candle filter, leaf
filter, cartridge filter, etc. The classification of these filters are rather contextual.
12.4.9.1 Plate and frame filter (diatomaceous earth filter)
Plate and frame filtration is classified under powder filtration, cross-flow filtration,
etc., depending on the context. Powder filtration connotes filtration with kieselguhr
or perlite as the filter aid. Kieselguhr is the term used for fossils of diatoms of which
there are more than 15000 types in the sea. Kieselguhr usage can vary between 80
and 200 g/HL. Perlite is a material of volcanic origin and consists principally of
aluminum silicate.
Kieselguhr filtration is widely used for the preliminary plate and frame filtration of
beer. It is performed using a fine wire mesh, having a mesh gap of 70-100 m, or
other filters having a much larger pore size than the guhr particles (2-4 m). This
means that the guhr can pass unhindered through the mesh or filter (thereby making
the beer even more turbid than it was before) unless some special techniques are
used.
To obtain a perfect filtration effect a filter cake of filter aid is applied in three
coating layers, viz., (i) base, primary, or precoat layer, (ii) second coat or safety layer,
and (iii) continuous dosing. See Fig. 12.32 for the principle of kieselguhr
filtration.
199
For the base layer, degassed water or filtered beer containing a concentrated
suspension of a coarse guhr is circulated at an overpressure of 2-3 bar throughout
the filter. A pressure-stable primary layer is thereby built up which will prevent the
finest filter aid entering the filtrate. The primary layer forms the most important
element for the further build-up of the cake and for filtration itself. The particles of
this primary layer bear against one another and mutually prevent each other from
flowing any further (through the wire opening or plate pore).
For primary coating, guhr is used at the rate of about 700 to 800 g/m2. This is 70%
of that used in the total precoat.
Suporting layer (wire mesh,

shaped wire or specialized tissue)
Continuous
dosing
Second
First precoat with precoat
coarse guhr
Fig. 12.32 Principle of kieselguhr filtration
The second coat ensures that even the first filtrate after precoating runs clear. This
layer is again applied with degassed water or filtered beer but a finer, more effective
kieselguhr mixture is used. This is adequate to retain the haze and prevent blocking
of the filter. It is very important to have a very uniform distribution of the precoat
over the entire filter surface. Thinner regions or edges in the precoat cause
unevenness in flow and possibly also allow haze to pass through.
Continuous dosing serves mainly to maintain the permeability of the precoat after
the change over to filtration with a constant volume flow rate. The filter aid is
applied as body feed, which means that it is continuously mixed with the beer bulk
(in regulated amounts) throughout the filtration process. The constant flow rate is
necessary since with pressure surges or flow irregularities the bridges formed on the
sieve may be broken through and the beer would run turbid. This must be prevented
in all circumstance between inflow and outflow. It is desirable that this pressure
increase occurs slowly and continuously up to an excess pressure of 2-5 bar. The
pressure difference should on average increase at most 0.2 to 0.3 bar per hour.
Normally, the continuously dosed mixture consists of 2/3 medium guhr and 1/3
fine guhr. Kieselguhr usage during continuous dosing is between 60 and 120 g/HL.
200
During filtration, entry of oxygen is very damaging and this should be minimized by
removing air from pipes, beer, and kieselguhr before filtration. Only degassed water
must be used. Usually, flushing with CO2 before running the device is advantageous.
Kieselguhr filtration alone is not sufficient to give final clarity to beer. Therefore the
beer is further filtered in “polishing” filters (e.g., membrane filters) to give the
former a polished appearance.
Flow of liquid in plate and frame filter
Plate and frame filter consists of a unit in which alternating, usually square, plates
and frames are suspended. Filter sheets are hung over the plates on both sides and
these form seals between the frames and plates. The filter sheets are made of
cellulose and ion exchange resins. The sheets are stabilized by mass hardening so
that they are washable and can therefore be used for a long time. After filtration the
kieselguhr is spayed off and the filter sheet can then be used again. See Fig. 12.33 for
a schematic of the plate and frame filtration.
The kieselguhr is circulated (as a slurry) from the precoat tank to produce ~ 1.5 mm
thick coating. Recirculation is continued until the liquid becomes clear. Without a
drop in pressure, a second coat is applied. The actual filtration entails continuous
metering of the earth into the beer (as body feed, explained earlier) before filtration.
A normal length of time can be 8-12 hrs.
Frame
Feed
Channel inside
the frame
Filter sheet coated

with kieselguhr
Plate Tap Grooved surface for
guiding the filtrate
Filtrate
Fig. 12.33 Component parts of plate and frame filter
12.4.10 CARBONATION
After the beer has reached the final degree of clarity, it is stored in a pressurized
buffer tank called Bright Beer Tank (BBT). CO2 is injected through an orifice in the
pipe during the transfer. The whole is rested at 0°C for 24 hrs for CO2 dissolution.
The final concentration of CO2 is around 0.5%. CO2 fulfils various functions, viz.:
 Produces characteristic flavor (bite) of a carbonated beverage
201
 Produces fizz and head
 Prevents in-bottle gushing and foaming
 Creates anaerobic condition, which in turn prevents oxidation and microbial
growth
The pressure inside the bottle is around 20 psig. The O2 level in the beer should be
less than 0.1 ppm.
12.4.11 BOTTLING
Packaging can be done in kegs, cans, barrels, and bottles. In bottle- and can filling,
the fillers are always under counter- or hyper-pressure. There are two basic methods
of filling, viz., (i) Equal pressure filling (isobarometric method), and (ii) Differential
pressure method.
In the equal pressure method, the beverage inlet is opened and because there is the
same pressure in the bottle and the filler, the bottle is filled as a result of the
difference in height. In the differential pressure method, the higher pressure of the
filler forces the liquid in.
The metering of beer into the bottle is based on height or level. Beer filling machines
are always built as rotating machines with up to 200 filling valves. The bottles are
delivered on a conveyor belt, separated to a predetermined spacing by a separating
device, and positioned on a lifting platform under the filling elements by a star wheel
loading device.
The filling is accomplished in phases. The bottle is first positioned for receiving the
liquid, raised, and CO2 injected to displace the air. Air can also be displaced with
water (deaerated) jet at high pressure. The bottles are then filled with fillers that
operate at three variable speeds: first at a slow speed (to avoid frothing), then rapid speed
(bulk filling), and finally slow speed (to achieve fine filling) again.
The bottles are crown-corked to withstand the internal pressure. Since beer is
sensitive to light, colored bottles (amber or green) are used. Upon exposure to
sunlight, beer tends to become sunstruck or skunky. Skunk is a polecat known to
emanate very bad smell. The UV radiation of the sunlight and iso-α-acids of the
hops undergo photochemical reaction to produce prenyl mercaptan, the compound
responsible for the off odor. The compound is also called isopentenyl mercaptan or 3-
methyl-2-butene-1-thiol. The scheme of reaction is shown in Fig. 12.34.
4-methyl-3-pentenoyl Photo-catalyzed -scission

side chain of 3-methyl butenyl radical
UV light
isohumulone
H2S from fermentation
Prenyl mercaptan
Fig. 12.34 Formation of skunky odor
202
Selective reduction of the isomerized α-acid with sodium borohydride is one
alternative to prevent this defect. The α-acid, however, is less bitter. Reduced iso α-acid
does not take part in photochemical reaction and hence colorless bottles can also be
used. This, however, could not be successful commercially. The beer packed in
colorless bottles was reminiscent of horse urine in color!
12.4.12 PASTEURIZATION
Pasteurization of beer can be done by three main methods:
1. Heat treatment
2. Sterile filtration
3. Chemical treatment
Heat treatment includes (i) Hot Filling (no longer used), (ii) Flash Pasteurization, and
(iii) Tunnel Pasteurization (slowly being replaced by flash pasteurization)
12.4.12.1 Flash pasteurization
In flash pasteurization the beer is heated by a plate heat exchanger to at least 68-
75°C and held at this temperature for about 50 s. Then it is cooled down again. A
regeneration section (similar to milk pasteurization) is used for economizing energy.
It is important that the CO2 saturation pressure is lower than the applied pressure at
all times and that pressure at the beer inflow side is greater than that at the outflow
side. High pressure pumps for pressures up to 12 bar are necessary for this. The CO2
saturation pressure follows Henry’s law. Keeping temperature constant, the solubility
of CO2 (%) at different pressures is obtained by simply multiplying the solubility of
CO2 at atmospheric pressure (1 bar) with the desired absolute pressure (bar). For
example, at a constant temperature, if the solubility of CO2 is 0.321%, the solubility
at 5 bars will be (0.5315)%.
In beer pasteurization, a term called pasteurization unit (denoted by PU or PE) is used.

Pasteurization unit is defined as the time in minutes necessary at a particular
temperature (for a defined effect). The temperature of 60°C is set as the standard
and it is calculated that:
Number of PU (or PE) = time (min)  1.393(temperature in the heater – 60°C)
Thus, when heated for 1 min, the PU at different temperatures is as follows:
For 60°C, PU = 1.393(60-60) = 1.3930 = 1
For 61°C, PU = 1.393(61-60) = 1.3931 = 1.393
For 62°C, PU = 1.393(62-60) = 1.3932 = 1.94
For 63°C, PU = 1.393(63-60) = 1.3933 = 2.70
For 64°C, PU = 1.393(64-60) = 1.3934 = 3.76
For 65°C, PU = 1.393(65-60) = 1.3935 = 5.24
For 66°C, PU = 1.393(66-60) = 1.3936 = 7.30
and so on
203
For beer pasteurization, 5-6 PU are reasonably adequate but 14-30 PU are used to
allow margin of safety. If 15 PU is chosen as an example of what is necessary, the
time needed at different temperatures is calculated as follows:
At 66°C for 15 PE, the time needed (in min) = 15 PU/7.03 PU = 2.06 min
and so on
12.4.12.2 Tunnel pasteurization
Tunnel pasteurizers are meant for bottled and canned beers. At present, tunnel
pasteurization is being replaced by flash pasteurization or sterile filtration.
In the pasteurizer, the filled bottles are heated in stages, subjected to pasteurization
temperature for a fixed time, and then cooled again. The residence time at the
pasteurization temperature must be selected such that the core part (which is about
1.5 cm above the bottom of the middle of the base of the bottle) is also heated for
an adequate time-temperature regime. Because of the poorly conducting glass
material of the bottle, uniform heating is not easy. The space in the bottle during
pasteurization must not be less than 5% of the volume of the bottle otherwise the
pressure in the bottle can cause breakage. In modern pasteurizers, the temperature is
monitored by a recorder. Heating and cooling of the bottles and cans is performed
using various water circulation paths in order to utilize recovered heat. Passage
through the tunnel takes about an hour.
The main components of the tunnel pasteurizer consist of (i) the transporting drive
mechanism and (ii) the spraying and water circulation system. The transportation is
done by conveyer chains or “walking beam” conveyor system.
12.4.12.3 Sterile filtration (filter stabilization)
There is an increasing trend to pasteurize beer by sterile filling method. This method
of stabilization helps retain the beer flavor to a significant extent. Consequently, beer
that has been sterile-filtered is considered organoleptically superior.
Membrane filters and modules are available for such a filtration. The filtration is
carried out after kieselguhr filtration followed by a series of 3 additional stages of
filtration. The first stage employs module- or cartridge filter with pore size of about
5 m. The second stage filter (polishing filter) employs cartridge filter with pore size
of 1 m. The third stage filter (sterilizing filter) employs cartridge- or module filter
with pore size of 0.45 m. The installed view of a typical membrane filter (spiral
wound) used for sterile filtration is given in Fig. 12.37. The exploded view of the
module (functional unit) is given in Fig. 12.35. Spiral wound module consists of thin
film composite (TFC) system. A flexible, porous sheet is placed between two flat
membranes. This “sandwich” thus produced is sealed on three of its four edges. The
folded side is sealed to a cylindrical collector tube on both sides of a distributor with
holes drilled in it. Several sandwiches are thus fastened and separated from one
204
another by a spacer of flexxible plastic. The
T fluid to be treated circuulates in the spacer
and the poroous sheet ensures the drainnage of permeeate towards the axial colleector.
The wound components
c are
a snugly fit into
i a housingg (Fig. 12.36). The dimensioon of
a spiral wounnd module is: 10-20 cm (diaameter) and 300-180 cm (lenngth).
Permeate pipe
coontaining Permeate flow
w (after passagge
Feed coollection holes through mem brane into
solution permeate colllection materiaal)
Retentate
Permeate Permeatte
Retentate
Feed Permeate
solution collection matterial
Membranne
Feed
F flow across
feed
f spacer
channel
c
Feed channel Covering
spacer
Figg. 12.35 Spirall wound filter packing
Fig. 12.36 A typical hoousing for spirral wound filteer
Fig. 12.37
1 Installatiion of spiral wound
w filter
w intended for draft beer / draught beerr. The term dr

Initially, sterrile filtration was draft is
nowadays ussed in a very looose sense annd so the distinnction betweeen draft- and other
beers is becooming increasingly blurred. Draft beer iss usually packeed in bulk (inn kegs
and barrels). It has compaaratively short shelf-life.
205
12.4.12.4 Chemical pasteurization
It entails addition of one the agents listed in Table 12.4. The use of SO2 in the final
stage is not uncommon (70 ppm in UK). Sodium benzoate and sorbic acid are also
permitted.
Table 12.4 Chemicals used in pasteurizing beer
Chemical agent Level (ppm) Remarks

n-heptyl-p-hydroxybenzoate Up to 12 Added immediately after filtration
Diethyl pyrocarbonate (DEPC) 70-100 Persistent fruity aroma
Octyl gallate 10-50
12.5 HIGH GRAVITY BREWING
There are several methods used for the production of special beer types. One
particular brewing method of significance is the High Gravity Brewing. High gravity
fermentation involves worts of up to 18 °P and even higher. Following
fermentation and maturation, the beer is diluted with cool, carbonated water to a
prescribed original gravity or to a prescribed alcohol concentration. There are a
number of advantages associated with high gravity brewing. It results in beers that
are more consistent (% alcohol, original gravity, etc.) and more physically stable
since the compounds responsible for haze are more easily precipitated at higher
concentrations. Handling more concentrated wort results in increased utilization of
equipment and lower energy costs. The disadvantages are longer fermentation times,
different flavor characteristics, and poorer hop utilization than normal gravity
fermentations.
The reason for introducing the process may be an insufficient brewhouse capacity
which can be overcome in this way. However, the chief reason is that the water
which is used later in a cold state for dilution does not have to be heated up and
boiled with the wort. This gives substantial economic advantage.
12.6 QUALITY CONTROL
The quality control of beer includes both organoleptic and physicochemical analyses.
Some of the important parameters to be tested are alcohol content, dissolved O2,
CO2, haze, bitterness, gravity, diacetyl level, pH, taste, color, head retention, etc.
Despite extreme care, batch-to-batch variations do occur. Alcohol, bitterness and

color are generally adjusted. Deareated water can be used for reducing the alcohol
content. Caramel may be used for adjusting color. Iso α-acid extract may be added
for maintaining the desired level of bitterness.
Finished beer must appear fresh, bright, and without faults to the customer and
hence the quality is a matter of great concern. The beer must also be free from
microorganisms to ensure wholesomeness and biological stability. The ethanol
content must obey fiscal rules but is also of major importance for the flavor of the
206
beer. This is further influenced by a wide range of compounds that may be present
in even very small amounts. Visually the finished beer must form a nice foam on
pouring and it must have an attractive color.
Despite use of the choicest raw materials and careful brewing performance the beer
is a fragile liquid, especially when not stored cold. The fine, balanced aroma of fresh
beer is eventually replaced by a less attractive smell and likewise the taste
deteriorates. The basis for this decay is a matter of intense research.
Sensory analysis is the most powerful test of beer quality. Beer flavor is of course the
single most important parameter in sensory analysis. Beer flavor is a very complex
subject. Over 800 compounds have been identified that contribute to the
characteristic flavor of beer. The main flavor characteristics are the bitter taste
derived primarily from the hops, an alcoholic note from ethanol, and a carbonation
mouthfeel from CO2. Secondary flavor notes include fruity-estery flavors, alcoholic
notes from higher alcohols, and various sulfur components. However, most of the
flavor compounds are present in very small quantities (below their individual taste
threshold) and act synergistically to provide the balanced and refreshing taste of
good beer. Occasionally undesirable flavor components may appear, giving the beer
various off-flavors.
The flavor components and their precursors originate from the raw materials,
namely malt, adjuncts, water or hops, or are produced by the yeast during
fermentation of the wort. Consequently, the selection of raw materials and/or yeast
strain has great impact on the flavor. However, the flavor is also influenced by
technological factors which affect the composition of the wort, and the conditions
during fermentation, maturation, filtration, and bottling.
Flavor assessment is therefore very important in quality control of beer. One of the
most important tools is the sensory analysis by a panel of well-trained tasters. To
enable a precise description of a beer sample a common terminology has been
elaborated. Each flavor impression is quantified on a scale of 0-10 (Fig. 12.38).
In recent years the methods for chemical analysis have improved dramatically. It is
now possible to monitor the concentration of many flavor-active components.
Quantification of higher alcohols and esters by headspace analysis using a gas
chromatograph is now a routine analysis in many breweries. At the Carlsberg
Research Laboratory, analyses for a wide range of yeast secondary fermentation
products have been established. These analyses are used to study the effect of
different raw materials, yeast strains, or brewing processes on the profile of flavor
components in the beer.
Beer will deteriorate rapidly unless stored under cold and dark conditions. The stale
"cardboard" flavor which may appear upon storage is mainly due to very small
amounts of trans-2-nonenal, a lipid degradation product formed during malting and
wort production. At the Carlsberg Research Laboratory the brewing process - from
malting to beer pasteurization - is examined to pinpoint the critical steps of lipid
oxidation.
207
body
9
sweet 8 sour, acid
malty, grainy 7
bitter
6
5
malty, grainy sulfury
4
3
estery, fruity 2 sulfidic
C
cooked, vegetable
solvent-like
metallic
resinous,
grassy
salty
caramel,
burnt
phenolic malty, grainy
fatty diacetyl
acids
Fig. 12.38 A typical flavor wheel for the sensory analysis of beer
12.7 SOME TERMINOLOGIES
12.7.1 HEAD
Formation of an attractive head of foam is a very crucial aspect of beer quality. A

good beer foam can be described as follows:
 Stable head consisting of small, tight, white bubbles

 Adhesion of foam to the side of the glass (so-called lacing) whilst the beer is
being drunk
Appearance of foam is due to the rising CO2 but the stability is a combined result of
a host of factors. Beer foam is primarily stabilized by adsorbed proteins,
polypeptides and β-glucan derived from malt. Because of macromolecular
complexity and heterogeneity of the macromolecular components of beer, the
adsorbed layer is not dominated by single species or simple mixture of species. High
molecular weight glycoproteins and highly hydrophobic polypeptides are usually
effective in stabilizing foam, although iso-α-acids from hops and ethanol in beer are
also involved in some complex manner. Low ethanol content acts as foam
enhancing component by sharply lowering the surface tension, and consequent
formation of small bubbles. High alcohol concentration is detrimental to foam
stability: it precipitates the proteins.
Recent researches have shown that foam active proteins have molecular weight
higher than 8000. Lipid Transfer Protein 1 (LTP 1), a 10 kDa protein of barley origin,
has been identified as the major protein component in beer foam. The
transformation of LTP 1 into the more foam-promoting form takes place during
wort boiling and involves unfolding of the three-dimensional structure.
208
12.7.2 HAZE AND CHILLPROOFING
Even after filtration to clarity, beer may develop turbidity or cloudiness upon
storage. This turbidity, called haze, may be either biological or non-biological.
Infection of beer with bacteria and subsequent growth leads to biological haze. This,
however, is not of importance in pasteurized beers.
Non-biological haze is the most important index of physical stability of beer.

Colloidal materials such as polypeptides and polysaccharides, which form aggregate
with each other and with polyphenols, contribute to haze formation. The simple
phenolics in beer polymerize to yield active polymers (tannins) which complex with
beer proteins to form a reversible chill haze that precipitates from the solution at 0°C
but redissolves at 20°C. Upon further polymerization and complexing, a non-
reversible permanent haze is formed. It has been believed that protein serves as a
backbone of haze, and the most significant metal ion involved is calcium.
The polyphenols of importance in beer haze are proanthocyanidins from barley

grain and hops (see Fig. 12.39 for barley proanthocyanidins). The proanthocyanidins
in hops are generally termed tannins due to their greater complexity.
Proanthocyanidins show affinity towards certain proteins (hordein in particular)
which are characterized by high proline content. During mashing as well as wort
boiling, complexing between proteins and tannins occur to give precipitates.
Haze formation in beer is undesirable. It is one of the major factors that determine
the shelf-life of bottled or canned beer.
A common method to prevent haze is chillproofing. Chillproofing merely implies

removal by precipitation or adsorption of residual proteins or protein hydrolysate
products from beer, thereby eliminating the backbone of the haze. Some of the
methods of chillproofing are:
1. Use of cold cellar temperatures for prolonged period and good, tight
filtration. This is an expensive and time-consuming process
2. The use of proteolytic enzymes, such as papain, during aging. Papain
hydrolyzes the protein backbone
3. Removal of proteins with adsorbents such as tannic acid, bentonite, silica gel, etc
R
OH
H
HO O OH
H OH OH
H Procyanidin B-3: (R = H)
OH H
HO O OH Prodelphinidin B-3: (R = OH)
OH
H
HO
Fig. 12.39 Two dimeric forms of proanthocyanidins in barley/malt
209
Chillproofing can also be achieved by removing tannins (rather than proteins) using
polyvinylpolypyrrolidon (PVPP, Fig. 12.40) which closely resembles polyproline.
O H O
CH CH2
C N C C
N
H2C CH2 O C CH2
C
H2 H2C CH2
m n
Polyproline PVPP
Fig. 12.40 Schematic formulation of PP and PVPP
Polyproline and PVPP both possess a carbonyl group neighboring a proton-free

nitrogen atom, a very potent site for hydrogen bonding.
Having thus removed all the haze components, the beer becomes stable even when
cooled. That is, the beer is no longer susceptible to chilling, and is hence
childproofed.
Another attractive alternative for producing haze-free beer is the use of malt that is
free from proanthocyanidin (discussed earlier).
12.7.3 FLOCCULATION
Flocculation is simply a reversible aggregation of dispersed yeast cells into flocs (loose
clumps). Ideally, brewing yeast does not flocculate at the beginning of the
fermentation, but only after all the nutrients have been used up. However,
depending on the conditions, the yeast may initiate flocculation either too early or
too late, leading to either improper fermentation or the need of centrifugation,
respectively. In order to improve the control of flocculation during beer production
the genetic mechanisms of flocculation are being studied.
Yeast flocculation is a complicated process that is currently only partly understood.

It requires the presence of at least two types of molecules on the yeast cell surface.
One type is mannans (carbohydrate chains), which are produced by the gene
products of the MNN genes and are present on the cell surface at all times. The
other type is flocculins (sugar binding proteins), which are the gene products of the
FLO genes, that are activated only after depletion of nutrients. The flocculins bind
to mannans through calcium bridges on the surface of neighboring cells leading to
the cross binding of cells and ultimately the formation of flocs. Due to the reduced
surface-to-volume ratio of the aggregated cells, the flocs sediment much faster
relative to the free cells. At a microscopic level, yeasts capable of flocculation have
hairy outer surfaces due to the presence of these binding molecules. Flocculation
characteristics of yeast cells are of great technological importance. Since yeast
clumps tend to be carried to the surface with entrapped CO2, recovery of the former
becomes very easy. Flocculation can sometimes be problematic: it leads to hung or
stuck fermentation, thereby leading to incomplete attenuation of the beer. Hung
210
fermentations are invariably caused by two factors, viz., (i) premature flocculation,
and (ii) inability of yeast to metabolize maltotriose.
12.8 SOME FAMOUS BEERS OF THE WORLD
The qualitative ranking of beer cannot be completely objective. The list that appears
in Table 12.5 has been taken from Beer International (1997). The largest producer of
beer is England. The next largest producers are Germany, China, and Japan. See Fig.
12.41 for world beer consumption.
Table 12.5 Some famous beers of the world
Beer % Alcohol Type Producers

Tetley Mild 3.2 Ale England
Taylor Landlord 4.3 Ale England
Marsten Pedigree 4.5 Lager England
Samuel Adams Boston Lager 4.8 Lager USA
Budweiser Budvar 5.0 Lager Czech Republic
Schneider Weiss 5.3 Wheat beer Germany
Guinness Foreign Extra Stout 7.5 Ale Ireland
Africa (4.69%)
Middle east (0.64%)
Central and South Oceania (1.39%)
America 14.38%) Japan (4.35%)
Asia excluding
Japan (24.33%)
North America
(17.39%)
Europe (32.83%)
Total consumption worldwide = 150392000 kiloliters
Fig. 12.41 Leading beer consuming countries (2004)
12.9 BEER SPOILAGES
Beer is a poor environment for microorganisms. As such, five intrinsic factors

control the microbial growth. These factors are: (i) low pH, (ii) low redox potential,
(iii) low nutrient status, (iv) presence of ethanol, and (v) presence of hop
components and metabolites.
Any organism that grows in beer should overcome all these hurdles. Extrinsic
factors such as addition of preservatives, storage at refrigeration temperatures,
pasteurization treatment, etc., further reduce the possibility of beer spoilage.
Nevertheless, beers get spoiled, and when this occurs the main causes may be due to
few notorious microorganisms, some of which are given in Table 12.6.
211
Table 12.6 Some beer defects and the associated microorganisms
Spoilage organisms Defect in beer Other details

Acetic acid bacteria Rope formation,
acetification, turbidity,
discoloration, strange
flavor
Wort bacteria: Off flavor including These microorganisms
They include certain phenolics and vegetable normally survive the initial
members of taints stages of fermentation
Enterobacteriaceae, e.g.,
Citrobacter, Enterobacter,
Escherichia, Hafnia,
Klebseilla.
Obesumbacterium proteus Increased level of This is a fat, rod-shaped
higher alcohols bacterium that grows in wort
during alcoholic fermentation
Lactic acid bacteria Increased acidity, Their nutritional requirement
foreign flavor is similar to that of the yeasts
and hence compete for
growth
Wild yeasts Foreign flavor They are versatile and hardy
and thus survive
fermentation
Zymomonas mobilis Objectionable stench It produces acetaldehyde and
and heavy turbidity H2S. It uses the Entner
Deuodoroff pathway of
metabolism
12.10 PURIFICATION OF CARBON DIOXIDE
Commercial CO2 is obtained either by the combustion of carbonaceous materials or

as a by-product from fermenting processes as well as from a number of chemical
processes releasing large amounts of CO2. Extraction of CO2 from fermenting
processes is carried out in Recovery Based Units (RBU), which come in different
designs. Fig. 12.43 shows CO2 recovery unit designed by GEA-Tuchenhagen
Brewery Systems, Germany.
Breweries produce large amounts of CO2 as a by-product of fermentation. This CO2

is extracted, purified and used for the carbonation of beer. Details regarding the
operation and maintenance of the RBU may vary from manufacturer to
manufacturer but the principle involved in the extraction and purification of CO2 is
essentially the same. Through appropriate scrubbing, filtration and separation
technology the CO2 recovery plants meet the strictest CO2 quality requirements. The
key advantages of RBU are:
 Beverage quality CO2 from own source
212
 Low operating cost
 Environmental benefits
 Low installation cost
 User-friendly, fully automatic operation
The CO2 gas developed in the distillery and brewery contains a lot of impurities
(such as alcohols, aldehydes, H2S, NOx, etc.). The gas is therefore subjected to a
series of operations to recover and purify CO2. A design produced by Witteman
Company, India is described in the following paragraphs.
The CO2 generated from the closed fermenter is led through a vessel called “foam
trap” (Fig. 12.42). Here the gas is washed with water to get rid of gross impurities
like foam, cells, and denatured proteins. Thereafter the gas is passed through a water
scrubber (in some designs, an additional KMnO4 scrubber is also used) to remove
water-soluble impurities and aerosols. The gas is now compressed in a double-stage
compressor to about 16 bars (gauge), cooled, and passed through a pair of dual
tower (only one tower is shown in Fig. 12.42 due to space constraints) that contains
activated carbon and a desiccant.
Water
Feed gas Scrubber
Water
Foam
trap After-cooler Vent
Activated carbon
Desiccant
Inter- Dual tower CO2 -
Drain cooler deodorizer/Dryer
Air Vent
Drain
CO2 Liquid CO2
compressor CO2 stripping
condenser column
Reboiler
Storage tank
Liquid CO2 at -25oC
and 16 bars
Utilization
Fig. 12.42 Schematic of carbon dioxide recovery in brewery
Only one tower is operated at a time so that the other can be simultaneously
regenerated. Regeneration in this context refers to reactivation of activated carbon
by back-flushing the tower with hot air or steam. Activated carbon is used to remove
213
oodor compounnds such as alldehydes, H2S, NOx, etc. Desiccant
D is useed to dry CO2 to
leess than 10 ppm
p moisturee content. In some designss, a “balloon”” is installed just
bbefore the com mpressor. Ballloon is a tannk that serves as a reservoiir of CO2 forr an
uuninterrupted supply to thhe compressoor. Although the compresssed gas can be
ccondensed andd stored for use,
u an additional stripping column
c must be used if a vvery
hhigh purity COC 2 is requirred. The striipping processs involves vvaporization and
ccondensation cycles. The inncondensable gases like O2 and N2 are veented away w while
CCO2 is collecteed at the bottoom. The puriffied gas is subsequently storred as liquid C
CO2
inn thick-walledd cylinders at 15-20 bars (gaauge) and aboout -25°C. Thee volume of C CO2
iss reduced by about one-sixxteenth. A suittable evaporattor unit is useed when dry C CO2
iss required for the carbonatiion of beveragges.
TThe purity of CO
C 2 intendedd for soft drinkks and brewerry is very dem
manding. A typpical
sspecification for
fo food-gradee CO2 is given in Table 12.77.
TTable 12.7 Standard for food-grade CO2
PParameter Specificcation
PPurity 99.9%, v/v min.
MMoisture 20 ppm
m, v/v max.
OOxygen 30 ppm
m, v/v max.
CCarbon Monooxide 10 ppm
m, v/v max.
AAmmonia 2.5 ppm
m, v/v max.
NNitric Oxide / nitrogen dioxxide 2.5 ppm
m, v/v max. eaach
NNonvolatile reesidue 10 ppm
m, w/w max.
AAcetaldehyde 0.2 ppm
m, v/v max.
TTotal sulfur coontent (as S) excluding SO
O2 0.1 ppm
m, v/v max.
SSulfur Dioxidde 1 ppm, v/v max.
AAppearance inn water No coloor or turbidityy
OOdor and tastee in water No foreeign taste or oodor
Fig. 12.43 Carbon dioxide recovery plant (brewery CO

O2 )
2214
CHAPTER 13
MICROBIAL PRODUCTION OF ETHANOL
13.1 INTRODUCTION
Ethanol can be produced chemically (> 90%) as well as microbiologically. However,

chemically produced alcohol is not used for beverage purpose. The reason for this
can be many. For example, there is always some health risk in the use of chemically
synthesized alcohol. Another important reason is the lack of congeners. The quality
of spirits obtained by a microbiological process can never be obtained by chemical
means. Below is given an example of chemical synthesis of ethanol.
H PO , 300 C, 60 atm
Ethylene  H 2 O 
3 4
 CH 3CH 2 OH
The merits of microbiological production of ethanol can be listed as follows:
 No need of tremendous temperature, pressure, and a multitude of complex

reactions
 Uses cheap and readily obtainable raw materials
 Ecologically friendly
 It is the only option when ethanol is to be used for beverage purpose
13.1.1 MICROORGANISMS
Bacteria, yeasts, and molds are all capable of producing ethanol. For example,
Zymomonas mobilis (bacteria), Mucor species (mold), Saccharomyces cerevisiae (yeast),
Schizosaccharomyces pombe (yeast), etc., can be used for the same. However, for
industrial fermentations, only yeasts are used. Of them, S. cerevisiae and to some
extent, Schizosaccharomyces are the only commercially used yeasts. S. cerevisiae can
produce up to 18% alcohol by volume (abv). Schizosaccharomyces is a fission yeast
normally used in continuous process.
13.1.1.1 Desirable properties of yeasts
Not every type of yeast is useful or is used in alcoholic fermentation. The strains
used in industrial fermentation are of course highly improved strains. These
organisms must possess certain desirable properties, the important ones of which are
listed as follows:
 Ethanol tolerance
 Flocculation
 Resistance to killer activity
 Osmotolerance
Killer activity is due to the ability of the yeast to produce a toxin called zymocin.
Yeasts with ability to elaborate zymocin are not only resistant to other similar killer
strains but are also killers to sensitive strains.
13.1.1.2 Preservation and maintenance
Primary stock culture is preserved by low risk methods like lyophilization and
desiccation. Working stock cultures are normally prepared in slants or broths. The
conventional medium for the isolation and growth (also for maintenance) is MYPG
agar adjusted to pH 4.5. After growing for 2-3 days at 30°C, the culture is stored at
4°C. It will remain stable for about 6 months if drying up and/or contamination is
checked.
13.2 INDUSTRIAL PRODUCTION
13.2.1 RAW MATERIALS
As such, several raw materials can be used for the fermentation. The main
categories of basic raw materials are (i) saccharine materials, (ii) cellulosic materials, and
(iii) starchy materials.
Cellulosic and starchy materials require extensive treatment before actual use
because the organisms do not possess the suitable enzymes for hydrolyzing these
complex polysaccharides. Cellulosic materials are usually hydrolyzed by chemical
means, for example, with acids and alkalis. The cost of preparation often comes to
over 50% of the total production cost. Unless cheap methods of hydrolysis are
available the feasibility of ethanol production using cellulosic materials is remote.
One alternative could be development of source of cellulase, either from bacteria,
mold, or the yeast itself. However, this possibility has not yet been realized in so far
the commercial production of ethanol from cellulosic materials is concerned. The
few cellulose processes that exist today (for fuel alcohol) have been discussed later.
Starchy materials are nevertheless used in the production of a wide range of

alcoholic beverages and spirits. The process is more cost effective than the cellulose
process. The hydrolysis can be carried out conveniently using suitable diastatic
enzyme sources such as malt and molds. Starchy materials are used for alcoholic
beverage production (e.g., beer, whiskey, cereal wines) rather than the ethanol, which
has diverse end use.
For large-scale ethanol production, saccharine materials (sugar items) are still the
materials of choice. Of them, molasses is the most preferred material. Molasses
comes in many types, e.g., blackstrap molasses, high-test molasses, and refinery molasses.
High-test cane molasses is simply the concentrate of cane juice (sugar not extracted)
and is therefore relatively costly. Refinery molasses comes from the intermediate
stage of sugar manufacture. Since it contains significant amounts of crystallizable
sugar, refineries do not sell it. Blackstrap molasses is the final by-product of sugar
refinery. Nutrients and minerals are available in it in highly concentrated form. It
contains as much as 50% of fermentable sugars.
216
13.2.2 MEDIUM PREPARATION
Molasses as it comes is of about 80° brix (specific gravity: 1.40). Water is added to it
to make a solution of 15-16° brix (specific gravity: 1.06). The medium is seldom
pasteurized or sterilized. Since nitrogen source can be limiting, (NH4)2SO4 can be
added to the medium at the rate of 1 kg/50 HL (1HL = 100 liter). The pH is
adjusted to 4.5 with concentrated (98%) H2SO4.
13.2.3 INOCULUM BUILD-UP
The inoculum is prepared separately in a stepwise fashion (Fig. 13.1). Pure yeast
culture from the working stock is first propagated in a shaker flask in MYPG broth for
about 2 days at 28°C. It is next transferred to a vessel called yeast machine. The vessel
contains sterile medium (molasses medium), usually above 10 times the volume of
shake flask. There the yeast is grown aerobically for about 2 days at 28°C. The
contents are next transferred to a propagator called bub vat. It also contains sterile
medium, over 10 fold the volume of yeast machine. The vessels are closed ones. Air
is supplied at the rate of 1/8 vol/vol/min and propagation carried out as aseptically
as possible for 2 days at 28°C. The transfers are made in several stages until the
desired amount of inoculum is obtained. The number of yeast cells for pitching
should be around 30-50 million cells/ml of the medium in the final fermenter. Care
should be taken in the inoculum build up not to shift the yeast towards alcoholic
fermentation. Since alcoholic fermentation occurs only at higher sugar
concentrations the opposite may be done to shift the yeast towards respiratory
growth. The reverse should be the case in the final fermentation, though (i.e.,
glucose effect must be maintained).
13.2.4 PREPARATION OF THE FERMENTER
Conventionally, fermentation is carried out in batch mode. The vessels, which are
generally cylindroconical in configuration, have the capacity of 550 to 1000 HL (Fig.
13.1). The fermenter can be sterilized using live steam or disinfectants such as NaOCl
(sodium hypochlorite).
13.2.5 PITCHING
The prepared medium is transferred to the fermenter first. Active inoculum is then
added at the rate of 3-4% by volume. In certain cases, the pitching rate can be as
high as 20% by volume. Often, yeast is recycled after harvesting of the previous
batch, in which case the inoculum takes the form of yeast cream. The yeast should
be of good physiological quality, though. Whatever the method, the pitching rate is
optimally maintained at about (3-5) 107 cells per ml of the main fermentation
medium.
13.2.6 FERMENTATION
The fermentation starts a short while after pitching. The temperature of

fermentation is very critical. It must be maintained at 28-35°C and not more. This is
achieved by using internal cooling coils. In very small fermenters, water jacket or
217
even air-cooling (surface cooling) can be used. Heat generation is of the order of
11.7 kcal/kg substrate consumed. A change of 0.75-1°C/h is not unusual. High
temperature is undesirable for following reasons:
 Foaming occurs uncontrollably

 Pathogens may multiply
 Alcohol loss may occur (up to 1.5%)
 Yeast viability may decrease
 Premature flocculation may occur, leading to hung fermentation
 Higher alcohols may be produced
The fermentation is usually complete within 40-48 hrs after pitching. Depending on
the initial sugar concentration, the final broth (wash or beer) contains 7.5-8% abv.
The yield is about 92% of the theoretical conversion, because the yeast cells utilize
some amounts of sugar for their own cell build-up.
Off gas CO2
Overhead
tank absorber Water in
Dilute alcohol
Diluter
Cleaning
Main fermentor agents
Molasses
tank
Product
Slant Shaker Water out
flask Heat
Propagators Water in
exchanger
Fig. 13.1 Batch fermentation of ethanol
13.2.7 RECOVERY
The final broth can be treated in many ways. In the following paragraphs, brief
descriptions of three common methods are given.
1. Melle-Bionot Process
In this process the yeast is centrifuged (Fig. 11.9) and recovered. While the
supernatant is taken away for distillation, the yeast cream is washed, treated with
food-grade acid (to kill the contaminants) and returned to the main fermenter. This
process is preferred because it does not require repeated inoculum build-up. The yeast
is thrown away after a given number of cycles or after it becomes contaminated to
unacceptable level.
2. Half broth recycled
Only half of the fermented broth (wash) is withdrawn. The other half (along with the
yeast mass) serves as an inoculum for the next batch.
218
3. Yeast not recycled: See later
Whatever the method, the wash is first dropped into a vessel called beer tank so that
the main fermenter becomes empty for reuse. The wash in the beer tank can now be
processed (settling, centrifugation, distillation, etc.).
13.3 CONTINUOUS FERMENTATION
Where there is an inexhaustible source of raw material, continuous process is very

efficient. Although there are many variations of continuous fermentations, the cascade
process is the most successful one. It consists of series of tanks with inlet, overflow,
and stirrer facilities. The prepared medium is continuously fed and the
corresponding amount simultaneously withdrawn. The flow, medium concentration,
and the pitching rate are adjusted such that the fermentation is complete by the time
broth leaves the last stage. The ethanol in the final beer is as high as 14%. See Fig.
13. 2 for the scheme of continuous fermentation.
Medium
Medium
Medium
Medium
Air
To distillation
Fig. 13.2 Scheme for cascade process of ethanol fermentation
Like any other fermentations, continuous fermentation also has advantages and
disadvantages. Increased productivity, uniformity of operation, ease of automation,
etc., are the well-known advantages while the danger of contamination due to
prolonged periods of fermentation is the main limitation. The real economy in
continuous fermentation results from the elimination of down time, the time required
for emptying, cleaning, filling, etc., of the fermenter.
13.4 BIOCHEMISTRY OF ETHANOL FERMENTATION
Starting from glucose, yeast uses a set of 12 enzymatic steps. The stoichiometry of
the reaction is:
zymase
C6 H12 O6  2CH3CH 2OH  2CO2  Energy
The organism uses EMP pathway, generating 2 ATP per mole of glucose converted
to ethanol, plus CO2. Ethanol, which is the end product, is a primary metabolite. In
an industrial fermentation, the basic strategy is to maintain Crabtree effect during the
fermentation. A truncated form of the metabolic pathway for ethanol synthesis is
given in Fig. 13. 3.
219
2 ATP 2 ADP 4 ADP 4 ATP
Glucose 2[1,3-di P glycerate]

2 Pyruvate
2[NAD+] 2[NADH+H+]
2 CO2
2 Ethanol 2 Acetaldehyde
Alcohol dehydrogenase
Fig. 13.3 Simplified pathway of ethanol biosynthesis
The theoretical yield of ethanol from glucose is 51.1% (mass/mass). However,

because the yeast utilizes some sugar for its own growth (cell build-up), the practical
yield is 44-49%, which is 86-95.9% of theoretical value. The productivity of a typical
fermentation is 1.9 g/liter/h.
13.5 BIOCHEMISTRY OF HIGHER ALCOHOL PRODUCTION
Higher alcohols in ethanol are responsible for the characteristic aroma. When they are
in high concentrations, they can cause headiness and dryness. Technically, higher alcohols
are also called fusel oils. They have boiling points of 125-140°C. Unless severely
infected, the concentration of higher alcohols is les than 0.5% of ethanol.
The most important higher alcohols found in ethanol are propanol, butanol and pentanol
(amyl alcohol). The production is limited to exponential growth phase and dependent
on yeast strain, pH, and temperature. Fusel oils such as isobutanol, isoamyl alcohol,
amyl alcohol, and phenyl ethanol are produced by sequential reactions, viz.,
transamination, decarboxylation, and reduction of respective substrate amino acids.
However, propanol is produced from α-keto butyrate. The general sequence is
illustrated in Fig. 13. 4.
NADH+H+ NAD+
CO2
-amino acid -keto acid aldehyde alcohol
R CH COOH transamination O decarboxylation
R CHO reduction R COH
NH2 R C COOH
Fig. 13.4 Simplified pathway for higher alcohol biosynthesis
Examples:
Valine    isobutanol
Leucine    isoamyl alcohol
Isoleucine    amyl alcohol
α – ketobutyrate   propanol
13.6 METHANOL
In alcoholic fermentations, methanol is produced due to demethylation of pectin by

pectin esterase. Pectin esterase originates from the substrate or molds. There are
220
certain strains within yeasts also, which can elaborate pectin esterase. The activity of
the enzyme rises as pH increases from 1 to 6. When ingested, methanol is oxidized
to formaldehyde, which is toxic to living cells. Formaldehyde alters the biological
activity of proteins. As little as 30 ml can cause blindness, and even death. An
antidote of this poison is ethanol. Ethanol exerts a competitive inhibition and the
methanol is excreted away slowly through the urine.
13.7 DISTILLATION AND RECTIFICATION
There are many types of distillation and rectification systems available. Some of the
common systems used for producing 95% alcohol (rectified spirit) are:
 One-column system
 Two-column system
 Three-column system, e.g., (a) Barbet system and (b) Othmer system
 Vapor recompression
 Vacuum rectification
 Multiple effect distillation
 Six-column reagent alcohol system
For beverage purpose, 1 to 3 column systems are adequately satisfactory.
13.7.1 TWO-COLUMN SYSTEM
This system consists of two integral parts (a) Analyzer, and (b) Rectifier. The analyzer
is used to exhaust beer, i.e., remove alcohol from the beer. The rectifier is used to
purify the alcohol and bring it to a high strength (95-96% abv).
13.7.1.1 The analyzer
The analyzer is a cylindrical tank with about 18 plates stacked within. The beer
enters the analyzer at the top and follows a zigzag course down the column. Steam
enters at the bottom and travels countercurrently, depriving the wash of its alcohol.
The exhausted wash is run out. The vapors issuing from the top of the analyzer
enter the rectifier somewhere in the middle of the column.
13.7.1.2 The rectifier
The rectifier is composed of specially designed fractionation column with a number of

chambers (plate or tray ~ 40, see Fig. 13. 5). In practice, to overcome the shortcoming
that may result from under-designing, the number of trays is usually 10% more than
the theoretical requirement.
The tray comes in various designs (bubble cap tray, sieve tray, valve tray, etc.) and they
are placed at intervals of 60-75 cm up the height of the column. The bubble cap tray is
shown schematically in Fig. 13.5. Each tray has a conduit called downcomer. Liquid falls
through the downcomer by gravity from one tray to the one below it. The flow across
each plate is shown in Fig. 13.5.
221
A weir on the tray ensures that there is always some liquid (holdup) on the tray and is
designed such that the holdup is at a suitable height, e.g. such that the bubble caps are
covered by liquid. Being lighter, vapor flows up the column and is forced to pass
through the liquid, via the openings on each tray. The area allowed for the passage of
vapor on each tray is called the active tray area.
The feed is introduced somewhere in the middle part of the column. The section
above the feed is called enrichment- or rectification section. The section below the feed
line is called stripping section (see Fig. 13.6). The steam issuing from the reboiler (Fig.
13.6 and 13.7; several designs are available) takes the volatiles upwards along with it.
The vapors are condensed externally and returned to the same rectifier. As the
condensed vapors flow down they again meet the rising mixture of steam and
alcohol. The repeated condensation and evaporation make the vapor richer and
richer in alcohol. There is provision for separating the vaporized alcohol into 3
fractions, viz., Fraction I, Fraction II, and Fraction III.
Outlet weir Downcomer
Active tray area

Direction of flow
of condensed Vapor (rising)
ethanol Bubble cap
Inlet weir
Cap support Cap
Plate
Riser
Spider
Detailed structure of bubble cap
Fig. 13.5 Arrangement of plates in the column
Fraction I
This fraction is also called heads. It consists of low-boiling fraction. It is drawn from
the top or separately from an aldehyde-stripping column. The fraction consists
mainly of aldehyde, formic esters, and a small amount of uncondensed alcohol.
222
Fraction II
This is the main fraction and contains 96% alcohol (rectified spirit). See Fig. 13. 6
for an idea about the fractions.
Feed
Heads
Feed line
Condenser
Ethanol
Reboiler Reboiler Fusel oil

Waste
Heat exchanger Analyzer Rectifier
Fig. 13.6 Schematic diagram of ethanol distillation (two-column system)
Condenser
Enriching Reflux drum

Reflux
(rectification)
section
Feed Distillate
Stripping
section
Reboiler
Heat in
Heat out
Bottoms
Fig. 13.7 Schematics of rectification and stripping sections
223
Fraction III
This is a high-boiling fraction (125-140°C) and consists mainly of fusel oils (see page
220 also). A small stream is continuously bled from the bottom of the column and
condensed outside. The fusel oils become insoluble in ethanol when cooled. The
separated ethanol is recycled to the rectification column while the fusel oil is taken
out from the receiver (every two to three days).
13.8 USES OF ETHANOL
1. As a solvent: ranks second to water

2. Chemical intermediate: for the preparation of synthetic rubber, acetaldehyde,
acetic acid, ethyl acetate
3. Fuel: mixed with gasoline to produce gasohol
4. Laboratory use: as reagent, disinfectant, in spirit lamps
5. Beverage: blending and fortification
All the acohol which is drunk is absorbed in the stomach (20%) and the small
intestine (80%). About 10% of the ingested alcohol is eliminated in the exhaled air
and urine. The rest is rapidly diluted in the blood stream and body fluids. Alcohol is
metabolized into fat, H2O, and CO2 but this occurs at a very slow rate. An adult liver
can break down only about 10 g alcohol per hour.
The immediate effect of alcohol is its toxic action on the central nervous system.
The risk of alcohol drinking starts at a level of 0.3 g per liter in the blood stream.
The increasing amounts of alcohol in the blood stream and their effects are as
follows:
2 g/liter leads to drunkenness (intoxication)

3 g/liter results in complete weakness
4 g/liter induces coma
5 g/liter causes certain death
13.9 INDUSTRIAL ALCOHOL
Industrial alcohol (also called commercial alcohol) is ethanol produced and sold for non-
beverage applications. It is denatured to prevent its use as a beverage. Denaturing
involves mixing ethanol with small amounts of poisonous or unpleasant substances
to make the ethanol undrinkable. The removal of all these substances would involve
a series of treatments more expensive than the excise tax on alcoholic beverages.
Often, very uncharacteristic colors are added to industrial alcohol to differentiate it
from spirits intended for alcoholic beverages.
Industrial alcohol is available in three forms, viz., (i) Completely Denatured Alcohol
(CDA), (ii) Specially Denatured Alcohol (SDA), and (iii) Pure Ethanol. CDA contains
denaturants such as pyridine, wood naphtha (mainly xylene), etc. SDA contains
chloroform, acetic acid, ethyl acetate, formaldehyde, etc., as the denaturant. Pure
ethanol does not contain any denaturant but still should not be used for alcoholic
beverages.
224
13.10 PROOF AND PROOF SPIRIT
According to the US Official definition, Proof shall mean the ethyl alcohol content of a liquid
at 60°F (15.6°C) stated as twice the percentage ethyl alcohol by volume. Proof spirit shall mean
that alcoholic liquor which contains 50% ethyl alcohol by volume at 60°F as unity. In England
and Canada, proof spirits contain 49.25% alcohol by weight at 60°F. This is equal to
57.061% by volume. It is to be noted that the remaining percentage in both the
definitions (US or UK) is that of water. For alcohol contents below that of proof
spirit, the concentration may be expressed in terms of underproof (UP), and for above
50%, overproof (OP). Stated differently, a spirit of 125° proof is 25° above proof
spirit. It can therefore be written 25° overproof. Similarly, a spirit with 75° proof is
25° lower than proof spirit and hence can be written 25° underproof.
13.11 DEHYDRATED (ABSOLUTE) ALCOHOL
Dilute ethanol is readily concentrated by distillation because the volatility of ethanol

in dilute solution is much higher than that of water. At higher concentrations,
ethanol and water form an azeotrope at 89 mole % ethanol (95.7% by weight). The
volatilities of water and ethanol mixtures at this particular state are the same.
Continued boiling produces vapor of the same composition and no further
enrichment is possible. This mixture, which behaves like a pure chemical, thus boils
at a constant temperature. It distils over completely without change in composition.
Such a mixture is called constant boiling mixture or azeotropic mixture. It is because of
this nature, alcohol cannot be produced in anhydrous form by simple distillation.
The production of dehydrated (anhydrous) alcohol requires special techniques such
as azeotropic distillation, extractive distillation, pressure-swing distillation, etc. The former two
methods require the introduction of a third component into the system. Although
treatment of the distillation methods is out of the scope of this book, brief
descriptions on the former two methods are given in the following sub-sections.
13.11.1 AZEOTROPIC DISTILLATION
Azeotropic systems generate very intriguing vapor-liquid-equilibrium (VLE) curves.

The two VLE plots shown in Fig. 13.8 show two different azeotropic systems, one
with a minimum boiling point and one with a maximum boiling point.
1.0 1.0
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Liquid Liquid
Maximum boiling point Minimum boiling point
Fig. 13.8 Vapor-Liquid-equilibrium curve of homogenous azeotropic system

225
In both plots, the equilibrium curves cross the diagonal lines, and these are
azeotropic points where the azeotropes occur. In other words, azeotropic systems
give rise to VLE plots where the equilibrium curves cross the diagonals. Note the
shapes of the respective equilibrium lines in relation to the diagonal lines that bisect
the VLE plots.
Both plots shown in Fig. 13.8 are obtained from homogenous azeotropic systems. An
azeotrope that contains one liquid phase in contact with vapor is called a
homogenous azeotrope. A homogenous azeotrope cannot be separated by
conventional distillation. However, vacuum distillation may be used as the lower
pressures can shift the azeotropic point. Alternatively, an additional substance may
be added to shift the azeotropic point to a more ‘favorable’ position.
When this additional component appears in appreciable amounts at the top of the
column, the operation is called azeotropic distillation.
When the additional component appears mostly at the bottom of the column, the
operation is called extractive distillation (described shortly).
The third component added here is called entrainer or material separating agent (MSA).
This component (usually benzene) forms a ternary azeotrope of benzene-ethanol-
water in the ratio 24:54:22, which boils at 64.85°C, a boiling point lower than that
for alcohol-water binary mixture (78.13°C).
Anhydrous alcohol is produced from rectified spirit. The ternary azeotrope that
readily distils over is collected and condensed. Upon cooling, water readily separates
out from benzene. Alcohol is miscible in water as well as benzene. The dilute
ethanolic water can be rectified before recycling while ethanol-benzene mixture is
recycled. Since the water needed for forming the ternary azeotrope must come from
the 5% water present in the rectified spirit, the rectified spirit that progressively
becomes dehydrated remains at the reboiler from where it is later recovered. See Fig.
13. 9 for the schematic diagram of azeotropic distillation.
Condenser
B+A
W+A
Feed
Azeotropic Stripping
column column B = benzene
A = alcohol
W = water
Anhydrous
Condenser Water + Ethanol
ethanol
Fig. 13.9 Outline of azeotropic distillation
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13.11.2 EXTRACTIVE DISTILLATION
The third component added here increases the relative volatility between the two
original components. Ethylene glycol (CH2OH)2, has been extensively used for this
purpose. It associates chemically with water and then causes the volatility of ethanol
to increase.
Sometimes, salts (such as potassium acetate) are used to form complex with water
and break the azeotrope. At salt concentration of 5 mole%, the azeotrope can be
completely broken.
13.12 ETHANOL FROM CELLULOSE
Cellulosic ethanol or cellanol is a general term for ethanol fuel produced from
lignocellulose, a structural material that comprises much of the mass of plants.
Cellulosic ethanol is chemically identical to ethanol from other sources (such as corn,
starch, or sugar) but has the advantage that the lignocellulose raw material is
available in a great diversity of biomass including waste from urban, agricultural, and
forestry sources. Lignocellulose requires a greater amount of processing to make the
sugar monomers available to the microorganisms that are typically used to produce
ethanol by fermentation.
There are at least two methods of production of cellulosic ethanol, viz., (i) Cellulolytic
method, and (ii) Gasification method.
The cellulolytic method entails hydrolysis of cellulose followed by fermentation of

the generated free sugars. The gasification method is a chemical process which
produces synthesis gas that can be converted to ethanol by fermentation or
thermochemical catalysis (e.g., the Fischer-Tropsch process).
Because of the relevance, the first method will be described here. The preparation of
ethanol from cellulose-, starch-, and sugar-containing raw materials involves the
following general steps:
1. Pretreatment: the physical or chemical conversion of the raw material to a

hydrolyzable substrate
2. Hydrolysis: the enzymatic reaction that converts the starch or cellulose to
sugars
3. Yeast fermentation: the conversion of sugars to ethanol and carbon dioxide
4. Purification: the separation of ethanol from the by-products and wastes
The treatment as well as fermentation can be carried either in a batch- or a

continuous process. The continuous process has not reached full degree of
perfection. In the paragraphs to follow, both the methods will be briefly described.
13.12.1 PRETREATMENT OF LIGNOCELLULOSE
For acid hydrolysis, wood chips are adequate, but for enzymatic hydrolysis, some
chemical or physical pretreatment will usually be required to achieve a reasonable
227
rate and extent of hydrolysis. The objectives of pretreatment are to reduce
crystallinity (of cellulose) and to increase available surface by maximum destruction
of fiber structure and interaction between the cellulose molecules.
13.12.1.1 Chemical pretreatment
Considerable attention has been given to agents that will cause swelling of the
cellulose and disrupt the crystalline structure. There are two ways in which this
occurs:
 Intercrystalline swelling, because of uptake of water between the crystal

units, which causes a reversible volume change of up to about 30%
 Intracrystalline swelling, which involves penetration of the crystalline
structure by water, leading to unlimited swelling or complete solution of the
cellulose.
Current chemical processes include alkali treatment and treatment with sulfur
dioxide.
Alkali Treatment
The treatment of cellulose-containing residues with low concentrations of alkali

makes them considerably more susceptible to enzymatic- and microbiological
conversion. An example of alkali treatment using straw as the cellulose source is
described below.
The straw is treated with 20% alkali and, after standing, is neutralized with acetic
acid or simply by mixing with silage (which contains lactic acid). This technique has
also been applied to wood residues. Straws and hardwood residues, with lignin
contents generally less than 26%, respond to the treatment, while softwood residues,
with lignin contents higher than 26%, do not.
An alkali treatment method that is claimed to be very effective for straw and bagasse
is dilute alkali treatment (the Beckmann process). This method involves treatment of
the raw material with 1% sodium hydroxide at 45°C for 3 hrs.
Sulfur Dioxide Treatment
Disruption of lignin-cellulose bonds by treating moist wood with gaseous sulfur

dioxide under pressure at 120°C for 2-3 hrs appears to offer potential for large-scale
processing, though the economics are as yet uncertain. This method increases
digestibility to around 60%.
13.12.1.2 Physical pretreatment
Steam explosion, ball- or attrition milling, and two-roll compression milling are
effective for many substrates and provide a product of high bulk density, permitting
use of 20-30% slurries in the saccharification reactor. This is important if
concentrated sugar solutions are to be produced.
228
The best pretreatment currently available is the high pressure and high temperature steam:
Stake Technology and Iotech (both in Canada) have pretreatment processes that
produce animal feed from straws or hardwoods by steam treatment.
13.12.2 SACCHARIFICATION OF CELLULOSE
Saccharification is the process by which the pretreated cellulosic substrate is

converted into a sugar solution. These simple sugars in turn can be used as a
substrate by the yeast for alcohol fermentation. The saccharification process can be
carried out chemically (by dilute acid hydrolysis) or by enzymic hydrolysis.
13.12.2.1 Acid Hydrolysis
Dilute acid hydrolysis is very effective in breaking the glycosidic linkages between
component hexoses, but it also breaks down the sugar hexose units. As a result, the
product's acidity must be neutralized, and the amount of sugar is less than quantitative
because of sugar degradation. The yield of sugar depends on the relative rates of two
reactions that occur when cellulosic materials are treated with dilute acid:
k k
Cellulosic material 
1
 Sugar 
2
 Breakdown products
In simple batch processes, the rates of reactions k1 and k2 are approximately equal,
so that maximum yields are limited and the hydrolyzate contains as much breakdown
products as contaminants. For starch, which is amorphous, the rate of hydrolysis is
much faster than degradation, and sugar yields approach the theoretical level. Lignin
has apparently little effect on the rate, as most woods hydrolyze faster than cotton or
ramie (Asian shrub used for fiber). Crystallinity of the cellulose is thus the governing
factor in dilute acid hydrolysis.
13.12.2.2 Enzymatic Hydrolysis
Pretreated substrate, neutralized to approximately pH 4.8, is mixed with enzymes at

the required level of activity (see Fig. 13.10 for the assay of cellulose activity). In the
commercial fermentation, the highest substrate concentrations that can be stirred
(10% or more) are used, and the mixture is incubated at 45-50°C and pH 4.5-4.8.
The enzymes are destroyed by even brief exposure to high temperatures (60°C or
greater), or by a pH below 3.0 or above 8.0.
The costs of both pretreatment and saccharification are functions of scale: for
systems in which mechanical or chemical pretreatment is followed by fermentation,
large-volume operation would be attractive in industrialized countries as a means of
reducing unit cost. It may, however, be possible to have comparatively cheap
methods at the other end of the scale. One such simple process developed by
Toyama et al. (Fig. 13.11) is described below.
Mixed chopped, pretreated substrate, such as alkali-treated straw, bagasse, or

sawdust (adjusted to pH 4.0) is mixed with either crumbled Trichoderma koji (solid
culture), preferably made from the same substrate as the saccharification substrate,
or with commercial cellulase in a Shocho jar - a large ceramic jar with a narrow neck
to keep out air and prevent contamination (Shocho is a sweet-potato liquor). The
229
substrate can be in the form of a very thick slurry, since once it is mixed well it will
not be stirred. Citric acid (0.5%) is added as a preservative and the jar stored at 45°C.
In 5-6 days this should yield 15-20% sugar syrup.
culture + broth
1/2 ml 1ml buffer add 1x6cm

enzyme (pH 4.5) filter paper
filter strip (50mg) stir incubate 1hr
50oC
filtered
enzyme
broth
boil 5 min
read color value 16ml H2O 3ml DNS
at 550 nm reagent
Fig. 13.10 The filter paper assay for determining cellulase activity of enzyme
buffer cellulase
20-25% pH 4 15-20%
cellulose 5-6 days sugar
45 oC
Fig. 13.11 Simplified saccharification process of cellulosic substrates with cellulase
13.12.3 FUTURE OF CELLULOSE FERMENTATION TECHNOLOGY
In its current state, cellulose fermentation technology - except perhaps for limited,
small-scale applications such as the Toyama koji method of saccharification - has
little likelihood of contributing to the production of alcohol fuels in the immediate
future. The processes are either too complex or expensive, or require too much acid
and alkali or energy for pretreatment, to be able to compete with other potential
sources. However, given the increasing need for liquid fuels other than petroleum,
the development of cheap and reliable saccharification technologies is necessary and
will undoubtedly be achieved. The research and development will be better done in
the industrialized countries, and the developing world should monitor progress and
take advantage of improvements.
230
13.12.4 NEW TECHNOLOGY
Developing technologies that may decrease the cost of ethanol production can be
considered in terms of pretreatment, fermentation, alcohol recovery, by-product
recovery, and waste treatment.
Much of the research on ethanol processes is aimed at improving pretreatment for

lignocellulose feedstocks to enhance the efficiency and reduce the cost of their
hydrolysis to sugars. Some of the processes currently being examined are discussed
next.
13.12.4.1 Purdue Process
This is a unique process for hydrolyzing crop residues and wood. Hemicellulose is
first removed with dilute acid and then the cellulose and lignin are dissolved in
concentrated sulfuric acid. The cellulose and lignin are then precipitated from the
acid by addition of methanol. Since the precipitated cellulose is in an amorphous
form, it is readily hydrolyzed by the appropriate enzymes.
13.12.4.2 General Electic Process
In this process, wood chips are first heated in alkaline aqueous butanol to separate
the hemicellulose, cellulose, and lignin. The hemicellulose dissolves in the aqueous
phase, the lignin dissolves in the butanol, and the cellulose remains undissolved.
The degraded hemicellulose can be fermented to additional butanol or converted to

the sweetener xylitol. The lignin-butanol fraction can be cooled to separate the lignin
or used as a fuel. The cellulose can be washed and hydrolyzed to glucose for
fermentation to ethanol.
13.12.4.3 Natick Process
The Natick process consists of five steps:
1. Selection of an abundant and inexpensive cellulosic substrate such as

municipal waste or aspen chips
2. Pretreatment of this substrate to enhance its enzyme susceptibility,
preferably by ball-milling or two-roll compression milling
3. Production of active cellulase. As a result of screening thousands of
organisms over the past 40 years, the Natick group has selected Trichoderma
reesei as the best source of active cellulase
4. Utilization of the cellulase and -glucosidase to saccharify cellulose. The
treatment yields 5-15% glucose syrups in continuous hydrolysis or 10-30%
glucose syrups in batch hydrolysis
5. Fermentation of the resulting glucose syrups to ethanol with Candida or
Saccharomyces yeasts
231
All steps of the Natick process have been carried out at 200-400 liters, pilot-plant
scale and a complete description and economic analysis is available from the Natick
laboratory
13.12.4.4 Iotech Process
In this process, wood chips are exposed to high-pressure steam for several seconds,
followed by explosive decompression.
At the Georgia Institute of Technology, samples of untreated and steam-exploded

poplar chips were extracted with water and solvents. Results indicated a fivefold
increase in ethanol extractables in the steam-exploded chips over the untreated
samples - from about 5% to about 25%. Since lignin is the major component in the
extractables, this steam treatment may facilitate degradation of the remaining
cellulose.
13.12.5 CONTINUOUS PRODUCTION OF CELLULOSIC ETHANOL
The description given below is the continuous cellulosic fermentation method

developed by Bio-Process Innovation (BPI), Inc. (USA). It uses a continuous
cascade fermentation system for producing ethanol from waste sugars. BPI is
currently working towards applying the technology to waste paper, cornstalks, straw,
or sawdust.
As shown in Fig. 13.12, biomass feed is introduced continuously into the first of 3-5
stirred reactors placed in series, with the outflow of one reactor flowing into the next
reactor. The liquid stream that moves from reactor to reactor is contacted with a
stripping gas to remove the ethanol.
Biomass input Soluble Xylanase

hemicellulose,
lignin
Xylan, cellulose
pretreatment process Xylose fermentation
Cellulose
Cellulase
Ethanol or other
Cellulase fermentation
treatment product
Cellulose/Cellulase
fermentation
Liquid recycle
Pump
Lignin, ash, protein
Fig. 11.12 Continuous production of cellulosic ethanol
232
A low-energy solvent absorption/extractive distillation system extracts ethanol from
the stripping gas and recovers the gas for reuse. Separating the ethanol product as it
is formed increases the rate of ethanol production. BPI, Inc. has also developed a
highly flocculent yeast that further speeds the fermentation by maintaining high cell
densities while operating continuously. The final effluent from the fifth reactor
consists of waste lignin and ash, which is dewatered so that the water can be
recycled.
A five-stage, 40,000-gallon unit has been operating at Permeate Refining Company

in Hopkinton, Iowa, since June 1996 on waste starches and sugars. A small pilot unit
operating on cellulosics is currently being tested at BPI, Inc. in West Lafayette,
Indiana. A small plant in Spring Green, Wisconsin (Spring Green Ethanol), is now
using BPI’s technology for converting permeate mother liquor to ethanol. Several
companies are currently evaluating BPI technology for whey- and molasses-based
ethanol plants to be sited and built in the near future.
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CHAPTER 14
WINE TECHNOLOGY
14.1 INTRODUCTION
Wine is one of the world’s oldest alcoholic beverages, dating back to 6000 BC. It is
believed to have originated in Egypt and Mesopotamia (Iraq and Eastern Syria).
14.1.1 DEFINITION
Wine without qualification means the end product of complete or partial alcoholic
fermentation of fresh grape juice. Wines produced by the fermentation of juices of
fruits, berries, honey, etc., are required to indicate source (the raw material) on the
label. For example, orange wine, pear wine, apple wine (cider/cyder), etc.
14.1.2 WORLD PRODUCTION
Around 80% by volume of wine is produced by European countries. There are 7.742
million hectares of vineyards in the world producing 58.681 million MT of grapes
each year. About 27 billion liters of wine are produced from these grapes. In 1996,
the biggest wine producers worldwide were: France (21.9%), Italy (21.6%), and Spain
(12%). The United States produced about 6.8% only. See Fig. 14.10 and Table 14.4
for an idea on world wine statistics.
14.1.3 CLASSIFICATION OF WINE
Wines can be classified on various bases viz., (i) color, (ii) relative sweetness, (iii) effervescence,
(iv) alcohol content, and (v) the system used by Wine Advisory Board, USA. However, three
basic groups of wines are most easily distinguishable for the consumer. They are
(i) table wines, (ii) sparkling wines and (iii) fortified wines. A summary of the classification
scheme is given in Table 14.1.
1. Color
Based on color, wines can be classified into (a) red wines, (b) white wines, and
(c) pink wines (rosé). Red and white wines are the more important ones. Rosé can be
prepared by blending red- and white wines or by using lesser color extracts from
grapes.
2. Relative sweetness
Wines may be classified as either sweet or dry depending on the presence or absence
(respectively) of residual sugar in wine. Dry wines contain sugar below 0.12% while
sweet wines may contain sugar as high as 7%. They may be red or white. Off-dry or
semi-dry wines are also available.
Table 14.1 Classification of wines
Basis of
Class/Type Description Example
classification
Color Red wine Contains the red coloring Burgundy
matter of skin, pulp and seeds
White wine Does not contain the red Rhine wine
coloring matter of skin, pulp
and seeds
Pink wine Low concentration of red Rosé
coloring matter is maintained
Relative Sweet wine Contains up to 7% sugar Sherry (sweet)
sweetness Dry wine Contains less than 0.12% sugar Sherry (dry)
Alcohol Natural Contains 8.5-16% alcohol by Table wines
content volume (% abv)
Fortified Contains 17-21 % abv Sherry
Effervescence Still Does not contain CO2 Chianti
Sparkling Contains CO2 (natural or Champagne
added)
Wine Advisory Dessert wine Contains sugar; taken after Sherry (sweet)
Board, USA meal
Appetizer wine Dry; fortified; taken before Sherry (dry)
meal
Sparkling wine Contains CO2 Champagne
Red Table wine Natural; red in color Chianti
White Table wine Natural; pale yellow to straw Rhine wine
color
______________________________
There is considerable overlapping among wine types in the classification shown above. For example, a Red
Table wine can at the same time be sweet, sparkling, fortified or natural. Similarly, a fortified wine can be
sweet, sparkling, red, or white.
3. Alcohol content
Wines may be classified either as natural or fortified. Fortified wines are those that
have received additional distilled spirit (wine spirit or brandy) to bring the alcohol
content in the range 17-21% by volume. Due to high alcohol content, fortified wines
may be stable even without pasteurization. Natural wines do not contain added
spirits. Normally, they have alcohol content up to 16% by volume. Natural wines
with alcohol contents in the range 8-10% are called light wines.
235
4. Effervescence
Wines may be classified as still or sparkling. Still wines do not contain CO2 while
sparkling wines contain CO2, either natural (resulting from fermentation) or artificial
(externally added).
5. System used by Wine Advisory Board
The board has divided wines into five main groups, viz., (i) White Table Wine, (ii) Red
Table Wine, (iii) Dessert Wine, (iv) Aperitif Wine (Appetizer Wine), and (v) Sparkling Wine.
See Table 14.1 for the properties of these wines.
14.1.3.1 Red Table Wine
It contains the red coloring matter of the grapes. It is mostly light and dry or semi-
dry (up to 14% abv). Red wines usually contain more flavor and aroma components.
They are served at room temperature to release aroma characters. Red wines being
more robust may be served in a glass with a generous, wide bowl and a narrower
mouth. The bowl enables the wine to be easily swirled in the bowl without spilling to
encourage evaporation of some of the volatile compounds. The smaller mouth of
the glass concentrates the ensuing bouquet. Some of the more important examples
of red wine are Claret, Burgundy and Chianti (pronounced as: kee áantee). See Fig.s 14.7-
14.9 for an idea about the glasses.
14.1.3.2 White Table Wine
It is colorless, still, light, dry to sweet. A white table wine is not in the true sense
white: the color actually ranges from straw through brown. White wines are usually
served chilled because at warmer temperatures they quickly lose their volatile
characters and become flat and tasteless. Examples: Rhine wine, Sauterne.
14.1.3.3 Dessert Wine
It is a still wine fortified in the range 17-21% abv. It is, as the name implies, taken
after meal and is sweet in taste. Examples: Tokay, Muscatel, White port, Angelica, Sherry
(sweet).
14.1.3.4 Appetizer Wine
This wine is taken before meal as an aperitif. It is still and fortified. Examples: Sherry
(dry), Vermouth (pronounced as: vər moóth), Port.
14.1.3.5 Sparkling Wine
This wine contains CO2, either natural or artificially added, and can be natural or
fortified. It is usually sweet. Sparkling wines are served in tall, narrow glasses that
clearly display the beautiful bubbles as they rise to the surface. Examples: Champagne
(natural), Madeira (fortified).
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14.1.4 SOME NOTED WINES
Wines from France are considered to be among the highest-quality wines in the
world. The most renowned wine-producing regions are Burgundy and Bordeaux.
 Burgundy: any of various types of red or white wines from Burgundy area of
Eastern France. Burgundy implies dark purplish color.
 Chianti: (particular type of) red or white wine from central Italy.
 Sherry: A type of yellow or brown, uniquely processed fortified wine,
originally from southern Spain. It may be sweet or dry. In the sweet type,
sugar may be present at concentrations up to 7%.
 Claret: (any of various types of) dry red wine, especially from Bordeaux area of
France.
 Port: Strong, sweet, red wine made in Portugal
 Champagne: (any of various types of) sparkling straw-colored wine from eastern
France
 Madeira: White dessert wine from the island of Madeira. It can be still or
sparkling
 Vermouth: Strong, white wine flavored with herbs, drunk as an aperitif wine
(often in strong cocktail).
14.2 WINE YEASTS
Wines can be prepared using either natural yeast flora of the grapes (spontaneous
fermentation) or pure cultures (culture yeasts). Many manufacturers still depend on
spontaneous fermentation. Spontaneous fermentation has following advantages and
limitations:
Advantages:
The wine will be of unique quality in terms of bouquet because the end product is
the result of interaction of diverse yeast types. Each yeast type will contribute unique
flavor to the wine.
Disadvantages:
 Since the yeast profile is diverse, spontaneous fermentation may sometimes lead
to failure
 Most strains (and also genera) do not produce large amounts of alcohol
while a few strains produce undesirable organic compounds such as organic
acids, H2S, higher alcohols, etc., that may affect the flavor.
Grapes harbor both desirable and undesirable yeasts, e.g., Hansenula, Kloeckera, Pichia
(film formers), Saccharomyces, Candida, Metschnikowia, to name a few. The most
important in winery, however, are Saccharomyces spp especially, S. cerevisiae and S.
uvarum. In general, they are not present in dominant numbers initially. However, as
the fermentation progresses, they quickly outnumber other groups thereby creating
environment for vinification.
237
14.2.1 PURE CULTURES
Pure cultures can be obtained either from commercial suppliers or by isolation in the
laboratory from grapes. In the latter case, isolation can be done by streaking on grape
juice agar. At present, a number of wine yeasts (pure culture) are available
commercially: champagne, burgundy, sauterne, pommard, to name a few. Each
confers a distinctive flavor to a particular wine. The all-purpose wine yeast is, as
the name implies, suitable for many wines. Bakers and brewers yeast are suitable
only for home wine-making: they yield low amounts of alcohol. Of the two yeasts,
viz., S. cerevisiae and S. uvarum, the former contains strains that yield much larger
amounts of alcohol (18-20% abv) than the latter (7-8% abv). In industry, often a
combination of natural flora and pure culture is used. While the natural flora
contributes to flavor, pure culture controls the direction of fermentation.
14.2.1.1 Desirable properties of wine yeasts
For producing a good quality wine, the choice of yeast is as important as the choice
of grapes. The desirable characteristics of an ideal wine yeast are:
 High alcohol production

 Appropriate settling characteristics
 Osmotolerance
 Minimal by-product formation
 SO2 tolerance
14.3 GRAPES
For wine production, Vitis vinifera, Vitis labrusca, and to a smaller extent, Vitis
rotundifolia are used. Vitis vinifera is the European wine grape and is by far the most
important. There are over 5000 varieties of Vitis vinifera today. Natural factors make
wine from a particular region unique. Known in the wine industry as terroir, these
factors include local climate (temperature, rainfall, sunlight), location of grapevines
(altitude and slope), and soil (structure, composition, and water drainage).
The single most important factor that contributes to wine’s character is the grapes that
are used. Grapes influence the wine’s flavor, alcohol content, acidity, and even its color.
Grape juice is highly variable in composition. Its major constituents are: water,
carbohydrates (glucose, fructose, pentose, pectins), nitrogenous compounds (protein and
protein-split products), vitamins, enzymes, and aroma compounds. The color may range
from white, green, pink, red, to purple (see Fig. 14.1 also). Some of the more important
physicochemical characteristics of matured grapes are given in Table 14.2. Grape
juice with very high sugar concentrations are used in the production of strong or
sweet wines.
238
Table 14.2 Physicochemical parameters of grapes
Variable Rannge Commments

Sugar 120-250g/L Penntose: 1g/L; gllucose:fructosse =1:1
Acid 5-15gg/L As tartaric
t acid
Pectin 0.02-00.6%
pH 3-44
Fig. 14.1 Purple grapees
14.4 PRODU
UCTION OF
F RED TABLE WINE
The outline of
o red table wine
w productioon is given in Fig. 14.2. Thee main steps iin red
table wine making
m are as described
d in thhe following paragraphs.
14.4.1 GRAPPE SELECTION
The grapes usedu for the production

p of red table winnes are selecteed varieties off Vitis
vinifera. Sincee the color off red table winne comes fromm the grapes, the berries shhould
be of purple or red color (see
( Fig. 14.1).
14.4.2 CRUSSHING AND

D SULFITING
G
w productioon, the grapess are harvesteed from the viineyards and taken
In modern wine
to winery whhere they are passed
p thoughh destemmer-ccrusher machiine. Three typpes of
crushers are generally usedd: (i) Roller typee, (ii) Disintegraator, and (iii) G
Garolla type. Thhe last
one is more generally usedd. The crusheed grapes fall in i the sump beeneath the cruusher.
The whole iss then taken too fermentationn tanks using special plungeer pumps.
Sulfiting (adddition of sulfuur dioxide) is done during crushing,

c and this is primarrily to
(i) inhibit thee growth of unndesirable miccroorganisms (acetic acid baacteria, some lactic
acid bacteriaa, and some grape
g yeasts) and (ii) bind acetaldehyde (acetaldehyde has
undesirable organoleptic
o properties).
p
The toxic efffect of SO2 is due to the diissolved moleccular SO2. Thhe effect of SO
O2 on
yeasts variess from species to species, and even wiithin strains. IIn general, SO2 is
239
seldom used at a rate above 150 ppm. Moldy grapes may need 200 ppm, though.
Higher concentrations of SO2 markedly delay fermentation (sometimes as long as 2
months). The most commonly used source of SO2 is potassium metabisulfite (KMS).
14.4.3 MUST TREATMENT
The grape juice meant for wine fermentation is called must. For consistent wine
quality, the quality of must should also be consistent. If the must does not meet the
requirement, grape juice concentrate, sugar, acid, etc., must be added for the
adjustment. This manipulation to standardize the must is called amelioration. Addition
of sugar is supposed to produce substandard wine and is prohibited in some countries.
Gallization is a term used to imply addition of water and sugar prior to fermentation
in order to (i) increase alcohol content, (ii) increase total volume, and (iii) decrease
acidity. Chaptalization is another term used to imply addition of sugar only.
14.4.4 WARMING
If the weather is cold, the must is warmed to 27°C. This facilitates color extraction
and yeast action.
14.4.5 STARTER PREPARATION AND ADDITION
Typically, the pure culture kept in a slant is filled with sterile grape juice and
incubated at 27°C for 12 hrs with occasional shaking. This is then transferred to a
large flask containing sterile grape juice and again incubated for 3-4 days with
occasional shaking. The whole is then transferred to a propagator (about 25 liter
capacity). After aerobic growth for 4-5 days, this in turn is transferred to a final
propagator that contains 25 HL of sterile grape juice. Aeration is accomplished by
supplying sterile air (filtered air). Three to four hours after inoculation, 100-125 ppm
(parts per million) SO2 is added for acclimatization. The propagators are all closed
vessels. The vigorously growing yeast is now used to inoculate the main
fermenter at a rate of 2-3% by volume.
14.4.6 FERMENTATION AND COLOR EXTRACTION
The fermenters are mostly of stainless steel or wood. The type of container used and
the temperature of fermentation influence the characters of the wine. During
fermentation, the temperature is kept at about 27°C. Low temperatures slow down
yeast activity and color extraction, and promote lactic acid bacteria. High
temperatures lead to alcohol and aroma loss due to evaporation. High temperatures
may also lead to stuck or hung fermentation in which the yeasts flocculate
prematurely: the yeasts are weakened and will leave 1-6% sugar unfermented.
Temperature control is achieved through cooling coils. The fermentation is
monitored by periodic testing of temperature and taking Balling degree (1° balling =
1% sucrose solution in terms of gravity). The test is done 2-3 times a day.
The skins, pulp, and seeds rise to the surface during fermentation to form a cap of
one to several feet in thickness. The cap is thoroughly punched and mixed with the
juice using a pole, several times a day. Alternatively, the juice may be drawn from the
240
bottom by a pump and sprayed over the entire surface of the cap, again several times
a day. The color is extracted due to the solvent action of alcohol (generated during
fermentation) and the temperature of fermentation. Most pectic substances are
destroyed or precipitated by alcohol. Tannins are also extracted. The duration of
fermentation can be judged by the amount of color and tannins extracted. It
generally takes 3-4 days.
Color can also be extracted by HTST heating of crushed grapes (to 85°C). The color
is primarily anthocyanin. Since the yeast flora is killed during heating, pure culture
must be used for fermentation.
Another interesting process for color extraction-cum-fermentation is maceration

carbonique. In it, the injured berries are kept for 8-10 days in a vessel under layer of
CO2. During this time, intracellular fermentation occurs in grapes thereby forming
1.5-2.5% ethanol by volume. In the meantime color will be extracted. The berries are
later on pressed and fermentation carried out for additional 48 hrs. Following
advantages have been claimed for this method:
 The aroma of wine is more intense

 The concentration of polyphenols is low
 Bacterial fermentation of malic acid is favored
14.4.7 DRAWING OFF
Drawing off is done to remove pomace from the primary-fermented must. It consists
in allowing the free run wine to flow into tub through a large bronze spigot
(trap/valve/plug) and pumping the wine to secondary fermentation tanks. The
pomace is strained off in stainless steel screens during pumping.
14.4.8 PRESSING
Pressing is done to remove residual wine from the pomace and this is done in a
vertical or horizontal basket or Williams press, the pressure being applied by a
hydraulic ram. This wine is normally kept separately to produce press wine (low-grade
wine: contains tannins, sediments, etc.). Mixing press wine with free-run wine lowers the
quality of the latter.
14.4.9 AFTER-FERMENTATION
Primary fermentation normally leaves some amounts of residual sugar. This is

fermented in storage tanks by the surviving yeasts. Aeration is done to invigorate the
yeast. Temperature control is critical again because low temperatures also lead to
stuck fermentation. To avoid this, temperature control and bottom aeration (to
intermix the contents) is done. Chemical analysis is done until sugar content falls
below 0.2% (in case of dry wine).
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PURPLE GRAPES SO2
(75-125 ppm)
Yeast Destemming
Crushing
Propagation Must
Primary fermentation
Drawing off and pressing

SO2
(to make 75 ppm) Free run wine Press wine Pomace
Secondary fermentation and filling
Racking, blending, fining, malo-

lactic fermentation
Aging
Filtration and tartrate stabilization
Polishing
Pasteurization
Bottling, labeling, casing
RED TABLE WINE
Fig. 14.2 Industrial production of Red Table Wine
14.4.10 FILLING AND MALO-LACTIC FERMENTATION
Wine from secondary fermentation is carefully drawn out and filled in tanks of table
wine, and sealed. Final phase of fermentation takes place here also. In very obstinate
cases, it may be necessary to add yeast foods: ammonium phosphate or urea. The
stage is taken care to protect wine against vinegar bacteria by means of a fermentation
bung. This may be prepared by boring a small hole in a cellar bung and inserting
through it a U-tube. The bung is inserted in the bunghole in the tank and one arm of
the tube is immersed in a jar or bottle containing dilute KMS solution. This helps
build a slight positive pressure of CO2 in the tank, which prevents the growth of
vinegar bacteria or film-yeasts. After bubbling in the fermenter bung practically
ceases, indicating that the fermentation is nearly complete, the bung is replaced by a
plain, soled, cellar bung. The process is complete within about 6 weeks at most after
crushing of berries. The wine is now assumed to be dry.
14.4.10.1 Malo-lactic fermentation
Malo-lactic fermentation refers to secondary fermentation in which lactic acid

bacteria are allowed to metabolize malic acid to (lactic acid + CO2). This
fermentation is particularly useful if the titrable acidity of the wine is to be reduced.
Wines with low levels of acidity should be protected from malo-lactic fermentation:
wine quality decreases if the acid level falls too low. Malo-lactic fermentation can be
242
easily prevented by early racking, cool storage, and maintaining 100 ppm or more of
SO2. On the other hand, if such a fermentation is desired it can be facilitated by
leaving the wine on the lees (yeast sediments) for prolonged periods at higher
temperatures. This storage causes lysis of yeast cells and releases amino acids and
other nutrients needed for the growth of the ‘contaminant’ lactic acid bacteria. The
biochemistry of fermentation is given in Fig. 14.3.
COOH
CH2
L-malic acid
CHOH
COOH
L-malate dehydrogenase Malo lactic enzyme
Pyruvate + CO2 Pyruvate + CO2

L-lactate dehydrogenase
CH3CH(OH)COOH
Lactic acid
Fig. 14.3 The malo-lactic pathway
Malo-lactic fermentation has an important bearing in the quality of wine. It is a

natural way of reducing acidity in wine. Besides, the fermentation also results in
wines with greater softness and mellowness. The bacteria implicated for malo-lactic
fermentation are Leuconostoc oenos, Lactobacillus, and Pediococcus, the first one being the
most important.
14.4.11 BLENDING, RACKING, AND FINING
After the yeasts have settled down well in storage tanks the clear wine portion is
carefully siphoned away from the lees. After carrying out routine chemical and
organoleptic tests, blending is carried out. Blending is one of the most important
cellar operations the main purposes of which are to: (i) develop specific types, and (ii)
maintain character and quality of wine types. Usually, the wine of a given vintage is blended
and earlier this is done, the better. Wines labeled vintage should not be blended.
Following blending, the wine is amended with a total of 100 ppm SO2. Sediments
are allowed to settle and the clear wine siphoned to yet another vessel. This process
is called racking. Racking serves many purposes, viz., (i) removes considerable amount of
CO2, (ii) raises redox potential, and (ii) clarifies wine.
One danger of leaving the new wine in contact with its yeast sediment is that it may
lead to yeast autolysis and, at low redox potential, formation of H2S. Normally, wine
should be racked within a month of the end of fermentation. Racking process
normally entails a sacrifice of 2-3% wine in lees.
Clarification by conventional racking is a long process. To hasten this, certain agents,

commonly called fining agents, are added during racking. Fining is a traditional method
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of bringing about clarification. Fining agents may be used during aging as well. They
not only clarify the wine (by physical adsorption) but also help remove excess
tannins. Some of the more important fining agents are gelatin, tannins, isinglass, and
bentonite. Typically, bentonite can be used at a rate of 1.5 g/liter. However, it is
essential that the fining agents be tested for dosage optimization before use.
14.4.12 AGING
This is one of the most interesting and one of the most important, yet one of the
most complex processes of wine making. Newly fermented wine is cloudy, harsh in
taste, yeasty in flavor and odor, and without the pleasing bouquet that develops later
in its history. Aging (also spelt ageing) can be done in oak barrels as well as in bottles,
the latter being called binning. Depending on the type of wine, aging can be done for
6 months to several years. If fining agents are used in the aging, racking is done at
least twice a year. Aging, however, can be hastened by aeration, refrigeration, and
pasteurization. Since aging is simply a maturation or mellowing process it can occur
throughout the storage period. Aging can be done after clarification, post-
pasteurization, or even after final bottling.
The principal changes in flavor and bouquet during aging in the wood are generally
believed to be due to slow oxidation process: wood extractives also have a material role
on flavor. Several changes, e.g., formation of esters, etc., also occur.
14.4.13 FILTRATION
Filtration is carried out in plate and frame filter using filter aid such as Hyflo super cel,
diatomaceous earth, etc. This helps remove suspended solids, clouding agents, etc.
14.4.14 TARTRATE STABILIZATION
New wines are supersaturated with respect to potassium tartrate. Storing in cold
temperatures allows crystallization and sedimentation of this compound, which can
finally be removed. The crystals are called wine diamonds and are not hazardous. In
commercial practice, tartrates are removed by passing wine through ion-exchange
resin.
14.4.15 PASTEURIZATION
Wines with alcohol contents less than 17% are susceptible to spoilage. The shelf-life
can be increased by pasteurization. Pasteurization of wine can be done by following
techniques:
1. HTST: the wine is heated at 80°C for 1 sec, cooled, and filled
2. Fill Cold: the wine is first filled and heated in water bath or by hot water
spray to an internal temperature of 60°C
3. Hot Bottling: the wine is heated to 55-70°C and filled hot.
In some wines, the quality is reduced by pasteurization while that of others may be
improved. The response is related to grape variety.
244
The alcohol content of wine
w has a signnificant effect on the heat rresistance of yyeasts
and has beenn found to bee more importtant than otheer variables likke pH and ressidual
sugar. High level
l of alcohool content redduces D and Z values.
An alternativve to pasteurrization is filtrration sterilizatiion. In this mmethod, membbrane

filters of porre sizes 0.65-1.2 μm are useed, the former being more ggeneral in use.. This
filter removves bacteria also.
a Howeveer, the wine should be hhighly polisheed by
prefiltration thhrough variouss types of deptth filters prior to using this m
method.
14.4.16 BOT
TTLING, LAB
BELING, AN
ND CASING
These are thhe final operattions that the wine receivess at the cellar.. The objectivves of
bottling are to
t protect winne against spoilage organismms, oxygen, ligght, and to proovide
bottle-aged wines
w (Fig. 14..4).
Fig. 14.4 Wine Cellarr
A wine cellar in the Bordeauux region of Frrance contains thousands of bbottles of winee. Fine
wines are bestt stored under dark,
d cool, and moderately dryy conditions. TThe bottles are sstored
horizontally too prevent the coorks from dryinng out.
14.4.17 SOM
ME MAJOR COMPONEN
C NTS OF WINE
E
Being produuced from coomplex materiial, wine conttains innumerrable componnents.

The most im
mportant (qualiitatively) of thhem are given in Table 14.3.
245
Table14.1 Some major components of wine
Component Concentration
Ethanol 14%
Methanol Traces to 0.6 g/L
Higher alcohols 0.15-1.0%
Acetaldehyde 200-500 ppm
Esters 200-400 ppm
Volatile acidity 0.03 g/100 ml as acetic acid; maximum limit = 0.14 g/100 ml
Pectins and gums 0.3-0.5 g/100 ml
Water and sugar Variable
Glycerol Trace
The dominant higher alcohols in wine are 1-propanol, 1-butanol, 2-methyl-1-

butanol, 3-methyl-1-butanol, 1-pentanol, and 1-hexanol.
14.5 PRODUCTION OF WHITE TABLE WINE
White table wine is a grape wine which does not contain the red coloring matter of the
skins, juice, or pulp of grapes. The wine color ranges from straw to brown.
White table wines differ fundamentally from red wines in production, composition,
and sensory quality. Since white wines are not produced by fermentation on skins,
the tannins and extract contents are lower. White table wines are more delicate in
flavor and are usually sweet. On the other hand, red table wines are more flavorful
and are usually off-dry to dry.
14.5.1 GENERAL PROCESS OF WHITE TABLE WINE PRODUCTION
Except for some steps, the technique of white table wine production is similar to
that of red table wine. The process starts with the selection of white grapes. During
crushing, sulfiting is done at the rate of 75-150 ppm SO2. The crushed grapes are
allowed to stand in the vat at cold temperature overnight. A small amount of tannin
gets extracted and sliminess is lost. The loss of sliminess, which is due to the action
of pectic enzymes, is advantageous because it increases the flow of free-run juice.
Without sulfiting, particularly during warm weather and long standing, oxidation as
well as other enzyme-catalyzed reactions may occur. These can affect the color and
organoleptic properties of wines unfavorably. Further, sulfiting is particularly
important for vinification of grapes with low concentration of acids. Sulfiting helps
prevent bacterial fermentation of malic acid to lactic acid.
The settled juice is separated from the sediments and skins by many different
methods, including drawing off, centrifugation, and filtration. The sediments may be
pooled and fermented separately for low-grade wines. Settled-and-drawn off must
produces earlier-clarifying wines but some believe that the wines are less flavorful.
Settling is particularly desirable with musts intended for sweet table wines. Must
246
from grapes infected by Botrytis cinerea (a mold) should always be settled. Natural or
artificial infection with Botrytis is sometimes desirable. This mold develops on grapes
exposed to a rainy and foggy climate. Its action on grapes causes a rapid evaporation
of moisture and consequent increase in sugar content. The infected berries are said
to be botrytized. The mold produces glycerol (up to 2%), which gives a unique
botrytized flavor in wine.
14.5.2 AMELIORATION
Acid, if needed, may be added. Addition of acid before fermentation promotes

cleaner fermentation and apparently gives smoother wines than if added later during
aging: but losses of acid in the lees are greater if the acid is added before
fermentation. Tartaric acid is preferred. Citric acid should not be used if acidification
is to be done before fermentation. Some wine makers add a small amount of tannins
also. It aids in stabilization but the wine will usually be slightly darker. Sugar
concentration can be adjusted as described for the red wine.
14.5.3 STARTER ADDITION
Starters can be added with or without heat treatment of the must. Usually, HTST
(85°C) is carried out where heating is used. Starter is added at the rate of 2-3%
vol/vol. The yeast should preferably be of granular (i.e., agglomerating) type, such as
champagne- or burgundy strains. The propagation method is similar to that
described for red table wine.
14.5.4 FERMENTATION
White wine fermentation is done at 10-15°C, which is lower than that for red wine.
At temperatures exceeding 20°C, the fermentation not only becomes stuck but the
bouquet, aroma, and flavor are also damaged. Fermentation is carried out in lined
steel, concrete- or wooden tanks, ovals, or puncheons.
After fermentation, the wine is removed from the lees as soon as possible before
yeasts begin to autolyze. The rest is the same as for red wine. The wine is bottled for
a storage period, which may extend up to 2 years for high-quality wines.
14.6 FORTIFIED AND SWEET WINES
Production of fortified wines and sweet wines does not need special procedures.
Fortification can be carried out by addition of distilled spirits either to a fully
fermented or partially fermented (or fermenting) wine. The final alcohol
concentration is made 17-21% by volume. The high alcohol content makes the wine
more stable and may keep well even without pasteurization. Fortification of stuck
wines is another way of producing fortified sweet wines. Sweet wines can also be
produced by adding appropriate amounts of sucrose solution (commonly called
dosage) to dry wines.
247
14.6.1 SWEET TABLE WINES
Example: sauterne
Sauterne (pronounced: sō túrn) is prepared from sweet grapes and fermentation

arrested at some point by racking and adding a heavy dose of SO2 (250 ppm or
more). Another method of producing sweet sauterne is to add, after aging, the
required amount of grape juice preserved in unfermented condition with about 1000
ppm SO2. This preserved juice is called mute (a French term).
14.6.2 SHERRY
The origin of sherry is Spain. Sherry is a type of wine containing fortifying grape
spirits or added alcohol, having the taste, aroma, and characteristics generally
attributed to this product, and an alcohol content of not less than 17% by volume.
Sherry is the most important Californian wine type. There are three methods of
sherry production:
1. The solera process: bouquet is due to flor yeasts

2. Submerged fermentation: bouquet is due to flor yeasts
3. The baking process: bouquet is due to baking
Sherry can be either sweet or dry. Dry sherry is used as an appetizer while sweet
sherry is used as a dessert.
14.6.2.1 Solera process
Selected grapes are first sun-dried to concentrate the sugar. They are then crushed
and plastered by the addition of CaSO4 in the form of finely powdered gypsum in
order to increase the acidity (and lower pH). CaSO4 reacts with potassium tartrate
with the formation of free tartaric acid. This reduces susceptibility of the must to the
growth of lactic acid bacteria.
Fermentation is carried out as with other wines. The base wine, which has alcohol
content between 14.5-15.5% by volume, is partially filled in horizontal barrels. The
contact of the wine with air allows the development of flor yeast, Saccharomyces
bayanus by today’s designation. The yeast metabolizes wine components, principally
alcohol, and produces characteristic flavor compounds, notably acetaldehyde and
some other compounds such as acetals, esters, and higher alcohols.
Sherry flavor develops in an interesting manner. Barrels are arranged in ranks, usually
3-6. Flor formation occurs about two weeks after the filling step. Some amount of
matured wine is drawn from the last rank without disturbing the flor. This rank is
now filled with wine from the preceding rank. The sequence goes on and the first
rank receives young wine. In a five-rank system, by the time wine leaves the last rank
it will have an average age of 12 years. Refilling is done at fixed intervals. The bottom
row is called solera (which means floor). The other layers are called criadera (which
means nursery). The wine that is drawn from the bottom row is now blended,
248
fortified, clarified, and filtered. See Fig. 14.5 for an idea about the arrangement in
ranks.
Criadera 3rd
Criadera 2nd
Criadera 1st
Solera
Fig.14.5 Example of arrangements of barrels in solera process
14.6.2.2 Submerged fermentation
This method is used for the production of cheap sherry. The quality does not
approach that of solera process. In this method, the sherry process is accelerated by
developing flor yeast in submerged culture. Air requirement is met by aerating the
base wine.
14.6.2.3 Baking process
The sherry bouquet is developed by baking the base wine at 50-60°C for 10-20
weeks. The base wine, after fortification, should be left in tanks for 24 hrs. This
allows the separation of yeast cells and colloids that flocculate during fortification.
The process brings about slight browning and certain degree of oxidation. The
quality is improved in oak barrels. Fortification is done before baking and sugar can
be added at the rate of 1-10%. Baking is followed by cooling and stabilizing (with
bentonite), racking, filtering, polishing, and aging. Light colored sherry is produced
by charcoal treatment. The whole may finally be blended before bottling. The wine is
stored for 6 months to 3 years.
14.7 SPARKLING WINES
Sparkling wines are those which readily foam because of high concentration of CO2,
either natural or artificially introduced. The CO2 pressure is 4-5 atm at 20°C.
However, in the US, wines containing a pressure of slightly more than 2 atm may be
called sparkling wines.
Sparkling wines may be prepared by various methods:
1. Champagne process: bottle fermentation, yeast removal by disgorging

2. Transfer process: bottle fermentation, transfer to a tank, and removal of yeast
by disgorging
3. Bulk fermentation: fermentation in large tanks
4. Carbonation: carbonation of base wine obtained by normal fermentation.
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14.7.1 THE CHAMPAGNE PROCESS
In the classical bottle fermentation, a dry wine (cuvé) is taken for secondary
fermentation. The cuvé has 10-12% abv. Blending is done to give an acidity
between 0.7 and 0.8% as lactic acid. Before fermentation, the cuvé receives some
sugar (18 g/bottle), typically at the rate of 25 g/liter and champagne yeast (0.3
g/bottle). The secondary fermentation takes place in thick-walled, tightly corked
bottles at 9-12°C. The fermentation requires several months. After that, the wine
remains on yeast for several months or years (2-3 years). All the bottles are kept half-
way inverted (~ 45º) so that the yeasts collect in the neck of the bottles. The settling
down of yeast is aided by gradual shaking and by increasing inclination of the bottles
so that they reach a vertical position after several days. This shaking-and-inclination
process is called riddling or remuage (Fig. 14.6). Riddling is done daily until all the
sediments come to the cork. The bottles are then cooled to -1.1 to -5.5°C.
Yeast
sediment
Cork
Hole for
inserting
bottles
Riddling rack
Bottle
Fig.14.6 Example of riddling in champagne making
The neck of the bottle (containing the yeast) is then frozen by placing it in brine or
other freezing solution. When the cork is removed the solid plug containing the
yeast is ejected. This is called disgorging. When skillfully done, only 1-3% of wine is
lost. The lost amount is replaced by adding sucrose solution (dosage). The sucrose
concentration depends on the end product desired. The bottle is again tightly closed
with a wooden cork.
14.7.2 THE TRANSFER PROCESS
In this process the fermentation is carried out in a manner similar to that for classical
champagne process. Riddling may not be done. The yeast is removed by transferring
the wine from the bottles to a tank under nitrogen pressure (closed system). Dosage
is added and filtration carried out in a closed system under CO2 or nitrogen counter
pressure. Bottling is done as in champagne process.
250
14.7.3 THE BULK FERM
MENTATION
N PROCESS
This methodd is suitable forf the mass production

p off sparkling wiines. The winne has
somewhat poorer
p qualityy. Secondary fermentationn is carried oout in pressuurized
vessel. A cerrtain amount of unfermented residual sugars is retainned in the winne so
that there is no need for addition of dosage.
d After filtration,
f the wine can be filled
into bottles. In this processs the CO2 geenerated durinng the secondaary fermentatiion is
also retainedd.
14.7.4 THE CARBONAT

TION PROCE
ESS
This methodd entails impreegnation of thhe base wine with CO2. Thhe quality is laargely
determined by b the qualityy of the base wine.
w In conttrast to seconndary fermentaation,
artificial carbbonation allow
ws only weakk binding of CO
C 2 in the wi
wine. Consequuently,
the gas escappes more quickkly when the wine
w is opened.
Fig. 14.7 Champagne

C Fllute Fig. 14.8 White Winne Glass Fiig. 14.9 Snifter
Sparkling winnes are often served in tall, flute-shaped glasses called champagne fflutes.
White wine glasses are typically
t smalller than red wine glasses.. Distilled liqquors,
particularly brandy or coognac, are traaditionally served in ballooon-shaped snnifter
glasses.
Table 14.4 Major

M wine-prooducing counttries of the woorld, 1996
Wine Wine Wine Total grrape, Area of

production, exports, consumptioon, '000 M MT vines, ''000
million L million L million L ha
France 5965 1229 3479 77011 9177
Italy 5877 1511.5 3562 94599 9222
Spain 3267 672.9 1475 48466 12244
USA 1864 163.8 2046 49355 3111
Argentina 1268 125.4 1355 20400 2111
S. Africa 1000 99.6 406 14400 1066
Portugal 953 200 580 12700 2599
Germany 830 300.8 1866 12977 1055
251
14.8 WINE SPOILAGES AND DEFECTS
As with other alcoholic fermentations, wine fermentation is also a robust process.

Because of the alcohol content, low pH, and low nutrient status, most spoilage
organisms fail to thrive in wine. The extrinsic factors such as pasteurization and
storage under cold condition further reduce the chances of spoilage in wine. Some
of the more important spoilages that may occur in wine are given in Table 14.5.
70
60
50
40
30
Romania
20 Hungary
Belgium 23.5
Denmark 29.0 25.0
Uruguay 29.3
Spain
10 Greece 34.8
34.0
Switzerland 34.9
Luxembourg 43.5
0 Italy 52.0
France 53.5
Portugal 60.0
61.0
Fig. 14.10 World Wine Consumption in 1997 (in liters per capita)
Table 14.5 Wine defects
Defects Organism involved Remarks

Acetification Acetobacter sp Formation of vinegar
Mousy Gluconobacter suboxydans Formation of gluconic acid from glucose
Tourne Lactic acid bacteria Production of lactic acid
Amertume Fructose and glycerol Fermentation of glycerol and fructose.
fermenters Fermentation of fructose to mannitol
gives bitter taste and this particular
fermentation is called mannitic
fermentation.
Malo-lactic Lactic acid bacteria Conversion of malic acid to lactic acid,
fermentation thereby reducing the titrable acidity
252
CHAPTER 15
BRANDY AND WHISKEY
15.1 BRANDY
Brandy is a distillation product of grape wine. Brandy can also be prepared from
other wine types. Some of the more important brandies are given in Table 15.1. The
name brandy comes from the Dutch word brandewijn, meaning "burnt wine."
Table 15.1 Some important brandies
Brandy type Country Fruit used

Cognac France Grapes
Armagnac France Grapes
Kirsch (pronounced: kee.əsh) France/ Switzerland/ Black morello cherry
Germany
Slivovitz Balkans Plum
Perry Britain and Sweden Pear
Applejack/ cider brandy America Apple juice
Framboise France Raspberry
Fraise France Strawberry
Mirabelle France Yellow plum
15.1.1 GENERAL METHOD OF BRANDY PRODUCTION
Generally, brandy is produced from white grapes. Brandy from red grapes is
somewhat inferior to that from white grapes and contains larger concentrations of
higher alcohols
It is desirable to avoid treatment of the must with SO2. Among other things, SO2
corrodes the copper distillation column. The fermentation temperature is kept below
24°C. Distillation can be done in batch or continuous mode. The distillate is aged in
oak barrels for several years. Some prefer to add caramel to adjust the color. The
color of brandy comes from the wood in which it is aged.
15.1.1.1 Cognac
Cognac (pronounced: con.yak) comes from the Cognac region in France, and is
double distilled using pot stills. Popular brands include Hine, Martell, Rémy Martin,
Hennessy, Ragnaud-Sabourin, Delamain, and Courvoisier.
SSelected grapees are pressed immediately and a only slighhtly for the prroduction of hhigh
qquality cognacc. Emphasis iss given on clean wine whichh has been stoored for as shhort
titime as possible, and which must not be oxidized. Tradditionally, disttillation is carrried
oout in direct-fireed copper pot stiills, 150-500 liiter capacity. See
S Fig. 15.1 ffor an idea abbout
tthe distillation pot.
TThe new winee (with lees, but not more than t 8% of addded lees) is pplaced in the still
aand brought too boil. Distillaation is carriedd out along with
w lees to proovide bouqueet to
tthe brandy. Distillation
D coontinues until the vapor contains
c negliggible amountt of
aalcohol. This takes
t 8 hrs or more and thee main distillatte (wash) conttains 24-32% abv.
A tail fraction may be separated (it contaiins fusel oils). A second disttillation is carrried
oout after pooliing the distillaates from threee other simillar batches off distillates. TThis
ddistillation lastts for 14 hrs. About 1-2% heads (contaiining aldehydees) are separaated.
TThe main disttillate averages 60-70% abvv. The new brrandy is placeed in limousin oak
ccasks. The oakk is well driedd before beingg made into caasks (see Fig 55.8 and 5.9). TThe
ccasks are washhed several tim mes with wateer and once with
w brandy. BBefore too m much
ttannin is extraacted the branndy is transferrred to used casks
c or tankss. It acquires bbest
qquality after 15-20
1 years inn the wood. The
T matured cognac is dililuted with waater
bbefore bottlingg so that the alcohol
a contennt is above 40%% by volume.
Fig. 15.1
1 Copper stills
s for batchh distillation off whiskey and brandy
115.1.1.2 Armaggnac
AArmagnac is made
m from grapes
g of the Armagnac reegion in Soutthwest of Fraance
((Gers, Landess, Lot-et-Garonne). Some of the popuular brands oof armagnac are
DDarroze, Baroon de Sigognacc, Larressinglee, Delord, Lauubade, Gélas, aand Janneau.
IIn contrast too cognac, armagnac is obtaained in singlee distillation ffrom wine whhich
ddoes not contaain lees. The brandy
b has an alcohol conteent of 52-53% % by volume. IIt is
pproduced geneerally by contiinuous distillattion.
mplex aroma and

BBrandy possessses a wide raange of flavoor componentts. It has com
fflavor, with at least 546 idenntified compoounds. The main componennts of brandyy are
ggiven in Table 15.2.
2254
Table 15.2 Components of brandy
Components Concentration
Ethanol >40%
Methanol Traces to 0.188%
Fusel oil 0.3%
Aldehydes 10-107 ppm
Esters 400-700 ppm
15.2 WHISKEY
Whiskey is also spelt as whisky in Scotland, England and Canada. According to US

definition, whiskey is a category of distilled alcoholic beverage obtained from a
fermented mash of grain.
15.2.1 KINDS OF WHISKEY
Whiskeys are broadly divided in to two categories, (i) straight, and (ii) blended. The
term straight is somewhat misleading.
Straight whiskey can be a mixture of whiskeys, so long as the same distiller produces
it during the same period, or it may be made from any mixture of grains, provided at
least 51% of the total is accounted for by the grain with which the finished product
is later identified. Thus, corn must make up at least 51% of the mash bill to be called
straight bourbon. The same is the case with rye, another important mash ingredient.
Straight scotch is a pure malt whiskey. This is sparingly produced since 1853.
Some of the principal whiskey types are:
 Scotch whiskey: malt is the basic raw material

 Irish whiskey: uses malt plus 5 other grains
 American whiskey: mainly rye whiskey, corn whiskey, and bourbon whiskey
 Canadian whiskey: uses a blend of cereals
 Japanese whiskey: uses blended grains, including some amount of rice.
Corn whiskey is distilled at less than 160° proof. It is made from mash containing
80% or more of corn. It is primarily aged in used, charred oak barrels. Bourbon
whiskey also contains corn as the main ingredient but the quantity is lesser than that
for corn whiskey.
15.2.2 GENERAL PRODUCTION METHOD
Corn and malt, and very often rye, are the principal ingredients of the mash bill. Malt
is an indispensable ingredient of whiskey. It serves as an enzyme source, substrate
for yeast, and provides characteristic flavor. Wheat and rice are also used in some
whiskeys.
255
SSee Fig. 15.2 for the outlinne of whiskey production. Distillation
D off whiskey cann be
ddone either in pot stills or continuous stillls, the formerr being classicaal.
AAfter the maltt and other inggredients havee been obtainned, the main production stteps
aare: (i) Mashiing, (ii) Fermeentation, (iii) Distillation,
D (iv) Filling andd maturation, and
(v
(v) Bottling
115.2.2.1 Mashinng
TThe malt is grround to grist in a mill and is then fed innto mash tunss (Fig. 15.2) aas in
bbrewing. Mash filtration produces
p worrt (that contaains assimilabble nutrients for
yyeasts) and speent grain called draff. The wort
w is taken foor fermentatioon while the ddraff
iss used as cattlle feed.
Fig.
F 15.2 The innside of mashh tun
115.2.2.2 Fermenntation
TThe finished wort

w is cooledd and fermented in vessels called washbaccks (which cann be
oof wood or steeel, capacity raanging from 6000
6 to 450000 liters; see Figg. 15.4). The ppH,
ttemperature, anda sugar cooncentration, all are imporrtant. The feermentable suugar
ccontent is maiintained betw ween 12 and 20% to give allcohol contennt of 5-8% in the
wwash (fermenteed mash). Thhe pH is mainttained by addiing food gradde acids like laactic
aacid. Alternatiively, a bacterrial souring caan also be emmployed. Fig. 115.7 gives a fflow
ddiagram of whiskey
w produuction using bacterial
b ferm
mentation for maintaining the
aacidity. Wheree bacterial ferm mentation is used,
u pasteurization is esse ntial to kill thhese
bbacteria before the next staage of yeast fermentation.
f Whatever be the process, the
wwashbacks aree never filledd to the top since the woort froths siggnificantly durring
ffermentation. The
T fermentaation usually laasts for 2-5 daays at 27°C.
TThe fermentation is done using improvved strains of Saccharomycees cerevisiae. Yeeast

mmanagement is an importannt part of the process
p and thhis is similar tto other alcohholic
ffermentations.. The yeast is propagated
p firrst in the labooratory. Thereaafter it is builtt up
inn a series of propagators
p called dona tubb and yeast tankk (Fig. 15.3). The propagattion
pproceeds asepttically under aerobic
a condittion. The finall fermenter (FFig. 15.4) receiives
2256
the culture at the rate ofo 2-3% of the t fermenterr volume. Yeeast tanks coontain
mount of yeastt to pitch severral fermenterss.
sufficient am
Dona
D tub Yeast tank
Fig.
F 15.3 Yeasst propagationn tanks
15.2.2.3 Distiillation
Distillation can
c be carriedd out by conntinuous- (in towerst similarr to those used in
ethanol distilllation) or battch method (copper stills). Continuous
C diistillation is caarried
out mostly foor bourbon prroduction in the
t US.
The copper pot

p stills in which the washh is distilled haave become thhe ultimate syymbol
of whiskey distilleries
d (Figg. 15.5 and 15.6). The stills are
a made from
m copper sincce it is
a material thhat is easy too work with, it does not rust, r and it iss an efficientt heat
conductor.
Fig. 15.4 Main

M fermentter
257
IIn general mallt whiskey is distilled
d twicee. The stills ussed for the firrst distillation are
ccalled wash stilllls. The resultinng low wines sppirit has an alccohol contentt of 20-26%. TThe
loow wines spirrit is distilled second
s time inn spirit stills. Thhe stillman (perrson in chargee of
ddistillation) shhould be very careful duringg the second distillation (thhe spirit run). TThe
mmore volatile heads called foreshots are collected sepparately so thhat they can be
rredistilled togeether with the next batch off low wines.
Swan neck Pre-heater
Head
C
Coiled pipe
C
Condenser
Boiler
Fire
B
Barrel
Fig. 15.5 Schematic of open-fired

o ketttle distillation
Copper pot still Alcoohol safe (Spirrit safe)

Fig. 15.6 Equipment requuired in whiskkey distillationn
TThe desired sppirit (the part of the spirit to

t be used forr making whiskey) is calledd the
mmiddle part or the heart of thee run and startts to come thhrough as thee alcohol conttent
rreaches about 75%. The stiillman has to regularly meaasure the alcoohol content w with
aan alcohol metter during the distillation. For
F this, the diistillate is led tthrough a secttion
ccalled spirit saffe (where the alcohol
a is conntinuously meeasured; Fig. 115.6) and thenn to
tthe collection tanks.
AAfter the alcoohol content in the distilllate decreasess to about 644%, the stillmman
pperforms whaat is called cutting on spirit. He now diveerts the spiritt into a separrate
2258
container. This fraction of distillate is called fients or tail and contains appreciable
amounts of higher alcohols.
Corn and other cereal grains
Coarse milling Milling Malt
Mashing Mashing
(66oC/30 min, pH 5.2-5.5) (46-49oC)
Filtration
Lactobacillus delbrueckii Wort

(thermophilic homofermenter)
Spent grain
Cooling (draff)
(63oC)
Culture yeast
Bacterial fermentation
(53oC/6-10hr, or pH~3.8-3.9)
Propagation
(dona)
Pasteurization
Propagation (82oC/30 min)
(plant dona)
Cooling
2-3% by volume (23-28oC)
Yeast fermentation
(27oC/2-5 days)
Straining
Distillation
Maturation
Bottling
WHISKEY
Fig. 15.7 Outline of whiskey production
15.2.2.4 Filling and maturation
All casks used to store whiskey are made from oak. Most distilleries use oak casks
(Fig. 15.8) that have contained sherry or bourbon. Whiskey receives its natural
amber color from interaction with the wood, although it has become increasingly
common to artificially add color by using the E150 additive.
The spirit is not legally considered to be whiskey until it has been stored in wood for
at least three years. Some of the whiskey evaporates through the wood during
storage (Fig. 15.9). About 1-2% of the whiskey evaporates each year in a natural
process which is called the angel’s share. Since the alcohol content must be at least
40% in order for whiskey to be called whiskey, this means that there is a theoretical
limit to how many years a whiskey can be stored before it has to be bottled. For
example, if a whiskey looses 1.5 % of its alcohol content each year it may only be
stored for 32 years before the alcohol content drops below 40%. Because of this it is
unusual for whiskey to be stored much longer than 30 years. Yet another reason for
259
tthe limited maaturation periiod is that whhiskey constanntly picks up tannin from the
wwood, and tooo much tanniin ruins the whiskey.
w The greater part oof all single m
malt
wwhiskey is storred between 8 and 12 years..
Figg. 15.8 Filling whiskey in baarrels
Fig. 15.9 Whiskeyy being aged inn casks
115.2.2.5 Bottling
ng
BBefore the whhiskey is bottlled it is usuallly filled into large

l tanks too be cut with de-
mmineralized water
w to 40, 433 or 46% alcoohol content. After the whhiskey is cut iit is
ccommon to chhill-filter it. Thhis is done too remove slighht impurities ffrom the whisskey
wwhich otherw wise would cause a cloudding effect at low tempeeratures. Not all
ddistilleries praactise chill-filttering since they
t believe that
t it removves some of the
ccharacter of thhe stored whisskey. See Fig. 15.10
1 for botttling line and bbottled whiskeey.
2260
Fig. 15.10 Whiskeey being filled in bottles
15.2.3 SCOT
TCH WHISKE
EY
Barley malt isi the key com

mponent of Scotch
S whiskey. Special metthods are useed for
malting. Dryying of barley isi accomplisheed in burning peat. This im mparts characteeristic
peatiness to the
t whiskey. TheT five prinnciple steps of o scotch whhiskey makingg are:
(i) Malting, (ii) Mashing, (iii) Fermentation, (iv) Distiillation, and (vv) Maturation.
15.2.3.1 Maltting
As usual, thiis entails steepping, germinaation, kilning, and milling. TThe sproutedd green
malt is curedd in a tower-shaped kiln thhat has a furnnace at the baase. Anthracitee and
peat are burrned in the fuurnace and the upward draaught of hot aair is assistedd by a
powered fann. Among othher things, killning on peatts provides a very characteeristic
peaty flavor to the malt. Peat
P is formedd from decom mposed vegetaable matter annd the
peat reek or smoke given off from com mbustion is immparted to thee malt. In the early
drying stagess whilst the grain
g is in a soft moist conndition the peeat reek permmeates
into the barlley. In the lattter drying staages the outeer skin of the grain will alsso be
flavored.
The malt is stored for a week

w or two and a then milleed in a four-rooller mill. An ideal
grist should have a very coarse appearrance (this assists in mashihing and filtraation).
Formation ofo flour shouldd be minimizeed because thiss impairs masshing and filtraation.
A typical com
mposition of the
t grist is: 144% coarse matterials and hussks, 78% fine grits,
and 8% flourr.
15.2.3.2 Mashhing
Mashing is done
d in mash tuns as in thee case of otheer whiskey typpes. The qualiity of
water is impportant here also.
a The proccess involves the applicatioon of three w waters.
The first water is introducced at aroundd 70°C to get a striking temmperature of 665°C.
After mixingg, the mash iss allowed to sit
s for 1 hr foor the enzym matic conversioon of
carbohydratees into simple forms. Thereeafter the worrt is drained, ccooled to 22°C
C and
261
pumped to washbacks (fermenting vessels). Second water is applied at 78°C so that
the mash attains a temperature of 70°C. Quantitatively, the second water is usually
about half that used for the first water. After mixing, the mash is left for 30 min. The
wort is processed as earlier. Third water is applied at 90°C in the form of sparge to
recover the residual sugars. The extract is again pooled to the washbacks while the
draff grain is fed to cattle.
15.2.3.3 Fermentation
Highly improved strains of Saccharomyces cerevisiae are used for the fermentation. The
pitching rate is about 1% by volume. This means that the yeast must be propagated
separately before the main fermentation. The pH and sugar concentration of the
wort must be carefully maintained at 3.8 and 16-20% respectively. A temperature of
22°C is optimum for the fermentation. The fermentation may proceed up to 50 hrs.
See Fig. 15.11 for the outline of scotch whiskey preparation.
GRIST
Milling
Mashing
(66-65oC/30 min in tuns provided with rakes)
Filtering Residue Mashing (3 times)
Liquor Liquor
Yeast culture
Cooling Spent grain (draff)
(25oC)
Fermenter (termed backs; made of steel, wood, etc)
Fermentation
(27oC)
Wash
(8-9.5% abv)
Primary distillation
(5-6h; non-selective; 3-fold concentration achieved; product
termed low wine)
Second distillation Recycle

* First fraction (foreshot)
Residue (pot ale) * Third fraction (fients)
* Second fraction (25-30o overproof)
Aging and bottling
SCOTCH WHISKEY
Fig. 15.11 Outline of Scotch whiskey production
262
15.2.3.4 Distillation
The fermented mash (wash) is pumped along with the yeast sediments and
distillation is carried out in copper stills (called wash stills) with tall swan-necks. The
stills are either direct-fired or electrically heated. The solids present in wash are
prevented from settling during distillation by a rotating arm called rummager. The
alcoholic vapors are condensed in a series of pipes in a cold water jacket to give a
distillate of about 21%. This distillate is termed low wines.
The wine then passes through the second still, the low wine still or spirit still. The same
process is repeated in the second distillation, but the stillman must be careful to
separate foreshots (early part of the run, which is pungent and impure) and the fients
(latter part of the run that contains higher alcohols). Only the middle part of the
distillate (at about 70-75% alcohol) is collected for whiskey making. The timing for
removing different fractions is based on the alcohol content, which is monitored
regularly with an alcohol meter. The foreshots and fients are once again mixed and
redistilled.
15.2.3.5 Maturation
The distillate thus produced must be matured in oak casks for at least three years to
be legally termed Scotch whiskey. For single Malt Whiskey, at least 8-10 years of
maturation is needed.
263
CHAPTER 16
VINEGAR PRODUCTION
16.1 INTRODUCTION
The word vinegar originated from the French word vyn egre whose literary meaning is
sour wine.
Vinegar is the product obtained by acetic acid fermentation of alcohol-containing

solutions. It must contain at least 4 g of acetic acid per 100 ml at 20°C and may not
contain more than 0.5 ml (v/v) ethanol. The pH is usually 2-3.5. It is customary to
express the strength of vinegar in terms of grains where 1% (m/v) is equal to 10
grains.
Technologically, vinegar is the product of a two-stage fermentation. In the first step

yeast converts sugar to ethanol anaerobically, while in the second step ethanol is
oxidized to acetic acid (ethanoic acid) aerobically by bacteria of the genera Acetobacter
and Gluconobacter.
16.1.1 TYPES OF VINEGAR
Vinegar may be classified on the basis of raw material from which it has been
prepared. In fact, anything that contains enough sugar or alcohol and is in no way
objectionable as food may be used to make vinegar. As a result, vinegar can be: cider
vinegar, whey vinegar, alegar (from ales), malt vinegar, grain vinegar, spirit vinegar, and so on.
By far the largest percentage of vinegar is the spirit vinegar. It is more commonly
called distilled vinegar and less usually, white vinegar or alcohol vinegar.
16.2 PRODUCTION OF DISTILLED VINEGAR
16.2.1 THE MASH
The raw material for vinegar production is called vinegar stock. The vinegar stock for
distilled vinegar can be (i) dilute, purified ethanol, or (ii) fusel oil containing pure
spirit.
It is customary in almost all countries to denature the ethanol that serves as a raw
material for vinegar industries. One of the common denaturant is ethyl acetate. This
is split into acetic acid and ethanol during fermentation. Denaturation can also be
done with distilled vinegar.
All mashes must contain ethanol, water, and some nutrients for the growth and
metabolism of acetic acid bacteria. The water used should be bacteriologically clean
and chlorine-free. Most natural raw materials (e.g., in the case of rice vinegar, malt
vinegar, etc.) do not need the addition of extra nutrients. The composition of the
vinegar stock (besides ethanol) for distilled vinegar is given in Table 16.1.
Table 16.1 Composition of vinegar stock (besides ethanol) for distilled vinegar
Material Amount (g/liter)

Glucose 0.5-1.0
CaCO3 0.1g/L (for demineralized water)
NaCl 0.1 (for demineralized water)
K, Mg, (NH4)2HPO4, SO4¯ 0.3 (total)
Commercial nutrient mix* 0.07-0.2
*Commercial nutrient mix refers to supplements such as malt extract, dried yeast, Acetopep®,
etc.
16.2.2 THE ORGANISM
Acetification, the oxidation of ethanol to acetic acid, is performed by members of

the genera Acetobacter and Gluconobacter. These are Gram-negative, catalase positive,
oxidase negative, strictly aerobic bacteria. Acetobacter species are better acid producers
and are common in commercial vinegar production. However, their ability to oxidize
acetic acid to CO2 + H2O (called overoxidation), a property that distinguishes them
from Gluconobacter, can cause problems in some circumstances. Fortunately,
overoxidation can be repressed by ethanol and this fact can be used for controlling
it. In practice, ethanol content of the stock is not allowed to fall to the level whereby
overoxidation is induced.
Most acetic acid bacteria associated with commercial acetification are difficult to
culture on conventional solid media. They are very unstable, subject to variation, and
die rapidly under cultural conditions other than specific for them.
To preserve and maintain them, they are always maintained continuously in

laboratory fermenters. Under correct condition, the organism can retain their
property indefinitely. This is possible even in large fermenters. Therefore, despite the
propensity of Acetobacter towards variation, it is quite possible to perform vinegar
fermentation year-in year-out without interruption.
16.2.2.1 Biochemistry of acetification
Acetic acid bacteria, depending on species and cultural conditions, can use several
pathways to utilize sugar for energy. The oxidation of ethanol to acetic acid,
however, is the relatively simpler pathway by which acetic acid bacteria derive their
energy. It occurs in two stages, mediated by alcohol dehydrogenase and aldehyde
dehydrogenase. Both the enzymes are associated with the cytoplasmic membrane and
265
have pyroquinoline quinone (PQQ) as a coenzyme. PQQ acts as a hydrogen acceptor,
which then reduces a cytochrome. The consequent electron transport establishes a
proton-motive force across the membrane which can be utilized to synthesize ATP.
See Fig. 16.1 for the biosynthetic pathway of acetic acid during vinegar production.
CH3CH2OH
PQQ
Alcohol dehydrogenase 2e
PQQH2
CH3CHO ATP Proton motive force Aerobic electron transport
H2O PQQ
Aldehyde dehydrogenase 2e
PQQH2
CH3COOH
Overall: CH3CH2OH + O2 CH3COOH + H2O
Fig. 16.1 Biosynthesis of acetic acid in Acetobacter
From stoichiometry of the equation (Fig. 16.1) it can be calculated that 1 liter of
ethanol should yield 1.036 kg of acetic acid and 0.313 kg of water. This leads to the
approximate relationship that 1% v/v ethanol will give 1% w/v of acetic acid, and this
relation is used to predict the eventual acidity of vinegar and to calculate
fermentation efficiency. It implies that, in absence of overoxidation, evaporative loss
and conversion to biomass, the sum of concentration of ethanol (%v/v) and the
concentration of acetic acid (%w/v), known as the total concentration or GK (from
German term, Gesammte konzentration), should remain constant throughout
acetification. The GK yield is the GK of final vinegar expressed as % of the GK at
the start of the fermentation.
16.2.3 FERMENTATION PROCESS
There are three main methods of vinegar production, viz., (i) The Orleans Process (open
vat process), (ii) The Quick Vinegar Process, and (iii) Submerged Fermentation (bubble
process). These are described in the subsections to follow.
16.2.3.1 The Orleans process
This is also called French method, slow process, etc. This is a surface culture
method. The process is no longer used and the equipment can be seen only in
museum of factories. The equipment consists of a barrel, about 200 liter capacity,
with several openings (see Fig. 16.2). The vinegar produced by this method is
supposed to be of the finest quality.
For starting, one-fourth to one-third of the barrel is filled with a good-grade active
vinegar, preferably from previous fermentation. This serves as an inoculum. 10-15
liters of wine may also be introduced along with the vinegar. Vinegar stock is then
introduced through the top of the barrel via a pipe whose one end rests at the
bottom of the barrel. The barrel is about half filled. Air passes freely though the
266
muslin-covered hole. The whole is left at 21-29°C for 14 days to a month, or until
the acetification reaction reaches the predetermined GK.
Muslin-covered
funnel
Air-hole covered
Air-hole covered with muslin cloth
with cotton wool
Viewing port
Level indicator
Tap
Fig.16.2 Vinegar production by Orleans process
A film of Acetobacter, called mother of vinegar, forms on the surface. Now, about one-
third of the vinegar is slowly drawn out through the tap (at the bottom) and
equivalent amount replaced with vinegar stock (from the funnel) without disturbing
the film. The cycle can be repeated several times.
16.2.3.2 The quick vinegar process
This method is also called German process, Fring’s generator process, Trickling process (solid-
state), etc. The process was discovered by Boer and later modified by Schutzenbach.
The quick vinegar process derives its name from the faster rate of acetification
achieved by increasing the area of active bacterial film and improving O2 transfer to
the acetifying stock. The acetic acid bacteria grow as a surface film on an inert
support material packed into a false-bottom vat. The size of the vat varies from 20
to 60 m3.
The acetifying stock is sprayed onto the packing material. The stock trickles down
against a current of air, which is either pumped through the bed or drawn up by the
heat of reaction within it. The packing material normally consists of some
lignocellulosic materials such as birch twigs, vine twigs, beech wood, etc. The vinegar
stock is collected in a sump at the bottom of the vat and recirculated until the
desired level of acidity has reached. The faster rate of reaction means that wash heats
up during passage through the bed and, depending on the size of the fermenter,
some cooling may be required. Fring’s generator is an improvement over the older
trickling process. Automatic systems are used here to control the temperature at 26-
28°C. The recirculated stock is sprayed back through a sparger onto the beech wood
shavings. The new stock usually contains 10.5% ethanol, 1% acetic acid, and 284
ppm special nutrient mix, e.g., acetopep®. The new stock gets mixed with vinegar
absorbed in the shavings to produce a microbiologically favorable condition (8%
acetic acid + 4% ethanol) for the production of acetic acid. The operation is semi-
continuous. Addition of stock is carried out in 2-3 steps over a period of several
days. The total time is between 4 and 10 days. The tank is largely emptied when the
residual ethanol reaches 0.3%. The yield is between 85-90% and 5 liter vinegar/m3
of shaving can be obtained per day. Fring’s generator can also be run in tandem. See
Fig. 16.3 for schematic diagram of Fring’s generator.
267
Fig. 16.3 Schematic
S diaagram of Fringg’s generator
D
Disadvantages off quick vinegar process
p over subm
merged process
 Uniform distributioon of stock is not possible. Due to ethannol concentrattion

gradieent, overoxidation is possiblle at certain seections of the bed
 Mashees high in nuttrient content and low in etthanol concenntration aggravvate
this problem
p by forming
f slimee deposits onn the carrier material therreby
pluggiing up the column
 Non-hhomogeneity of o the carrier column makees it impossiblee to distributee air
 Variattions in tempeerature cannott be corrected
 Low yield
y
A
Advantages of quuick vinegar proocess over submerrged process
PPerformance isi less affectedd because the acetification rate

r is slow, annd it rarely stoops.
TThis has one important coonsequence: bacteriophage-att
b tack (attack bby bacteria-eatting
vviruses) is less severe. The possible
p reasons could be thhe heterogeneeity of the cultture
ppresent, whichh allows organnisms with diffferent phage--susceptibilitiees to take oveer in
tthe event of phhage attack.
116.2.3.3 Submeerged process (Frrings acetator)
TThis method isi the most wiidely used proocess for the production
p off distilled vineegar.
NNot only is thee acetificationn rapid, the tottal concentration that can bbe handled is aalso
ssignificantly hiigher (≈ 15%)).
TThe acetator veessel (Fig. 16.4) can be of wood or stainless steel wiith sizes that can
hhandle 75-12000 liters ethanol in 24 hrs. The
T acetic aciid bacteria groow suspendedd in
2268
the medium which is oxygenated by sparging air. The process requires uninterrupted
and a very high rate of aeration. Aeration is carried out using a special device called
Frings aerator. The aerator is a hollow body turbine surrounded by a stator. As the
turbine rotates, air is sucked in and distributed through outlets. The equipment does
not need compressed air and is therefore called self-priming or self-respiratory. Baffles
are provided for uniform mixing.
The Frings aerator is specifically designed for submerged mode of vinegar

fermentation keeping in view the extreme sensitivity of the acetic acid bacteria to
lack of air. At a pH of 2.5 and 10-14% acidity, a stoppage of mere one minute is
sufficient to completely arrest the acetification: the effect is so extensive that the
acetification will not resume when aeration is resumed.
The Frings aerator has fully automatic systems. A mechanical defoamer is provided
at the top of the fermenter. Foams occasionally form due to dead cells. The
defoamer consists of a horizontal, rotating device that sucks in foam through a port
and throws it through a pipe leading to the tank-bottom.
The method is semi-continuous. The temperature is closely controlled at around

29°C. Before the alcohol level falls below 0.2% (and 13.3 g acetic acid/100 ml) about
40% of the fermenter content is pumped out. Without interrupting aeration, the
fermenter is refilled with new mash of total concentration 15% (1% acetic acid and
14% ethanol). The vinegar stock is added in such a way that it is rapidly mixed by the
aerator. Concentration gradient should be avoided, as this is detrimental to acetic
acid bacteria.
The method is advantageous in that it is rapid and high concentration of acetic acid
can be produced. A semi-continuous run takes 24-48 hrs.
Defoamer Exhaust
Foam recirculation pipe
Cooling water out
Alkograph
Air Cooling water in

Vinegar
Feed
Fring's aerator
Fig. 16.4 Schematic diagram of Fring’s acetator
269
16.2.4 DOWNSTREAM PROCESSING OF VINEGAR
Vinegar produced from natural raw material (e.g. cereals, fruit juice, etc.) is turbid
due to suspended solids. A rest period of several months is therefore recommended
for such vinegar. The insoluble materials will precipitate out over this storage period.
In the distilled vinegar, turbidity in the raw form is due to bacteria. The bacteria
settle down on standing the vinegar. An aqueous suspension of bentonite may be
added to the vinegar for hastening the clarification. This particular step is called
refining. The upper supernatant is now easy to filter and this is carried out with
suspensions of diatomaceous earth. Filtration must remove all suspended materials
such as vinegar bacteria or occasionally appearing vinegar eels. Simple plate-and-frame
filter is satisfactory but membrane ultrafiltration has also been used.
Vinegar is often pasteurized by short heating, e.g., 60-70°C for a few seconds before
bottling. In some countries, 50 ppm SO2 is permitted.
16.2.5 USES OF VINEGAR
 Vinegar is used in the home for the preparation of salads and vegetables.
 In food industry, it is used for the production of pickles, other vegetables,
fish, mustard, mayonnaise, and salad dressings.
 Occasionally, it is also used as antiseptic. The acid restricts the spoilage
microflora such as yeasts, molds, and lactobacilli. The mold Moniliella
acetoabutens is very resistant to acetic acid, though.
It is well established that, although addition of strong acids has a more pronounced
effect on pH pro rata, they are less inhibitory than weak lipophilic acids (e.g., acetic-
and lactic acid) at the same pH. This is because microbial inhibition by weak acids is
not solely due to the creation of a high extracellular proton concentration, but is also
directly related to the concentration of undissociated acid (Fig. 16.5).
Plasma Neutral pH Low pH

membrane
HA A+ + H+
ATP
ADP
HA A+ + H+
Fig. 16.5 Microbial inhibition by weak organic acids
Unlike protons and other charged particles, undissociated lipophilic acid molecules
can pass freely through the membrane; in doing so they pass from an external
environment of low pH (where the equilibrium favors the undissociated molecule)
to the high pH of the cytoplasm (around 7.5 in neutropliles). At this higher pH, the
equilibrium shifts in favor of dissociated molecule, so the acid ionizes producing
protons which will tend to acidify the cytoplasm and break down the pH component
270
of the proton motive force. The cell will try to maintain its internal pH by expulsion
of the protons leaking in but this will slow growth as it diverts energy from growth-
related functions. If the external pH is sufficiently low and the extracellular
concentration of acid high, the burden on the cell becomes so great, the cytoplasmic
pH drops to a level where growth is no longer possible and the cell eventually dies.
16.2.6 DIFFERENTIATION OF SYNTHETIC AND NATURAL VINEGAR
Artificial (synthetic) vinegars are made by diluting and coloring concentrated acetic
acid. They are also labeled non-brewed condiment. Biologically produced vinegar can
be distinguished from artificial vinegar by measuring the 14C content with a
scintillation counter. The basis is that fossil fuels have lower 14C content.
16.2.7 MICROBIAL SPOILAGE OF VINEGAR
Sometimes, undesirable strains of Acetobacter, e.g., Acetobacter acetii sub-species

xylinum, can cause sliminess. Other organisms such as film yeasts, molds, and algae
may sometimes cause problem by oxidizing acetic acid and/or producing foreign
flavors.
271
CHAPTER 17
MICROBIAL PRODUCTION OF CHEMOTHERAPEUTIC AGENTS
17.1 INTRODUCTION
Despite being diverse, microorganisms exist together. This association is termed

symbiosis. Basically, microbial associations are of three types: (i) positive, (ii) neutral, and
(iii) negative. The last association can be further subdivided into (a) competition,
(b) predation, (c) parasitism, and (d) antagonism. Antagonism is a classical example of
antibiosis. Antagonism in microorganisms refers to the phenomenon whereby one
microorganism adversely affects the environment of the other by killing, injuring, or
inhibiting growth.
Antibiotics are a special category of chemotherapeutic agents that are administered

to fight infections (usually microbial) of humans and animals. Although the classical
definition of antibiotics associates it with secondary metabolite, the term antibiotic
today includes many similar but chemically synthesized chemotherapeutic
compounds also. Over 8000 antibiotics are known and several hundred discovered
yearly. Nearly 3000 antibiotically active substances have been detected in lichens,
algae, higher animals, and plants. Each year about 300 new antibiotically active
substances are detected of which 30-35% are secondary components from
fermentation with known antibiotics. Of the large number of known antibiotics of
microbial origin, only 123 antibiotics are currently being produced by fermentation.
In addition, some 50 antibiotics are produced as semi-synthetic antibiotics. Three
antibiotics, viz., phosphonomycin, pyrrolnitrin and chloramphenicol are produced completely
synthetically. It generally takes 7-10 years of time and investment of 40-80 crores of
rupees for a new antibiotic to touch market from its first discovery in laboratory.
Bacteria as well as fungi elaborate antibiotics. Some of the common examples are
given in Table 17.1.
Not all antibiotics qualify as chemotherapeutic agent. Some of the fundamental

requirements of an antibiotic to be recognized as a chemotherapeutic agent are:
1. Wide spectrum: it must be active against a wide range of pathogens

2. Prevent the development of resistant forms: pathogens should not easily gain
resistance to the antibiotic in question
3. Selective nature: it must act only against the target and not the host organism
4. Not disturb the normal gut flora when orally administered
Table 17.1 Examples of organisms capable of producing antibiotics
Organism type Organism Antibiotic Property Use

Fungi Penicillium chrysogenum Penicillin aG+Pneumonia,
pharyngitis
Penicillium griseofulvum Griseofulvin Antifungal Skin and hair
lesions
Cephalosporium acremonium Cephalosporin G and G cUTI,
+ b –
pneumonia
Bacteria Bacillus polymyxa Polymyxin Antitumor UTI,
gastroenteritis
Streptococcus lactis Nisin G+ Non-medical
use
Actinomycetes Streptomyces griseus Streptomycin G+ and G– Tuberculosis,
UTI
Streptomyces aureofaciens Tetracycline G and G UTI, cholera
+ –
Streptomyces erythrus Erythromycin G+ Cholera

Streptomyces norsei Nystatin Antifungal Skin lesions
Micromospora pupurea Gentamicin UTI, abscess
aG+: active against Gram positive organisms; bG–: active against Gram negative organisms;
cUTI: active against urinary tract infection
17.1.2 CLASSIFICATION OF ANTIBIOTICS
Antibiotics can be classified in several ways. The important schemes for the
classification are given in Table 17.2. Table 17.3 gives the addresses of some
pharmaceutical companies that produce antibiotics.
Table17.2 Schemes for the classification of antibiotics
Scheme Examples of class Examples of antibiotics

Based on extent of Bacteriostatic Penicillins
effect Bactericidal Tetracycline
Fungicidal Griseofulvin
Fungistatic Nystatin
Based on chemical β-lactams Penicillin, Cephalosporin
nature Aminoglycoside Streptomycin
Tetracycline (Anthracycline) Tetracycline
Polypeptide Gramicidin
continued….
273
….. continued
Target Antiviral Interferon

Antitumor Nalidixic acid
Antibacterial Streptomycin
Antifungal Nystatin
Mode of action (the Inhibition of cell wall synthesis Penicillin, Cephalosporin
manner in which the Inhibition of protein and Tetracycline,
effect is manifested) nucleic acid synthesis Streptomycin,
Gentamicin,
Erythromycin,
Chloramphenicol
Inhibition of specific enzymes Sulphonamides
Damage to cytoplasmic Nystatin
membrane
Table 17.3 Pharmaceutical companies that produce antibiotics
Antibiotic Company Country

Penicillin G Hoechst AG FRG
Pfizer Inc USA
NOVO Industri A/S Denmark
Merck & Co USA
Sarabhai Chemicals Ltd India
Hindustan Antibiotics Ltd India
Penicillin V Hoechst AG FRG
Pfizer Inc USA
NOVO Industri A/S Denmark
Glaxo Laboratories UK
Semi-synthetic penicillins Pfizer Inc USA
Merck & Co USA
CIPAN Portugal
Bristol Myers Co USA
Streptomycin NOVO Industri A/S Denmark
Pfizer Inc USA
Glaxo Laboratories Ltd UK
Merck & Co USA
Sarabhai Chemicals Ltd India
Tetracyclines CIPAN Portugal
Pfizer Inc USA
Hindustan Antibiotics India
Hoechst AG USA
274
17.2 STREPTOMYCIN
Streptomycin is an aminoglycoside antibiotic produced by selected strains of Streptomyces

griseus. The antibiotic works by inhibiting the synthesis of DNA and proteins. It was
first isolated in 1944.
17.2.1 CHEMISTRY
It is basic in nature, with solubility in water at the rate of 20 g/liter. It is stable to pH

changes. It can withstand boiling temperature. Being a base, streptomycin is usually
produced as salt, normally of HCl and sulfate. One unit of streptomycin is equal to 1
μg of free base.
Streptomycin is composed of 3 subunits: (i) aminocyclitol (= streptidine, which is an

inositol derivative), (ii) L-streptose, and (iii) N-methyl-L-glucosamine. See Fig. 17.1 for the
general structure of streptomycin. Some of the common examples of this antibiotic
and the respective R groups are shown in Table 17.4. Streptidine is most accurately
characterized as a guanidinocyclitol (rather than aminocyclitol).
Dihydrostreptomycin is a semi-synthetic streptomycin. It is produced industrially by

catalytic reduction of streptomycin. It is more suitable than streptomycin.
NH2
C NH NH2
NH C NH
NH
L-Streptidine
O O
R1
L-Streptose
R2
R3 CH2 O Streptobiosamine
R5O O
CH2OH
R4 NH N-Methyl-L-glucosamine
Fig. 17.1 General structure of streptomycin
17.2.2 USES
The antibiotic is used in the treatment of tuberculosis, urinary tract infection, and systemic
infection by Gram positive bacteria, and against bacteria that have gained resistance to
penicillin. Non-medical uses include preparation of selective media, in cloning
experiments, and as laboratory standard for quantitative analysis of streptomycin.
275
Table 17.4 Some streptomycins and their R groups
R group
Antibiotic
R1 R2 R3 R4 R5
Streptomycin CHO OH H CH3 H
Hydroxystreptomycin CHO OH OH CH3 H
Dihydrostreptomycin CH2OH OH H CH3 H
Deoxydihydrostreptomycin CHO H H CH3 H
Mannosidostreptomycin CHO OH H CH3 M*
M*: Mannose moiety
17.2.3 LIMITATIONS
The antibiotic exerts a neurotoxic reaction upon prolonged use. It can lead to hearing
loss and loss of balance (that is, it is ototoxic). Streptomycin may sometimes damage
kidney also. Dihydrostreptomycin has lesser side effects than streptomycin.
The drug may lead to development of streptomycin-resistant forms of pathogen. It

is therefore advisable to use the drug along with p-aminosalicylic acid or isoniazid. The
resistant forms of microorganisms can develop through mutation giving rise to: (i) strains
with chromosomal resistance (immune to streptomycin), and (ii) strains with
enzymes capable of inactivating the antibiotic.
Medical use of streptomycin has diminished in the recent decades due to widespread
use of other aminoglycoside antibiotics (e.g., gentamicin, tobramycin) and is now
generally reserved for medical treatment (via intramuscular injection) in combination
with other antibiotics (e.g., penicillin).
17.2.4 MODE OF ACTION
All aminoglycosides affect protein synthesis. The target of this antibiotic is the 30S
subunit of the 70S ribosome of the prokaryotes. It strongly inhibits initiation and
elongation of peptide chains. It also causes misreading of mRNA thereby leading to
insertion of wrong amino acids (and therefore production of faulty polypeptides).
Finally, under the influence of streptomycin, some molecules of nucleic acids (e.g.,
rRNA, tRNA, and denatured DNA) also act as mRNA although they ordinarily do
not have this property. Accumulated evidence shows S12 (a small ribosomal protein
of 30S subunit) is the ultimate target. See Fig. 17.2 for the explanation.
The notations P and A in Fig. 17.2 refer to peptidyl-and aminoacyl site respectively. 30S
and 50S refer to physical characteristic of prokaryotic ribosome. The suffix S refers
to sedimentation coefficient expressed in Svedberg unit. Prokaryotic ribosome (70S)
can be broken down into 30S and 50S subunits. The 30S subunit contains 21 small
proteins designated s1, s2,…s21. Each of these proteins has very important functions
in protein synthesis and some of them are actually enzymes. The 50S subunit
contains 33 large proteins, designated L1, L2, L3….L33 (see page 57 also).
276
50S ribosome
New aminoacyl tRNA
30S ribosome fMet tRNA
Tetracycline
5' 3' 5' P A 3'
3' 3'
mRNA peptidyl site Streptomycin 5' P A 5' P A
aminoacyl site Initiation

Chloramphenicol
New aminoacyl tRNA
Erythromycin
Gentamicin
5' 3' 5' 3' 5' 3' 5' P A 3'
Transpeptidation Translocation Transpeptidation
Fig. 17.2 Inhibition of protein synthesis by antibiotics
17.2.5 BIOSYNTHESIS OF STREPTOMYCIN
Study in cell-free system shows involvement of several enzymes for the synthesis of
streptomycin. All the three subunits of streptomycin (Fig. 17.1) originate from glucose
but entail distinctly different pathways. According to available experimental evidence
the assembly of the streptomycin molecule can be envisaged to include the following
major steps:
1. Transfer of dihydrostreptose to streptidine-6-phosphate to form dihydro-

streptose streptidine-6-phosphate
2. Transfer of N-methyl-L-glucosamine to the pseudodisaccharide with the
formation of dihydrostreptomycin-6-phosphate
3. Oxidation of dihydrostreptomycin-6-phosphate to streptomycin-6-phosphate
4. Hydrolysis of streptomycin-6-phosphate to streptomycin
The final intermediate of the pathway, streptomycin phosphate, is biologically inactive

but becomes active following removal of the phosphate group. See Fig. 17.3 for the
outline of biosynthesis.
Many organisms synthesize mannosidostreptomycin (which has much lower antibiotic

activity and is therefore undesirable) before the actual formation of streptomycin.
This is not an obligatory intermediate, though. In the course of synthesis,
mannosidostreptomycin is degraded by the organism’s own enzyme
mannosidostreptomycinase to yield streptomycin. In a typical fermentation, the
concentration of mannosidostreptomycin can reach up to 40%.
Biosynthesis of streptomycin is regulated by an inducer called A-Factor (-

butyrolactone). In Streptomyces griseus, A-Factor is an autoregulatory factor that at
extremely low concentration triggers streptomycin biosynthesis and cell
differentiation by binding a repressor-type receptor protein. The antibiotic is
synthesized in the idiophase and this occurs only after A-Factor has reached a critical
concentration.
277
D-glucose
Nucleotide sugars
metabolism
2.7.1.1 2.7.1.2
Myo-inositol-1-P D-glucose-1-P
5.5.1.4 5.4.2.2
D-gucose-6-P 2.7.7.24
3.1.3.25
dTDP-glucose
Myo-inositol
4.2.1.46
1-amino-1-deoxy-
scyllo-inositol dTDP-4-oxo-6-
deoxy-D-glucose
2.7.1.65
5.1.3.13
1-amino-1-deoxy-
scyllo-inositol-4-P dTDP-4-oxo-L-
Polyketide sugar rhamnose StrF
2.1.4.2 unit biosynthesis StrG
1.1.1.133
1-guanidino-1-deoxy-
scyllo-inositol-4-P dTDP-L-rhamnose
dTDP-L-
dihydrostreptose
Streptidine-6-P
NDP-N-methyl-
2.4.2.27 L-glucosamine
O-1,4--L-
dihydrostreptosyl-
streptidine-6-P
Aminoglycoside
Streptomycin Streptomycin-6-P
3.1.3.39 Dihydrostreptomycin-6-P
2.7.1.72
Fig. 17.3 Biosynthesis of streptomycin
2.7.1.1 = hexokinase; 2.7.1.2 = glucokinase; 5.5.1.4 = inositol 3-P-synthase; 3.1.3.25 = inositol-

P-phosphatase; 2.7.1.65 = scyllo-inosamine kinase; 2.1.4.2 = inosamine-P-amidotransferase;
5.4.2.2 = phospho-glucomutase; 2.7.7.24 = dTDP-glucose synthase; 4.2.1.46 = dTDP-glucose-
oxo-reductase; 5.1.3.13 = dTDP-L-rhamnose synthetase; 1.1.1.133 = dTDP-4-
dehydrorhamnose reductase; 2.4.2.27 = dihydrostreptosyl transferase; 3.1.3.39 = streptomycine-
6-phosphatase; 2.7.1.72 = streptomycin kinase; StrF and StrG = streptomycin biosynthesis
proteins; dTDP = deoxythymidine diphosphate.
17.2.6 GENERAL PRODUCTION METHOD
17.2.6.1 Microbial strain
Improved strains of Streptomyces griseus are used for the industrial production of
streptomycin. From Walksman’s discovery up to the present day, the productivity of
Streptomyces griseus has increased by over 100 fold. Classical mutation programs are
used for the improvement of the strains.
278
17.2.6.2 Culture medium
Glucose is the carbon source of choice. This has to be so because glucose is the
precursor of streptomycin. The preferred nitrogen source is soybean flour meal.
Ever since Rake and Donovick’s experiment with soybean flour, no better nitrogen
source has been found. Minerals are automatically inclusive because of the complex
nature of the medium. Sodium chloride is always thought necessary, though. A
typical composition of the medium is given in Table 17.5. It has been shown that
streptomycin production is inhibited by phosphate concentration as low as 1.5 10-2
M. So, phosphate is not normally added in complex media containing glucose.
Table 17.5 Typical composition of production medium for streptomycin

Glucose 60 + (10)a
Soybean meal 30
Cornsteep (100%) solids 4
(NH4)2SO4 9 + (1.5)a
NaCl 2.5
KH2PO4 0.025
CaCO3 0.5
Soybean oil 7
Figure in parenthesis with a superscript (-)a denotes complementary addition during main
fermentation. This means that the above composition can also be used for inoculum build-
up.
17.2.6.3 Production
The inoculum is built up in a stepwise manner at 27°C. The process starts with the
plate-culturing of lyophilized spore cultures in soy flour agar medium. Incubation is
done at 27°C for 2-3 weeks. The spores are then transferred to a shaker flask. After
growth for some time the contents are again transferred to propagator for biomass
build-up. The medium is sterilized as usual. The fermenter is inoculated at the rate of
5-10% vol/vol. The process is aerobic. Inadequate supply of air (O2) leads to
accumulation of lactate and pyruvate, which is undesirable. The pH is maintained at
around 7 and the main fermentation is carried out at 27°C.
The fermentation is triphasic. Trophophase lasts for 24 hrs. The pH increases

because of preferential utilization of soybean meal. Growth and concomitant
accumulation of A-Factor (also written, Factor A) is also rapid. Idiophase lasts for 2-
7 days during which streptomycin is rapidly synthesized. Glucose utilization is very
rapid. The third phase marks the cessation of antibiotic synthesis. Cells begin to lyse,
and pH rises due to NH3 liberation. Harvesting is done before the third phase
commences. The yield is about 1200 μg/ml.
279
Filtered broth Water
Dilute broth
Adsorption
(in AMBERLITE ICR, in sodium form)
Removal of ions
(washing with EDTA of pH 8)
Elution
(with 2.5N H2SO4 until pH drops to 5)
Decolorizing
(with DARCO-G6)
Antigen removal
(by filtration through polyacrylamide gel,
cellulose acetate, or by dialysis)
Concentration
(by evaporation)
Streptomycin sulfate
(98% pure)
Fig. 17.4 Recovery of streptomycin
17.2.6.4 Recovery
The broth (beer) is filtered in rotary vacuum filter to remove mycelia (Fig. 17.10a and
17.10b). Water is added to the liquor in the ratio 1:1 and passed through adsorption
column. Through the same column, EDTA solution is passed to remove metal ions.
The adsorbed, pure streptomycin is eluted from the column with 2.5 N H2SO4. See
Fig. 17.4 for the outline of recovery process. Further processing entails decolorizing
with carbon, antigen removal by filtration, concentration, and drying. The final
product is either sulfate- or hydrochloride salt of streptomycin. The purity will be of
the order of 98%.
During fermentation, streptomycin can be found both in the culture fluid and bound
to the mycelia. The bound antibiotic is released from the cell walls by treatment with
acids, alkali, or ionizable salts or sonication.
17.3 PENICILLIN
Penicillins are a class of β-lactam antibiotics of related structure with slightly

different properties and activities. They are produced by Penicillium molds. All
penicillins have a common nucleus called 6-aminopenicillanic acid (6-APA). 6-APA
(Fig. 17.5) is a fused β-lactam-thiozolidine ring. The side chain attached to this ring
gives each penicillin its unique characteristic.
280
Side chain
S CH3
R CONH CH3 6-APA
N COOH
O
-lactam ring
Fig. 17.5 6-aminopenicillanic acid
17.3.1 THE ORGANISM
Although many Penicillium molds are capable of producing penicillin, not many of
them are of commercial value. The trade fermentation today employs mutated
strains of Penicillium chrysogenum Wisconsin Q 176. See Fig. 17.6 for the genealogy.
Penicillium chrysogenum 1951

Plating and selection
NRRL 1952 B25

X-ray
X 1612
UV rays
Q Wisconsin 176
Intermediate strains
Commercial strains
Fig. 17.6 Genealogy of commercial strains of Penicillium chrysogenum
17.3.2 CLASSIFICATION OF PENICILLINS
Penicillins can be classified on two main bases: (i) application point of view, and (ii)
production point of view. From application point of view, penicillins can be divided
into 4 categories:
1. Penicillin G type: These are used to combat streptococcal and staphylococcal

diseases
2. Ampicillin and its relatives: These are active against Gram-positive- and some
Gram-negative bacteria
3. Penicillinase-resistant types: These are used to combat bacteria that have
developed resistance to penicillin G
4. Extended-spectrum antibiotics: These are active against pseudomonads (a
group of proteolytic bacteria)
From production point of view, penicillins can be categorized as (i) natural

penicillins, (ii) biosynthetic penicillins, and (iii) semi-synthetic penicillins.
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1. Natural penicillins
Penicillins obtained by fermentation in the usual medium are called natural

penicillins. The side chain, R, comes from precursors present in the fermentation
medium. Some of the common natural penicillins are penicillin-G, V, X, F, K, and
dihydropenicillin. Penicillin G and V are by far the most important natural penicillins
(See Fig. 17.7). Since the fermentation medium is complex in composition, a wide
variety of precursors may be available to the organism. This can result in several
chemically different penicillins in a single fermentation.
Penicillin Structure Precursor

Penicillin G CH3
S CH2COOH
(benzyl penicillin) CH2 CONH CH3
N COOH Phenyl acetic acid

O
Penicillin V CH3
S OCH2COOH
(phenoxypenicillin) OCH2 CONH CH3
N COOH Phenoxy acetic acid

O
Fig. 17.7 Chemical structure penicillin G and V
2. Biosynthetic penicillins
Biosynthetic penicillins are in fact natural penicillins. The only difference is in the
manipulation of fermentation so that the desired type of natural penicillin is
obtained in large amounts. In essence, the production of biosynthetic penicillins
entails external addition of the precursors of side chain so that the fermentation
becomes more selective. For example, addition of phenoxyacetic acid during natural
fermentation produces penicillin V in large amounts.
3. Semisynthetic penicillin
Production of semisynthetic penicillin entails fermentation as well as enzymatic

and/or chemical steps (discussed earlier, page 133). The 6-APA is produced by
fermentation and the side chain is later added by chemical reaction.
17.3.3 USES OF PENICILLIN
Penicillin finds wide use in medical as well as other fields. Its therapeutic application
in general includes treatment of syphilis, gonorrhea, meningitis, anthrax, pneumonia,
and pharyngitis. The side effects are relatively rare but can include immediate or
delayed allergic reactions – specifically skin rash, fever, and less frequently,
anaphylactic shock. Anaphylactic shock refers to allergic reaction in which the release
of histamine can be widespread, leading to edema. In extreme cases, heart and
circulatory failure can occur.
Penicillins also find wide use in cloning experiments, especially for selecting clones
on the basis of reporter or marker genes. In general microbiology laboratory, it is used
for auxanography and for the preparation of selective media.
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17.3.3 PROPERTIES OF PENICILLIN
Penicillins are markedly sensitive to pH, temperature and penicillinase enzyme. The
sodium and potassium salts are freely soluble in water and alcohol but only slightly
soluble in benzene and chloroform. They are colorless in crystalline form.
17.3.5 BIOSYNTHESIS OF PENICILLIN
Penicillin is synthesized as a classical secondary metabolite from three amino acid

precursors, viz., L--aminoadipic acid, L-cysteine, and L-valine. L--aminoadipate is
an intermediate of the L-lysine biosynthetic pathway and is also provided by
catabolic conversion of L-lysine. However, the importance of the catabolic
degradation of lysine for penicillin has not been clarified yet.
Starting from these amino acids, penicillin biosynthesis is catalyzed by 3 enzymes,

namely: (i) -(L--aminoadipyl)-L-cysteinyl-D-valine synthase, denoted by ACVS,
(ii) isopenicillin N synthase, denoted by IPNS, and (iii) Acyl-SCoA: isopenicillin N
acyl transferase. The outline of penicillin synthesis (Brakhage, 1997) is shown in Fig.
17.8.
-ketoglutarate Acetyl-SCoA
Glutamate
-keto adipate
-amino adipate
L-lysine
L-cysteine
Metabolic block ACV synthetase
L-valine
ACV
(Aminoadipyl cysteinyl valine)
IPN synthase
IPN
(Isopenicillin N)
Acyl-SCoA:isopenicillin N acyl transferase
6-APA
(6-Aminopenicillanic acid)
Phenylacetyl-SCoA
CoA-SH
Benzyl penicillin
(Penicillin G)
Fig. 17.8 Penicillin biosynthesis by aminoadipate pathway
Thus, in Penicillium chrysogenum, L-lysine and penicillin share the same biosynthetic
route up to α-aminoadipic acid. The mutated strains can be supposed to be lysine
auxotrophs. Alpha aminoadipic acid route is one of the two routes used by
283
microorganisms for the synthesis of lysine. The other route, called diaminopimelic acid
pathway, is the main pathway used by commercial strains of lysine-producing bacteria.
In P. chrysogenum, L-lysine is known to be a potent inhibitor of penicillin synthesis.

Since L--aminoadipic acid is a branch point between the lysine and penicillin
biosynthetic pathway, lysine inhibition seems to operate at one or more steps of
lysine pathway.
17.3.6 MODE OF ACTION
Gram-positive bacteria, particularly Staphylococcus aureus, are sensitive to penicillin.

The antibiotic works by inhibiting biosynthesis of peptidoglycan of the bacterial cell
wall: it obstructs the cross-linkage between N-acetyl muramic acid and N-acetyl
glucosamine. The antibiotic also interferes with the interpeptide cross-links. The
bacterial growth is arrested as a result of weakened cell wall. See Fig. 17.9 for the
mode of action.
Site of interference
AGA AMA AGA AMA AGA
Peptide chain
AMA AGA AMA AGA AMA
Fig. 17.9 Mode of action of penicillin on cell wall of Gram-positive bacteria
The notations AMA and AGA refer to N-acetyl muramic acid and N-acetyl
glucosamine, respectively. In particular, the antibiotic inhibits the enzymes
transpeptidase, transglycosylase, and D-D-carboxypeptidase.
17.3.7 PRODUCTION OF NATURAL AND BIOSYNTHETIC PENICILLINS
17.3.7.1 Medium consideration
The medium formulation depends on whether the fermentation is carried out by

surface liquid culture method (classical) or the fed-batch culture method (modern).
In the classical method (which is still used in many places), lactose is the universally
preferred carbon source. Glucose is also included in the medium but this is primarily
for mycelial growth. Abundant growth is very essential for the main fermentation.
Glucose is a readily assimilable carbon source and so the organism does not use it
for the production of penicillin, which is a secondary metabolite. The production of
penicillin occurs in the idiophase. The biosynthesis is the result of depletion of one or
more nutrients. The mold produces penicillin only when it is forced to live on
lactose (which is not readily metabolizable). Stated differently, the organism should
experience a condition of starvation for the penicillin to be synthesized. A typical
fermentation medium for batch fermentation of penicillin is given in Table 17.6.
284
Cornsteep liquor (CSL) is universally used as nitrogen source. Nitrogen is often
supplied as NH3 because the cornsteep nitrogen may not be adequate. CSL also
contains growth factors, minerals, and above all, precursors of side chains. CSL is a
concentrated form of steepwater in which corn has been steeped for the
manufacture of starch, gluten, and other corn products. CSL contains 50% solids.
Although the composition is highly variable, the concentrations of total nitrogen,
lactic acid, amino nitrogen, reducing sugars and ash are around 7.4-7.8%, 12-27%,
2.6-3.3%, 1.5-14%, and 18-20%, respectively (on dry basis).
Table 17.6 A typical fermentation medium for penicillin production
Components Amount (%)

Cornsteep liquor 3.5
Lactose 3.5
Glucose 1
CaCO3 1
KH2PO4 0.4
Edible oil 0.25
Penicillin precursor --
For the production of biosynthetic penicillins, the desired precursors must be added
externally. The addition is continuous and in regulated amounts (0.5-1.5 g/liter).
Usually, the uptake of precursors is 80-90% because the attachment process does
not take place with absolute specificity. Addition of a good amount of precursor is
desirable for directing the synthetic reaction towards the desired direction but the
toxicity of excess precursor that may result on the microorganisms must also be
seriously considered. This calls for regulated feeding of precursors.
Following sterilization, the medium is transferred to the main fermenter, which may
be as big as 250 m3 in capacity. The initial pH is kept at 6.5. Inoculum is built up the
normal way and 10-20% vol/vol is added to the main fermenter. The pure culture
(which normally comes as lyophilized spores) is initially grown in special sporulation
agar. This is then transferred to shaker flask, which contains a supplement of 2%
sucrose. Incubation is done at 25°C for some days. The culture is then transferred to
propagator of considerable size. Aseptic- and aerobic fermentation ensues here also.
The main fermenter may be inoculated with either spores or mycelium. The
methods of inoculum build up, as also the method of inoculation, may vary here. A
spore concentration of about 5  105/ml of medium has been used in various
studies.
Classical fermentation
In the classical method, the main fermentation is triphasic in nature. During the
active growth phase, glucose and cornsteep components are rapidly utilized. The pH
remains constant. At the end of this phase, however, glucose and cornsteep
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components deplete. Ammonia is liberated due to deamination reaction.
Consequently, pH rises to 7-7.5. This pH is optimum for the synthesis of penicillin.
The antibiotic is very sensitive to pH changes. The latter can be controlled by adding
suitable amounts of H2SO4 or CaCO3. The late growth phase is followed by
idiophase, the phase in which penicillin synthesis occurs. Growth ceases in this
phase because the nutrients have depleted. The organism begins to utilize lactose.
This condition forces the mold to synthesize penicillin. Care must be taken not to
allow the late idiophase to proceed too far, as this leads to cell lyses and consequent
degradation of penicillin.
The duration of fermentation is typically 6 days. Since the organism is aerobic,

aeration should be done at the rate of 0.5-1 vol/vol/min. Turbine agitators are used
to facilitate aeration because the rheology of the medium becomes very complex as
the mycelial growth progresses. Precursors are batched or added constantly in
regulated amounts. The fermentation is normally carried out at 26°C.
During fermentation, periodic checks must be carried out for penicillin yield and
contamination. The penicillin yield (harvest titer) for a typical fermentation is about
40-50 g/liter.
Fed-batch fermentation
Today, trade fermentations are carried out in fed-batch mode, and lactose is replaced
by sucrose, glucose, or other crude sugars. The process involves 2-3 initial seed
growth phases followed by a fermentation production phase having a time cycle
ranging from 120 to 200 hrs. High dissolved O2 levels are crucial, especially during
the peak growth period that occurs at 40-50 hr time-period cycle.
About 65% of the carbon is metabolized for cellular maintenance, 20-25% for
growth, and 10-12% for penicillin production. Sugar and precursors are fed
continuously and the sugar is also used to help regulate the pH of the fermentation
between 6.4 and 6.8 during active penicillin fermentation phase.
Cornsteep liquor and cottonseed- or soybean meal, ammonia and ammonium sulfate
represent the major nitrogen sources. The essential precursors are phenylacetic acid
(for penicillin G) and phenoxyacetic acid (for penicillin V) that are either fed or
batched.
Mini-harvest protocols are often used in penicillin fermentations. This “batch-fill

and withdraw” system involves the removal of 20-40% of the fermenter contents
with replacement with fresh sterile medium. This procedure can be repeated several
times during the fermentation without yield reduction and, in reality, can enhance
the total penicillin yield per fermenter. Thus, although a single batch can be
complete in about 50 hrs, the extended culture protocol requires a time cycle of 120-
200 hrs.
286
17.3.7.3 Harvvesting
Harvesting iss done before the mycelia begin

b to lyse. The
T mycelia arre filtered in rrotary
vacuum filteer (Fig. 17.100a and 17.10bb). Washing isi done repeaatedly to elutte the
antibiotic. Fiilter aids suchh as hyflow maay be used duuring the filtraation. The brooth is
cooled prom mptly to 0-4°C C to minimizee enzymatic- and a chemical degradation oof the
antibiotic.
17.3.7.4 Puriffication
Penicillin cann be purified by

b two methodds, viz., (a) carbbon process, and (b) solvent extrraction.
The carbon process is an a obsolete process.
p Solveent extractionn is now usedd for
purification//concentrationn of penicilliin. This latter method is thus an inddustry
standard.
Sttationary automaatic
vaalve ring
V V V = Vacuum
P = Pressure
P
V
Level of material rotating
element
to be filtered P
V
V V Filter cake
Feed
Fig. 17.10a Rotary vaccuum filter of mycelial brothh
Fig. 17..10b Industriaal-scale rotary vacuum filter
287
The solvent extraction process
Penicillin is very sensitive to temperature and pH. At pH 2 and 20°C, the half-life is
only 15 min. It is therefore very important that extraction cycles be very fast. In
practice, each extraction cycle takes not more than 60-90 sec.
The temperature during the extraction is maintained at 0-3°C. The most common
solvents used for the extraction are amyl- and butyl acetate. Basically the process
entails extraction with organic solvent followed by back-extraction in alkaline
aqueous phase.
To begin with, the pH of the penicillin broth is adjusted to 2.5-3.5 with H3PO4 or
H2SO4 (dilute, 10% vol/vol). Emulsifiers may be added at the rate of 0.003-0.1% in
the organic solvent. At this pH, penicillin is preferentially soluble in the solvent.
The extraction takes place in continuous, countercurrent, multistage, centrifugal

extractor (Podbielniak D-36 is normally used). The ratio of broth to solvent is kept
at around 5-7:1. A single extraction will produce 5-7 fold concentration and the
concomitant 5-10% loss of penicillin.
This extraction in organic solvent is immediately followed by back-extraction in

water. Water is added to the penicillin-rich solvent in the ratio water:solvent = 0.1-
0.2. The pH of the mixture is adjusted to 5-7.5 by adding NaOH or KOH in the
water. At this pH, penicillin shifts its selectivity towards water. The resulting aqueous
extract is rich in Na- or K-salt of penicillin. At neutral pHs in water, penicillin is
ionized. In acid conditions this ionization is suppressed and the penicillin is more
soluble in solvents.
The extract is again dissolved in organic solvent by adjusting the pH to 2.5-3.5. This
time the amount of solvent is reduced. This is again followed by aqueous extraction.
The cycle is repeated until the required degree of concentration is achieved. The final
extract will necessarily be an aqueous extract of Na- or K-salts of penicillin. The
spent solvent is recovered for reuse.
The resulting aqueous solution is treated with 0.25-0.5% carbon to remove pigments
and other impurities. The solution is subjected to filtration to remove trace
impurities, bacteria, and pyrogens. Crystallization is done by salting out using sodium
acetate as the salt. The crystals are washed with water and dried in anhydrous
isopropanol, butanol, etc. The final drying is done in warm air, vacuum, radiant heat,
or large horizontal bed driers.
The penicillin thus produced must conform to the FDA standards. The strength and
dosage of penicillin is expressed in terms of international units. Each of these units is
equal to 0.0006 g of the crystalline fraction of penicillin G.
The yield from modern strains can be as high as 40-50 g/liter. The loss figure
(during handling, filtration, extraction, crystallization, drying) is about 22%.
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17.3.8 SEMI-SYNTHETIC PENICILLINS
Semi-synthetic penicillins are produced by a combination of microbial and enzymatic

or chemical process. 6-APA is the principal intermediary in the manufacture of semi-
synthetic penicillins. See immobilized enzyme (Section 10.19.2, page 133) for a full
treatment of the topic. Approximately 75% of the total bulk penicillin is used for the
production of semi-synthetic penicillins and cephalosporins.
17.4 TETRACYCLINE
Tetracyclines are a group of broad-spectrum antibiotics which are closely related

chemically and very similar in biological action. They all have hydronaphthacene
skeleton. They can be prepared microbiologically as well as chemically. Some well-
known members of this family are given in Table 17.7. The chemical structure is
given in Fig. 17.11. Tetracycline has low toxicity and good oral absorption. It is
bacteriostatic and requires high dosage. This antibiotic is used in the treatment of
shigellosis, salmonellosis, typhoid fever, brucellosis, etc. It is also used in feed to
eliminate parasites (and thus help gain weight in animals). The antibiotic also finds
use in the preservation of fish (the ice in which the fish is kept is treated with
tetracycline).
R1 R3 N(CH3)3
R2 OH R group
OH Antibiotic R1 R2 R3
Tetracycline (TC) H CH3 H
CONH2
OH Chlortetracycline (CTC) Cl CH3 H
OH O OH O Oxytetracycline (OTC) H CH3 OH
Fig. 17.11 General structure of tetracycline
Because of the development of strains of microorganisms resistant to the

tetracyclines, these antibiotics have lost some of their usefulness. They are no longer
the drugs of first choice for treatment of staphylococcal-, streptococcal-, or
pneumococcal infections. Nevertheless, they are still useful for the treatment of UTI,
trachoma (chronic eye infection), gonorrhea, and Rocky Mountain spotted fever.
Tetracyclines can be taken orally or applied topically. The recommended maximum
dose is 4 g/day.
Table 17.7 Some members of tetracycline
Antibiotic Organism
Tetracycline Streptomyces aureofaciens
Chlor(o)tetracycline (= Aureomycin, Biomycin) Streptomyces rimosus
Oxytetracycline (= Terramycin, Geomycin) Streptomyces antibioticus
289
17.4.1 BIOSYNTHESIS OF TETRACYCLINE
Biosynthesis of tetracyclines, particularly in the latter stages, has been studied with
the use of mutant cultures. It has been established that pretetronid (Fig. 17.12)
intermediates are involved in the synthesis. These intermediates are converted by
Streptomyces aureofaciens to tetracyclines.
Acetyl-SCoA
L-Asparagine Acetyl-SCoA
carboxylase
Asparagine-oxo-acid
transaminase Malonyl-SCoA
2-Oxosuccinate
CH3 Malonamoyl-SCoA
OH
6-Methylpretetramide
CONH2
OH OH OH OH
4-Hydroxy-6-methylpretetramide
6-Methylpretetramide
4-Dedimethylamine-4-oxo- 4-Demethyl-6-dehydro-
anhydrotetracycline anhydrotetracycline
Tetracycline chlorination- Anhydrotetracycline

responsible enzyme Anhydrotetracycline
monooxygenase
4-Dedimethylamine-4-oxo-
anhydro-7-chlortetracycline 12-Dehydrotetracycline
Chlortetracycline Oxytetracycline
Fig. 17.12 Biosynthesis of tetracycline
17.4.1 PRODUCTION OF TETRACYCLINE
Tetracycline can be produced chemically as well as microbiologically. The microbial

production of all tetracyclines is similar. Chlortetracycline production, however, is
comparatively simpler than the production of other tetracyclines. In particular,
production of tetracycline is very sensitive to chloride content in the medium: it
leads to the production of chlortetracycline rather than the tetracycline as shown in
the examples and figures elsewhere.
For trade fermentations, UV mutants of Streptomyces aureofaciens are used. The

organism comes in the form of lyophilized spores. The inoculum preparation
requires several stages. Starting from the spores dried on sand or lyophil vials, one or
more shaker flask stages may be used and then one or two inoculum tank stages.
The sporulation medium, inoculum build-up medium, and the production medium
have different compositions. A summary of the composition is given in Table 17.8.
290
Table 17.8 Medium used in tetracycline production
Purpose
Spore production Inoculum build-up Production
Meat extract: 2% CSL: 2% Sucrose: 1%
Asparagine: 0.05% Sucrose: 3% CSL: 1%
Glucose: 1% CaCO3: 0.5% (NH4)2PO4: 0.2%
Component KH2PO4: 0.05% KH2PO4: 0.2%
Agar: 1.3% CaCO3: 0.1%
MgSO4.7H2O: 0.25%
ZnSO4.7H2O: 0.005%
CuSO4.5H2O: 0.00033%
CSL = Cornsteep liquor MnCl2.4H2O: 0.00033%
During inoculum build-up, the organism remains in the shake-flask for 24 hrs at
28°C. The final propagator uses medium of the same composition. About 5%
inoculum is added and propagation carried out for 19-24 hrs at pH 5.2-6.2. The
main fermenter receives 2-10% of inoculum from the final propagator. The
fermenter has a nominal capacity of 25,000 to 75,000 liters. Fermentation is carried
out in sterilized medium (121°C for 1-2 hr). The main fermentation runs for 60-65
hrs at 28°C. The pH is around 5.8-6. It is a submerged fermentation requiring 0.5-2
vol/vol/min of aeration. Agitation is carried out with mechanical agitators. Lard is
used as an antifoam. Glucose is generally not used in the main fermentation as this
exerts catabolite repression. The yield is around 15000 units per ml.
17.4.1.2 Harvesting and purification
The mycelium in the beer is removed in a rotary vacuum filter (Fig. 17.10a and
17.10b). The liquor can be purified and concentrated in several ways, including
adsorption in activated charcoal, solvent extraction (amyl alcohol), etc. An example
of extraction is given in Fig. 17.13.
Alkyl sufonic acid Filtered liquor
Alkyl sufonate salt

Triethylamine (insoluble)
(bring pH to 8: the antibiotic
Filtration
dissolves at alkaline pH)
Insoluble impurities
Suspend in 2-ethoxy ethanol
Filtration
Charcoal treatment
HCl treatment (to bring pH to 0.8)

(the antibiotic is insoluble at acidic pH)
HCl salt of tetracycline
Fig. 17.13 Purification of chlortetracycline
291
17.4.2 MODE OF ACTION OF TETRACYCLINE
Tetracyclines inhibit a lot of enzyme reactions essential for the vital processes of
bacterial cells. The most sensitive biochemical reaction that is inhibited is the
synthesis of proteins. Tetracyclines work by binding specifically to the 30S ribosome
of the bacteria, preventing attachment of the aminoacyl tRNA to the RNA-ribosome
complex. It simultaneously inhibits other steps of protein biosynthesis. Tetracycline
can also alter the cytoplasmic membrane and this in turn causes leakage of
nucleotides and other compounds out of cell. This does not directly kill the bacteria
but instead inhibits them.
292
CHAPTER 18
MICROBIAL PRODUCTION OF FLAVORS AND FRAGRANCE
18.1 INTRODUCTION
Microorganisms are capable of producing a wide range of compounds that have

flavor and fragrance value. These compounds can be used in food items as well as
non-food items, such as perfumes. Not all of the potential of microorganisms in this
regard has been fully tapped. Even for those that have apparently reached
commercial success, details are not available. Some of the important flavor and
fragrance materials that can be obtained from microorganisms are diacetyl, lactones,
butyric acid, etc.
18.2 DIACETYL
Diacetyl (= 2,3-butanedione) is a naturally occurring chemical characterized by a

powerful and diffusive odor resembling butter when dilute. It is extensively used in
imitation butter and other dairy flavors and in other numerous flavors where butter
notes are desirable. Diacetyl also finds limited use in perfumes, primarily in
reconstituting essential oils. The chemical structure of diacetyl is shown in Fig. 18.1.
18.2.1 PRODUCTION
Diacetyl can be formed via two different methods. The first one involves chemical
synthesis. For example, diacetyl can be synthesized from methyl ethyl ketone by
conversion to an isonitroso compound which is then decomposed by hydrolysis with
hydrochloric acid to diacetyl.
A second method for producing diacetyl is by bacterial fermentation. For example,

glucose can be fermented to methylacetylcarbinol which is then oxidized to form
diacetyl.
In the microbial process, diacetyl can be produced from two groups of

microorganisms, viz., bacterial starters, and bakers- or brewers yeast. When brewers-
or bakers yeast is used, the organism is grown aerobically on sucrose. Bacterial
process is probably more widely practiced. Good candidates in this group are:
Streptococcus lactis, Streptococcus diacetylactis, and several Leuconostoc species. The easiest
method for natural diacetyl production is probably from starter culture (lactic
cultures from dairy industries). The outline given by Reed and Peppler is shown in
Fig. 18.2. Lactose or citrate can be used as the substrate in this process.
Citric acid is a precursor for both diacetyl and acetoin (see Fig. 18.1), and often, the
final fermentation product is a mixture of diacetyl and acetoin (= acetyl-methyl-
carbinol). In addition to the precursor citrate, the production of diacetyl is enhanced
by a pH below 5.5, low temperature, and aeration. A pH below 5.5 favors citric acid
permease activity and restricts diacetyl reductase activity. Aeration promotes both the
formation and accumulation of diacetyl by increasing oxidation-reduction potential
of the culture. This results in enzymatic stimulation and spontaneous oxidative
decarboxylation of α-acetolactic acid to diacetyl. Addition of humectants (e.g.,
glycerol and sucrose) to lower the water activity (aw) to about 0.96 has been found to
increase the amount of diacetyl (Toller, 1981).
Citric acid
Citratase
Acetic acid Oxaloacetate
CoA-SH Oxaloacetate
decarboxylase CO2
Acetyl-ScoA Pyruvate -acetolactate
CoA-SH TPP
Diacetyl Active acetaldehyde Diacetyl

Reducing environment
-acetolactate
Oxidative
decarboxylation
Acetoin
CO2
(3-hydroxybutanone)
Diacetyl O OH
O O CH3 C C CH3
CH3 C C CH3
Fig. 18.1 Microbial synthesis of diacetyl
Liquid skim milk or blends of

cheese whey (9-10% solids)
* Citric acid
* Yeast extract
* Phosphates
Pure culture Sterilize
(85oC/40 min)
Fermentation
* pH: 4.9
* Temp.: 22-25oC
* Aeration: adequate
* Duration: 16-18 hours
Distillation
Diacetyl
Fig. 18.2 Diacetyl production from starter culture
18.3 LACTONES
A lactone is a cyclic compound containing an ester group in the ring, and those
having 3, 4, 5, 6 and 7 members are referred to as -, -, -, -, and -lactones,
respectively. Lactones are useful as starting materials or solvents for the synthesis of
various compounds, such as pharmaceutical preparations, flavors and fragrance, and
294
agricultural chemicals, etc. Lactones are ubiquitous in food, contributing to taste and
flavor nuances. Numerous odor and taste characteristics have been attributed to
lactones. Among these are oily-peachy, creamy, fruity, nut-like, coconut, honey, and
so on. γ-octalactone and γ-nonalactone possess coconut aroma and are highly
desired by flavorists.
Many strategies are known for synthesis of lactones, including those using chemical
methods and fermentation. Because of relevance in industrial microbiology, only the
microbial process will be discussed here. The use of microbial process to produce
lactones would appear to have advantages over synthetic methods because the
former combines into a single step the multiple reactions required by a synthetic
method. Moreover, the microbial process would satisfy the desire to obtain flavor
and fragrance material from natural sources.
There are several methods (patented or in industrial operation) available for the
microbial production of lactones. An example for the production of -decalactone is
described next.
Boog and coworkers (1993) have described a method for the production of -
lactone from 11-hydroxy fatty acid or its ester by aerobically culturing Saccharomyces
cerevisiae. The conversion of 11-hydroxypalmitic acid into -decalactone is given
below.

 
OH OH
15 13 9 7 5 3 1 9 7
11  O O
16 14 12 10 8 6 4 2
O 10 8 6
11-Hydroxypalmitic acid -Decalactone
According to the description, the production of -decalactone from 11-

hydroxypalmitic acid or its ester is as follows:
The medium consists of 2% (w/w) soy peptone, 0.5% (w/w) yeast extract, and 1%
(w/w) 11-hydroxypalmictic acid. The medium is steam-sterilized at 121°C for 20
min. The hydroxy fatty acid used as the substrate may be conveniently added as the
only carbon source. To facilitate the dispersion of the substrate in the culture
medium, a suitable emulsifier may be added in an amount up to 1% (w/w) of culture
medium. The substrate may also include an ester of 11-hydroxypalmitic acid but this
will require introduction of lipase (for hydrolysis) during the fermentation.
The fermentation is carried out either in batch or fed-batch mode. In the latter case,
11-hydroxypalmitic acid is the component for fed-batching. Yeast cells are added at
the rate of 1000 cells per kg of medium. The pH of the medium is adjusted to 6.5
with 1 M NaOH. Aeration and agitation is carried out such that the oxygen level is
above 10% of saturation level. Fermentation is carried out at 28°C. Foaming of
fermentation broth may be prevented by the addition of conventional antifoaming
agents (e.g., silicone oils, diglycerides, etc.). If an ester of 11-hydroxypalmitic acid has
been used, a suitable lipase is added during the fermentation. The fermentation
295
duration is dependent on the metabolic characteristic of the organism. If the
organism is itself able to metabolize the -lactone, the fermentation must end before
this degradation occurs. The general duration of fermentation in such a case is about
10 days, but the amount of -lactone in the broth must be regularly checked to
ensure that the degradation phase is not initiated. Usually, pasteurization of the
broth is done to arrest the degradative reactions. On the other hand, if the organism
does not have the ability to metabolize -lactone, the fermentation duration is not
crucial.
The reaction mixture usually consists of -hydroxy alkanoic acid and the
corresponding -lactone. For lactonization, some additional conversion steps must
therefore follow the main fermentation. Lactonization can be done in the fermenter
itself by acidifying the broth to pH 3 with glacial acetic acid. The lactone is then
extracted with butyl acetate. Finally, after removal of the solvent, the residue is
distilled to obtain -decalactone. The yield is about 85%. For this particular protocol,
the amount of -decalactone formed is 1-1.5 g/kg of broth.
The lactone thus produced is suitable for adding to flavorings or foodstuffs (e.g.,
margarine, vegetable fats and oils).
18.4 BUTYRIC ACID
Butyric acid at low concentrations is used to supply butter-like note to flavors. It

finds particular application in natural cheese flavors. The esters of butyric acid may
also contribute to the flavors of various products. Pentyl butyrate provides a strong,
ethereal, fruity odor reminiscent of apricot, banana and pineapple; isobutyl butyrate
supplies an ethereal, fruity, somewhat pungent odor suggestive of pear, pineapple,
and banana.
Although natural butyric acid as an ester may be found at concentrations of 2-4% in

butter, its isolation is an expensive and difficult process. As a result, the fermentative
production of natural butyric acid is a valuable alternative.
Butyric acid is produced primarily by obligate anaerobes (bacteria of genera

Clostridium, Butyrivibrio, and Eubacterium) and Fusarium (mold). The clostridia,
particularly Clostridium acetylbutyricum (= acetobutylicum), have been studied in detail.
The latter’s ability to produce organic solvents such as acetone and butanol has led
to commercial processes which may be modified and adapted to produce butyric
acid. The mechanism for butyric acid synthesis in microorganisms has been
summarized by Gottschalk (1979) as in Fig. 18.3.
Besides proper selection of microorganisms, it is necessary to maintain the pH of the

medium above 5.0 in order to direct the fermentation away from solvent formation
and towards butyric acid formation. Calcium carbonate may be used to control the
pH above 5.0. CaCO3 plays another role as well, possibly that of serving as a point
of attachment for the microorganisms. Using a simple medium consisting of cerelose
(commercial dextrose), dried yeast, and CaCO3, yield of 1% butyric acid was
obtained.
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Glucose
2 Pyruvic acid
2 Acetyl-SCoA
Acetoacetyl-SCoA
-hydroxybutyryl-SCoA
Butyryl-SCoA
Crotonyl-SCoA
O
Butyric acid CH3CH2CH2C OH
Fig. 18.3 Biosynthesis of butyric acid
Additional work by Sharpell and his co-workers showed that increased yield of
butyric acid could be possible with further modifications of the medium and
operation conditions. The toxic nature of butyric acid towards Clostridium species,
however, appears to be a limiting factor.
One of the derivatives of butyric acid that have found use as flavoring compounds is
-keto butyrate (= 2-oxo-butyrate). Its production using a mutant strain of
Neurospora crassa (a mold) has been described by Zurbriggen and coworkers (2004).
The mutant strain is so chosen that it is capable of accumulating -keto butyrate in
the medium and exhibits very low or no acetolactate synthetase activity.
In the preferred embodiment, the fermentation is carried out in two stages, the first
one for the biomass build up and the second one for the production of -keto
butyrate.
First of all, abundant spores of Neurospora crassa are produced on an agar plate. The
spores are suspended in sterile water and used as a starter culture to inoculate a
synthetic medium (e.g., Vogel-saccharose medium; composition not given here) for
the production of abundant vegetative cells (not spores) and relevant enzymes. The
fermentation (the first stage) takes place at a pH of 4.5-6 for 2-5 days at 20-30°C.
The biomass is collected by filtration and the second stage of fermentation carried
out in a different medium. For the second stage fermentation, the cells are
suspended in phosphate buffer (0.1 M) of pH 9-11. The buffer is supplemented with
threonine (0.2-4%, as a substrate) and valine (0.01-0.5%, as an enzyme trigger) to
convert threonine into -keto butyrate. The fermentation is carried out at a
temperature of 23-30°C and it proceeds for 18-48 hrs. The bioconversion of
threonine to -keto butyrate is up to 90%.
The medium is filtered to remove the fungal biomass. The broth is then subjected to
suitable ion-exchange resin for purification. When -keto butyrate is to be used for
the synthesis of other flavor compounds, such as emoxyfurone, the filtrate may be
used directly (without further purification).
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CHAPTER 19
MICROBIAL PRODUCTION OF POLYSACCHARIDES
19.1 INTRODUCTION
Depending on function, microbial polysaccharides are of 3 main types:
1. Intracellular: represented by reserve polysaccharides

2. Structural: represented by cell wall polysaccharides
3. Exopolysaccharides (EPS): represented by capsule- and slime polysaccharides
19.1.1 EXOPOLYSACCHARIDES (EPS)
Because of relevance, only EPS will be described here. EPS are basically water-
soluble gums of microorganisms. EPS in microorganisms occur in the form of
capsules or slimes. Commercially important EPS are those that readily diffuse in
fermentation broth and can be subsequently recovered. EPS have great commercial
value because of their novel and unique physicochemical properties. They have
found a wide range of applications in the food, pharmaceutical, and other industries.
Some of the more important EPS are xanthan gum, alginates, and dextran.
19.2 GENERAL PROCESS FOR MICROBIAL EPS PRODUCTION
Processes for EPS production are characterized by extreme rheological changes of

the fermentation broth, low product concentration, and the diversity of subtle
structural changes which occur throughout fermentation and the marked effect these
changes can have on the product’s end application performance. The success of EPS
production therefore depends on controls at two levels: (i) environmental, and (ii)
equipment design and operation. It is difficult to generalize EPS production process
but the more general aspects of a typical EPS production process can be outlined as:
 Fermentation medium: defined glucose salts or sucrose, inorganic nitrogen

supplement, and minerals
 Duration of fermentation: 40 hrs
 Temperature: 30°C
 Process: batch
 Yield: 0.7 kg dried product/kg of glucose
 Moisture: 5%
 Product loss: 5%
19.2.1 GENERAL RECOVERY PROCESS
As with many microbial metabolites, the recovery process entails:
1. Concentration
2. Purification
3. Deactivation of contaminating enzymes (cellulase, pectinase, etc.)
4. Modification (to alter functional properties such as handling and dispersion
characteristics)
The steps involved, in general, are:
1. Cell removal (dilution followed by centrifugation/ filtration)

2. Isolation (precipitation, liquid-liquid partitioning, etc.)
3. Dewatering (drying in forced air or vacuum dryers)
4. Milling (to regulate size for dispersibility, flowability, etc.)
5. Additional processing (physical- and chemical treatment to affect purity,
cosmetic appearance, moisture pick-up and clumping, handling characteristics,
etc., depending on the intended end application)
6. Packaging
19.3 GENERAL USES OF EPS
EPS are generally used as emulsifiers, stabilizers, binders, gelling agents, coagulants,
lubricants, film formers, thickening agents, molecular sieves, etc. In the following
sections, some EPS and their uses will be discussed.
19.4 XANTHAN GUM
Xanthan gum is an anionic, branched heteropolysaccharide. Its molecular weight is

greater than 106. This macromolecule has a cellulose backbone linked to trisaccharide
residue, viz., β-D-glucose, α-D-mannose, β-D-glucuronate and variable amounts of
acetate and pyruvate (see Fig. 19.1).
The gum has very interesting properties in aqueous solution. Its viscosity is independent
of temperature over the range 10-70°C and is fairly constant over the pH range of 6-9.
Xanthan gum is commercially produced from mutant strains of Xanthomonas

campestris. Xanthan gum is the only microbial EPS which constitutes an important
component of total world polysaccharide market.
19.4.1 PRODUCTION OF XANTHAN GUM
The medium for the production of xanthan gum from Xanthomonas campestris consists
of dextrose, sucrose and cruder forms of carbohydrates, the choice being dependent
on the type of end product desired. Nitrogen, phosphate and magnesium are also
supplied.
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O O O O Cellulose backbone
O O O O O (-glucose units)
n
O
CH2OCOCH3
O
Acetate of - - D-mannose
COO- M+
COO- M+ OCH2 O
O
O Salt of glucuronic acid ()
O
CH3 O
Complex of mannose () M+ refers to Na+, K+, 1/2 Ca++, etc.
Fig.19.1 Molecular structure of xanthan gum
The inoculum is built up from lyophilized culture. Series of serial transfers and tests
are needed before the culture is transferred to shaker flask. The contents of the
shaker flask are in turn transferred to a seed tank for the final inoculum. The growth
is highly aerobic. The seed tank has provisions for aeration. The main fermentation
medium contains glucose, distiller’s solubles, KH2PO4, and trace elements. Because
of the high viscosity attained by the culture fluids, only about 3-5% concentration of
glucose can be utilized efficiently. Fermentation is carried out at 28-32°C. The pH is
controlled at 6-7.5 using suitable neutralizing chemicals. The fermentation is
controlled by frequent checks for contamination and polysaccharide production
(colorimetric). The duration of fermentation is about 2 days. At the end of the
fermentation, the culture fluids attain a viscosity of as high as 7000 cps.
After the fermentation has completed, the broth is heated to kill the cells. Water is
added to dilute the broth. The purification/concentration of xanthan gum is carried
out by precipitation with alcohol. Dewatering, drying and milling can be done by any
of the conventional methods. See Fig. 19.2 for the flow diagram.
19.4.2 USES OF XANTHAN GUM
Xanthan gum finds both food- and non-food uses:
1. Food applications
 Dressings (high oil, low oil, no oil), relishes and sauces, syrups and
toppings, starch-based products (canned desserts, sauces, fillings,
retort pouches), dry mix products (desserts, gravies, beverages,
sauces, dressings), farinaceous foods (cakes), dairy products (ice
cream, cheese), and confectionary. Deacylated polysaccharide has
an excellent film-forming property. The derivative is prepared by
controlled treatment with alkali
2. Non-food applications
 Flowable pesticides, liquid feed supplements, cleaners, abrasives,
polishes, metal workings, ceramics, foundry coatings, texturized
coatings, slurry explosives, dye- and pigment suspensions
 Oil field application
300
 Drilling fluids, polymer flooding, work-over and completion fluid
Medium
Lyophilized culture
Fermentor
* pH: 6-7.5 Slant
* Temp.: 28-32oC Shaker flask
* Sterile air
* Contamination check Agitated seed fermentor
* Production check
Recovery
* Heat treatment to kill cells
Precipitation with alcohol Alcohol
Washing Spent alcohol Distillation
Dewatering/ Drying/ Milling
Fig. 19.2 Outline of microbial production of xanthan gum
19.5 DEXTRAN
Dextrans are branched, neutral homopolysaccharides composed exclusively of α-D-

glucose residues. 95% of these residues are linked through carbon 1 and 6. About
5% are linked by 1-3 bonded side chain containing about 3 units. The molecular
weight is between 30106 and 39106. See Fig. 19.3 for the structure of dextran.
CH2 CH2 CH2 CH2 CH2

O O O O O O O O O O O
CH2OH n
O
O
CH2OH
O
CH2OH
O
O
O
Fig. 19.3 Molecular structure of dextran gum
19.5.1 PRODUCTION OF DEXTRAN
Dextran is produced by two methods, viz., (i) whole culture method, and (ii) enzymatic
method. Two species of bacteria are used for the fermentation: they are Leuconostoc
mesenteroides and Leuconostoc dextranicum.
19.5.1.1 The Whole Culture Method
The main substrate is sucrose. A typical composition of the fermentation medium is:
10% sucrose, 0.5% KH2PO4, 0.25% yeast extract, 0.1% NaCl, 0.06% (NH4)2SO4,
and 0.02% MgSO4.7H2O.
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The medium is taken to a slurry tank (1,500 gallon). The medium is heated (with
agitation) to 60°C, and finally pasteurized in plate heat exchanger at 142°C.
Thereafter the medium is cooled instantly to 25°C. This medium is used for the
main fermentation as well as the seed vessels. The inoculum is built up in several
stages at 25±0.5°C with an aeration of 0.5 vol/vol/min. The final inoculum tank,
called bazooka has 10 gallon capacity. The seed tank has 206 gallon capacity. The
main fermenter has 1,300 gallon capacity. Fermentation is done at 25°C. The initial
pH is kept at 7 but is allowed to fall to 4.5 during fermentation. This final pH is also
an indicator to the completion of fermentation. No sucrose will be left in the
medium at this stage. Fermentation is usually carried out until the viscosity of the
culture fluid increases to 400-700 cp.
19.5.1.2 The Enzymatic Method
The enzyme responsible for the production of dextran is called dextran sucrase. It is
elaborated by strains of Leuconostoc mesenteroides. The enzyme helps polymerize the
glucose fraction of the sucrose molecule that has been supplied as the substrate.
The production is carried out in two stages: (i) enzyme production by growing the
microorganism, and (ii) enzymatic conversion with the enzyme thus formed. The
initial fermentation is for enzyme production and therefore requires, in addition to
sucrose, a balanced medium that contains nutrients adequate enough to support the
growth of the enzyme producer. Typically, the growth medium contains 2% CSL,
2% sucrose, 0.1% KH2PO4, and trace amounts of inorganic salts. Fermentation is
carried out at 24°C. The pH is kept at 6.7 and the fermentation carried out for 6 hrs.
The enzyme produced in the broth is recovered by centrifugation and the crude
extract used for the second stage fermentation (enzymatic reaction). See Fig. 19.4 for
the outline of dextran production.
Leuconostoc Medium * 2% CSL

mesenteroides * 2% sucrose
* 0.1% KH2PO4
* Inorganic salts: traces
Fermentation
* Temp.: 24oC
* Duration: 6 hours
* pH: 6.7
Filtration * Sucrose
* Primers
Broth with enzyme
Reaction vessel
* Temp.: 30oC
* Duration: 8 hours
* pH: 5.1
Recovery
Fig. 19.4 Production of dextran by enzymatic method
The second stage reaction is carried out at 30°C, at a pH of 5.1, using 5-10% sucrose
solution as the substrate. Manipulation can be done here to regulate the molecular
302
weight of dextran. Increasing the concentration of sucrose will produce low-
molecular weight dextran. Primers are universally used to initiate the reaction.
Primers, as mentioned here, refer to hydrolyzed dextran at a concentration of about
2%. Primers come mainly from methanol taken away for recovery (see later). The
reaction proceeds for 8 hrs. Stirring can be done to bring about uniformity in
reaction. Dextran sucrase uses only the glucose portion of sucrose for
polymerization. Thus, approximately 50% of the original weight of sucrose solution
remains as D-fructose at the end of the reaction.
19.5.2 RECOVERY OF DEXTRAN
Dextran broth is first of all precipitated in tanks by adding an equal volume of

methanol. The supernatant contains low molecular weight dextrans and this is taken
away for methanol recovery while the precipitate is dissolved in double-distilled,
pyrogen-free water at 60-70°C. It is then hydrolyzed with HCl at 100-105°C to
produce clinical dextran (average mol wt 75,000 ± 25,000). Vacuum is applied at this
stage to remove residual methanol. The liquor is then mixed with diatomaceous
earth and polish-filtered in a plate-and-frame filter. The filtrate undergoes several
purification stages to obtain clinical dextran. That is, the liquor is again precipitated
with methanol. This time, methanol is added in calculated amount so that the clinical
dextran is selectively precipitated while the low molecular weight dextrans remain in
the solution. The temperature, pH, etc., must be closely controlled. The precipitate is
again purified, concentrated, and spray-dried.
19.5.3 USES OF DEXTRAN
Some of the varied uses of dextran are listed as follows:
 Oil well drilling: It was used as oil drilling fluid additive until 1950’s. It is
now considered uneconomical
 Blood plasma extender: Sterile, pyrogens-free, approx. 6% solution of
dextran having molecular weight in the range 50,000-100,000 (clinical
dextran) can be used in emergency to restore blood volume in cases of
shock due to blood loss
 Iron-dextran complex, which can be used as a source of nutritional iron
 Molecular sieves: Molecular sieves are prepared by cross-linking dextran
with epoxy compounds and NaOH. The degree of cross-linking determines
the pore size and water regain value of the molecular sieves (and thus their
molecular exclusion characteristics)
19.6 ALGINIC ACID
Although most of the commercial alginate produced today is derived primarily from
the sea kelp Macrocystis pyrifera, microbiologically derived alginates are under
development and their future is very promising. The wide variety of products
obtained from Azotobacter vinelandii have been found to possess physical properties
similar to the alginates derived from marine algae. The biopolymers derived from
this organism have a wide range of molecular weights and it has been postulated that
303
the extracellular enzyme alginate lyase may play an important role in the molecular
weight of alginates.
19.6.1 CHEMISTRY OF ALGINATE
Alginate is a general term used to describe the salts of alginic acid, the most notable
of which is sodium alginate. Alginic acid is a weak organic acid, which readily forms
salt with different bases. The acid is a linear polysaccharide composed of varying
proportions of β(14) linked D-mannuronic acid and α(14) linked L-guluronic
acid residues in blocks and alternating sequences in their linear chain. The presence
of L-guluronic acid in alginic acid is important because increasing its content
improves gelling characteristics of alginate in the presence of calcium ions. See Fig.
19.5 for the chemical structure.
-D-Mannuronic acid block -L-Guluronic acid block

COOH 6 COOH 6 COOH COOH COOH
5 5
O O O O O
4 1 1 O 4 1 4 1 O 4 1 O 4 1 O 4 1
O O
3 2 3 2
COOH COOH
-1,4-linkage -1,4-linkage
Fig. 19.5 Partial structure of alginate (note the orientation of bonds at C1 and C4)
19.6.2 USES OF ALGINIC ACID
Sodium alginate is widely used in research as a gelling agent to immobilize a wide

variety of cells, such as microbial cells, and plant and mammalian cells. Alginates
have ion exchange properties similar to ion exchange resins. The relative affinity of
divalent metal ions depends on the relative amounts of D-mannuronic and L-
guluronic acid units that are present in the macromolecule. The gum also finds use
as film former, emulsifier, restructuring agent (in fruit gels), and stabilizer (in ice
cream).
19.6.3 FERMENTATION
Some of the bacteria capable of producing alginate are Azotobacter vinelandii,

Pseudomonas aeruginosa, etc. Pseudomonas aeruginosa is not preferred because it has
association with pathogenic infection in humans.
The fermentation is carried out in continuous fermenters. Since the bacterium is

fastidious the medium is probably very complex. Oxygen is closely monitored. Too
high levels of aeration increase respiration, leading to conversion of substrate to
CO2. Too low levels of aeration, on the other hand, lead to accumulation of poly-β-
hydroxybutyrate (reserve lipid).
There is not much detail about the recovery of bacterial alginate. The recovery
method of algal alginate is probably used here also. The method, in essence, entails
precipitation with CaCl2, regeneration with acid-water, and neutralization with
304
sodium carbonate to obtain alginic acid in the form of sodium salt. See Fig. 19.6 for
an outline of the fermentation and recovery.
Complex medium
Lab culture
Pretreatment
Inoculum build-up Cell removal Cells
Temp.: 30oC CaCl2 Precipitator and

separator
Air Waste
Acid and water Contactor and
separator
Na2CO3 Contactor and

separator
Holding tank
Sodium alginate
Drying, milling, and packaging
Fig. 19.6 Production and purification of alginate
19.6.4 MICROBIAL BIOSYNTHESIS OF EPS
Very little is known about the biochemical pathways involved in the biosynthesis of
different EPSs. The majority of the EPSs are assumed to be synthesized within the
cell in a mechanism analogous to that involved in cell wall synthesis. Very few EPSs
have been reported to be synthesized outside the cell. A simple sequence for the
synthesis of a homopolysaccharides of glucose units can be shown as in Fig. 19.7.
Mutase ADP
Glucose-6-P Glucose-1-P
UTP ATP
PPi
UDP-glucose UDP
Synthetase
[Glucose]n [Glucose]n+1
Fig. 19.7 Biosynthesis of glucose polymer
Branches in the linear chain occur under the influence of branching enzymes.
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CHAPTER 20
MICROBIAL PRODUCTION OF FATS
20.1 INTRODUCTION
Microorganisms have long been known to produce lipids and therefore to be

potentially useful for the production of oils and fats. Such organisms may be termed
oleaginous in keeping with the terminology used for oil-bearing plant seeds. For the
most part, oils produced by oleaginous strains of eukaryotic microorganisms
approximate to that of conventional oilseeds (with respect to physicochemical
properties).
A definition of what constitutes an oleaginous organism poses some difficulty. A

pragmatic definition would suggest that a microorganism containing more than 20-
25% oil would be deemed a suitable candidate for commercial consideration.
The lipid-containing microorganisms include bacteria, algae, and fungi. The range of
fatty materials produced by the microorganisms can be conveniently classified as:
1. Simple lipids: represented by triglycerides

2. Compound lipids: represented by phospholipids, glycolipids, etc.
3. Miscellaneous: represented by vitamin A, sterols, carotenoids, etc.
A short list of microbial lipids, the organism involved, and the substrate used is
Table20.1 Representative groups of lipid producing microorganisms
Representative organism Substrate Product (lipid)

Chlorella pyrenoidosa (algae) Carbohydrate Triglyceride
Blakeslea trispora (algae) Carbohydrate β-carotene
Saccharomyces cerevisiae (yeast) Wort Ergosterol
Acinetobacter sp HO1-N (bacteria) Hydrocarbon Waxes
Alcaligenes eutrophus (bacteria) Glucose Poly-β-hydroxybutyrate
A brief description of each of the above lipid types is given in the following
paragraphs:
20.1.1 TRIGLYCERIDES
These are natural oils, the hydrolysis of which gives fatty acids and glycerol only. The
general structure is shown in Fig. 20.1.
CH2OCO-R1 CH2OH R1-COOH
R2-COOCH + 3H2O HOCH + R2-COOH
CH2OCO-R3 CH2OH R3-COOH
Triglyceride Glycerol Free fatty acid (FFA)
Fig. 20.1 Triglyceride and its hydrolysis
20.1.2 POLY-β-HYDROXYBUTYRATE
Poly--hydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA) that consists of

repeating units of -CH(CH3)-CH2-COO- (Fig. 20.2). PHB is an energy storage
material produced by a variety of microorganisms in response to environmental
stress (limitation of nitrogen or phosphorus in the growth medium). Although many
bacteria are capable of producing PHB, Alcaligenes eutrophus strains have remained the
organisms of choice for industrial production. They can produce up to 96% PHB
(based on dry cell mass). Commercial PHB is sold under the name Biopol®.
Because PHB is biodegradable, there is considerable interest in using PHB for

packaging purposes (such as edible packaging) in order to reduce impact of human
garbage on environment. PHB has application in antibiotics, drugs delivery, medical
suture, and bone replacement.
CH3 O CH3 O CH3 O CH3 O

HO CH CH2 C OH HO CH CH2 C O CH CH2 C O CH CH2 C OH
  n=100-30000
-hydroxybutyrate Poly--hydroxybutyrate
Fig. 20.2 Structure of poly-β-hydroxybutyrate
20.1.2.1 Biosynthesis of PHB
Poly--hydroxybutyrate is synthesized by the bacterium (Alcaligenes eutrophus) utilizing

three enzyme systems, viz., (i) -keto thiolase, (ii) reductase, and (iii) PHA synthase.
The natural producers also have PHA depolymerase that degrades the polymer and
uses the breakdown metabolites for cell growth. A summary of PHB synthesis is
given in Fig. 20.3.
PHB is insoluble in water and is relatively resistant to hydrolysis. It sinks in water,

has good oxygen permeability, is non-toxic, and is biodegradable. Despite these
interesting properties, PHB is used only in limited areas because of two fundamental
reasons, viz., (i) prohibitively high cost of PHB, and (ii) brittleness. Researches are
being done to overcome these limitations. The cost can be brought down by large-
scale production, strain improvement, and improvement in the fermentation
process. The brittleness can be reduced by incorporating other alkanoates in the
material to form copolymers with a wide range of plasticity and other
physicochemical properties.
307
2 Acetyl-SCoA
-keto-thiolase
Acetoacetyl-ScoA
Reductase
-hydroxybutyrate
PHA synthase
Poly--hydroxybutyrate
Fig. 20.3 Biosynthesis of poly--hydroxybutyrate by Alcaligenes eutrophus
20.1.2.2 Microbial production
Using Alcaligenes eutrophus, PHB can be produced by two general methods, namely, (i)
Parallel process, and (ii) Serial process. In the Parallel process, cell growth and PHB
formation occurs together in a single fermenter. In the Serial process (which is more
common), microorganisms are first grown on carbon source (usually glucose) to
obtain large biomass. Then the medium is depleted of an essential nutrient (nitrogen)
and polymer-forming substrate is added (Fig. 20.5). This is converted directly to
polymers and essentially only little growth occurs. An outline of a typical PHB
production is given in Fig. 20.4.
Seed culture
Extraction
Inoculum tank (with propylene carbonate)
Medium Fermenter Centrifuge
Centrifuge Filter press
Acetone wash Repeated washing

(with hot water)
Centrifuge
Acetone wash
Freeze dryer
Freeze dryer
Mechanical pulverizer
PHB
Fig. 20.4 A typical outline of microbial production of PHB
Substrate PHA-forming substrate
Cell growth PHA formation
Fig. 20.5 Serial process of PHB production
308
20.1.3 WAXES
The composition of wax is of a simple ester of a fatty acid with a fatty alcohol:
CH3 (CH2 ) x COOH  HOCH 2 (CH 2 ) y CH3  CH3 (CH2 ) x COOCH 2 (CH 2 ) y CH3  H2O,
where x and y are usually either 14 or 16, although shorter chain alcohol with y =1 to
3 have been reported.
20.1.4 BETA CAROTENE
It is a precursor of vitamin A. It contains 40 carbon atoms (or 8 isoprenoid units). The

simplified structure is given in Fig. 20.6. For microbial production, see page 391.
H3C
H3C CH3 CH3 CH3 2
1 CH3 CH3 H3C CH3

CH3 ionone
-carotene ionone
Fig. 20.6 Simplified structure of β-carotene
20.1.5 ERGOSTEROL
Ergosterol occurs in yeast. Yeast requires this sterol for the synthesis of essential
membrane components. It is also regarded as an anaerobic growth factor for yeast. The
chemical structure of the compound is given in Fig. 20.7.
CH3
H3C CH3
H3C
CH3
H3C
8
5
7
HO 6
Fig. 20.7 Structure of ergosterol
20.2 SOME MICROORGANISMS CAPABLE OF PRODUCING LIPIDS
20.2.1 BACTERIA
Only a few bacterial species are known which can produce appreciable amounts of
extractable neutral lipids (the triglycerides). The Mycobacteria-Nocardia group of
organisms is well known for cellular lipid contents but these lipids are complex-
structured, often occurring in a bound form as part of the cell envelope structure.
Some of these species do contain triglycerols but their exploitation is not sensible as
the co-extraction of toxic or allergenic substance from them is highly likely. For
309
example, cell walls of Corynebacterium, Mycobacterium, and some other nocardioforms
contain mycolic acid. Mycolic acid is a high molecular weight α-branched β-hydroxy
fatty acid of the general formula:
R1 CH CHCOOH
OH R2
The mycolic acid derivative, called cord factor (= Trehalose dimycolate) is toxic and
plays an important role in the diseases caused by Corynebacterium diptheriae and
Mycobacterium tuberculosis. The cord factor inactivates the mitochondrial membranes of
the phagocytes.
The only bacterium which has been reported as producing significant amounts of
triglycerol is Arthrobacter AK-19. This organism is unlike any other bacteria in that it
can contain up to 80% of its biomass as lipid; this lipid, moreover, is predominantly
composed of triglycerols and would thus seem an excellent candidate for commercial
exploitation. The only drawback in this organism is its slow growth rate. However,
there is possibility of growing the bacterium as a symbiont along with an algal culture.
The provision of an external carbon source, other than CO2, would then be obviated.
20.2.2 ALGAE
Notable oleaginous algae, (fat content in parenthesis) are Chlorella pyrenoidosa (70%),
Botryococcus braunii (53%), Dunaleilla salina (47%), Monalanthus salina (70%), etc. The
major limitations in their use are production of lipids other than triglycerol type and
markedly slow growth rate.
Chlorella pyrenoidosa and Chlorella vulgaris appear worth investigating among the
oleaginous algae. The algal lipids are marked by their exceptionally high proportions
of polyunsaturated fatty acids (PUFA). Unfortunately, as these are the types found in
fish oil, the frequent complaint against algal lipids is about their unpleasant fishy
odor. The desirability of including polyunsaturated fatty acids in the diet might
suggest that a proportion of algal oils could be mixed with a more saturated or
monounsaturated oil, viz., palm oil or rapeseed oil, to give nutritionally acceptable
blend.
20.2.3 YEASTS AND MOLDS
The number of oleaginous microorganisms in this class is not very large. Some 16
classified species have been reported as producing better than 25% lipid. The
prerequisite for lipid accumulation in these microorganisms is the possession of
ATP:citrate lyase; this enzyme is present only in lipid-producing yeasts and therefore
is an extremely powerful determinant for lipid production. Some examples of
oleaginous yeasts (lipid content in parenthesis) are: Cryptococcus terricolus (55-65%),
Rhodotorula glutinis (syn. gracilis) (74%), and Candida curvata (51-58%).
The major accumulating lipid of yeasts and fungi is the triglycerol fraction, which
accounts for up to 92% of the total lipid of the cell. The fatty acids of yeasts are
usually in the approximate order of abundance: oleic > palmitic > linoleic > stearic
310
acid. Modifying the cultural conditions, however, can vary the order of abundance of
fatty acids. Such conditions would include variation in O2 tension, choice of growth
substrate, growth temperature, as well as the growth rate of the organism itself.
The fatty acids from molds show a greater range and diversity than those from
yeasts. Members of Entomopthoraceae are characterized by the presence, often in
substantial amounts, of short-chain fatty acids (C10-C14).
20.3 GENERAL CULTURAL CONDITIONS
A wide range of substrates has from time to time been considered for the
production of oils and fats. These include various starchy crops and wastes,
molasses, whey, peat (compost) hydrolysates, and ethanol. The use of hydrocarbons,
e.g., methanol, has fallen out of favor.
As lipid represents a reserve storage material, the medium should have a high carbon-
to-nitrogen ratio (usually, 50:1). In a batch culture the microorganism grows until the
nitrogen is consumed but thereafter it continues to take up the excess carbon and
convert this to lipid. Thus, a biphasic growth pattern can be expected.
With some of the slower growing molds, the rate of lipid accumulation appears to
coincide with the growth rate. Although this is probably fortuitous, the result is that
the lipid content of the cells increases at the same rate as growth proceeds.
In a continuous culture, lipid accumulation is achieved by growing microorganisms

under nitrogen-limiting conditions at a dilution rate of about 30% of maximum specific
growth rate. The build-up of lipid is dependent on the correct balance between
growth rate and the specific rate of lipid biosynthesis so that the optimum amount
of carbon can be diverted into lipid and the minimum into other cell components.
A conversion of carbohydrate to lipid of 20% would appear near to possible

practical limit because the theoretical maximum is about 33 g triglycerol from 100 g
of glucose, assuming that all the carbon of the medium is converted into lipid
without the synthesis of any other cell components. The low conversion is apparent
from the following fact: synthesis of one mole of palmitic acid in a eukaryotic cell
such as yeast needs, theoretically, 145 ATP as against 130 ATP obtained by β-
oxidation of the same. A total of 8 acetyl-ScoAs are diverted for the biosynthesis.
Assuming that all the acetyl-ScoA come from carbohydrate, 4 moles of glucose are
wasted for energy production alone. Since there are 3 fatty acids plus a glycerol
moiety in a triglyceride, the energy requirement in terms of glucose is very high.
Only after this energy has been furnished can the conversion of carbohydrate to fat
occur.
As far as lipid production from yeast is concerned, the only current commercial
enterprise utilizing non-carbohydrate carbon source is that using gas oil to yield the
protein called Fermosin. This biolipid is produced as a byproduct.
311
20.4. MICROBIAL PRODUCTION OF PUFA-RICH OIL
This discussion relates to production of high-value product, namely essential fatty

acid, using microbial technique. Reference is given to the patent filed by Streekstra
and Brocken (2005). These workers have described a method for the production of
microbial oil rich in polyunsaturated fatty acids (PUFA), arachidonic acid (which is
an essential fatly acid) in particular. The process involves a two-stage fermentation
using Mortierella alpina (a fungus). In the first stage, the organism is provided with
excess of carbon and nutrients so that lipids (including arachidonic acid) are
accumulated in large amounts. The second stage of fermentation involves restricted
feeding of the carbon source, with the result that lipids except arachidonic acid are
preferentially utilized to meet the metabolic need. This event leads to increase in the
proportion (35-40%) of arachidonic acid in the lipid bulk. The fatty acid is
predominantly in the triglyceride form (about 99.5%).
Briefly, the production process is as follows:
The organism is propagated in the order: 100 ml  500 ml  2400 liter by

aerobically growing it at 25°C for 24-48 hrs in each stage. The culture medium
consists of glucose (2%), yeast extract (1.2%), and silicone antifoam (0.02%). The
medium is sterilized after adjusting the pH to 7. Aeration in the propagators is
achieved by bubbling air and agitation.
The first-stage fermentation medium contains 3.5% glucose, 0.5% yeast extract,
0.03% antifoam, 0.1% NaH2PO4.2H2O, 0.2% KH2PO4.2H2O, 0.05% MgSO4.7H2O,
0.06% citric acid monohydrate, and 0.0001% ZnCl2. The pH is maintained at 6±0.1
before fermentation. Aeration is carried out at a rate of 0.5 vol/vol/min.
Fermentation is carried out for 170 hrs at 25°C. This phase is characterized by
accumulation of lipids by the organism.
The second-stage fermentation starts 5-6 hrs before the termination of the
fermentation (the total fermentation time for both the stages being about 175 hrs).
In this stage, a 50% aqueous solution of sterile glucose is fed to maintain glucose
content at about 0.5 g/kg of medium/hr. A 25% solution of yeast extract is also fed
to maintain ammonia level at around 30 mg/liter. During this stage, the
microorganism experiences starvation because of carbon source restriction and to
overcome this state it preferentially falls back on lipids other than arachidonic acid.
After the fermentation has completed, the microbial biomass is harvested by

filtration. The filtered cake is pasteurized (to kill the cells and inactivate lipid-
degrading enzymes), extruded/crumbled/kneaded, and dried for extraction of oil
using organic solvents (e.g., hexane) or supercritical fluid (liquid CO2).
Although the oil can be used as such, it can also be further purified to meet the
exacting criteria by refining, bleaching, deodorization and polish filtration.
This microbial oil (which is rich in essential fatty acid, arachidonic acid) is suitable
for including in infant formula, human foodstuffs, feed supplement, and
pharmaceutical preparations.
312
20.5 OUTLINE OF BIOSYNTHESIS OF SIMPLE LIPID IN EUKARYOTES
Acetyl-ScoA is the precursor of fatty acids. However, it does not spontaneously

polymerize; it must first be activated to a highly reactive form, malonyl-ScoA, (a
three-carbon unit). The carboxylation requires biotin-dependent enzyme and so the
requirement of biotin for oleaginous microorganisms is obvious. The biosynthesis
takes place in somewhat complex manner, but the essential steps for one cycle that
allows lengthening of the existing chain of fatty acid by 2 carbon units are: Activation,
Attachment to binding sites, Condensation, Dehydration, and Reduction (see Fig. 20.8).
Attachment to
binding sites
Acetyl-SCoA
CO2 Activation
Condensation Reduction
CoA-SH Acetoacetyl- D--hydroxybutyryl-
Malonyl-SCoA S-enz-complex
S-enz-complex
2H
CO2 Dehydration
Attachment to binding sites
H2O
Crotonyl-S-enz-complex
2H Reduction
Butyryl-S-enz-complex
Malonyl-SCoA Elongation
Fatty acid
Fig. 20.8 Biosynthesis of fatty acid in eukaryotes
20.6 FUTURE PROSPECTS
The future prospects for microbial oils might be seen to lie in three possible areas:
1. As a substitute for high-value plant oils

2. As novel material not available from other sources
3. As an alternative to SCP production in waste-processing systems
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CHAPTER 21
RIBOFLAVIN PRODUCTION BY YEAST
21.1 INTRODUCTION
Riboflavin (= vitamin B2, or vitamin G, or lactoflavin) has the empirical formula

C17H20N4O6 and the structural formula is:
CH2(CHOH)3CH2OH
H3C N N O
NH
H3C N
O
Riboflavin (oxidized state)
It is a crystalline yellow-orange powder. It is bitter in taste and practically odorless. It

is sensitive to alkalis and is decomposed by UV radiations. This vitamin is an
essential growth factor for animals and humans.
Riboflavin can be produced by two methods, viz., (i) Chemical, and (ii) Microbiological.
The chemical synthesis is carried out by Kuhn’s process (Fig. 21.1) or modification
of it. The basic material needed for chemical synthesis are D-ribose, 6-nitro-3,4-
xylidine, and alloxan. Condensation of D-ribose and 6-nitro-3,4-xylidine followed by
catalytic reduction gives phenylenediamine. The latter reacts with alloxan to give
riboflavin.
H 3C NH2
H 3C NO2
6-nitro-3,4-xylidine
H OH
C H3C NHCH2(CHOH)3CH2OH H3C NHCH2(CHOH)3CH2OH
(CHOH)2 O
Condensation Catalytic reduction
HC H3C NO2 H 3C NH2 H
CH2OH Phenylenediamine O N O
D-ribose NH
O
O
Riboflavin Alloxan
Fig. 21.1 Chemical synthesis of riboflavin
Since the purest form of riboflavin is produced by chemical means it is this type that
is valued therapeutically.
21.1 USES OF RIBOFLAVIN
 Therapeutic purposes
 Fortification of cereal products (e.g., enriched bread, flour, etc)
 In animal feed (2-8 g/metric ton)
21.2 MICROBIAL PRODUCTION OF RIBOFLAVIN
Riboflavin can be produced by a number of microorganisms, including bacteria,

yeasts, and yeast-like microorganisms. The most important (commercially) of these
are Eremothecium ashbyii, Ashbya gossypii, and certain Clostridium species. A short list of
the microorganisms of potential or realized value is given in Table 21.1.
Table 21.1 Some noted producer microorganisms of riboflavin
Bacteria Yeast Yeast-like

Clostridium butyricum Candida flareri Ashbya gossypii
Clostridium acetobutylicum Candida guilliermondia Eremothecium ashbyii
The bacteria Clostridium butyricum and Clostridium acetobutylicum are commercially used
for acetone-butanol fermentation but riboflavin can be recovered as a byproduct of
the process.
The yeast-like organisms, Ashbya gossypii and Eremothecium ashbyii, are plant pathogens
that cause disease in cotton and other plants. This attribute makes it mandatory that
sterilization be carried out before discarding the cultures and fermentation residues.
Of the two microorganisms mentioned above, Ashbya gossypii has greater stability
with respect to riboflavin-producing capacity: it does not degenerate as readily as
Eremothecium ashbyii.
21.2.1 TRADE FERMENTATION USING Ashbya gossypii
21.2.1.1 Stock culture
The culture may be transferred at weekly intervals on a medium containing peptone

(0.5%), yeast extract (0.3%), malt extract (0.3%), commercial glucose (1%), and agar
(2%). Incubation may be done at 27-30°C.
21.2.1.2 Inoculum development
A loopful of 24-hr old culture of Ashbya gossypii NRRL-Y-1056 is placed in 100 ml of

the following medium in 500 ml flask and incubated for 24 hrs in a reciprocating
shaker at 26-30°C: glucose 2%, peptone 0.5%, cornsteep liquor 1%, and water to
make 100 ml. The pH of the medium is adjusted to 6.5 before sterilization.
The contents of the flask are used to seed 6 liters (in a 9-liter flask) of medium
having following composition: glucose 2%, cornsteep liquor 1%, animal-stick liquor
315
0.5%, and water to make 6 liters. The pH of the medium is adjusted to 6.5 before
sterilization.
The organism is grown for 24 hrs with aeration provided by passing sterile air. The
culture can now be inoculated in 1000-1500 liters of medium for the main
fermentation.
21.2.1.3 Main fermentation
The medium for main fermentation can be semi-purified sugar, glucose, plus
additional crude organic nutrients such as peptone, cornsteep liquor, etc. In certain
instances, however, glucose may be totally replaced by lipid such as corn oil. The
medium can be sterilized (continuous sterilization) at pH 4.5 and 135°C for 5 min.
In batch sterilization, 15 psig can be used for 3 hrs.
Iron content of the medium above 5 ppm is detrimental. To regulate it to 1-3 ppm,
which is the optimum, iron or steel fermentation equipment is avoided. Plastic or
cobalt-coated fermenters can be used instead.
The temperature is maintained at 28-30°C. The initial pH is 6-7.5. The process is a

submerged aerated fermentation but excess air inhibits mycelial production and
reduces the riboflavin yield. An aeration rate of about 0.25 vol/vol/min is
satisfactory. If foaming becomes excessive, it can be controlled by the initial addition
of emulsified silicone antifoam and later by soybean oil. During the fermentation,
the riboflavin content must be periodically determined (by fluorimetry).
Contamination checks are made by growing the samples in malt-yeast extract agar.
The fermentation lasts for 4-7 days.
21.2.1.4 Recovery
The final beer is heated for an hour at 60-120°C to free riboflavin from the
mycelium. The solids may be dried to a crude product for animal feed
supplementation, or processed into a refined grade. In either case the pH is adjusted
to 4.5. For feed-grade product, the broth is concentrated to about 30% solids and
dried on double drum driers.
When a crystalline product is desired, the broth is heated for an hour at 121°C to
solubilize riboflavin. Insoluble matter is removed by centrifugation and the broth
treated to recover the vitamin. The broth is reduced by bacterial or chemical means
to precipitate riboflavin from the broth. The precipitated riboflavin is then dissolved
in water or polar solvents, or an alkaline solution, oxidized by aeration, and
recovered by crystallization from the aqueous or polar solvent solution or by
acidification of the alkaline solution.
21.2.1.5 Changes occurring during the fermentation
The fermentation progresses through 3 phases as described in the following

paragraphs:
316
Phase-I
Rapid growth, little production of riboflavin, rapid utilization of glucose, lowering of

pH due to pyruvate accumulation
Phase-II
Glucose depletes, growth ceases, sporulation starts, pyruvate decreases, ammonia

increases due to increase in deaminase activity, pH value increases, rapid synthesis of
cell-bound riboflavin (as FAD and FMN) occurs, rapid increase in catalase activity
and a disappearance of cytochromes
Phase-III
Cellular regulatory mechanisms for FAD synthesis break down. Autolysis occurs,
releasing free riboflavin into the medium as well as some riboflavin in the nucleotide
form.
From these observations, it can be concluded that at about the same time of
sporulation there is a shift from the initial cytochrome-type terminal respiration to a
terminal respiration utilizing flavoproteins, and that this flavoprotein respiration is
accompanied by an overproduction of the flavin prosthetic group.
Certain purines, but no pyrimidines, stimulate riboflavin production without

simultaneously stimulating growth. This is because purine or purine precursors are
used by the microorganisms to construct the middle and right hand rings of the
riboflavin molecule.
21.3 BIOSYNTHESIS
The starting material is the guanosine derivative (Fig. 21.2). This is transformed to 6-
hydroxy-2,4,5-triaminopyrimidine derivative. The nature of R1 and R2 is unknown.
O O O
N H2N H2 N
NH NH -NH3 NH
N N
NH2 HN NH2 +H2O HN O
N N
R1 R2 H R H
Guanosine derivative
O H O
N Riboflavin N
H3 C NH synthetase H3 C NH
H3 C O H3 C O
N N N N
R R
Riboflavin
R = Ribitol residue 6,7-dimethyl-8-ribityl lumazine
Fig. 21.2 Microbial synthesis of riboflavin
317
The latter derivative is reduced to corresponding ribityl derivative. Replacement of an
amino group by a keto group yields 5-amino-2,6-dioxy-4-(1'-D-ribityl amino) pyrimidines.
The steps leading to 6,7-dimethyl-8-ribityl lumazine are unknown. Formation of
riboflavin is completed by riboflavin synthetase.
318
CHAPTER 22
MICROBIAL PRODUCTION OF ORGANIC ACIDS
22.1 CITRIC ACID
Citric acid is a tricarboxylic acid with the molecular formula:
CH2COOH
HO – CCOOH
CH2COOH
It was first isolated by Scheele from lemon juice in 1784. Microbial production of
citric acid started in USA in 1923. In 2000, the annual production of citric acid
worldwide was 736,000 MT.
22.1.1 PRODUCTION METHODS
Citric acid can be produced using molds, yeasts, or bacteria. Except for patents and
few research articles, literature details on yeast- and bacterial processes are still
scarce. Candida, Pichia, etc., are the main organisms studied in the yeast process. In
the bacterial process, mutated strains of Corynebacterium species have been reportedly
used. The mold process is by far the most important from trade fermentation point
of view. Today, improved strains of Aspergillus niger (mold) are universally used for
citric acid production.
22.1.1.1 The mold (fungal) process
Based on the fermentation differences, there are three main types of mold process
for citric acid production: (i) solid substrate fermentation (koji process), (ii) surface
culture, and (iii) submerged culture. Aspergillus niger strains used for the commercial
processes are very efficient, producing above 80 g citric acid per 100 g glucose. The
organism in general exhibits marked sensitivity to iron and zinc, particularly to iron,
in the medium. These elements promote growth of the organism but at the cost of
citric acid.
1. The koji process
This method is widely used in Japan. The method accounts for 1/5th of total citric
acid produced in Japan. The fermentation is carried out in moist wheat bran. Since
wheat bran is rich in minerals, only special, iron-tolerant strains of Aspergillus can be
used. Although wheat bran contains ~ 66% carbohydrate, it is probably
supplemented with suitable amounts of sugar for the fermentation. Ferrocyanides or
copper may also be supplemented. After sterilization of the substrate (which is in the
paste form with ~ 70% moisture content), the fermentation is carried out in batches,
in shallow trays or rotolouver-type drum fermenters, at a pH of 5.5. Temperature,
humidity, and air supply can be maintained in a manner similar to that for other koji
processes. Inoculation is done with pure mold spores. Mold spores are usually
suspended in 0.1% Tween 80 (a wetting agent) before mixing with the substrate. The
concentration of the spore is, of course, of prime importance, and is typically
maintained at 107 spores per ml of suspension. Fermentation is carried out at around
30-32°C until the pH of the bran extract falls to 1.8-2.0, which corresponds to about
6 days. Aeration rate is optimally maintained at 0.8 vol/vol/min. After fermentation
is over, the extract is recovered by maceration in water followed by filtration. The
liquor is purified by precipitation with lime followed by regeneration with H2SO4.
The final liquor is treated with activated carbon, concentrated, and crystallized. The
yield is low because of the difficulty in controlling trace metals and process
parameters.
2. Surface culture method
Production
The medium can be either synthetic (refined sugars) or complex (cane molasses).
When cane molasses is used, the excess minerals must be removed prior to
fermentation. Several treatment options are available for the same, for example,
deionizing, use of sequestrants, etc. In trade fermentation, the deleterious effect of
iron is counteracted by dosing alkali ferrocyanide (e.g., K4Fe[CN]6) as iron-chelating
agent. This compound becomes toxic if present in the medium in excess. It is
therefore customary to keep the free K4Fe[CN]6 below 20 μg/ml in the medium.
Molasses medium should be adjusted to a pH of 5-6 (usually with H2SO4) although
this is not the optimum pH for the mold. The main reason behind this is the
presence of acetic acid in molasses. Unionized acetate (which occurs at lower pH
values) represses spore germination. The pH is therefore brought to 5-7 to ionize
acetic acid and make germination of spores favorable. This is not a problem when
synthetic media are used. A pH as low as 2.5-3.0 can be used in the case of refined
media. The sugar concentration of the medium is maintained at 15%. This level
represents a good compromise. Sugar contents above 15% lead to increased residual
sugar and accumulation of oxalic acid in the beer while sugar contents below 15%
result in reduced yield.
Additional nitrogen requirement for the fermentation can be met by supplying

(NH4)2SO4. After normal sterilization procedure the medium is inoculated with
spores of Aspergillus niger grown separately. The concentration of spore is typically
105-107 per ml of medium.
The spores of the culture are prepared in a stepwise manner from lyophil vials. The
first step is to grow the organism in special sporulation agar. Thereafter it is
transferred to other suitable medium for either mycelium build up or spore
production. Both the forms (spores and mycelia) can be used for the main
fermentation. When spores are to be used, they are usually suspended in suitable
wetting agents, such as Tween 80, as the carrier.
320
Fermentation is carried out in very high-purity shallow aluminum trays. The ratio of
medium volume to surface is maintained 1.22 ml/cm2 for maximum performance.
The organism is highly aerobic and hence must by supplied with adequate amount of
oxygen. This is met by supplying air at the rate of 0.5-1.5 vol/vol/min. The
inoculated trays are stacked in racks kept in rooms or compartment with provision
for ventilation and continuous aeration. The temperature is maintained at around
30°C and the duration of fermentation ranges from 7 to 14 days. The final broth
contains about 7% citric acid.
The fermentation must be continuously controlled. Laboratory analysis must be

periodically carried out for citric acid and residual sugar contents.
Recovery
The first step in the recovery is filtration of mycelia in rotary vacuum filter (Fig.
17.10a and 17.10b). The mycelia must be repeatedly washed with water but without
bringing about excess dilution. The citric acid in the liquor is precipitated out as
calcium citrate followed by regeneration with H2SO4. For detail of recovery, see
submerged fermentation described shortly.
3. Submerged culture method
Production
The preparation of medium is the same as that for surface culture. The inoculum is
normally in the mycelial form.
Fermentation is carried out in very high-grade stainless steel fermenters. The

fermenters can be of two main types, (i) stirred, aerated, baffled tank, and (ii) aerated tower
tank. The cultural condition is also similar to that of surface culture. Aeration is done
at a rate of 310-6 gram mole O2/ml/min. Aeration is very critical during the final
stage of fermentation: lack of O2 leads to remetabolism of citric acid. The temperature
is maintained at 30°C and the fermentation usually lasts for 4-5 days.
Stimulants are universally added in submerged fermentation. One of the most

important stimulants used in citric acid fermentation is methanol. It is added at the
rate of 3-4 % during fermentation. Methanol has been assumed to delay spore
formation and alter cell wall permeability in Aspergillus niger, thereby increasing the
yield. Antifoams (such as silicone oil, octadecanol, etc.) are also added during the
fermentation.
There are some variations in the submerged culture process. In one variation, the
fermentation is carried out in two stages, viz., growth stage and production stage in
separate vessels. In another variation, the mycelium is reused up to 3 times: this
eliminates the costly inoculum build-up stage.
Recovery
The beer is filtered in rotary vacuum filter to remove mycelia. The liquor can be
treated by any of the two different methods, viz., (i) classical method and (ii) solvent
extraction method, for refining citric acid. There are many other patented methods also.
321
The classical method, which entails precipitation of citric acid with lime and
regeneration with H2SO4, is probably the most widely used method. The solvent
extraction method uses combination of solvents for extraction of citric acid and
back-extraction in alkaline aqueous phase as calcium citrate. The most commonly
used solvents are mixtures of tributyl phosphate plus kerosene, and tributyl phosphate plus
butyl acetate (100: 5-30).
The classical method of recovery
The filtered liquor is first treated with lime to selectively precipitate out oxalate. As a
rule, lime is added to the liquor in calculated amount to allow spontaneous rise in
temperature to 80-90°C. The precipitated calcium oxalate is removed by filtration and
the liquor treated again with hydrated lime. Hydrated lime is added to the liquor at
controlled rate (1 part hydrated lime per 2 parts liquor) over a period of 1 hr until the
temperature of the liquor reaches ~ 95°C. This event precipitates out citric acid as
calcium citrate. The insoluble calcium citrate is separated from the liquid portion by
filtration and the precipitate treated with equivalent amount of H2SO4 to regenerate
citric acid. The gypsum (calcium sulfate) resulting from this treatment is thrown away.
The final steps of purification consist of decolorization, crystallization, drying, and
packing. See Fig. 22.1 for an outline of recovery by classical process.
Filtered broth Lime

Decolorizing
Oxalate precipitation
Concentration
Filtration Hydrated lime
Ca-oxalate Crystallization Mother liquor
Ca-citrate precipitation
Centrifugation
Filtration H2SO4
Waste liquid Drying
Regeneration
Gypsum Sieving
Citric acid
Packing
Fig. 22.1 Recovery of citric acid by precipitation-regeneration method
At 40°C, citric acid crystallizes out as the anhydrous acid, and below 36.5°C as the
monohydrate.
Yield of citric acid
Under practical condition, the yield of citric acid is 70-90 g per 100 g of glucose. The
theoretical yields from sucrose and glucose anhydrous are 123% and 117%
respectively.
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22.1.2 BIOSYNTHESIS OF CITRIC ACID
Citric acid produced by commercial strains of Aspergillus niger is typically an overflow

product due to faulty operation of citric acid cycle. The major route of formation is
the condensation of acetyl-ScoA and oxaloacetate. Aspergillus niger uses 78% of the
sugar via EMP (Embden-Meyerhof-Parna) pathway. Oxaloacetate, which is
constantly needed in the synthetic step, is regenerated by carboxylation of triose
compounds or by glyoxylate cycle. It has been found that the enzymes leading to the
synthesis of cis aconitate and isocitrate is rather inefficient in citric acid producing
strains. This could be one of the reasons for overproduction of citric acid.
22.1.3 USES OF CITRIC ACID
Citric acid is used in food, confectionery and beverages, in pharmaceuticals and in

industrial fields. Its use depends on three properties: acidity, flavor, and salt
formation. A summary of the uses of citric acid is given in Table 22.1.
Citric acid forms a wide range of metallic salts including complexes with copper,
iron, manganese, magnesium, and calcium. These salts are used as sequestering
agents in industrial processes and as anticoagulant blood preservative. Citric acid also
exhibits antioxidant properties in fats and oils where it reduces metal-catalyzed
oxidation by clelating traces of metals such as iron. There are two components to its
use as a flavoring: the first is due its acidity, which has little aftertaste; the second to
its ability to enhance other flavors.
Table 22.1 Application of citric acid
Industry Property Market share

Food About 75%
Beverages Acidulant
Jellies, jams, etc. Flavoring
Fats and oils Antioxidant
Frozen foods Antioxidant
Pharmaceuticals About 10%

Effervescent Acid
Vitamins Antioxidant
Anticoagulants Sequestering
Iron preparations Salt formation
Cosmetics Buffering
Industrial About 15%
Cleaning (metals) Sequestering
Detergents Buffering
Photographic Buffering
Primer binding Sequestering
Polymerizations Sequestering
323
A process to remove sulfur dioxide from flue gases has been developed where citric
acid is used as a scrubber, forming a complex ion which then reacts with H2S to give
elemental sulfur, regenerating citrate. This may become more important with
increased environmental pressures.
Citric acid esters of a range of alcohols are known: the triethyl-, butyl- and
acetyltributyl- esters are used as plasticizers in plastic films and monostyryl citrate is
used instead of citric acid as an antioxidant in oils and fats.
22.2 FUMARIC ACID
Fumaric acid was formerly produced by fermentation, but now it is produced by

hydrocarbon oxidation. The microbial process has fallen out of favor. The molecular
structure of the acid is:
H C COOH
HOOC C H
Fumaric acid is crystalline in nature. It is sparingly soluble in water (0.7 g/100 ml).
Sodium- and potassium salts are readily soluble in water. The acid finds use in
acidification of beverages, manufacture of aspartic acid, aspartame (a dipeptide
artificial sweetener composed of aspartic acid and phenylalanine), and polyester
fabrics. Trade fermentation utilizes improved strains of Rhizopus nigricans.
The acid is biosynthesized in the TCA cycle but can also be formed via glyoxylate
cycle. Since ethanol can also serve as a carbon source, the pathway appears to be
more than one.
22.2.1 MICROBIAL PRODUCTION
22.2.1.1 Cultural condition
Hexose sugars are the raw materials of choice. Molasses can also be used but the
invertase activity is not possessed by all strains. The most common nitrogen source
is ammonia or urea. Minerals play a very important role in fumaric acid
fermentation. Zinc in particular should be kept at suboptimal level. Excess zinc
allows the formation of acids other than fumaric acid. Methanol is generally used as
a stimulant but the effect of methanol is quite different from that in citric acid
production: the methanol molecule gets incorporated in the fumaric acid molecule.
No details are available for the fermentative production of fumarate. The inoculum
is probably prepared as in the case of citric acid. The fermentation is carried out
either as submerged- or surface culture, at 28-33°C. The pH is maintained at around
5-6 with constant addition of Na2CO3 or K2CO3. Continuous neutralization is
essential because the organism is very sensitive to acidity resulting from fumaric acid
accumulation. The acid being sparingly soluble in water tends to crystallize out in
324
mycelium. Addition of Na2CO3 or K2CO3 makes it soluble. CaCO3 is not used for
the neutralization because it also crystallizes. Aeration is a crucial aspect of fumaric
acid fermentation. Deficiency of O2 supply leads to accumulation of ethanol in the
medium, which is undesirable.
22.2.1.3 Recovery
The first step is the familiar rotary vacuum filtration (Fig. 17.10a and 17.10b). The
broth that contains the salt of fumaric acid is acidified thereby rendering the acid
insoluble in water. The final step entails crystallization of the acid from hot water.
22.3 GLUCONIC ACID
D-gluconic acid or more correctly, pentahydroxy (α, β, γ, δ, ε) caproic acid, is an

oxidation product of glucose. Its molecular formula is:
HOOC-(CHOH)4-CH2OH
Gluconic acid can be produced by microbial as well as chemical means. Both the
methods are fully competitive.
22.3.1 USES OF GLUCONIC ACID
Gluconic acid finds wide use in food as well as non-food items, for instance:
1. Glucono-δ-lactone is used as a latent acid in baking powders, and in instant,

chemically leavened breads
2. Sodium gluconate is used as calcium- and iron sequestrants
3. Gluconic acid is used as an ingredient in chemicals for removing milk stone in
glass bottles
4. It can be used for the treatment of calcium deficiency diseases. Gluconic
acid being non-toxic and metabolizable, it can be used for introducing
cations like Ca++ into the body
5. Gluconic acid finds use as an additive in cement mixes
6. It is used in foliar formulation (for supplying trace minerals)
7. It is also used for the preparation of chlorheximide (a disinfectant)
22.3.2 BIOSYNTHETIC PATHWAY
The organism uses a mixture of pathways. The main pathway, which directly leads to
gluconic acid, utilizes glucose oxidase, an FAD-dependent enzyme:
O
β-glucose  2
oxidase
 Glucono- -lactone  H 2 O 2
Gluconic acid tends to form internal linkages to form δ, and γ lactones. All the three
species exist in equilibrium. See Fig. 22.2 for the outline of biosynthesis.
325
-D-glucose
Mutase
-D-glucose
Glucose oxidase
D-glucono--lactone HMP enzymes
D-glucono--lactone
D-gluconic acid 6-P-Gluconate
Pyruvate
Pyruvate + Glyceraldehyde
Pentose sugars
Fig. 22.2 Biosynthetic pathway of gluconic acid
22.3.3 MICROBIAL PRODUCTION OF GLUCONIC ACID
Commercial production of gluconic acid utilizes mold (Aspergillus niger) or bacteria

(Gluconobacter suboxydans).
22.3.3.1 Cultural condition
Glucose is the main carbon source in the mold process. CSL, ammonium salts and
urea are used for nitrogen source. Minerals must also be balanced. An outline of the
production process (mold) is given in Fig. 22.3. The glucose concentration is kept at
around 22%. The inoculum build-up process starts by sporulation in solid agar
medium. The main inoculum is in the form of mycelia. About 10% by volume is
used for the final inoculation. The pH is maintained at 6.5 with NaOH.
Fermentation is carried out in stirred, baffled tanks at 30-33°C for a period of about
19 hrs. Aeration is done at the rate of 1.5 vol/vol/min (a back- pressure of 2 bars).
NaOH is continuously added to maintain the pH: this also prevents the
crystallization of gluconic acid in the pipelines.
Harvesting is preceded by a short rest period (30 min) to allow the mycelia to settle
at the bottom of the fermenter. Later on the mycelia is forced out along with about
20% of the broth. This fraction is used again as inoculum, thereby reducing the
downtime. After certain cycles, the mycelia is separated in a rotary vacuum filter and
used as a source of glucose oxidase.
The broth contains all the three species of gluconic acid, viz., gluconic acid, glucono-
δ-lactone, and glucono-γ-lactone. These species are separated by selective
crystallization by preparing supersaturated solution and seeding with appropriate
species. For example, gluconic acid can be crystallized out at temperatures below
30°C, glucono-δ-lactone at 36-57°C, and glucono-γ-lactone at temperatures above
70°C. Pure gluconic acid can be produced by full neutralization with Ca(OH)2,
followed by regeneration with H2SO4. The final step entails removal of residual
calcium ion by ion-exchange resin.
The theoretical yield of gluconic acid from anhydrous glucose is 109% (m/m).
Under practical conditions, the yield is 90% for a good fermentation.
326
Spores of Aspergillus niger
(NRRL 3)
Sterilized fermentation medium Solid sporulation medium
(22% glucose + other ingredients)
Propagator
(spore germination and mycelia formation)
NaOH 10% vol/vol
(to bring pH to 6.5) Fermentor
(baffled, aerated, stirred)
Fermentation
* Temperature: 30-33OC
* Aeration: 1.5 vol/vol/min
* NaOH: continuously added to maintain pH at 6-7
- This also prevents the problem of crystallization
of the acid in the pipes
* Duration: 19 hrs
Harvesting
* Stop aeration and allow the mycelia to settle for 30 min
* Force out mycelia along with 20% volume of broth
* Transfer the remaining broth for further treatment
* Reuse mycelia (along with broth) for up to 3 times
Broth
(contains mixture of gluconic acd,
glucono--lactone and glucono--lactone)
Recovery
Fig. 22.3 Mold process for gluconic acid production
Gluconic acid can also be produced by bacterial fermentation. See Fig. 22.4.
Starch
Cornsteep liquor
Ammonium sulfate Hydrolysis by bacterial
-amylase till 20-25 DE
Gluconobacter Sterilize
suboxydans
Fermentation
CaCO3 * Air: 1 vol/vol/min
for neutralization * Temp.: 30oC
* Initial pH: 6
* Final pH: 3.2
* Duration: 24 hrs
DE falls to 10
(all sugars utilized)
Crystallization
Fig. 22.4 Bacterial process for gluconic acid production
The basic raw material is starch. Cornsteep liquor and ammonium phosphate are
added to meet nitrogen- and growth factor requirements. CaCO3 is used during
the fermentation for neutralization of the acid. The duration of fermentation is
about 24 hrs.
327
CHAPTER 23
MICROBIAL PRODUCTION OF AMINO ACIDS
23.1 INTRODUCTION
Amino acids are building blocks of proteins. In mammals, and especially in man, a
number of amino acids cannot be formed by generally known biosynthetic
mechanism. This is basically because man cannot synthesize the α-keto acids needed
for the synthesis of corresponding amino acids. Such amino acids are called essential
amino acids (it is more correct to call them indispensable amino acids), and they must
be supplied externally, for instance, through diet.
23.2 PRODUCTION ASPECT
Amino acid can be synthesized quite economically by chemical means. Chemically

synthesized amino acids are usually racemic mixtures of D- and L-isomers. It is to be
noted, only the L-isomer is of value for flavor application or for food/feed
supplement. The D-isomers are biologically inactive. To date, chemical resolution of
DL-racemic mixtures has been relatively expensive, although a Japanese process is
apparently in commercial use.
Microbiological production of amino acids on the other hand does not involve
operational difficulties of high temperature and pressure often encountered in
chemical catalytic processes. The microbial process can be carried out under ambient
conditions. Besides, the end products produced by them can be obtained in a pure
form because the enzyme systems in an organism are known for high selectivity.
23.3 PRODUCTION OF L-GLUTAMIC ACID
In 1995, the annual worldwide production of glutamic acid was 370,000 MT. By
2002, the production reached 1 million MT. The main producers of glutamic acid are
Japanese companies: Ajinomoto Co., and Kyowa Hakko Kogyo Co.
Glutamic acid is a negatively charged dicarboxylic acid having the structure:

HOOCCH2CH2 CHCOOH
NH2
23.3.1 USES
Monosodium salt of glutamic acid (MSG) is used as flavor enhancer. It enhances the
flavor of meat and meat products. Glutamic acid is also the starting material for a
variety of specialty chemicals. N-acyl glutamate is used in cosmetics, soaps, and
shampoos. Oxypyrrolidone carboxylic acid is used as a natural moisturizer. Amides of
glutamates can be used as gelatinizing agents: it can gelatinize mineral oil spilled in
the ocean. In particular, this property can be gainfully utilized for marine
antipollution purposes.
23.3.2 MICROORGANISMS
Most glutamic acid producing bacteria are Gram-positive, non-spore forming, non-
motile, and biotin dependent. Examples of some of the more important glutamic
acid bacteria are given in Table 23.1. Overproduction of glutamic acid is possible
through the use of organisms dependent on biotin, oleic acid, or glycerol (they are
auxotrophic mutants).
Table 23.1 Examples of commercially employed glutamic acid bacteria
Genus Representative organism

Brevibacterium B. divericatum, B. flavum
Corynebacterium C. glutamicum, C. lilium
Microbacterium M. flavum var glutamicum
Arthrobacter A. globiformis
23.3.3 BIOSYNTHESIS OF GLUTAMIC ACID
Glutamic acid is synthesized through (i) glyoxylate cycle as an oxaloacetate generating

system (without CO2 fixation), and (ii) through Phosphoenol pyruvate (PEP) to form
oxaloacetate with CO2 fixation. See Fig. 23.1 biosynthetic pathway.
Glucose
CO2
Glucose-6-P
Triose-P Pentose-P
CO2
CO2 Acetyl-SCoA
CO2
CO2
CO2
Oxaloacetate Citrate
Malate Isocitrate
Glyoxylate CO2
Fumarate -ketoglutarate
NH3, NADH + H+
Succinate
H2O, NAD+
L-glutamic acid
Fig. 23.1 Biosynthesis of glutamic acid
329
The bacteria use both EMP and HMP pathways. Compounds from these pathways
are fed into TCA cycle. In all, the bacteria use about 16 enzymatic steps. The final
product is formed by reductive amination of α-ketoglutarate.
Two enzymes play very important role in the biosynthesis of glutamic acid. They are
(i) PEP carboxylase, and (ii) α-ketoglutarate dehydrogenase. The efficiency of CO2- fixation
depends on PEP carboxylase activity. α-ketoglutarate dehydrogenase can transform
α-ketoglutarate to glutamic acid as well as CO2 + water via succinyl-ScoA. Bacterial
α-ketoglutarate dehydrogenase is such that it carries out the preferential synthesis of
glutamic acid at a rate several times faster than that for the oxidation.
23.3.4 GLUTAMIC ACID EXCRETION AND CELL WALL PERMEABILITY
The overproduction of glutamic acid is in fact a function of cell wall permeability of the
bacterium. Under normal condition, the cell wall is impervious enough to block the
flow of glutamic acid that has been synthesized inside the cell. Accumulation of
glutamic acid inside the cell soon exerts product inhibition and the organism, in response
to this, stops the synthetic reaction. This is an undesirable aspect when it comes to
overproduction of glutamic acid. The elucidation of biochemistry of glutamic acid
biosynthesis has made it possible to overcome this effect. The basic strategy used in
this case is to weaken the cell wall of the bacterium. This can be achieved by adding,
during the growth phase, agents capable of inhibiting cell wall synthesis, e.g., penicillin,
cephalosporin, detergents, etc. An equivalent effect can be achieved by limiting biotin
content in the medium or by supplying saturated C16 and C18 fatty acids. The
explanation for the last sentence runs as follows: biotin is a cofactor of acetyl-ScoA
carboxylase, an enzyme responsible for the conversion of acetyl-ScoA to malonyl-
ScoA (the starting compound for the synthesis of fatty acid, viz., oleic acid). Good
supply of biotin in the medium leads to normal production of oleic acid. Oleic acid in
turn combines with inositol, mannose, etc., to form phospholipid, which is the
component of cell membrane (see Fig. 23.2). Limiting biotin in the medium leads to
synthesis of weak cell membrane, thus producing leaky cells. Leaky cells cannot
withhold the glutamic acid synthesized inside the cell. The addition of saturated fatty
acids also has similar function. They repress the synthesis of oleic acid.
The elucidation of biochemistry of glutamic acid biosynthesis has been a turning

point. It has made possible to use molasses for glutamic acid production. The initial
failure was, of course, due to high biotin content in the medium.
Phospholipids
Cell wall (inhibition)
Cell membrane
Penicillin Biotin (promotion)
Cephalosporin
Glucose Acetyl-SCoA Oleic acid
Glycerol C16 and C18
Glutamate Mannose (suppression)
Inositol
Excretion
Fig. 23.2 Relation between cell wall and excretion in glutamic acid bacteria
330
Glutamic acid can be produced by four methods:
1. By the hydrolysis of wheat gluten, soybean cake, or other proteinaceous

materials
2. By cleavage of pyrrolidone carboxylic acid (5-oxoproline)
H 
O N COOH HOOCCH2CH2CHCOOH
NH2
5-oxoproline Glutamic acid
3. By one-stage fermentation (involving one organism)
4. By two-stage fermentation process (one organism produces α-ketoglutarate

and another organism produces glutamic acid from α-ketoglutarate)
Because one-stage fermentation is universally used, discussion on the glutamic acid

production will be limited to one-stage fermentation, which may be of batch-, fed-
batch-, or continuous type.
23.3.5.1 One stage fermentation process
The most widely used carbon and energy source is the carbohydrate, such as
molasses and starch hydrolysates. Certain strains can also utilize non-carbohydrate
materials such as acetic acid. When molasses is used, most of the growth
requirements are met. The nitrogen requirement is met normally by supplying
gaseous ammonia. Gaseous ammonia fulfils several objectives: (i) supplies nitrogen,
(ii) maintains pH by neutralizing the accumulated acid, and (ii) avoids unwanted
dilution of the medium.
Whatever the mode of operation (batch, fed-batch, or continuous), the basic strategy
during the production phase (which is different from the active growth phase) is the
creation of a condition unfavorable for further growth of the organism (either by
limiting key components or by adding agents that interfere with their growth).
The fermentation is a highly aerobic one. Air pressure in the fermentation vessel is
critically maintained slightly above the actual requirement for cellular respiration.
Inadequate air supply leads to accumulation of lactic and succinic acid while excess
air supply promotes α-ketoglutarate accumulation.
Batch process
In the batch process, the sugar concentration is maintained at 10% glucose

equivalent. pH is maintained near neutrality with ammonia gas. The biotin content in
the medium should be suboptimal: it has been worked out that biotin should be 1-5
μg/liter of medium. Penicillin and similar other agents are added (to weaken the cell
wall) during the growth phase. The duration of fermentation is around 3 days at 30-
331
35°C. Glutamic acid excretion starts after the intracellular concentration of glutamic
acid has reached 50 mg/g of dry cell.
Nakashi and coworkers (1981) have patented a method which uses Corynebacterium
glutamicum that has been mutated to acquire temperature-sensitivity remediable with
an unsaturated higher fatty acid. The organism can readily overproduce glutamic acid
even in biotin-rich medium. Additionally, the process does not require addition of
agents for counteracting effects of biotin.
In this method, the mutant is initially cultured at a low temperature (28°C)

environment wherein the strain shows adequate growth until multiplication proceeds
to a certain desired extent. The temperature is then elevated to 40°C wherein growth
is reduced without an unsaturated fatty acid. This near-starving condition leads to
overproduction of glutamic acid whether the medium contains unsaturated fatty
acids or not.
The pH preferred for the fermentation is 6-9. A duration of about 72 hrs appears to
be sufficient.
Fed-batch fermentation process
The medium composition is basically similar to that for a batch process. At the
beginning of the fermentation, 0.65 ml oleic acid/liter of medium is added. The pH
is set at 8.5 with ammonia and automatically maintained at 7.8 during the course of
fermentation. After about 14 hrs, the temperature is increased from an initial 32-
33°C to 38°C due to growth. After the initial glucose is metabolized down to the
level of 0.5-2% (from an initial of about 12% glucose equivalent in the molasses
medium) glucose feeding is done until the fermentation is complete. On an average,
160 g of glucose is fed per liter of medium. Glutamic acid content is analyzed hourly.
Aeration is controlled in such a way that CO2 of the exhaust gas does not exceed
4.5% by volume. Fermentation is stopped after 30-35 hrs after the glutamic acid
production reaches about 100 g/liter. In general, the fermentation titer of glutamic
acid in industrial production is about 88 g/ liter.
Continuous process
Reference will be made here to the invention made by Tatsuya and coworkers
(1999), the work being assigned by Ajinomoto Co., Japan. Their system of
continuous fermentation allows simultaneous growth (of glutamic acid bacteria) and
accumulation of glutamic acid. Fermentation is carried out in a single fermenter.
There is no provision for cell recycle. Unlike in fed-batch or batch process, there is
no requirement for terminating the cell growth for inducing glutamic acid
accumulation. The schematic of the system published by the investigators is given in
Fig 23.3.
In this system, medium containing adequate nutrients for growth of the bacteria is
continuously fed but the growth is controlled through temperature, surfactants,
antibiotics, biotin concentration, and the like. After addition of the inoculum to the
fermenter, feeding and extraction of the medium is started from an appropriate
stage, intermittently or continuously. An example of the experimentation published
by the investigators is as follows:
332
The organism used for the study was glutamic acid producing bacteria Brevibacterium
lactofermentum ATCC 13869. The organism was grown in a shaker flask containing
sterilized (115°C for 15 min) medium of following composition at 30°C for 24 hrs:
3% glucose, 0.01% KH2PO4, 0.004% MgSO4.7H2O, 0.4% urea, 0.002%
FeSO4.7H2O, 0.002% MnSO4.4H2O, 1.5% liquid soy protein hydrolysate, and 300
g biotin/liter.
The seed culture was transferred aseptically to a small fermenter containing

following medium composition and having provision for agitation, aeration, feeding,
extraction, etc., needed for the continuous fermentation: 6% glucose, 0.01%
KH2PO4, 0.01% MgSO4.7H2O, 1.5% liquid soy protein hydrolysate and 300 g
biotin per liter of medium. The pH was adjusted to 7.5 with NH3 and aeration done
at the rate of 1.1 vol/vol/min. Fermentation was carried out at 30°C.
After 5 hrs of culturing, polyoxyethylene sorbitan monopalmitate (a surfactant) was

added to a final of 500 mg/liter. To the fermenter, a feeding solution containing
18% glucose, 0.01% KH2PO4, 0.01% MgSO4.7H2O, 1.5% soy protein hydrolysate
and 500 mg/liter of polyoxyethylene sorbitan monopalmitate was continuously
added at the dilution rate of 0.11 per hour (that is, the same volume was
continuously extracted). The sugar concentration of the extracted culture was
maintained at 5 g/liter or less.
The outcome of the cultivation for 40 hrs was such that the yield of glutamic acid
was 56% and the productivity 5 g/liter/hr. Compared to fed-batch method (control
used in this experiment), the productivity in the continuous culture was found to be
2-fold higher. The productivity was calculated as follows:
Glutamic acid concentration


Flow rate of drawn-out
Productivity in drawn-out solution (g/liter)  solution (liter/hr) 
(g/liter/hr)  Working volume in fermenter (liter)
Pump
Sugar, nutrient
Extracted broth
Air
Fig. 23.3 Schematic of continuous fermentation
23.3.6 COMMERCIAL YIELD OF GLUTAMIC ACID
After the growth phase, an ideal fermentation should proceed as:

C6H12O6 + NH3 + 1.5 O2  C5H9O4N + 3 H2O
333
This represents a 100% molar conversion, or 81.7% weight conversion of glucose
anhydrous to glutamic acid. Under practical condition, the molar conversion is 50 to
75% by resting cells.
23.3.7 PURIFICATION OF GLUTAMIC ACID
Before commercial methods of glutamic acid purification are discussed, a brief

mention of the polymorphism exhibited by the acid will be made here.
Polymorphism, contextually, is the existence of a chemical compound to adopt
different crystalline arrangements. Although chemically identical, different
polymorphs display a variation in physical properties (e.g., crystal morphology,
density, solubility and color) which exert an influence on the performance of the
product, for example, the bioavailability and shelf-life of pharmaceutical compounds.
L-glutamic acid exhibits two polymorphs, viz., (i) the metastable -form that is
prismatic or granular in nature, and (ii) the stable -form that is needle-shaped.
When a saturated solution of glutamic acid is heated to 70°C and cooled rapidly, -
crystals are formed. When the saturated solution at 90°C is cooled gradually to 40-
50°C, -crystals are formed. The -form is not preferred because it hinders
subsequent filtration or centrifugation associated with the purification steps.
In the industrial processes, the -form of glutamic acid is generally preferred

because of the ease in subsequent downstream processing. However, because of its
metastable nature, the crystals will slowly transform into the stable -form at
elevated temperatures (above 55°C). During the process, dehydration of glutamic
acid inevitably takes place thereby leading to the formation of appreciable amounts
of pyrrolidone carboxylic acid. Various methods have been developed to increase the
purification rate and minimize loss. These developments have largely been based on
the manipulation of temperature-, solvent addition-, and solution concentration
regimes. A method developed by Gallagher (1976) is as follows:
The fermentation broth containing glutamic acid not less than 60 g/liter (if it is less,
evaporation can be carried out) is adjusted to pH 4.5 with H2SO4 at room
temperature (25°C). If the pH is already low, it is adjusted with NH3. The broth is
agitated constantly for about 20 min until seed crystals begin to appear. Thereafter,
the broth is acidified to pH 3.2 (isoelectric pH of glutamic acid) and heated to 50-
55°C (heating above 55°C is avoided because this leads to formation of -crystals).
Next, continuing agitation, the temperature of the mixture is brought down to 20°C.
Crystals that consist of 70-80% -form will now be formed. The crystals are
recovered by centrifugation or filtration. The product can now be further purified to
meet the exacting criteria (e.g., recrystallization, ion exchange, decolorizing, etc). If
needed, the acid can be neutralized with NaOH to obtain monosodium glutamate.
Recently, Yoshiki and coworkers (2005), have filed a patent (assignee: Ajinomoto
Co., Japan) of glutamic acid purification. The method involves rapid transformation
of primary crystals into -form (which is later recovered) by the application of
activated carbon. The principle of the method is as follows:
334
Broth containing about 150 g glutamic acid per liter is mixed with 1% activated
carbon (w/w, based on the amount of glutamic acid) and heated to 90°C. The
mixture is stirred at this temperature for sufficient time interval until at least 30% of
the -crystals of glutamic acid are converted into the -form. The addition of
activated carbon has a very prominent effect in increasing the rate of transformation.
The experimental finding showed that the transformation time for carbon-treated
glutamic acid broth was 75 min against a control (containing no carbon), which was
588 min. Similarly, the level of pyrrolidone carboxylic acid formed was 4 mole% for
the treatment and 28 mole% for the control.
23.4 MICROBIAL PRODUCTION OF L-TRYPTOPHAN
L-tryptophan is an aromatic, indispensable amino acid. Its chemical name is α-

amino-β-indole propionic acid. The condensed structure is:
CH2CHCOOH
NH 2
N
H
This amino acid is produced by Japanese companies, viz., Showa Denko, Ajinomoto
Co., and Tanabe Seiyaku. The annual world production in 1995 was 400 MT. By
2002, the production volume reached 600 MT.
23.4.1 USES
Therapeutically, it is used as a component solution for transfusion. Since it is an

indispensable amino acid, it can also be used for the fortification of food
commodities like corn (maize) that contain limiting amounts of tryptophan.
23.4.2 MICROORGANISMS USED
Microorganisms used for the production of tryptophan are highly improved strains
of bacteria. Some of the industrially exploited or tested microorganisms are:
Corynebacterium glutamicum, Brevibacterium flavum, Bacillus subtilis, Candida fumicola,
Achromobacter liquidium, Pseudomonas putida, etc.
23.4.3 BIOSYNTHETIC PATHWAY
The microorganism uses chorismic acid pathway for the synthesis of tryptophan. Since
the synthesis of phenylalanine and tyrosine also share the same pathway, it is
obvious that tryptophan-producing strains are auxotrophic mutants. The outline of
the biosynthetic pathway followed by Corynebacterium glutamicum strain is shown in
Fig. 23.4.
335
Erythrose-4- COOH
phosphate 3-Deoxy-2-Keto-D-Arabino-
Heptulosonic acid-7- Shikimic acid HO OH
Phosphoenol phosphate (DHAP) OH
pyruvate
COOH
Chorismic acid O
Metabolic block
OCCOOH
due to mutation
OH
Feedback COOH
inhibition Prephenic acid Anthranilic acid
NH2
Phenylalanine Tyrosine Repression
L-tryptophan
Fig. 23.4 Biosynthetic pathway of tryptophan in C. glutamicum
There are three main methods for the production of tryptophan:
1. Production by fermentation
2. Production by microbial conversion
3. Production by enzymatic method
Detailed information on any of the above methods is not available. Literatures are
therefore based only on classical researches and patents filed for the method.
Because of relevance, only two of the above methods will be described here.
23.4.4.1 Production by fermentation
In the overproduction of tryptophan by fermentation, the basic strategy is to obtain

auxotrophs and/or analog resistant strains by mutation. Mutants carrying multiple
markers are more suitable as they are more stable. The classical work carried out by
Nakayama (1976) is used here as an example. He used Corynebacterium glutamicum KY
9456, a double auxotroph of phenylalanine and tyrosine for further mutation. A
stepwise mutation finally produced a strain called Px-115-97 that produced
significantly higher amounts of tryptophan. The parent strain was mutated in a
stepwise manner to develop resistance to 5-methyl tryptophan (5MTr), tryptophan
hydroxamate (TrpHxr), 6-fluoro tryptophan (6FTr), 4-methyl tryptophan (4MTr),
parafluoro phenylalanine (PFPr), paraamino phenylalanine (PAPr), tyrosine
hydroxamate (TyrHxr), and phenylalanine hydroxamate (PheHxr). The genealogy of
the bacterium used in the study appears in Table 23.2.
The auxotrophy produced metabolic block while the analog resistance released the
bacterium from repression by tryptophan. The yield gradually increased from a mere
0.15 g/L to final of 12 g/L in a nutritionally balanced cane molasses medium of
following composition: cane molasses (10% glucose equivalent), MgSO4.7H2O
(0.025%), KH2PO4 (0.05%), K2HPO4 (0.05%), (NH4)2SO4 (2%), CaCO3 (2%), and
Cornsteep liquor (2%). The pH was kept at 7.2. The organism was still sensitive to
336
phenylalanine and tyrosine, which implied that there was further scope for the
development by building multiple analog resistances.
Table 23.2 Genealogy of C. glutamicum mutated for tryptophan production
Genealogy/mutation Production (g/liter)
KY 9456 Phe,Ty 0.15

5MTr, TrpHxr, 6FTr, 4MTr
4MT-11 4.9
PFPr
PFP-2-32 5.7
PAPr
PAP-126-50 7.1
TyrHxr
Tx-49 10
PheHxr
Px-115-97 12
Following Nakayama’s work, several workers (Kino and coworkers, 1988; Ozaki and
coworkers, 1989; Ishida and coworkers, 1989, etc.) have patented improved methods
for the microbial production of tryptophan. Most of the works are based on
genetically engineered strains of Corynebacterium glutamicum. In the commercial
fermentation, the fermentation titer is about 58 g/liter.
23.4.4.2 Production by microbial conversion
Various microorganisms including auxotrophic- and regulatory mutants were

selected by different workers. Candida fumicola, Corynebacterium glutamicum, Bacillus
subtilis, and E. coli were studied. In this method, precursor of tryptophan such as
anthranilic acid or indole (which is toxic to microorganisms at higher concentrations)
is used in the medium. These precursors are chemically synthesized. The conversion
is catalyzed by tryptophanase (= tryptophan synthetase). A bioconversion method
developed by Stephen and coworkers (1990) utilizing recombinant E. coli will be
described next.
These workers have described a multi-stage microbial process for the production of
tryptophan where biocatalyst and bioconversion stages are segregated. In the
biocatalyst production stage bacterial host cells are transformed with a vector
containing a DNA sequence coding for tryptophanase wherein the expression of
tryptophanase gene is directly controllable; the transformed cells are induced to
synthesize tryptophanase; and then in a subsequent bioconversion stage the reaction
substrates for tryptophan synthesis are added and tryptophan which accumulates in
the reaction mixture is optionally isolated.
In an example, the workers used recombinant E. coli MD33 (in which plasmid
encoding for tryptophan was used, e.g., PIMS1015) as a tryptophanase producing
337
host. The engineered cells were first grown in a suitable broth at 30°C to maintain
the plasmid in low copy number state. Once the desired cell density was reached, the
temperature was raised to 37°C to induce an increase in the copy numbers of the
plasmid, with a concomitant rise in the amount of tryptophanase produced. The
tryptophanase levels reached 15-90% of total cell protein.
The cells were harvested and transferred to bioconversion vessel which contained
cosubstrates consisting of ammonium acetate (870 mM), sodium pyruvate (620 mM),
KH2PO4 (22 mM), pyridoxal-5′-phosphate (0.7 mM), and ethanol (870 mM). The pH
and temperature were maintained at 8.5 and 30°C, respectively. Fermentation was
carried out in a fed-batch mode by continuously feeding 5 M indole (suspended in
ethanol) such that the concentration of indole in the fermenter was less than 10 mM.
After a fermentation time of 75 min, the tryptphan titer was 24 g/liter, with a
productivity of 19 g/liter/hr. More than 99% of the feed indole was found to be
converted to tryptophan.
23.4.4.3 Production by enzymatic method
This method utilizes the enzyme tryptophanase, which catalyzes reversible synthesis of
tryptophan. The enzyme is produced by bacteria such as Achromobacter liquidium,
Pseudomonas putida, etc. Depending on the organism used, the major substrate can be
indole or indole derivatives.
Interesting as the above two methods appear, they have not gained commercial
importance because the costs of precursors such as indole and anthranilate are
prohibitively high at the present time.
23.4.5 RECOVERY
The culture broth is subjected to strongly acidic cation exchange resin. The adsorbed
tryptophan is then eluted from the resin with 0.5 N aqueous ammonia, and
crystallized to obtain crude crystals. The latter is dissolved in a small amount of hot,
50% aqueous ethanol, decolorized with activated carbon, and recrystallized to obtain
pure tryptophan crystals.
A method patented by Kono and coworkers (1991) is claimed to be simple and

economical. In this process, the crude tryptophan is dissolved in hot (~ 90°C) water-
acetic acid solution (1+1) and heated for 2 hr. Activated carbon and filter aids may
also be added in the mixture. The mixture is now cooled to 5-10°C to crystallize
tryptophan (tryptophan crystallizes at concentrations exceeding 25 g/liter). The
crystals are filtered, washed with cold water, and dried under reduced pressure. The
purity and yield claimed for this process are 99.1% and 98.9%, respectively.
23.5 MICROBIAL PRODUCTION OF L-LYSINE
L-lysine or α, ε-diaminocaproic acid is indispensable to man. It is a limiting amino acid

in cereal grains. Over 80% of the lysine commercially produced is used in animal
feed, and the annual demand is rising at the rate of 10%.
338
The condensed formula of lysine is:
 
CH2CH2CH2CH2CHCOOH
NH2 NH2
Commercially, lysine is available as lysine monohydrochloride. The amino acid can be

produced by chemical as well as microbial method. The annual world production of
lysine in 1995 was 70,000 MT. By 2002, the production volume reached about
600,000 MT.
23.5.1 BIOSYNTHESIS OF L-LYSINE
Lysine biosynthesis can occur by two different pathways, viz., (i) Diaminopimelate
pathway, and (ii) Aminoadipate pathway. The former pathway is found in bacteria,
certain lower fungi, algae, and higher plants while the latter is found in classes of
lower fungi, higher fungi, and Euglena (flagellated protozoa).
In the diaminopimelate pathway, the carbon chain is synthesized from pyruvate and
aspartate (and thus categorized as member of aspartate family). Other members of
amino acids that share diaminopimelate pathway are threonine, isoleucine, and
methionine. The important steps of diaminopimelate pathway are given in Fig. 23.5.
Isofunctional Metabolic block in mutants

enzymes Feedback
inhibition Feedback
inhibition
Aspartate Aspartatyl Aspartic -semialdehyde Homoserine Threonine
phosphate
Pyruvate
Dihydrodipicolinate
Repression
Diaminopimelate
Feedback Methionine Isoleucine
inhibition L-lysine
Fig. 23.5 Diaminopimelate pathway for lysine synthesis
Great developments have been made in lysine production technology since the first
discovery of microbial production of lysine in the 1950s. Today, lysine can be
produced by chemical-, microbial-, or enzymatic processes. The microbial process,
which is still at the forefront, utilizes genetically improved microorganisms. The
improvements are based on overproduction by the improved strains, development in
fermentation protocols (media formulation, optimization of process variables, etc.),
and refinement in purification techniques.
Microbial methods of production can be classified as:
1. Production by homoserine auxotrophs
339
2. Production by multiply improved strains
3. Production by enzymatic method
Although several methods of lysine production exist today, their details are still
being closely guarded. Because of relevance, only a few methods will be described in
the following sections.
23.5.2.1 Production by homoserine auxotrophs
The microorganisms used here are auxotrophic mutants of C. glutamicum. Double

auxotrophs, which require in addition to homoserine at least one of amino acids, viz.,
thre, met or ile for growth have been found to be highly stabilized. It may be stated
that the overproduction is due to the release of aspartokinase from concerted
feedback inhibition by branch end products due to the metabolic block (Fig. 23.5).
Fermentation is carried out in batch-, fed-batch-, or extended fed-batch mode.
Patents are appearing for continuous fermentation also.
In batch fermentation, microorganism grows until one or more of essential nutrients

is (are) exhausted or until fermentation conditions become unfavorable. In fed-
batch- or extended fed-batch fermentations, one or more nutrients is (are)
continuously or intermittently supplied to the culture medium, either from the
beginning of fermentation or after the culture has reached a certain stage, or when
nutrients are exhausted. The microorganism grows at a growth rate dictated by the
rate or timing of nutrient feed. In general, a single nutrient (very often a carbon
source such as glucose) is fed into the fermenter in order to overcome substrate
inhibition and high osmotic pressure.
An interesting variant of extended batch or fed-batch fermentation is the repeated

batch or fed-batch or fill-and-draw fermentation. In this method, a part of the
fermentation broth is removed at a certain time of operation, while feeding
continues. This extends the fermentation operation and leads to high product
concentration.
The carbon sources for the production can be molasses, starch hydrolysates, and in
some cases, acetic acid and ethanol. Cane molasses is the most important, though.
The fermentation occurs at neutrality. NH3 can be added to control the pH and
meet nitrogen requirement. Biotin is very important for growth and production: it
must be greater than 30 μg/liter. The requirement of biotin is variable and so is the
explanation behind it. In biotin-dependent strains, the excretion results from the
leaky cell wall. On the other hand, biotin is a coenzyme needed for the
decarboxylation-conversion diaminopimelate to lysine.
The seed culture is prepared in stages (see Table 23.3). The final seed culture
necessarily contains cane molasses for acclimatizing the organism with future
environment. The production medium contains 20% glucose (from cane molasses)
and 1.8% soybean meal hydrolysate. The amounts of growth factors (homoserine or
threonine and methionine) are added in suboptimal levels. Since the most important
intermediate is aspartic acid, its inclusion in the medium increases the yield of lysine.
340
Fermentation is carried out at about 28°C. Aeration is a crucial aspect of lysine
fermentation. It is kept at greater than the actual requirement for respiratory growth.
Oxygen deficiency may lead to lactic acid production at the cost of lysine, although
not as significant as in the case of glutamate production. The duration of
fermentation is about 3 days. The yield is about 40-50% based on sugar consumed.
Table 23.3 Composition of the seed culture medium for lysine production
Seed culture 1 Seed culture 2 Main culture

Glucose 2% Cane molasses 5% Molasses (20% glucose-
equivalent)
Peptone 1% (NH4)2SO4 2% Soybean meal 1.8%
Meat extract 0.5% Cornsteep liquor 5% hydrolysate
NaCl 0.25% CaCO3 1%
23.5.2.2 Production by enzymatic method
This method is used by Toray Company, Japan. The method in principle utilizes two
enzymes, viz., racemase and hydrolase to transform DL-α-aminocaprolactam (DL-ACL) to
lysine. In the industrial process, DL-ACL is produced synthetically using chemicals
such as NOCl, cyclohexane, NH3, HCl, etc., in a series of reaction steps (Fig. 23.6).
ACL is a compound industrially used in the prepraration of synthetic fibers, such as
Nylon-6.
Racemization and hydrolysis are the final reactions for producing L-lysine. The
reactions may be outlined as:
Racemization Hydrolysis
DL-ACL   L-aminocaprolactam  L-lysine
NOCl
Cyclohexane -amino- DL--amino caprolactam
cyclohexane oxime Beckmann O
NH3 NOH rearrangement
Hydrolase HN  NH2
 NH2
Racemase
L-lysine
Fig. 23.6 Outline of enzymatic synthesis of L-lysine
Aminocaprolactam racemase (EC 5.1.1.15) is produced by Achromobacter obae using

DL-ACL as an inducer. This enzyme specifically acts on neutral cyclic amides.
Cryptococcus laurentii, another organism, produces hydrolase inductively in a medium
containing L-ACL, glucose and other components. A similar optimum pH values for
both the enzymes allows efficient conversion, which appears to be a single step.
Industrially, resting cells of the above two organisms are used for the production of
enzymes. There is no detailed information about the use of enzyme but literatures
341
on its study are available. It has been reported that incubation of 100 ml of 10% DL-
ACL (pH adjusted to 8 with HCl) with 0.1 g of acetone-dried cells of C. laurentii and
A. obae nov. sp. at 40°C for 24 hrs resulted in 99.8% conversion of DL-ACL to L-
lysine.
The amino acid produced by enzymatic means is relatively free from debris. It is
therefore much easier to purify the amino acid. A very high-grade lysine can be
obtained by carbon treatment and crystallization.
23.5.3 PURIFICATION OF LYSINE
Several methods are available for lysine purification, and still more are being
developed. The extent of purification is dictated by the intended end use of the
product. For animal feed supplement, the product can be produced in the form of
liquid concentrate, powder, or grains. A general method for obtaining lysine of very
high degree of purity (for food- and pharmaceutical use) is described in the
following paragraphs.
The fermented broth (containing lysine) is treated with Ca(OH)2 to bring the pH to
11. It is then heated to 100°C for 30 min and aerated at 1 vol/vol/min for 2 hrs.
The resulting mixture is acidified to pH 5 with HCl or H2SO4 to precipitate the
calcium (as CaCl2 or CaSO4). The precipitate and the cells are removed by filtration
or centrifugation and the filtrate/supernatant is passed through cation exchange
resin (IR-120, NH4+ type) in one or more stages. Then the ion exchange column is
washed with distilled water. Next, lysine is eluted with 3% NH4OH. The resulting
product is concentrated by evaporation and the pH is again adjusted to 5 with HCl.
The mixture is cooled to 20°C to obtain crystals of lysine-HCl (~ 98% purity). The
crystals are recovered by centrifugation and then dried in fluidized bed to less than
1% moisture content.
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CHAPTER 24
YEAST ENZYMES AND MINOR PRODUCTS
24.1 INTRODUCTION
Yeast enzyme production differs from other yeast processes, viz., bakers yeast
production and SCP production in two ways:
1. Yeast enzymes are low volume-high value products

2. Fermentation process is largely secondary to downstream process
Yeast enzymes are of two types: (i) intracellular, and (ii) extracellular. Intracellular
enzyme production poses certain problems: (i) enzymes are produced in very small
amounts (due to feedback inhibition, repression, etc.), (ii) being inside the cell, they
cannot be taken out unless a mechanism for disrupting the cells is used. This calls
for added cost. Besides, there is the problem of separating nucleic acids that come
along as contaminant. Unless in the case of very high value enzyme, production of
intracellular enzyme is not cost-effective. This is the basic logic why emphasis has
almost always been on the production of extracellular enzymes.
24.1 JUSTIFYING THE DESIGN OF SPECIALIZED EQUIPMENT
For intracellular enzyme production, construction of specialized equipment is not

always justified. Since the fermentation is largely secondary to downstream processing,
the former can be totally circumvented if a ready-source of biomass that contains the
enzyme is available. In the case of extracellular enzymes, however, fermentation is a
must, as it is during the course of fermentation that extracellular enzymes are
secreted. Even in such cases fermentation economics must be thoroughly
considered. The equipment constructed must be of multipurpose type to ensure
flexibility in use. Thus, taking everything into account, it is essential that the
equipment designed be small but flexible, for example, stirred tank with aeration.
Examples of some of the important yeast enzymes are: Invertase or sucrase(= β-D-
fructosidase), lactase (= β-D-glactosidase), lipase, etc.
24.2 INVERTASE
The enzyme is also called sucrase. The scientific name is β-D-fructofuranoside

fructohydrolase (EC 3.2.1.26) but can be called β-D-fructosidase in short. Both
intra- and extracellular invertases are present in yeasts. Extracellular invertase is more
abundant than the intracellular counterpart. Extracellular invertase is a glycoprotein
(containing 50% mannose) with a molecular weight of 127,000. Intracellular
invertase is non-glycosylated and has a molecular weight of 120,000.
The natural substrates of invertase are sucrose, raffinose, and stachyose. The enzyme
works optimally at 60°C, has an optimum pH of 5.5, and acts selectively on the
fructosidic linkage on the fructose side of the oxygen bridge. See Fig. 24.1.
Invertase was the first enzyme to be immobilized for use on an industrial scale. This
was developed in the UK by Tate and Lyle during the early 1940s for syrup
production.
CH2OH CH2OH CH2OH

O Site of action O O Site of action
of invertase of invertase
O
CH2OH O CH2OH O
O O
CH2OH CH2OH
Sucrose Raffinose
Fig. 24.1 Action of invertase on its substrate
24.2.1 PRODUCTION OF INVERTASE FROM BOTTOM YEAST
Today, invertase is produced from selected strains of Saccharomyces cerevisiae. The

commercial production of invertase usually starts with an accumulation step. For this
purpose, pressed bottom yeast is suspended in 20-fold amount of nutrient broth
containing 4 parts (NH4)2HPO4, 4 parts KH2PO4, 1 part Mg(NO3)2, and 1 part
KNO3. The mixture is aerated for 3-8 hrs while the pH and temperature are
maintained at 4.5 and 28-30°C, respectively. During the same period, 3-20% sucrose
solution is added continuously, a procedure that ensures reduced catabolite
repression. At the end of the process the invertase activity of yeast increases by 15-
fold.
For the most part, only a small amount of the invertase is produced intracellularly in
the cytoplasm. The rest is extracellular, located within the cell wall or between the
wall and the membrane. In fully repressed cells, all the enzymes are intracellular, thus
indicating that the extracellular form is subject to more repression.
24.2.1.2 Recovery
First of all, yeast cells are concentrated by centrifugation. The release of invertase
from yeast is achieved by destruction of the structures responsible for the retention
of the enzyme. One method is autolysis with chloroform, toluene, or ethyl acetate at
30°C for not over 3 hrs. Alternatively, yeast cells can be disrupted in a homogenizer
(Gaulin M3, at 550 bar) until about 75 g protein is obtained per kg of moist cell
mass). The cells can be suspended in 0.1 M K2HPO4 buffer (pH 7.25) for the
homogenization. Following extraction from yeast, comparatively high purification of
344
invertase is necessary for its application in foods because the enzyme preparation
usually has an undesirable, irritating taste originating from yeast.
A method developed by Helmut and coworkers (1990) for the purification of

invertase is described next. This method has been claimed to be more efficient than
the conventional method.
The ruptured or lysed mixture (obtained as above) is cooled to 15°C, acidified with
acetic acid or phosphoric acid (pH 4) and agitated for half an hour. Then the mixture
is heated at 48-50°C for 9-10 min in a heat exchanger, the heat treatment being
necessary to denature the proteins other than the invertase (which is relatively heat
resistant). Next, the mixture is again cooled to 15°C, diluted in acetate buffer (pH 4)
and the whole centrifuged to remove undesired proteins and cell debris. Finally, the
supernatant is ultrafiltered to get permeate of invertase activity 3 U/ml.
Commercial invertase is available in liquid form that is stable for a year at low
temperatures. The enzyme is generally stabilized with glycerol, which is added in
amounts exceeding 55%.
24.2.2 BIOSYNTHESIS OF INVERTASE
In Saccharomyces cerevisiae, the ability to hydrolyze sucrose is conferred by any one of

the six (or more) polymeric sucrose genes (denoted SUC 1 to SUC 6), which reside
at loci distributed throughout the genome. Strains unable to ferment sucrose can
arise either by segregation during crossing over in strains containing SUC genes or
by mutation of a known SUC gene. Segregated negatives are termed SUC 0 to
distinguish them from the mutational non-fermenters suc 1, suc 2, suc 3, …..suc 6. The
presence of any one of the SUC genes in the genome leads to the production of any
one of the two forms of invertases (intra- or extracellular, that is).
Invertase mRNA is continuously synthesized under repressive conditions and the

level of this mRNA is regulated by the presence of glucose. The hexoses regulate
them at the level of transcription. The expression of invertase mRNA present in the
cell under repressive conditions is also regulated by glucose at the level of translation
or secretion, or both. Consequently, under repressive conditions, invertase is
destroyed before secretion occurs.
The two forms of invertases are encoded by two differently regulated mRNAs,
which differ only at their 5' ends. One mRNA, which is glucose-repressible, encodes
a signal-peptide containing precursor to the secreted invertase. However, another
mRNA, which is constitutively synthesized, does not encode a complete signal-
peptide sequence, and hence the translational product remains intracellular.
24.2.4 USES OF INVERTASE
 Analysis of sucrose in food products

 Manufacture of soft-centered chocolates, fondants, etc.
 Inversion of sucrose (to a limited extent)
345
24.3 LACTASE
Lactase is the trivial name for β-D-galactoside galactohydrolase (EC 3.2.1.23). It is

an intracellular dimeric enzyme and is responsible for the hydrolysis of lactose into
glucose and galactose subunits. The mechanism of hydrolysis is shown in Fig. 24.2.
Maintenance of sulfhydryl status is required for enzymatic activity. The estimated
molecular weight of lactase is 20300. It requires K+ and divalent cations (Mn2+,
Mg2+, etc.) for the activity.
Lactase is widely distributed in microorganisms, including the bacterium E. coli.

However, relatively few species of yeasts are able to assimilate lactose. The species
that have received interest to date are Kluyveromyces fragilis (syn: Saccharomyces. fragilis),
K. lactis, Candida pseudotropicalis (imperfect form of K. fragilis), etc.
In general, the ability to assimilate lactose by K. fragilis is due to a lactose permease

system (for the intact disaccharide) and a cytoplasmic β-galactosidase.
Site of action
of enzyme

O O O O
1 O 4  + 
Lactose -D-Galactose -D-Glucose
Fig. 24.2 Action of lactase on its substrate
Lactases from various microbial sources differ in properties such as pH optima. For
example, pH optimum for bacterial lactase is around 7.0; that of fungal preparations
near 5.0; and that from yeasts near 6.0. The lactase from Corticium rolfsii is
distinguished by its unusual activity and stability at pH 1.8-2.0. Yeast lactases are
activated by K+ and NH4+ but inhibited by certain metals such as copper and iron.
24.3.1 PRODUCTION
A number of methods have been patented. In a method patented by Myrs (1956),

the yeast may be grown on a whey medium. The whey derived from cheddar- or
cream- cheese manufacture is treated to remove heat-coagulable protein by adjusting
the pH to 4.5 and heating it at a temperature of 85-104°C, until coagulation is
complete. It is then filtered off. Ammonia is added at the rate of 0.1%. The whey
thus prepared is cooled to 30°C and inoculated with 10% by weight of actively
growing yeast. The temperature is kept at 30°C, and air is supplied to the medium at
the rate of 0.1 vol/vol/min during a period of about 24 hrs. The yeast cells
propagated under this condition are separated from the spent medium by
centrifuging and then washed with warm water. The cells are then quickly frozen at a
temperature close to -1°C to inactivate the zymase. The resultant lactase-active,
zymase-inactive product is dried under vacuum.
346
Where cell-free extracts are required, recovery can be affected by autolysis followed
by salt- or solvent precipitation. A typical method of autolysis for the release of
lactase from the cells entails pretreatment of cells with 80% ethanol for 1.5 hrs
followed by autolysis at pH 6.6 and 28°C for 15 hrs. The yield is approximately 90%
(Fenton, 1982). The diagrammatic representation of the production and recovery
process is given in Fig. 24.3 (production: Myers; recovery: Fenton).
During production of lactase, maximum rate of lactase induction can be achieved by

maintaining lactose concentration of above 2 mM/L in the medium. Glucose
repression can be minimized by maintaining glucose level below 1 mM/L.
PRODUCTION RECOVERY
Whey
* Centrifugation
Pretreatment * Washing with warm water
10% actively
* pH: 4.5 * Cooling to 0oC to
growing cells
* Heating to 85-104oC inactivate zymase
to coagulate proteins * Autolysis: 80% ethanol for
0.1% NH3 * Filtration to remove
proteins 1.5 hrs, standing at 28oC,
* Cooling to 30oC pH 6.6 for 15 hrs
* Salt (ammonium sulfate)
Fermentor/Fermentation
or solvent precipitation
* Temp.: 30oC
* Aeration: 0.1 vol/vol/min Pure lactase
* Duration: 24 hrs (Recovery = 90%)
Fig. 24.3 Production and recovery of lactase
Another method of lactase production patented by Fenton (1982) will be described

next. This method utilizes Candida pseudotropicalis UCD 55-31 and has been claimed
to be superior to conventional methods.
An inoculum of Candida pseudotropicalis UCD 55-31 was prepared by cultivating the

cells for 12 hrs at 28°C in 1 liter of nutritional medium of the following
composition: whey 4%, (NH4)SO4 0.5%, K2HPO4 0.5%, cornsteep liquor 0.1%,
MgSO4.7H2O 0.05%. The pH was adjusted to 4.5 with H2SO4 and sterilized at
110°C for 45 min.
Fermentation was carried out in fed-batch mode using an initial medium of

following composition: KH2PO4 0.18%, NH4H2PO4 0.1%, (NH4)2HPO4 0.1%,
MgSO4.7H2O 5 mg/liter, MnSO4 5 mg/liter. The medium was sterilized by autoclaving
at 121°C for 30 min. The inoculum was added at the rate of 10% (v/v).
A feed of following compostion was used: lactose 36.3%, yeast extract 0.6%,
cornsteep liquor 0.3%, nicotinic acid 6 mg/liter. Sterilization of the medium was
done at 110°C for 45 min. The medium was added continuously over the 60-hr
fermentation at a feed rate of 0.006/hr. Dissolved oxygen was maintained at > 10
ppm with aeration (0.5-0.75 vol/vol/min) and agitation. Fermentation was carried
out at 30°C and pH 4.5. After 60 hrs, the cell concentration reached 18 g/liter and
lactase content 4200 units/g.
347
The cells were harvested by centrifugation. The cell paste was treated with ethanol
(yeast biomass: ethanol = 1:3.5, m/v) for 90 min. Ethanol was later recovered by
filtration. The cells were resuspended in 0.1 M potassium phosphate buffer (pH 6.6)
so that the cell concentration in buffer was about 40 g/liter. Agitation was done at
30°C for 15 hrs.
The cells were next filtered and the lactase-enriched buffer collected. The enzyme
solution had 65 units/ml of lactase activity. The yield was 45% based on the
intracellular lactase content of the harvested cells.
Because lactase solution contained significant amounts of proteolytic enzymes, it was

removed by heating the aqueous solution (containing 30-90% glycerol) to about
60°C at pH 6.4. The protease activity was reduced by 90%.
24.3.2 USES OF LACTASE
The most important use of lactase relate to hydrolysis of lactose in whey and milk.
Hereditary intolerance to lactose precludes use of milk as a valuable protein source
in large areas of Asia and Africa. In addition, lactose causes a number of problems in
dairy and allied industries because of its poor solubility, resulting in crystallization in
concentrated dairy products. Enzyme hydrolysis is helpful in overcoming these
problems. Lactase is now utilized for accelerated ripening of cheese. The major
applications may be summarized as in the Table 24.1.
Table 24.1 Uses of lactase in food/feed industries
Raw materials Product Comments/advantages
Whey Animal feed Allows more whey to be incorporated in the

animal food. Prevents lactose crystallization
in whey concentrates
Whey Lactose-hydrolyzed Used as food ingredient in bakery,
whey syrup confectionery, and ice cream products
Deproteinised Lactose-hydrolyzed Properties similar to glucose syrups of

whey permeate syrup medium dextrose equivalent
Milk Lactose-hydrolyzed Improves digestibility in lactose intolerance.

milk Increases sweetness. Prevents crystallization
in milk concentrates
24.4 LIPASES
Lipases hydrolyze triglycerides to free fatty acids, partial glycerides, and glycerol.
Their natural substrates are triglycerides of long-chain fatty acids which are insoluble
in water. Lipases hydrolyze the ester bonds at the interface between aqueous phase
(in which the enzyme is soluble) and the insoluble substrate phase. Their relative
activity towards water-soluble fatty acyl esters is low. It is this ability to hydrolyze
insoluble fatty acyl esters which distinguishes lipases from esterases.
348
The general name of the enzyme is lipase (EC 3.1.1.3). The action of the enzyme on
triglyceride is shown in Fig. 24.4.
 CH2O CO R1  CH2O OH R1 COOH

Lipase
 HCO CO R2  HCO OH + R2 COOH
3H2O
' CH2O CO R3 ' CH2O OH R3 COOH
Triacylglycerol Glycerol Fatty acids
R refers to acyl group; , , and ' refer to position of attachment of fatty acids
Fig. 24.4 Action of lipase on its substrate
Commercial lipases are produced from porcine and bovine pancreas, Candida rugosa
(yeast), Rhizopus / Mucor species (molds), and Pseudomonas species (bacteria).
24.4.1 LIPASES FROM MICROORGANISMS
Depending on the positional specificity, i.e., the ester linkages of the triglyceride they
preferentially cleave, lipases can be divided in to two groups, specific- and non-specific.
Non-specific lipases release fatty acids from all three positions of the triglyceride
molecule. α, α'-specific lipases release fatty acids only from α and α' positions. The
fatty acids present in the β-position will be spontaneously converted to
monoglycerides in α or α' position, which will be broken down again by the same
enzyme. Some of the important commercial lipases have been tabulated in Table
24.2.
Table 24.2 Properties of commercial lipases
Enzyme source pH optimum Temp. optimum, °C Positional specificity

Pancreas 8.0 50 1,3 (i.e., α,α')
Candida cylindracea 7.5 50 Non-specific
Aspergillus niger 7.0 45 1,3
Mucor lipolyticus 7.5 50 1,3
24.4.1.1 Lipase from Candida antarctica
The lipase-producing yeasts of commercial interest are species of Candida,

particularly Candida rugosa and Candida antarctica. Lipase from Candida rugosa is
approved for food in Japan. Elsewhere, it is limited to use as processing aid: the
lipase should be in an inactivated form in the food. A method described by Michio
and coworkers (1987) for the production of lipase from Candida antarctica will be
described next. This lipase is considered to be novel because it is non-specific,
thermostable, and suitable for processing high-melting fats. They have reported the
occurrence of two types of lipases, viz., lipase A (mol wt 43 kD, thermostable and
isoelectric pH 8.0±0.2), and lipase B (mol wt 33 kD, more alkali resistant than lipase
A, isoelectric pH 6.0±0.2).
349
Production
The slant culture of Candida antarctica strain DSM 3855 was cultured in a shaker flask
at 26°C for 1 day in a sterilized, aqueous medium of following composition: peptone
0.6%, trypsin-digested casein 0.4%, yeast extract 0.3%, meat extract 0.15%, and
dextrose 0.1%.
The main fermentation was carried out aerobically in an agitated, batch fermenter at
26°C for 119 hrs without pH control. The aqueous medium had following
composition: pharmamedia® 4%, yeast extract 0.5%, sucrose 0.3%, soybean oil 3%,
K2HPO4 5 mg/liter, and MgSO4.7H2O 1mg/liter. The initial pH was kept at 6.2.
The lipase yield in the fermentated broth was found to be 157 LU/ml. The
abbreviation LU refers to lipase unit (1 LU = amount of enzyme which liberates 1
mole titrable butyric acid from tributyrate per min at 30°C and pH 7 with gum
arabic as an emulsifier).
Purification
The cells were removed from the broth by centrifugation. The supernatant was
collected and ultrafiltered. The permeate was mixed with 1 volume of 99% ethanol
and stirred for 30 min at 4°C to precipitate the enzyme. The precipitate was
recovered by centrifugation. The residual lipase in the supernatant was recovered by
again precipitating with 2.5 volumes of cold, 99% ethanol and subsequent
centrifugation. The precipitates were pooled (in the pellet form) and freeze-dried to
obtain concentrate with 16,200 LU/g. Further purification was done by
chromatographic method (hydrophobic interaction) and the resulting product
vacuum-dried to obtain lipase concentrate with 92,000 LU/g.
2.4.2 USES OF LIPASE
Lipases have a number of food- and non-food uses. They can be used in the fruit
juices, baked goods, and vegetable fermentation. Lipases find use in flavor
development (by splitting low molecular weight fatty acids) in cheese, margarine, and
butter. They are also used to improve emulsifying properties of ingredients (such as
lecithin and egg yolk). In the fat and oil industry, lipases are used as hydrolytic agent
and for interesterification. Lipases also have a number of non-food uses, such as in
the production of pharmaceuticals and pesticides, waste management, and
detergents. About 1000 MT of lipase is used in the detergent industry alone.
24.5 YEAST POLYGALACTURONASE
It is a soluble extracellular enzyme elaborated by Saccharomyces fragilis in complex as

well as synthetic protein-free media. The enzyme appears to be constitutive. Yeast
polygalacturonase (YPG) appears to be a single pectic enzyme produced by the yeast
in nearly pure form. It has a specific activity of 0.179 polygalacturonase units per
milligram of protein. Although it produces di- and galacturonic acid (monomer) by
the hydrolysis of pectic acid, it is unable to attack digalacturonic acid and hence
differs from mold polygalacturonase. Also, no esterase activity is associated with it.
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A crude solution of the enzyme may be prepared by growing Saccharomyces fragilis
strain 351 in synthetic yeast-nitrogen base (devised by Wickerham), containing 3%
glucose. The yeast is cultivated in 2.5-liter flasks, holding 1 liter medium each, at 23-
25°C for 4 days. The cells are separated from the medium by centrifuging. Toluene
is added to the solution (as a preservative) containing the enzyme. The product is
stored at 0°C. The enzyme may be concentrated to 13-fold by adsorbing it on pectic
gel at a pH of 3.0 and eluting it with 1N acetate buffer at a pH of 5.
The reaction concerned in hydrolysis of pectic acid by yeast polygalacturonase

follows 3 phases, viz., (i) Rapid linear phase, (ii) Slow linear phase, and (iii) Very slow phase.
1. Rapid linear phase: Pectic acid  tetra- + tri- + di- + galacturonic acid
2. Slow linear phase: Tetragalacturonic acid  tri- + galacturonic acid
3. Very slow phase: Trigalacturonic acid  di- + galacturonic acid
The optimum pH for the rapid linear phase is 4.4. The slow linear phase starts after
about 25% of the pectic acid is hydrolyzed. The optimum pH for this is 3.3 to 3.5.
The third, or very slow, phase commences after about 50% of the pectic acid is
hydrolyzed (also proceeds best at low pH). Yeast polygalacturonase is also called
endopolygalacturonase, as the action on the pectic acid chain is random rather than
regular. See Fig. 24.5 and 24.6 for an idea about pectic acid and pectin molecule. The
term pectic acid is applied to pectic substances most commonly composed of
polygalacturonic acids and essentially free from methyl ester groups. The salts of
pectic acid are either normal or acid pectates. The compound is a polymer of D-
galacturonic acids linked by α (14) glycosidic linkages.
COOH COOH COOH COOH COOH

O O O O O
O O O O
Fig. 24.5 Partial structure of polygalacturonic acid
Galacturonic Methyl galacturonate

acid residue residue
COOH COOCH3 COOH COOH COOCH3

O O O O O
O O O O
Fig. 24.6 Partial structure of pectin molecule
351
CHAPTER 25
MICROBIAL PROTEINS
25.1 INTRODUCTION
A major problem facing the world, in particular the developing nations, is the
explosive rate of population growth. The number of humans in the world now totals
6.3 billion (as of 2006). It is increasing approximately by 94 millions annually and
could well exceed 10 billions by 2050 if left uncontrolled. Conventional agriculture
may well be unable to supply sufficient food - in particular, protein - to satisfy such
demands. At least 25% of the world’s population currently suffers from hunger and
malnutrition; a disproportionate number of them live in the developing nations
where wars, arid or changing climates, and infertile lands hamper productive
agriculture.
To cope with the ever-expanding demand for proteins, several innovative

alternatives and processes have been developed. The use of microbes as protein
producers is one of them. Microbial proteins are more commonly called single cell
proteins (SCP, a term coined at the Massachusets Intstitute of Technology around
1966), referring to the fact that most of the microorganisms used as protein
producers grow as single cells or filamentous individuals rather than as complex
multicellular organisms such as plants and animals. SCP is a generic name which
refers to proteins, dry cells, or protein concentrates from microorganisms obtained
by growing in large amounts in a variety of abundant and inexpensive culture media,
and used as protein supplement for humans and animals. SCP must not be confused
with biomass and microbial biomass. Mushroom, for example is simply a biomass.
The incidental consumption of microorganisms by man in and as food can be traced

to prehistoric times but the realization or the microbe’s contribution to total diet
protein is only recent. The first conscious attempt to grow microbes for human food
was made in Germany with the drying of brewer’s yeast in 1910. Thereafter World
War II followed, which prompted many European companies towards commercial
production of SCP. Studies have multiplied after 1960 and today we have at our
disposal a wide range of substrates, processes, and microorganisms to choose from.
The three important concomitant events that contributed to progress in SCP
production were:
1. Realization of acute shortage of protein in food and feed (especially after the
war)
2. Realization of the fact that non-renewable resources could be
microbiologically exploited
3. Realization of the fact that organic wastes could be used for protein
production
There are several advantages of SCP over conventional crops used as protein source.
Some of the more important advantages are:
 Rapid succession of generation of microorganisms

 High protein content (43-85% dry basis)
 Production is ecologically friendly
 Wide range of substrates can be used
 Production can be carried out throughout the year (i.e., does not depend on
particular season as do the conventional crops)
 Consistent quality
 Little land requirement
 More easily modified (genetically)
25.2 SOME COMMERCIAL AND SEMI-COMMERCIAL PROCESSES
Several types of fermentation processes have been proposed for the production of
SCP products. Notable among them are the British (ICI, BP, Quorn, Pruteen),
American (Amoco), Japanese (Kanegafuchi), Finnish (Pekilo), Swedish (Symba),
Canadian (Waterloo), Russian, and Cuban processes.
Except for the Waterloo process, the rest of the processes named above are of little
attraction to developing or underdeveloped countries as these processes entail high-
technology operation and use of rare- or expensive substrates (e.g., methanol,
ethanol, etc.). Ironically, it is the third world where protein is needed most.
25.3 GENERAL CONSIDERATIONS
25.3.1 RAW MATERIALS
25.3.1.1 Carbon source
Two types of carbon sources can be used, viz., (i) renewable, and (ii) non-renewable. The
choice of carbon source depends on availability of raw material and the type of
microorganism.
Renewable resources include carbohydrates from agricultural and forestry products.

Notable among them are bagasse, paper mill wastes, manure, whey, and fruit-
processing waste. Although many bacteria can easily assimilate complex
carbohydrates, there are some industrially important bacteria that cannot do the
same: they need simple sugars. In such cases, the substrate must be supplied in
pretreated form. Pretreatment of complex carbohydrates can be done by physical
and/or chemical methods. A typical pretreatment used for bagasse from cane entails
hydrolysis of the material with 10% NaOH solution for 1 hr at 180°C.
Non-renewable resources include petroleum hydrocarbon, natural gas, and chemicals

derived from them (ethanol, methanol, chemical wastes). Methanol has received
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special attention because of its high solubility in water, lack of explosion hazards,
freedom from undesirable impurities, and ease of removal from the cell product.
25.3.1.2 Nitrogen source
Nitrogen sources include ammonia, ammonium salts, urea, etc. Chemical fertilizers
can also be used when non-renewable carbon sources are used. Animal wastes also
contain nitrogen in the form of urea, uric acid and other non-protein nitrogen. Meat
processing wastes contain collagen and other protein nitrogen.
25.3.2 MICROORGANISMS FOR SCP
A large number of algae, yeasts, molds, and bacteria have been studied as SCP
sources. Among the most promising genera and species are the following:
1. Algae: Chlorella sp. and Scenesdesmus sp., etc.

2. Yeasts: Candida guilliermondii, C. utilis, C. lipolytica, C. tropicalis, Saccharomyces sp.,
Kluyveromyces fragilis, Debaryomyces kloeckeri, etc.
3. Filamentous fungi: Agaricus sp., Fusarium sp., Chaetomium cellulolyticum,
Aspergillus sp., etc.
4. Bacteria: Aeromonas hydrophila, Alkaligenes eutrophus, Spirulina maxima,
Methylomonas sp., Pseudomonas sp., Hypomicrobium sp., Acinetobacter sp.,
Flavobacterium sp., and Methylophilus sp., etc.
25.4 BACTERIAL PROCESS
Non-photosynthetic bacteria are generally preferred. Notable among the bacteria

used are given in Section 25.3.2 (above). In general, these bacteria are required to
possess following properties:
 Ability to utilize cheap and readily available substrates for relevant

fermentation
 Low requirement for nutrient supplement (e.g., growth factors)
 High specific growth rate, productivity, and yield on a given substrate
 pH and temperature tolerance
 Practicable aeration requirement and foam control
 Culture stability, including freedom from bacteriophages
 Non-pathogenicity to humans and animals
 Absence of endotoxin
 Lack of potential to mate with known pathogens (e.g., members of
Enterobacteriaceae)
 Ease of separation from the growth medium by flocculation or
agglomeration.
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25.4.1 MEDIUM FORMULATION
The substrate should be nutritionally well balanced. The ratio of carbon to nitrogen
(C:N) is maintained in the range 10:1 or less to favor high cell protein content and
minimize the accumulation of lipids or cell storage substances (such as poly-β-
hydroxybutyrate). Phosphorus requirement is met by adding feed-grade (rather than
industrial grade) phosphoric acid to prevent contamination of the cell product with
arsenic or fluoride. Other minerals are usually present in sufficient amounts in the
dilution water. If needed, minerals should be supplied in the form of sulfates or
hydroxides. Mineral salts in the chloride form cause corrosion of the equipment.
25.4.2 PROCESS CONDITIONS
The bacterial strain should preferably have a cardinal growth temperature between
37 and 55°C. This saves the cooling cost. The pH is maintained at 6-7.2 by the
addition of ammonia (or ammonium salts) or phosphoric acid at regulated amounts.
The fermentation can be either batch, fed-batch, or continuous.
For a batch process, the medium normally consists of renewable carbon sources.
The concentration based on simple sugars such as glucose, sucrose, and lactose is
usually in the range 1-10%.
In the case of hydrocarbon substrates, including methane, methanol, ethanol, n-

alkane, etc., continuous processes are used for economic and process-control
reasons. The substrate concentration is maintained at close to zero to prevent
instability resulting from substrate inhibition. This is particularly important in
methanol substrate because it exerts methanol toxicity.
Aeration, cooling, and foam control can be achieved by any of the conventional
methods. With increasing cell yield, however, both oxygen demand and heat load
decrease.
Reported cell yields for various processes based on carbohydrate or organic nitrogen
substrate range from 0.25-0.61g dry matter/g substrate utilized. For hydrocarbons,
the value may reach as high as 1.2 g dry matter/g substrate utilized.
The specific growth rates vary widely, depending on the choice of microorganism,
substrate, temperature, feed rate, etc.
The often-cited example of bacterial protein product is that produced by Imperial

Chemical Industries (ICI) and British Petroleum (BP). The ICI Pruteen process for
feed-grade bacterial protein from methanol (using Methylophilus methylotrophus) has an
operating temperature of 35-42°C. Pressure Cycle air-lift fermenter design is used for
aeration. The design uses either air or air-and-agitator (combined) system for
agitation. The specific growth rate is 0.5/h, cell density 30 g dry matter (dm)/L, and
yield 0.5 g (dm)/g of substrate used. See Fig. 25.1 for an outline of the bacterial
process.
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25.4.3 PRODUCT RECOVERY
The product is recovered at a cell density of 30 g (dm)/L. Owing to the small size of
the cells (1-2 μm) and low density (1.003 g/cm3) conventional centrifuges cannot be
used. Filtration is also unsuitable as the filters get clogged very soon. And, of course,
one cannot use filter aids. Consequently, there has been considerable interest in
recovery by agglomeration and flocculation of cells. The collected mass, after
flocculation, can be centrifuged. The trade processes for recovering biomass are
probably closely guarded secrets: ICI proprietary process carries out concentration
without resorting to flocculation while Philips Petroleum Company relies on mixing
the biomass first with much larger yeast cells. The mixed cell product is finally
centrifuged for concentration.
Medium
(simple sugars: 1-10%) Methanol
Methylophilus Controlled feeding to
methylotrophus avoid methanol toxicity
pH adjustment
Inoculum (pH: 6-7.2)
Fermentor
Batch fermentor
Control Control
* pH: 6-7.2
* pH * Temp: 35-42OC
* Aeration
* Specific growth rate, : 0.5/h
* Temperature
* Aeration: air-lift
* Foam
Recovery and processing
Product recovery (Yield: 0.5g/g methanol
and processing
Batch process: from Continuous process: from
agricultural residues methanol (ICI Pruteen process)
Fig. 25.1 Bacterial process for protein production
25.4.4 WASTE TREATMENT
It must be noted that the spent growth medium and cell wash waters from recovery
processes have high BOD. Whenever possible, the spent medium should be
purified, sterilized, and recycled. In some cases, low concentrations of inhibitory
compounds may be very costly to remove from the spent growth medium. In any
event, the residues from the cell separation must be treated before discharge.
25.4.5 PRODUCT QUALITY AND SAFETY
Bacterial proteins have three potential applications, viz., in (i) animal feed, (ii) human
food, and (iii) functional protein concentrates and isolates. Nutritional and organoleptic
characteristics are of paramount importance in the first two applications while
functional effects in food products are important in the third application. Whatever
the use, the product must be acceptable from sensory standpoint. Also, the product
must be low in nucleic acid level and free from microbial toxins, pathogens, toxic
heavy metals, and chemical residues.
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25.4.6 NUTRITIONAL VALUE
The total nitrogen content in bacterial protein is as high as 13% or a protein content
of 83% (using N6.25). However, it must be appreciated that not all nitrogen is
derived from protein. An actively growing bacterial cell can have nucleic acid
content as high as 16% (on dry basis). Nucleic acids are detrimental to humans.
Bacterial proteins in general tend to be deficient in methionine content from the
standpoint of human and animal nutrition. From the data of some workers,
performances of selected bacterial SCP products in animal feeding studies are:
 Protein Efficiency Ratio (PER) = 1.88

 Biological Value (BV) = 62-67
 Protein digestibility = 90%
 Feed conversion ratio (kg/kg weight gain) = 0.0569-0.757
(Note: for casein, PER = 2.5, BV = 75)
25.4.7 FUNCTIONAL QUALITY
Bacterial protein concentrates and isolates have been prepared for evaluation of
functional effectiveness in food including:
 Water- and fat binding properties

 Emulsion and stability
 Gel formation and thickening
 Whippability
None of these functional proteins have been commercialized. Findings suggest that
the effectives of these proteins fall short of those from soybean protein. Besides, the
cost of obtaining safety data (for microbial protein products) to satisfy the regulatory
agencies is normally very high.
25.4.8 REDUCTION OF NUCLEIC ACIDS
The problem encountered in the removal or reduction of nucleic acids has been the
major limitation to the use of SCP products as human food. Some methods have
been developed to address this problem but the common setback in them all is the
high cost, which makes the process uneconomical. A typical process for reducing
nucleic acids in bacterial proteins entails subjecting the cells to heat-shock at pH 5 at
a temperature exceeding 60°C followed by raising pH between 6 and 10.
25.4.9 ECONOMIC CONSIDERATIONS
The capital cost of bacterial biomass production will depend upon the equipment
requirement (for storing, processing and handling substrates, sterilization and
cleaning operations, product separation, recovery and drying) and local land, site
preparation, and construction costs. Bacterial SCP production costs are highly
357
dependent upon the cost of carbon and energy source, and it may range from 13-
53%.
25.5 FUNGAL PROTEINS
Fungal proteins include proteins from filamentous fungi (molds), non-filamentous

fungi (yeasts), and mushrooms. Fungal proteins are collectively called mycoproteins.
25.5.1 FILAMENTOUS FUNGI
Mycoproteins from molds can be produced by various methods, such as the Finnish
Pekilo process, the Canadian Waterloo process, the Heurty process, etc. The
advantages of using molds for the production of proteins are:
 Good at breaking down a wide range of complex substrates, e.g., cellulose,

hemicellulose, pectin, etc.
 Can tolerate low pH values, which helps in resisting infection
 Few nutritional requirements for the culture
 Ease of recovery of the biomass by filtration
 Ease of handling and drying of biomass
 Structure conferred by mycelia can be used as a basis for food fabrication
There are disadvantages as well, for example:
 Comparatively poor growth rate

 Lower cardinal growth temperatures call for cooling requirements, and there
are comparatively few thermotolerant strains to choose from
 Protein content generally is unfavorable compared to that from yeast and
bacteria
 Fermentation broths are rheologically complex and difficult to aerate
 Production of a range of undesirable metabolites, e.g., oxalic acid
mycotoxins, etc.
 Poor nutritional properties compared to proteins from yeasts and bacteria
 Genetically unstable
25.5.1.1 The Pekilo process
This commercial process has received clearance for animal feed in Finland. In this
process, the substrate is a complex mixture of monosaccharides, acetic acid, and
aldonic acids. The pH is maintained at 4-5, and the temperature of fermentation is
around 38°C. The organism used is Paecilomyces variotii. Cell concentrations of
approximately 13 kg/m3 are normal at dilution rates of 0.14-0.3/h, giving a biomass
productivity of 2.7-2.8 kg/m3h. The carbon to nitrogen ratio can be 5:1 to 15:1.
Recovery can be done by filtration or centrifugation. For drying, tray or belt dryers
can be used. High temperatures are avoided as this markedly reduces the nutritional
value. Normally, a temperature of 75°C can be used for 20-30 min to bring the
moisture content below 10%.
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25.5.1.2 The Waterloo process
Waterloo process is probably the most versatile of all the mold processes thus far
used. It uses raw materials which occur universally in large quantities as waste
biomass. The materials include agricultural wastes such as animal manures and crop
residues (e.g., straw, corn stover, bagasse), and forestry residues (such as remnants
and pulpmill sludge). The process concurrently alleviates environmental pollution
frequently generated by these wastes.
Process description
The waterloo SCP process is based on the mass microbial cultivation of a new
cellulolytic fungus Chaetomium cellulolyticum, in solid-substrate systems. The basic
generic process uses a three-stage operation which involves: (i) thermal and chemical
pretreatment of cellulosic material, (ii) aerobic fermentation of the pretreated
material with nutrient supplements, and (iii) separation of the suspended solids (the
product) from the fermented broth.
Cellulose materials provide the main carbon source for the fermentation. The main
non-carbon nutrient supplements (N, P, K, etc.) are derived from synthetic chemical
fertilizer blends or animal manure. If manure is used, it is pretreated by anaerobic
fermentation to produce methane fuel gas as a byproduct. This fuel can be used to
supply processing energy. See Fig. 25.2 for the outline of Waterloo process.
Cellulosic
residues
Thermal/Chemical 2% solids Aerobic

Filtration
pretreatment fermentation
* Temp: 37oC
CH4 (1.2 Lit/Lit/h)
* pH: 5
* : 0.24 Waste liquid
* Yield: 3.2g solids/Lit/h
(BOD and CO2- free)
* Duration: 4hr
Fertilizer N, P, K Drying
and/or (optional)
Anaerobic
Manure 8%
fermentation
* Temp: 39oC
* pH: 7-8 SCP product
* Duration: 12 days
Fig. 25.2 Waterloo process for fungal protein production
The cellulosic material is pretreated with steam, hot water, or dilute alkali (depending
on the feedstock type) to sterilize it and enhance its fermentability by swelling
and/or partial delignification. A forage-grade carbohydrate co-product of
unfermented cellulose may also be produced admixed with the main SCP product.
This co-product is rendered digestible by the action of extracellular fungal cellulase,
which is generated during the fermentation. Any residual lignin serves as direct diet
roughage in the product. Because of the large mycelial growth forms, the SCP
product can be recovered by simple filtration methods. The effluents of the process
359
are essentially free from CO2 and BOD. The process is carried out in slurry or semi-
solid system, depending on the feedstock type. The growth rate efficiency of
Chaetomium cellulolyticum is 0.24/h, one of the highest known for cellulolytic fungi.
Product quality
The protein nutritional value of Waterloo SCP is comparable with the FAO
reference standard (soymeal and a well-known fodder yeast). The average protein
content of Waterloo SCP is 45% on dry basis, which is similar to that in commercial
grade soymeal and fodder yeast. Although SCP products are often compared with
soymeal, Waterloo SCP is more similar to meat in terms of protein quality and the
spectrum of other nutrients such as fat and vitamins. It should be noted that
Waterloo SCP is more attractive than the yeast for human use because of its lower
content of nucleic acids and higher content of sulfur amino acids.
The product is gray in color and mushroomy is odor. It can be fabricated into
granules, fibers, meat analogs and powder.
25.5.2 SCP FROM NON-FILAMENTOUS FUNGI
The organism in this category includes yeasts. Notable among them are Saccharomyces
cerevisiae, Saccharomyces fragilis, Candida utilis, Candida lipolytica, etc.
25.5.2.1 General process
Some detail has already been given in production of feed yeast. To enhance protein
accumulation, the carbon to nitrogen ratio is maintained at 7:1 to 10:1. The feed rate
must also be carefully controlled so that the substrate is not utilized for microbial
activities other than the accumulation of protein. Post fermentation treatment is
done usually after the cell concentration reaches 2%. Recovery does not create
problems because the cell size is large enough to be separated by centrifugation.
Yeast cells can be recovered by decantation-centrifugation (including washing)-
drying methods. After washing undesirable traces of medium, the biomass is further
subjected to rotary vacuum filter (Fig. 17.10a and 17.10b). The cake contains 20-40%
dry matter. This is again dried to 6-10% moisture content.
A method described by Marquez and coworkers (1989) for the production of SCP
from yeast is described next.
25.5.2.2 Reduction of nucleic acid content
In yeasts, total nucleic acid content varies from 8-12% on dry basis. DNA accounts
for about 1-2% of the total nucleic acid. Various methods are available for the
reduction of nucleic acids in yeast cells. One method, patented by Robinson, entails
alkali and/or heat treatment. Slurries of food-grade yeast cells, disintegrated cold at
8000 psig, are treated at pH 9.5 and 25-60°C for about 20 min after which they are
recovered by centrifugation. The alkaline extract is adjusted to pH 6-8 and heated at
temperatures of 110-120°C for 2-60 min. The treatment precipitates the protein, and
360
the RNA fraction is released into the supernatant. The heat treatment also sterilizes
the protein fraction. The final RNA level comes down to about 2%.
The protein content in yeast SCP is 45-49% (dry basis). Fat, carbohydrate and ash
contents are 4-7%, 26-36%, and 5-10%, respectively. Yeast SCP is rich in lysine but
poor in sulfur amino acids.
25.6 SAFETY ASPECTS OF SCP
Microbial proteins have high nucleic acid contents, reaching up to 16% (dry basis).
Nucleic acids present problem in man because they are only partially metabolized by
humans. Humans do not have uric acid oxidase that converts uric acid to readily
soluble (excretable) allantoin. Consequently, consumption by man of more than 2 g
nucleic acid per day leads to development of kidney stone and gout. This amount is
equivalent to 10 g of bakers yeast. Gout results from the precipitation of uric acid in
joints. Uric acid in turn results from the incomplete metabolism of nucleic acids in
man. Ruminants do not have this problem because they have uric acid oxidase
needed for the metabolism of uric acid. Microbial proteins are therefore more
suitable as feed than food.
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CHAPTER 26
TRADITIONAL FERMENTED FOODS AND BEVERAGES
26.1 INTRODUCTION
Almost without exception, fermented foods were discovered before mankind had
any knowledge of microorganisms other than as witness to the effects of their
activity. It was simply an empirical observation that certain ways of storing food
affected desirable changes in its characteristics. Much later, pure cultures were
isolated and improved for specific applications in processing foods and beverages.
Still later, purified enzymes and immobilized cells began to be used. More recently,
microbial biomass production has been developed into an industrial activity to
obtain protein-rich food/feed supplement.
Fermented foods and beverages have a significant role in all societies and result from
the action of microorganisms or enzymes on a wide range of agricultural materials
with associated desirable biochemical changes giving significant organoleptic
improvement to the final product. As a result of the fermentation process the
product is usually more nutritious, more digestible, has improved shelf-life, and is
toxicologically and microbiologically safer.
Fermented foods and beverages are an accepted and essential part of the diet in
almost all parts of the world. The preparation involves a wide diversity of raw
materials as substrates, using technology from the most primitive to the most
advanced, and achieving an astounding range of sensory and textural qualities in the
final product. Some selected fermented foods and beverages are given in Table 26.1
and Table 26.2.
Originally the most important of these changes have been an improvement in the
shelf-life and safety of a product. We now know that, in food fermentation,
conditions of treatment and storage produce an environment in which certain
organisms can flourish and these have a benign effect on food rather than spoiling it.
Fermented foods are of diverse nature: some resulting from lactic acid bacterial
fermentation; some, alcoholic fermentation by yeasts; some, mold fermentation;
some acetic acid bacterial fermentation; and many, by combination of these
fermentations. Today a large majority of individuals preparing foods by these
processes are practicing rule of thumb methods developed over a period of years by
their forebears.
Fermentation and drying are two of the oldest methods of preparation and
preservation of foods known to mankind. Even though the original physical and
chemical characteristics of the foods may be altered during fermentation, their
nutritive values are usually retained to a great extent. Many of the food preservation
practices antedate recorded history. Throughout the centuries fermentation has been
(and still remains) one of the most important methods for preserving foods.
Relatively few people, however, are aware that many food products consumed
regularly are prepared and/or preserved by fermentation process.
Table 26.1 Some selected fermented alcoholic beverages
Beverage Substrate Microorganism(s) Country

Wine Grape juice Saccharomyces sp. Temperate: N and S
hemispheres
Beer Malt and Saccharomyces sp. Industrialized countries
adjuncts
Sake Rice Aspergillus oryzae and Japan
Saccharomyces sake
Jand Millet/rice Wild molds, yeasts and Nepal
bacteria from murcha
Mead Honey Saccharomyces sp. United Kingdom
Perry Pear juice Saccharomyces sp. UK, France
Rum Molasses Saccharomyces sp. World wide
Table 26.2 Some selected fermented foods
Product Substrate Microorganism(s) Country

Dahi Milk Lactic acid bacteria India, Nepal
Kinema Soybean Mixed flora (yeast/mold/bacteria) Nepal
Natto Soybean Bacillus natto Japan
Tempeh Soybean Rhizopus oligosporus Indonesia
Sauerkraut Cabbage Lactic acid bacteria Germany
Sinki and Vegetables Lactic acid bacteria Nepal
gundruk
The preparation and preservation of foods by fermentation processes are dependent

upon the production by certain microorganisms of chemical substances that alter the
flavor of the food and are generally inhibitive to the growth of undesirable
microorganisms. The simplest example of such action is the inhibition of toxin-
producing bacteria by lactic acid produced in many fermented foods.
Fermented foods are the result of the metabolic activity of a few species of
microorganisms among the thousands of species of bacterium, yeast, and mold
known to mankind today.
According to Steinkrauss (1997), fermented foods are food substrates that are
invaded or overgrown by edible microorganisms whose enzymes (particularly
amylases, proteases, lipases) hydrolyze the polysaccharides, proteins and lipids to
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non-toxic products with flavors, aromas and textures pleasant and attractive to the
human consumer. If the products of enzyme activities have unpleasant odors or
undesirable, unattractive flavors or the products are toxic or disease-producing, the
foods are described as spoiled.
Fermentation plays at least five roles in food processing:
1. Enrichment of the human dietary through development of a wide diversity

of flavors, aromas and textures in food
2. Preservation of substantial amounts of food through the lactic-, alcoholic-,
acetic-, alkaline-, and high salt fermentations
3. Enrichment of food substrates biologically with vitamins, protein, essential
amino acids, and essential fatty acids
4. Detoxification during food fermentation processing
5. Decrease in cooking times and fuel requirements
26.2 CLASSIFICATION OF FERMENTED FOODS
Fermented foods can be classified in a number of ways. Steinkraus has classified

fermented foods based on following categories:
1. Fermentations producing textured vegetable protein meat substitutes in legumes/cereal

mixtures: Examples are Indonesian tempeh and ontjom
2. High salt/savory meat-flavored/amino acid/peptide sauce and paste fermentations:
Examples are Chinese soy sauce and Japanese miso
3. Lactic acid fermentations: Examples are sauerkraut, cucumber pickles, yogurt,
kefir, etc.
4. Alcoholic fermentations: Examples are wines, sake, palm wines, etc.
5. Acetic acid/vinegar fermentations: Examples are apple cider, tea fungus, coconut
water vinegar of the Philippines, etc.
6. Alkaline fermentations: Examples are kinema, natto, Nigerian dawadawa, etc.
7. Leavened bread: Examples are yeast and sourdough breads
8. Flat unleavened breads
26.3 SAFETY OF FERMENTED FOODS
Fermented foods generally have a good safety record even in the developing world.
Food fermentations that improve food safety are as follows:
1. Food fermentations involving lactic acid production

2. Food fermentations involving ethanol production
3. Food fermentations involving acetic acid production
4. Food fermentations involving highly alkaline conditions with liberation of
free ammonia
5. Food fermentations carried out in the presence of high salt concentrations
(above 13% w/w)
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26.4 MISO
Miso is the Japanese name given to paste-like, salty food made from varying
combinations and proportions of soybeans, barley or rye, and rice by a mixed
fermentation with molds, yeasts, and bacteria. Although miso is a seasoning, it is also
a traditional dietary staple used by the Japanese in preparation of soups for breakfast.
Similar products are made and consumed in other parts of the Orient also, for
example: chiang (China), tauco (Indonesia), doenjang (Korea), tao chieo (Thailand). Miso
has a distinctive pleasant aroma resembling that of soy sauce. Some varieties,
especially those that have greater proportion of soybean in the formula and have
been fermented for a long time, have a very meat-like flavor.
There are several types of miso found in Japan, for example, kome miso or rice miso,
mugi miso, sendai miso, mame miso, shinshu miso, edo miso, etc. The color of miso
ranges from light white to reddish brown. White miso is preferred in western Japan,
has a light color, a very sweet flavor, a low concentration of salt (5-6%), and a short
fermentation period of about a week at 23-33°C. Edo miso, preferred around
Tokyo, is reddish brown, has a low salt content, and requires two weeks of
fermentation. In general, the higher the proportion of soybeans in the recipe, higher
should be the salt content and longer the fermentation period. Overall, miso has 45-
50% moisture, 4.5-13% salt, 4-11% fat, 4-38% reducing sugars, and 5-5.4 pH values.
26.4.1 COMMERCIAL PRODUCTION
Miso production is a two-stage fermentation process, viz., aerobic, and anaerobic. The
essential steps are: (i) preparation of rice/barley, (ii) preparation of koji, (iii) preparation
of soybeans, (iv) mashing/mixing, and (v) anaerobic fermentation.
26.4.1.1 Raw materials
The basic raw materials for miso production are soybeans (yellow variety is
preferred), polished rice, rye or barley, salt and alcohol.
26.4.1.2 Preparation of rice/barley
Barley/polished rice is washed and soaked in large fiberglass or epoxy-lined steel

tanks holding 1000 kg or more of material. The duration is approx. 17 hrs. The
soaked mass is transferred continuously on stainless steel mesh belts through two,
long tunnel steamers. Steam is injected at 4.3 and 3.5 psig respectively in the first and
second chamber. The partially steamed rice/barley may be rinsed with water as it
passes from the first to the second chamber to remove free starch.
26.4.1.3 Koji preparation
The rice/barley is cooled in another conveyor belt and transferred to the koji
fermenter. The rice/barley is inoculated with a pure strain of Aspergillus oryzae (soyae)
spores selected for its ability to produce the required proteases, amylases, lipases,
and other enzymes in proper proportions and quantities. The inoculation rate is
0.1% (w/w), or equivalently, 109 spores/g.
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The koji may be fermented at 28-32°C for 3 days either in special rooms or
mechanically agitated drums or rectangular tanks. This step implies the aerobic phase
of miso production. Rotating drums are the easiest to explain here: they are about
1.8 m (diameter)  3.6 m (length) with temperature, humidity, and air flow controls.
The drums contain finger projections inside them for breaking up the developing
koji as the drum is periodically rotated.
By the time fermentation terminates, the rice/barley is fully covered with white
mycelia. It has a sweet, pleasant smell. Before sporulation occurs, further
development of mold is terminated by either subjecting it to anaerobic brine
fermentation, or cooled at low temperature, or mixed with 30% (w/w) of salt.
26.4.1.4 Preparation of soybean
Cleaned soybeans are hydrated to approximately double the weight by soaking them
overnight in large tanks. If dehulled soybeans are to be used, dehulling can be done
by either dry process (in burr mill) or wet process (in abrasive mechanical peeler).
The beans are thoroughly cooked in large batch-type or continuous cookers
(retorts/autoclaves) in steam or water at a temperature of 121°C for 30-40 min or
higher temperature equivalents. The batch retort can hold 1000 kg or more of
material. It is often mounted so that it can be rotated during the operation to hasten
cooking and facilitate emptying. The beans are now emptied onto conveyor belts
and rapidly cooled to prevent further darkening.
26.4.1.5 Mashing/mixing/preparation of green miso
The cooked beans are mashed and mixed with koji, required amount of salt added,
and the mixture inoculated with either miso (from previous batch) or pure cultures
of selected yeasts (e.g., Saccharomyces rouxii, Torulopsis) and bacteria (Pediococcus
halophilus, Streptococcus fecalis). This mixture is called green miso. The bacteria produce
the necessary acidity and yeasts produce alcohol, contributing to formation of esters,
and aroma and flavor compounds. Machines similar to large sausage grinders can be
used for mashing. Mixing continues in large mixing vats with heavy paddles. It is
important to mix the ingredients well so that variation in salt concentration in the
mash is less than 0.5%.
26.4.1.6 Anaerobic fermentation
The paste is then conveyed to temperature-controlled tanks or vats that can hold up
to 12000 kg. Spigots allow the removal of liquefied tamari sauce as the fermentation
progresses. The fermentation proceeds under anaerobic condition. The lower the
salt content and the higher the proportion of rice/barley koji to soybeans used, the
sweeter the resulting mixture. The more thoroughly the soybeans are cooked, the
darker the color and the higher the salt content, the longer the fermentation, and
also the more robust the meat-like flavor.
The duration of fermentation varies from 1 week (for white miso) to 2 years (for
mame miso). At the end of the fermentation, the miso is blended, pasteurized in tube
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heater, 2% alcohol added, and packed in unit sizes in plastic bags. See Fig. 26.1 for
flow diagram of miso production.
Barley/ polished rice

Soybeans
Soaking
Koji starter
Soaking overnight
Steeping (Aspergillus oryzae 0.1%, w/w)
Dehulling Cooling
Autoclaving
Koji-making
(121oC/ 30min)
(28-32oC/ 3 days)
Salt: to make 5-12% of final mash

Yeast: Saccharomyces rouxii
Bacterium: Pediococcus halophilus
Water: according to recipe
Mashing/ mixing
Alcoholic fermentation**
Aging*
Blending
Pasteurization
Packaging
* Aging is for heavily fermented miso
Miso ** Variable length of fermentation
Fig. 26.1 Commercial production of miso from whole soybean
For heavily fermented miso, aging can be done by allowing the miso to stand at
room temperature for about two weeks before pasteurization.
26.5 SOY SAUCE
Soy sauce is a light-brown to black liquid with meat-like salty flavor. It is prepared by
hydrolyzing soybeans, with or without the addition of wheat or other starchy
carbohydrate, in a strong brine (about 18% w/w) using enzymes produced by
Aspergillus oryzae (soyae). A two-stage fermentation is used. An aerobic solid-state
mold fermentation is followed by a mixed Lactobacillus-yeast submerged fermentation.
Soy sauce is the most widely consumed and the only oriental fermented product that
has become well known in the cookery of western countries. The product is called
shoyu in Japan, chiang-yu in China, kekap in Indonesia, kanjang in Korea, toyo in the
Phillippines, and see-iew in Thailand.
Soy sauce can also be produced by chemical hydrolysis of proteins and starch.
Although the hydrolysis is more complete the sensory quality is inferior to that
produced by fermentation.
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26.5.1 COMMERCIAL PRODUCTION OF JAPANESE KOIKUCHI SHOYU
Shoyu is the Japanese name for soy sauce. Koikuchi means dark in color. The
basic steps in commercial shoyu preparation entails: (i) Treatment of raw materials,
(ii) Koji production, (iii) Mash production and aging, (iv) Brine fermentation, (v) Mash
pressing, and (vi) Refining. See Fig. 26.2 for flow diagram of commercial soy sauce
production.
Soybeans Wheat grains
Soaking Sand-roasting
(180oC/ few min)
Autoclaving Seed culture
Crushing (tane koji, 1-2%, w/w)
Cooking (4-5 pieces)
Mixing
Koji-making Yeast: Saccharomyce cerevisiae
Bacterium: Pediococcus soyae
Mixing Salt: to give above 16% of mash
Brine fermentation
(30oC/4-8 months)
Pressing
Extract Cake
Fractionation/separation
Sediment Middle fraction Oil
Pasteurization
(70oC-80oC)
Packaging
Fig. 26.2 Commercial production of soy sauce from whole soybean
26.5.1.1 Treatment of raw materials
Clean soybeans (whole/defatted grits) are soaked for 10-15 hrs in water, which is
changed every few hours to prevent acidification by bacteria. The weight of the
beans increases by about 2.1-2.15 times. The hydrated beans are cooked for 1 hr in
steam at 10-14 psig in NK rotary cooker of capacity 1000 kg. The cooked beans are
then cooled rapidly.
Whole-wheat kernels (soft type/low protein content) are roasted at 170-180°C in

sand for several minutes (some workers mention roasting for less than a minute). A
rotary cylinder about 0.7 m in diameter and 2 m in length (capacity: 500 kg/h),
rotating at 25-30 rev/min is used. The sand is recycled and wheat grains crushed into
4-5 pieces in a roller mill. A slightly charred flavor is desirable.
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26.5.1.2 Koji production
Typically, cooked soybeans are mixed with roasted wheat particles in the ratio 1:1.
This mixture is inoculated with 1-2% (w/w) of seed koji, called tane koji, of
Aspergillus oryzae pre-grown in polished rice. Some workers mention use of 0.1-0.2%
koji. The mixture is transferred to porous stainless steel plates several meters in
length and width and the material is maintained to a depth of 30-40 cm.
Fermentation proceeds at 25-35°C. Careful control of temperature, aeration, and
moisture allows complete white mycelial growth in 45 hrs, help prevent development
of contaminants, and enhance development of proteolytic enzymes. Aeration is done
by feeding humidified air through the bottom holes. The resulting product is called
shoyu koji (or simply koji), which is a mixture of fungal hydrolytic enzymes and the
substrate. As the fermentation continues, the growth turns yellow and dark green. The
koji is now ready for brine fermentation.
26.5.1.3 Brine fermentation
The koji is mixed with 1.2-1.5 volumes of 23% salt solution. The mash, called
moromi, is transferred to concrete, wooden, or resin-coated steel tanks of 10-20 m3
capacity. It is important that the salt concentration be above 16%: this prevents
putrefaction. Pure cultures of Pediococcus soyae and Saccharomyces rouxii are added to
the moromi at the start and after one month of fermentation. The moromi is stirred
occasionally in the early stages to distribute heat and mix up the mash properly. The
fermentation normally continues for 4-8 months (1-3 years in traditional method). In
some variations, the moromi is transferred twice to other vats during the
fermentation period.
26.5.1.4 Pressing and filtration
The fermented moromi is filtered by pressing in hydraulic filter press through thick
clothes, at 100 kg/cm2 for 2-3 days. The cake, which typically contains 25%
moisture, is used for animal feed.
26.5.1.5 Refining/pasteurizing
The extract thus obtained is separated into three fractions: (i) sediments, (ii) supernatant
middle layer, and (iii) an oily layer at the top. The middle layer is further clarified by filtering
through keiselgel to obtain raw shoyu. This portion is standardized with respect to salt,
nitrogen level, etc., and pasteurized at 70-80°C in kettle or in heat exchanger.
Pasteurization helps remove heat coagulable materials as well as preserve the
product. The product is cooled, filtered again, and packed in 1-2-liter glass- or plastic
bottles. Benzoic acid or propyl- or butyl-p-hydroxybenzoate is sometimes used as
preservative. Shoyu can also be produced in the form of spray-dried powder. The oil
and sediments find other uses. For example, the oil fraction can be used in paints as
an antifreezing agent.
26.5.2 GENERAL PROPERTIES OF SHOYU
The chemical changes that occur in the production of shoyu and its flavor are
complicated. More than 100 compounds have been reported as flavor components
of soy sauce. The guaiacol compounds seem to have an important effect on overall
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flavor of Japanese shoyu. Typical chemical composition of soy sauce from whole
soybeans is given in Table 26.3.
Table 26.3 Some typical values of soy sauce from whole soybeans
Property/parameter Value
Baume’ 22.7
NaCl 18.5%
Total nitrogen 1.6%
Reducing sugar 1.9%
Alcohol ≈ 1%
pH 4.8
Shoyu and miso appear to be very similar both with respect to production, raw
material, and sensory quality. Nevertheless, there are some fundamental differences
between them, a brief mention of which appears in Table 26.4.
Table 26.4 Fundamental differences between miso and soy sauce
Description Miso Soy sauce

Raw material Soybean, rice, barley, rye Soybean, wheat
Koji material Starchy items Wheat and soybean
NaCl 5-12% 13-18%
Reducing sugars 10-38% 4-6%
Form of final product Paste Liquid
Yeast and bacteria for inoculation About 105 each About 106-8 each
Duration of fermentation Variable (1 week-2 years) 4-8 months
26.6 NATTO
Natto is a bacterial fermented oriental soybean product. It is popular in Japan

(except northern Japan) and Korea. In Japan, natto is seasoned with soy sauce, salt
or sometimes mustard, and served with rice.
Natto is characterized by persistent musty to ammoniacal flavor, and the presence of

viscous sticky polymers. The sticky substance is due to glutamic acid polymer.
26.6.1 COMMERCIAL PRODUCTION
The soybeans are first soaked in water at 15°C for 16-20 hrs and then steamed under
pressure (0.7 kg/cm2) for 30-40 min. The cooked beans are inoculated with Bacillus
subtilis (natto) and wrapped in paper-thin sheet of pinewood or packed in plastic
packages weighing 80-120 g. The fermentation lasts for 15-20 hrs at 40-43°C in the
package in which natto is sold. See Fig. 26.3 for an outline of commercial natto
production.
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Soybeans (100kg)
Soaking (16-20h/15oC)
Steaming (0.7kg/cm2) Pure culture

Bacillus natto
Cooling (37oC)
Packaging in unit packs
Fermentation in the packages

(15-20h/40-43oC)
Natto
(225kg, 60% moisture content)
Fig. 26.3 Commercial production of natto
Natto has a short shelf-life, partly because of high moisture content (60%) and partly
because it is usually prepared in small-scale plants with poor quality.
During fermentation, water-soluble nitrogen and ammonia nitrogen is greatly

increased. There is no change in fat and fiber content but the carbohydrates totally
disappear.
26.7 JAPANESE SAKE
Japanese sake is a clear, pale yellow, rice wine with an alcohol content of 15-16% (or
higher), a characteristic aroma, little acid, and slight sweetness.
26.7.1 INDUSTRIAL PRODUCTION
The industrial production of sake involves various steps. The essential steps are
described in the following paragraphs:
26.7.2 SELECTION/PREPARATION OF RICE
Rice of short-grained variety is considered best for sake production. The rice should
be finely polished to remove proteins, lipids and minerals. It is then washed and
steeped in water (until 25-30% water uptake), drained (4-8 hrs) and steamed (30-60
min). Steaming sterilizes and gelatinizes starch and the total water uptake amounts to
35-40%. The rice is cooled to 35°C for koji manufacture and 10°C for the
preparation of moromi.
26.7.3 KOJI PREPARATION
The rice prepared as above is inoculated with spores from tane koji (at the rate of 60-
100 g/100 kg prepared rice). Tane koji is prepared by culturing Aspergillus oryzae on
soaked, steamed, polished rice for 5-6 days or until abundant sporulation. The
inoculated rice is heaped on the floor of a room with controlled humidity and held at
26-28°C. The internal temperature of the heap rises to 31-32°C. After 10-12 hrs, the
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mold spores germinate. The rice is mixed and after 20-24 hrs the developing koji is
placed in 15-45 kg capacity wooden boxes. Mixing is done every 6-8 hrs to avoid
overheating. After 40 hrs, the temperature of the developing koji reaches 40-42°C,
and the mycelia will cover the grains. The mycelia contain sufficient hydrolytic
enzymes so that the koji can be used for saccharification of starch in the main mash.
26.7.4 PREPARATION OF KOONTOKA-MOTO
Koontoka-moto is a hot-mash, rapid-saccharification procedure used for the

preparation of yeast starter (moto). After a 6-hr starch hydrolytic step at 56-60°C
with koji amylases, the mash is cooled, acidified and filtered. The filtrate is used to
grow pure sake culture.
More recently, aerobically propagated compressed sake yeast has become available
commercially and can be inoculated directly as 7% (w/w) of the total rice used in a
moromi mash. Acidification of the mash is carried out with lactic acid. This method
eliminates the necessity of preparing moto.
26.7.5 MAIN FERMENTATION
For the main fermentation, mash (moromi), unsterilized koji, steamed rice, and
water are fermented in 6-20-kL tanks, each containing 1500-10,000 kg of rice. The
yeast population in moromi is built up in a stepwise manner over a period of 3 days.
Moto mash is combined with equal quantities of rice and water, reducing the yeast
count by two-thirds. After 2 days at 12°C, the yeast population rises to 108/g and
mash is diluted again by about one-half. The rice-koji-water mixture is added at 9-
10°C to suppress the growth of contamination microorganisms. The following day, a
third addition is made at 7-8°C, again reducing the yeast by one-half. In this way, the
yeast population of 2.5108 cells/g is reached after about 1 week of fermentation.
Such stepwise fermentation permits careful temperature control, important in
balancing saccharification and fermentation rates. With such control, ethanol
concentration approaches 20% abv in 20-25 days.
The moromi tends to form a rather viscous foam that may occupy one-third of
fermenter volume.
The mash is pressed, the liquor settled for 5-10 days, filtered, blended, and settled
again for 30-40 days. Everything needs to be carried out at low temperature. The
wine is pasteurized at 55-65°C and aged at 13-18°C with or without activated
carbon. Blending, dilution with water, filtration, and bottling marks the final steps of
sake preparation. 1000 kg of polished rice yields 3000 liters of sake (20% abv) and
200-250 kg of residue (sake-kasu).
26.8 KINEMA
Kinema is a fermented soybean food product indigenous to Nepal. It is mostly

prepared and consumed (and sometimes sold) by Limbus of eastern Nepal,
especially in the hills. The preparation is limited to household level. Methods
followed for its preparation are often subject to variation. Some of the more
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important factors contributing to variation are: locality, convenience, availability of
raw materials, and processing steps.
26.8.1 TRADITIONAL PROCESS OF KINEMA MAKING
Kinema preparation is a relatively simple process. The traditional process entails

cooking of soybeans (white or brown), cooling to room temperature, mixing with a
small amount of vegetable ash, wrapping with banana leaves or rice straw, and
leaving it for 2-3 days in a warm place for fermentation. The beans are usually
mashed in wooden pestle or macerated with hands so as to split them apart. A well-
fermented kinema has a slimy appearance (stringy when touched), tends to form
cake, and has a persistent nutty to musty flavor. Unless dried, the preparation has a
very short shelf-life (2-3 days) at room temperature.
Kinema preparation does not require any addition of microbial culture: it is a

spontaneous fermentation. The microbial flora present in the banana leaves or rice
straw (sometimes other leaves are also used) act as an inoculum. Wood ash, which is
so often considered an essential ingredient in kinema making, may not be
indispensable for the fermentation itself but can be considered desirable in that it
may furnish certain minerals to the organisms. The condition also becomes alkaline,
which favors fermentation. The use of ash can also be related to the development of
characteristic taste in kinema. See Fig. 26.4 for an outline of traditional kinema
making.
Whole soybean
Soaking overnight
Draining excess water
Cooking in fresh water

Wood ash
Draining dry
Mashing/Macerating
Wrapping in banana leaves
Packing in bamboo basket
Incubation at 35-40oC/2-3 days

(near the fire place)
Kinema
Fig. 26.4 Traditional preparation of kinema
With respect to properties, kinema falls in a position somewhere between the two
familiar oriental fermented soybean products, namely, tempeh kedele and natto.
Tempeh is a traditional mold-fermented food native to Indonesia (see later). The
most popular type of tempeh is produced from soybeans and is known as tempeh
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kedele. Natto is bacterium-fermented soybean food product of Japan (discussed
earlier).
Because of the nature of fermentation, the quality of kinema obviously never remains
consistent. Since rice straw as well as banana leaf is used as the source of inoculum
the quality of the final product can only be as good as the quality (and relative
proportions) of microorganism present in the source. The final product has the
characteristics of natto as well as tempeh but since the fermentation produces a
more ammoniacal odor, the characteristics of tempeh are usually masked. Overall,
kinema more resembles natto than tempeh. The sticky substance present in kinema
has been identified as exopolypeptides of D-isomeric glutamic acid having -
glutamyl peptide bonds. The organisms, according to T.B. Karki (1994), are: Bacillus
subtilis, Enterococcus faecium, Candida parapsilosis, and Geotrichum candidum
Natto, tempeh kedele, and kinema have many things in common, for instance:
1. Soybean is the basic raw material for preparation

2. They are traditional fermented foods of the orient
3. Traditional methods of preparation depend on spontaneous fermentation
Of the differences, the more important ones are as in Table 26.5:
Table 26.5 Fundamental differences between natto, kinema, and tempeh
Parameter Natto Tempeh kedele Kinema

Organism Bacteria Mold Mixed flora
Flavor/odor Musty to Nutty Nutty to musty
ammoniacal
Use Condiment, in Staple food, usually Condiments, in
soups, etc. deep fried soups, curry, and as
chutney substitute
Incubation 40-43C 35C 30-40C
temperature
26.8.2 NUTRITIONAL SIGNIFICANCE
Soybeans are fermented not primarily for preservation. In fact, the fermented
product has a very short shelf-life. Flavor development is the principal reason of
such fermentations. Nevertheless, among other things, fermentation of soybeans
also leads to following advantages:
1. Leaches out during cooking flatulent principles like stachyose and raffinose
2. Decreases/destroys antinutritional factors like trypsin inhibitors, lectins and
phytic acid
3. Increases soluble nitrogenous substances
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26.8.3 PALATABILITY
Palatability of fermented soybean products is largely a matter of food habit. What

can be mouth-watering to one can be offensive, or even revolting to another. For a
beginner, natto and kinema can be revolting but for a habitual consumer the same
flavor can be highly appetizing. Tempeh kedele has a much milder and nutty flavor
and is being slowly accepted in the West also.
26.9 TEMPEH
Tempeh is an oriental mold-fermented food indigenous to Indonesia. The most

popular type of tempeh is produced from soybeans (preferably white) and is known
as tempeh kedele.
26.9.1 TEMPEH ORGANISM
The fermentation is invariably a mixed culture of molds, yeasts, and bacteria but the
most important component appears to be Rhizopus oligosporus, although other
Rhizopus species and Mucor are also often isolated. One of the better producers of
tempeh has been identified by Hasseltine et al (1963) as Rhizopus oligosporus Saito
NRRL2710.
26.9.2 METABOLIC CHARACTERISTICS OF THE ORGANISM
Rhizopus oligosporus Saito NRRL2710 has a low amylase activity but high protease and
lipase activity. Fatty acids in soybeans are the principal source of carbon and energy.
Stachyose and raffinose are not utilized but common sugars such as glucose,
fructose, etc., support excellent growth. By virtue of high protease activity, the
organism can hydrolyze and utilize soybean proteins for nitrogen source. Proline,
glycine, aspartic acid, and leucine are excellent sources of nitrogen but tryptophan
supports no growth at all. It can utilize ammonium salts but not sodium nitrate.
Bacterial contamination is generally not encountered. Because the organism

produces an antibacterial agent, and also because it has the unique characteristic of
fast growth rate, there is little chance for bacteria to gain ground before the tempeh
fermentation is complete.
26.9.3 COMMERCIAL TEMPEH PRODUCTION PROCESS
Commercial tempeh preparation starts with dehulled, full-fat soybeans or soybean

grits. Grits yield better quality tempeh. The soaking time is usually 30 min to an hour
but some investigators consider this step superfluous. Commercial fermentations use
pure culture in regulated amounts. Banana leaves may be optional (see Fig. 26.5).
26.9.3.1 Processing loss
The dehulling, soaking, washing, cooking, and fermenting steps employed in the
preparation of tempeh all contribute to loss of soybean constituents. The total loss
of solids ranges from 24 to 48%, depending on the variety of soybeans and the
375
process used. The more significant losses are in dehulling and cooking. Loss can be
minimized by using less water during cooking but the fermentation as well as the
quality of tempeh will not be sound. In such cases, the tempeh shows less mold
development and much sporulation (and therefore discoloration). The flavor and
odor are also unpleasant and poor. The factor responsible for this is the presence of
heat-stable and water-soluble mold inhibitor in soybeans. This factor also inhibits
the formation of proteolytic enzymes by Rhizopus oligosporus. Therefore, soaking and
cooking of soybeans in excess water (which is discarded later) are essential to
tempeh making.
Soybean
[full-fat, 1000kg (900kg dry basis)]
Water
Soaking
(30-60 min)
Draining
Cooking
(with excess water)
Draining
Spreading/Cooling/Surface-drying
Inoculation Tempeh starter (ragi)

(106 spores/100g
Packing cooked soybean)
(in shallow trays or small packs)
Fermentation
(30-35oC/24-36hrs)
Tempeh kedele cake

(51kg dry basis)
Fig. 25.5 Commercial preparation of tempeh kedele
26.9.3.2 Mold management
Traditionally, small pieces of tempeh from previous fermentation serve as inoculum.

The fungus is then propagated mainly by means of fast-growing mycelia. The
disadvantage of this method is that it can lead to contamination by undesirable
microorganisms. Moreover, the inability of mycelia to survive adverse temperatures
and dehydration makes mycelia unsuitable for long-term preservation. For long-term
storage, either lyophilized cultures or other suitable modifications are prepared.
Spores may be produced in rice, soybeans, or wheat substrate. Soybean as a substrate
results in unpleasant flavor and odor while wheat bran results in poor sporulation.
Fermentation in rice of 40% moisture level for 4-5 days at 32C produces good
growth and spores. The whole can be made into slurry by blending with sterilized
water and then freeze-dried. The spores show comparable viability even after storage
for 6 months in plastic packages at 4C.
376
26.9.3.3 Inoculum size
The amount of inoculum required to make satisfactory tempeh is significant because

fermentation time becomes too critical if the amount of inoculum is too large. On
the other hand, too small an amount of inoculum provides chance for contaminating
bacteria to grow. A level of 106 Rhizopus oligosporus spores per 100 g of cooked
soybeans seems appropriate.
26.9.4SHELF-LIFE OF TEMPEH
Fresh tempeh has a shelf-life of only one to two days as sporulation of mold
discolors the product and a rich ammoniacal odor develops as proteolysis proceeds.
The release of ammonia makes the product noxious. Its shelf-life, however, can be
prolonged by various methods. In Indonesia, it is cut into slices and sun-dried. An
alternative method could be to first blanch the sliced tempeh to inactivate the mold
and enzymes and then freeze it.
26.9.5 PALATABILITY
Fresh tempeh has a pleasant nutty flavor and odor. It is free from beany flavor and
odor of raw soybeans and is therefore highly palatable. It is the only Oriental
fermented product that has been extensively investigated in the West. Many
countries in the West have now begun to consume tempeh.
26.9.6 NUTRITIONAL VALUE
Tempeh has a superior nutritional value over unfermented soybeans. Undesirable

soy components such as flatulence factors and trypsin inhibitors, etc., are removed
and/or destroyed during cooking and draining. Lipids become more resistant to
autoxidation. Niacin, riboflavin, Vit. B6, and pantothenic acid increase after
fermentation. The food also contains beneficial antibacterial components. At any
rate, it is a potential source of low-cost protein that can have significant role in
solving protein malnutrition.
26.9.7 CONSUMPTION PATTERN
Tempeh, in its various forms and types, is consumed in Java, Indonesia, and some
other Oriental and western countries. In Indonesia, the annual production of
tempeh is over 80,000 MT, which accounts for about 14% of the total soybeans
produced there. Indonesians consider tempeh to be a nourishing and easily digestible
food. They use it as a main dish. In Java, the per capita daily consumption of fresh
tempeh is in the order of 20-120 g.
The food is consumed in a variety of ways. The more common ways are:
1. Slicing, dipping in salt, and deep frying in coconut oil

2. Including pieces of tempeh in soups
377
26.10 TEMPEH BONGKREK
The preparation of this type of tempeh is principally similar to that of tempeh

kedele. It is popular in Java. The raw material used here is the coconut press cake.
Tempeh bongkrek has been associated with occasional outbreaks of food poisoning
due to the bacterium Pseudomonas cocovenenans growing in the product and elaborating
the toxins bongkrekic acid and toxoflavin. Since 1951, at least 1000 people are known to
have died as a result of this intoxication. Consequently, the Indonesian Government
prohibited the production of tempeh bongkrek in 1988.
26.11 ONTJOM
Ontjom is also mold-fermented product native to Indonesia. It is prepared from

peanut press-cake. The organism used is either Rhizopus oligosporus of Neurospora
intermedia. The preparation has a fruity/mincemeat character.
26.12 FERMENTED VEGETABLES
Most horticulture products can be preserved by lactic acid fermentation. In the West
the most important commercially are cabbage, cucumbers, and olives. Fermented
vegetables, commonly cabbage, in Korea is known as kimchi. The two most common
lactic acid-fermented vegetable products of Nepal are gundruk and sinki.
26.12.1 SAUERKRAUT
Sauerkraut production is thought to have been brought to Europe from China by

the Tartars. Initially, the shredded cabbage was deliberately made sour by adding
sour wine and vinegar. This produced a literally "sour cabbage". But as we know and
define now, sauerkraut is a clean, sound product of characteristic flavor, obtained by full
fermentation, chiefly lactic, of properly prepared and shredded cabbage in the presence of not less than
2%, nor more than 3% salt. It contains, upon completion of fermentation, not less than
1.5% of acid, expressed as lactic acid.
26.12.1.1 The technology
Like a number of other traditional vegetable fermentations, the commercial process

of sauerkraut production is technologically simple, but involves some interesting and
complex chemistry and microbiology.
Usually, where commercial sauerkraut production is practiced, special cabbage

cultivars are grown. These improved cultivars are well-adapted to mechanical
harvesting and at the same time inherently contain less water. The outer leaves are
removed mechanically and the cabbages decored before cutting into shreds of
about 1 mm (0.8-1.0 mm). The finely cut long shreds, called "slaw", are then
conveyed by belts or carts to the vats or tanks for salting and fermentation.
The level of salting is critical to obtaining a satisfactory product. It must be within

the range 2-3% w/w and is normally about 2.25%. Uniform distribution of salt
throughout the mass of cabbage is very essential. Too little salt (less than 2%) causes
378
product-softening to an unacceptable level. Too much salt (over 3%) interferes with
the correct microbial sequence, delays fermentation, and depending on the amount
of oversalting, may produce a product with a sharp, bitter taste, causing darkening of
the color, or favor growth of pink yeasts. The salt serves a number of purposes,
namely:
 It extracts moisture from the shredded cabbage by osmosis to form the

brine in which the fermentation will take place
 It helps inhibit some of the natural microflora of the cabbage, such as
pseudomonads (which would otherwise cause spoilage) and helps select the
lactic acid bacteria
 It helps maintain the crisp texture of the cabbage by withdrawing water and
inhibiting endogenous proteolytic enzymes which cause the product to
soften
 Finally, salt contributes to flavor of the product.
Commercially, sauerkraut fermentation is often carried out in concrete vessels with

synthetic polymer lining to protect the vessel from attack by the acid brine. After
salting and dumping in the vat, the whole is sealed by covering with plastic sheeting.
The sheet is then filled with brine on the top to press the sheeting on the cabbage,
thereby expelling the entrapped air. See Fig. 26.6 for an idea of the packing process.
Plastic sheet
Brine cover
Concrete tank
Fig. 26.6 Packing of slaw for fermentation
26.12.1.2 Fermentation condition
Temperature is a controlling factor in the sequence of desirable bacteria in the

sauerkraut fermentation. At the optimum of 18.3C or lower, the quality of
sauerkraut is generally superior in color, flavor, and ascorbic acid content because
the heterofermentative bacteria exert a greater effect. Under such a condition, the
fermentation is complete in 1-2 months. At a temperature of 32C, the fermentation
time is as short as 8-10 days, but at the cost of quality. Higher temperatures lead to
essentially homofermentative mode and the kraut is reminiscent of acidified cabbage.
Acidity, coupled with saturation of the mass of the product with CO2, is sufficient to
provide conditions necessary for the preservation of sauerkraut.
26.12.1.3 Microbiology of sauerkraut fermentation
Although commercial starter-cultures for sauerkraut fermentation are available, they

are not widely used: rather, spontaneous fermentation is generally employed. The
fermentation is initiated by Leuconostoc mesenteroides, which is among the less acid-and-
379
salt tolerant lactic acid bacteria (LAB) but grows the fastest during these early stages.
As a heterofermenter, it produces CO2, which replaces the entrapped air and helps
establish anaerobic conditions within the product. This event prevents the oxidation
of Vitamin C and loss of color. Since fructose is present as an alternative electron
acceptor, the bacterium also produces appreciable amounts of acetic (ethanoic) acid
acety-ScoA (the major contributor to sauerkraut flavor). As the pH drops due to acid
production, Leuconostoc is inhibited and replaced, first by heterofermentative
lactobacilli (such as Lactobacillus brevis), and then by homofermentative Lactobacillus
plantarum. Acid accumulation continues in the form of lactic acid although the pH
stabilizes somewhere around 3.6 (the pKa of lactic acid). At the end of fermentation
(which can last from 4-8 weeks) the total acidity of the product is 1.7-2.3%,
expressed as lactic acid, with the ratio of volatile to non-volatile around 1:4.
This succession of microorganisms produces interesting changes in the kraut during

fermentation. At the commencement, lactic acid bacteria comprise only about 1% of
the total microflora, but many of the non-lactics fail to grow. Two days later, lactic
acid bacteria account for more than 90% of the total microflora. During this time
they produce sufficient acid to decrease the pH to below 4, further inhibiting the
competing microflora. Underlying this overall dominance by lactic acid bacteria is a
natural succession of different species which contribute to the characteristic flavor
of sauerkraut. Fig. 26.7 shows some changes observed during sauerkraut
fermentation.
Total bacterial
6 8 count
Lactic acid
7 bacteria 2.0
5 6 1.6
Titrable
acidity
5 1.2
4 4 pH 0.8
3 0.4
Volatile acidity
3
0 2 4 6 8 10 12 14
Time (days)
Fig. 26.7 Chemical and microbial changes during sauerkraut fermentation
26.12.1.4 Defects in sauerkraut
Defects in sauerkraut arise mainly as a result of yeast and mold growth. They can
produce off-flavor/odor, loss of acidity, a slimy softened product as a result of
pectolytic activity, or a pink coloration due to the growth of Rhodotorula (yeast). In
the early stages of fermentation, Leuconostoc mesenteroides fermenting sucrose will
preferably utilize fructose, polymerizing the glucose moieties to produce dextran
380
slime. However, this is transient and the slime is later degraded and utilized by other
lactic acid bacteria.
26.12.2 GUNDRUK AND SINKI
Gundruk is a non-salted, lactic fermented vegetable product indigenous to Nepal. It

is made from vegetable leaves (e.g., mustard, radish, rayo). The fermentation is a
spontaneous one, caused by epiphytic LAB that inhabit the vegetable leaf (the raw
material).
Gundruk is believed to have existed in Nepalese culture since time immemorial. In

the rural areas (where gundruk is generally produced), gundruk is primarily used for
flavor reasons and as an alternative to green vegetables in the lean season. It is
valued for its uniquely appetizing flavor. In Nepal, about 2% of the gundruk
production has been industrialized. It is sold in the market in the dried form.
The quality of gundruk mainly depends on the balanced production of lactic acid and
acetic acid (ratio of about 10:7). Gundruk fermentation is primarily initiated by
heterolactic lactobacilli such as Lactobacillus cellobiosus and Leuconostoc mesenteroides. The
fermentation is subsequently completed by the more acid producing homolactic
bacterium Lactobacillus plantarum.
26.12.2.1 Technology
The methods of gundruk preparation tend to differ slightly, depending on the

geographical region, raw material, and the people preparing it. An oversimplified
method of gundruk preparation (suitable in small batches) is shown in Fig. 26.8.
Green leaves
(rayo, radish, mustard, etc.) Securing the lid
Cleaning Fermentation
(to remove foreign matters, (in an inverted position, under
mud, matured parts) warm condition for 7-8 days)
Withering
(for 24h to bring down Fresh gundruk
moisture to ~ 50%)
Sun-drying
Crushing/Fragmenting (to ~ 18% moisture)
Filling Packaging and storage
(in plastic jar)
Tamping Dried gundruk
(until the jar is almost full)
Fig. 26.8 Simplified process of gundruk preparation
A generalized method of gundruk production is rather straightforward: it entails

wilting of vegetable leaves in the sun for a few days, crushing, tamping in containers
(usually made out of bamboo stem, called dhungro), and fermented in a warm place
381
for a week to several days. The first sign of fermentation is the appearance of froth
(that oozes out of the container) and this is generally followed by leaching out of the
brown-colored juice. In general, it takes 10-15 days for the completion of
fermentation. The final operation in gundruk preparation consists of drying (to
moisture content around 10%) in sun and making ready (for example, packaging) for
sale, storage or distribution.
Gundruk is used as pickle or soup, alone or in some suitable combination with other
vegetables, and served generally in the main course.
Sinki is another similar lactic-fermented product in which radish is used as the raw
material. Sinki has a light- and more acceptable appearance. The production process
is more or less similar to that of gundruk. In the glut season, when large quantities of
surplus radish need to be fermented, the fermentation is carried out in pits (~ 1 m3
space) dug in the ground. The pit is first warmed up by burning dried leaves and
twigs and then lined (internally) with banana leaves. Radish pieces (which have been
previously wilted and crushed) are then tamped in. An outer covering of banana
leaves and an additional layer of other covering materials (e.g., straw) are given to
provide a facultative environment for the fermentation. The whole is left
undisturbed for 10-15 days for fermentation. Drying, storage, and consumption of
sinki are similar to that of gundruk.
26.13 JAND, NIGAR, AND RAKSI
Jand is an alcoholic beverage (undistilled) indigenous to Nepal. It is prepared by

solid-substrate fermentation of starchy cereals like corn, rice, wheat, and millet.
Millet is the material of choice because it is claimed to produce superior quality of
jand. Murcha, a starter culture, is used as the inoculum in traditional fermentation.
Murcha contains saccharifying molds, lactic acid bacteria, and fermenting yeasts. Jand
is therefore the result of concerted action of these microorganisms on the cooked
cereal. Fig. 26.9 shows a simplified biochemistry of jand fermentation.
MURCHA
molds
+
lactic acid bacteria yeast enzymes
+ (zymase) bacterial enzymes
yeasts
mold enzyme
(amylase) Ethanol
+
SUBSTRATE SIMPLE SUGARS Lactic acid
+
saccharification
Flavor compounds
aerobic reaction anaerobic reaction

(formation of jand)
Fig. 26.9 Simplified biochemistry of jand fermentation
The basic steps followed in the traditional jand making are: cooking of cereal, cooling
to room temperature, mixing with murcha powder, leaving it for a day or two for
biomass build-up (of essential organisms, viz., yeasts and molds), and fermenting in
tightly plugged containers. Originally, close-necked earthen pots were used for
382
fermentation but now plastic containers have largely replaced them. The duration of
fermentation may range from a week to several months, during which 4-15% alcohol
by volume (abv) may develop.
Due to continued saccharification and ethanol production, the mash gradually turns
limpid. At some point, a nearly clear supernatant is observed in the fermentation
vessel. This liquid is called nigar and is much prized by habitual jand drinkers. Nigar
can be categorized as a cereal wine (similar to Japanese sake) while jand (which contains
live yeasts and suspended particles) has been classified by various workers as a
category of cereal beer. See Fig. 26.10 for an outline of the traditional method of jand
preparation.
MILLET
Dehusking in wooden pestle

Cleaning/winnowing
Washing
Cooking to soft consistency

Murcha
Cooling
(to room temp. by spreading on bamboo
mats or trays)
Inoculation
Packing
(in loose packs)
biomass build up
(at room temp for overnight)
Packing
(in tight packs)
Fermentation
(at room temp./ 1 week-several months)
JAND
Fig. 26.10 Traditional preparation of jand
During serving, the mash is taken out, mixed with requisite amount of water,
squeezed, strained, and the cloudy extract drunk. Another variation of consuming
jand is as tongba (or tumba). In this variation, jand is loosely stuffed into a cylindrical
jug, lukewarm water poured in, a small bamboo tube (with a pair of eyeholes at one
end) inserted through the mash, and the extract sucked in (Fig. 26.11). This variation is
a more standard form of taking/offering jand in traditional ceremonies and ritual rites.
The traditional method of jand preparation has many shortcomings. The quality of
murcha is never consistent and so is the quality of jand. Optimum fermentation
conditions are difficult to maintain. Besides, sanitary conditions are not adequately
maintained in the tribal method. As of now, some studies are available that indicate
that use of pure cultures (isolated from murcha) can be an attractive alternative.
383
peepa for sucking the extract
lid
metal brace
barrel (~ 1/2 kg capacity)
Fig. 26.11 Tongba (a variation of serving jand)
When jand is pot-distilled, it becomes raksi, which is an unaged traditional spirit of

varying alcohol contents (15-50% abv). The product likens whiskey and is very
popular among the ethnic groups of Nepal. The pot distillation assembly used in the
locality for raksi production is shown in Fig. 26.12.
The traditional raksi making apparatus is rather inefficient. An improved design the
author has proposed for a 150-liter mash (the traditional still uses 15-20 liters of
mash per batch) is shown in Fig. 26.13. However, this design has not been tested so
far.
The scheme (Fig 28.11) utilizes low pressure steam to heat the fermented mash in a
steam-jacketed kettle. The boiler has two chambers, only one of which can be used for
steam generation at a time. The condensate from the still jacket is led through a non-
return valve into the boiler (compartment from where steam is not being led out). To
regulate the flow of steam, fire can be shifted alternately (manually) so that only one of
the compartments is heated (for steam) while the other compartment receives hot
water. The entire design does not use any pumps and is fully gravity-based.
cold water
copper pot for

batch condensation
earthen column
vapor
condensed ethanol
receiving pot
fermented wash
copper still
fire
Fig. 26.12 Raksi making apparatus
384
The rectification column is made up of a cylinder that is packed with broken glass
chips. The entire set is made of steel that is readily available locally. The equipment
can be completely fabricated in a local workshop that has facilities for rolling the
iron/steel sheet and welding. Valves can be purchased from retail stores.
Hot water
15
out
19
Condenser
Rectification 54
Cold column
water packed with
in glass chips
1/2'' pipe 17
Alcohol 19
Distillation pot
Vapor (200 liter capacity)
25 Insulation Steam pipe
Steam jacket 3-dimensional view of distillation pot

97
72 Fermented
Hot
material
1'' pipe condensate
pipe Non-return valve
Boiler
Mash
6
55
Supporting
drain beam Water
67
55
Drain Drain
Support
Fire
91
All measurements in centimeter

Drawn to scale
By Basanta K Rai
Program: Flash
2064-09-03 (Nov 18, 2007)
3-dimensional view of boiler
Fig. 26.13 Proposed improved distillation set for raksi making
385
CHAPTER 27
MICROBIOLOGICAL ANALYSIS OF NUTRIENTS
27.1 INTRODUCTION
Because of the similarity in nutritive requirements of microorganisms and experimental

animals, it is possible to use microorganisms to determine quantitatively many of the
substances that are known to be essential constituents of all living cells.
Microbiological assay involves the use of microorganisms as reagents for quantitative

determination of certain chemical compounds, particularly vitamins, amino acids,
growth factors, and antibiotics. Microbiological assay are highly specific and
unusually sensitive. For example, as little as 0.1 nanogram of biotin/ml can be
detected using the organism Lactobacillus casei.
The basic principle upon which microbiological assay depends is that in the presence
of limiting amounts of certain compounds, the amount of microbial growth is a
function of the amount of these compounds.
The response measured depends on the effect of the substance on the metabolism
of the organism. The responses are of two main types, viz., (i) growth response
and (ii) metabolic response.
27.2 GROWTH RESPONSE
The response is positive (promotion of growth) in assay of nutrients and negative

(inhibition) in assay of antibiotics. The growth response can be measured by
methods such as numerical counts, optical density, weight of cells, area (of the growth of cells
on the surface of the medium,) etc. Additionally, the growth response may be a
definite end point or an all-or-none response.
27.3 METABOLIC RESPONSE
In the case of metabolic response, whether positive or negative, metabolic products

or changes in some function may be measured. Among the measurable metabolic
responses are acid production, CO2 production, O2 uptake, reduction of nitrates,
hemolysis of red blood cells, antiluminiscent activity, and inhibition of spore
germination. Not all of these responses are easy to measure, though.
The microorganisms used for assay include bacteria, yeasts, fungi, and protozoa. The
use of bacteria generally poses fewer problems than the use of other groups of
microorganisms. They have been used to assay proteins, amino acids, carbohydrates,
and vitamins, and to evaluate antiseptics, disinfectants, and chemotherapeutic agents.
Lactic acid bacteria (including the genera Lactobacillus, Streptococcus, and Leuconostoc)
equal or surpass all other groups of microorganisms in the complexity of their
nutritional requirements (and hence their usefulness).
There are some requirements for an organism to be useful in microbiological assay.

The ideal test organism should:
1. Be sensitive to the substance being assayed

2. Be easily cultivated
3. Have metabolic function or response that is readily measurable
4. Not be susceptible during assay to variation in either its sensitivity or phase.
As an example of microbiological assay of nutrients, the use of Lactobacillus arabinosus

for the assay of niacin can be mentioned. This bacterium requires niacin for growth.
When it is inoculated into a medium containing all the necessary nutrients except
niacin, growth will not occur. If niacin is added to this medium the organism will
grow and the growth obtained, within limits, will increase as the amount of niacin is
increased. It is therefore possible to prepare a standard curve relating growth to the
amount of the vitamin. If a substance of unknown niacin content is added to the
medium and the test is carried out in the usual manner, the amount of growth
measured can be compared with the standard curve (of niacin), and from this the
amount of niacin in the unknown sample can be extrapolated (see Fig. 27.1 for an
idea). Some examples of the microorganisms used for the assay are given in Table
27.1. The amount of niacin can be obtained either from the graph (slope) or by
linear regression by first drawing a trendline (Excel program can be used) to yield an
equation of the form: y = mx + c, where: y = absorbance, m = slope, x = concentration
of nutrient, and c = intercept.
Amount of nutrient (vitamin or amino acid)
Fig. 27.1 The standard curve for microbiological assay
Table 27.1 Examples of microorganisms used in microbiological assay
Organism Compound assayed

Streptococcus fecalis (bacteria) Several amino acids
Tetrahymena gelei (protozoan) Folic acid
Neurospora crassa (mold) Biotin
Saccharomyces carlsbergensis (yeast) Pantothenic acid
Ochromonas malhamensis (alga) Vitamin B12
387
27.4 METHODS OF MICROBIOLOGICAL ASSAY
There are four main methods of microbiological assay by which the potency of
samples and standard solutions can be compared. They are: (i) diffusion, (ii) turbidity
or dilution, (iii) gravimetric, and (iv) metabolic response methods.
27.4.1 DIFFUSION METHOD
The assayed substance is allowed to diffuse through solid media (in which the
culture has been inoculated), and the zone of growth (or inhibition) of the test
organism formed around the application point (or area) of the substance is observed.
The size of the zone is a function of the concentration of the assayed substance. The
function can be expressed as a linear relationship between the size of the zone and
the logarithm of concentration of the substance. By measuring the distance the
substance diffuses and comparing it with that of a known standard preparation, the
potency of the sample can be assayed.
Diffusion may be of two types: (i) linear, which occurs when the substance is placed
in contact with a column of seeded agar in a capillary or test tube, and (ii) radial,
which occurs around a suitable reservoir containing the substance on a seeded agar
plate.
The horizontal (radial) diffusion can be carried out by two common methods: (i) cylindrical
method, and (ii) cup-plate method. In the former method, cylinders are embedded to a
fixed height in the solidified agar (see Fig. 27.2). In the cup-plate method, a depression
is made in the solidified seeded agar by removing a slug with a cork-borer. The test
material is placed in the cylinder (or depression, as the case may be) and the diameter
of the zone of growth or inhibition due to diffusion of the test substance is
compared with standard concentration of the assayed substance.
The drop-plate and paper disc method is a variation of the horizontal diffusion method.
In the drop-plate method, the test substance is placed directly on the medium
whereas in the paper-disc method the substance comes in contact with the medium
indirectly through paper disc (see Fig. 27.2).
capillary with
test sample paper disc
seeded plate zone of growth with test sample
Fig. 27.2 Linear and radial diffusion assay
27.4.2 TURBIDIMETRIC AND DILUTION METHODS
The distinction between dilution- and turbidimetric methods is that the former gives an
all-or-none end point in broth or agar, whereas the latter measures graded growth or
metabolic response.
388
The serial dilution method for the assay of antibiotics is important for food analysts.
In this method, several dilutions of the test-substance in small tubes is inoculated
with a test organism, incubated, and the lowest concentration of the substance that
causes apparently complete inhibition of growth of organism is taken as the
minimum inhibitory concentration.
In turbidimetric assay, graded concentrations of the test substance are added to a

series of test tubes or flasks containing a liquid nutrient medium. The medium is
inoculated with the test organism and incubated for a suitable time. The response of
the test organism is measured in a photometer, the scale reading of which may be
converted by calibration curve prepared with graded amounts of pure substance to
determine the potency of the assayed sample.
27.4.3 GRAVIMETRIC METHOD
The response of the test organism to graded concentrations of the analyzed

substance is determined after a suitable incubation time by measurement of the
amount of growth in terms of dry cell weight. Under the conditions of assay, this
weight is proportional to the concentration of the limiting factor. The majority of
the gravimetric methods use Neurospora crassa as the test organism.
27.4.4 METABOLIC RESPONSE
The response of the test organism to various concentrations of the assayed

substance is evaluated, after a suitable incubation period, as a change in specific
measurable metabolic parameters. Several parameters can be measured; however,
acid production is the only one used widely. The acid production is determined by
titration.
Although the basic principle of microbiological assay appears straightforward, in

practice the protocols can be quite involved. The complexity involved in sample
preparation can be taken as a good example. In this particular case, the sample must
not contain any components that may interfere with the response of the test
organism. Since the sample can come from a wide variety of sources, it becomes a
practical limitation to provide any generalized, foolproof method for the preparation
of the sample whereby it can be used for all assays. Another example of practical
complexity is in the measurement of cell response in terms of weight. In this case,
the cells must be meticulously washed free from the medium and the true dry matter
content determined.
27.5 EXAMPLE: ASSAY OF FOLIC ACID
Test organism: Streptococcus fecalis ATCC 8043
Inoculum: Prepare a stab culture using medium of following composition: Peptone 05g,
dextrose 1.0 g, anhydrous sodium acetate 0.6 g, kH2PO4 0.2 g, agar 2.5 g, distilled
water 100 ml. Maintain the pH at 6.8. The rest of the process is as follows:
 Inoculate from stab-culture a tube of broth of the same composition as

above but without agar.
389
 Incubate overnight at 37°C
 Transfer a suitable aliquot from this to 50 ml of sterile assay medium and
incubate at 37°C for 5 hrs.
 Use assay medium of following composition for the assay of folic acid:
Components Amount
Vitamin-free caesamino acid 5.0 g
Sodium citrate 26 g
Dextrose 10 g
K2HPO4 3.2 g
L-cystine 0.38 g
Distilled water 1 liter
 Dissolve L-cystine in dilute HCl and other ingredients in distilled water.

Adjust pH to 6.8, add stock solutions, and make to desired volume.
 Standard and dilutions: dilute standard folic acid and sample suitably to finally
contain 1 mμg per ml.

Stock solutions contain special compounds such as amino acids, vitamins, nitrogenous bases, etc.,
needed for the growth of the test organism. Usually, several stock solutions are used for a given assay
media. For folic acid assay, 5 different stock solutions are needed.
ASSAY PROCEDURE
1. Use 25 mm  200 mm lipless tubes with loose-fitting aluminum caps for the
assay
2. Prepare a series of tubes containing different aliquots between 0.5 ml and 5 ml
of the standard and sample dilutions
3. Raise the volume to 5 ml with distilled water
4. Add 5 ml of a double-strength assay broth (the one given above is a single-
strength broth)
5. Autoclave the medium at 15 psig/15 min and cool
6. Inoculate and incubate the tubes at 37°C overnight
7. Arrest growth of microorganisms by adding formalin
8. Measure turbidities (or percent transmittances) using suitable photoelectric
colorimeter
9. Draw a standard curve of % transmittance against the concentration of
vitamin on an arithmetic graph paper
10. Find the equivalent values of the standard for the responses of the sample
by interpolation and compute folic acid activity in the sample
390
CHAPTER 28
MICROBIAL PRODUCTION OF VITAMIN B12 AND ß-CAROTENE
28.1 INTRODUCTION
Microorganism can be used for the production of vitamins like thiamin, riboflavin,
and cyanocobalamin. Some other vitamins like Vit C can also be produced
microbiologically by Acetobactor suboxidans. So far, commercial fermentation has been
economical only for the production of riboflavin and Vit B12. Microbial production
of β-carotene is less cost effective than chemical synthesis but due to the rising cost
of raw materials, fermentation process may be more economic.
28.2 MICROBIAL PRODUCTION OF VITAMIN B12
Vit B12 is a vitamin that is synthesized in nature exclusively by microorganisms. The

Vit B12 needs of animals are covered by food intake or by absorption of Vit B12
produced by intestinal microorganisms. Humans obtain Vit B12 only from food since
the vitamin synthesized by microorganisms in the large intestinal tract cannot be
assimilated. Activated sludge from sewage treatment contains 4-10 mg Vit B12 per kg
but isolation from these sources is expensive. Vit B12 was first obtained
commercially as a by-product of streptomycin fermentation with yield of 1 mg/liter
of broth. As demand of Vit B12 increased, fermentation processes were developed
with high-yielding strains. Commercial production is currently carried out entirely by
fermentation. The current annual production (World) is over 15000 kg. Vitamin B12
production is based on media containing carbohydrate. Most Vit B12 fermentation
processes use glucose as carbon source. Several producing strains are known, some
of which are:
Microorganism Production, mg/liter

Streptomyces olivaceus 3.3
Micromonospora sp 11.5
Propionibacterium freudenreichii 19.5
Propionibacterium shermanii 23.0
Pseudomonas denitrificans 60.0
Propionibacterium and Pseudomonas are the commercially used genera.
28.2.1 PROCESS BASED ON PROPIONIBACTERIUM FREUDENREICHII
Propionibacterium freudenreichii as well as other mutant strains are used in a two-stage

process with added cobalt (10-100 mg/L). In the preliminary anaerobic phase (2-4
days), 5 deoxyadenosyl cobinamide is mainly produced. In the 2nd aerobic phase (3-4
days), the biosynthesis of 5,6-dimethyl benzimidazole takes place so that 5
deoxyadenosyl cobalamine (known as coenzyme B12) can be produced.
As an alternative to this two-stage batch process, both stages can also be operated
continuously in two tanks. During the recovery process, the cobalamins (which are
almost completely bound to cell) are brought into solution by heat treatment (10-30
min at 80-120°C, pH~ 6.5-.5). They are then converted chemically into more stable
cyanocobalamine. The raw product with about 80% purity is used as feed additive.
Additional purification is done (95-98% purity) for medicinal use.
28.2.2 PROCESS BASED ON PSEUDOMONAS DENITRIFICANS
Pseudomonas denitrificans has been found to be the most productive species among Vit
B12 producing microorganisms. In this one-stage process, the vitamin is produced
during the entire fermentation. Cobalt and 5,6-dimethyl benzimidazole must be
added as supplements. Sugar beet molasses is used as low cost carbon source, which
also contains betaine (which is assumed to cause activation of biosynthesis or an
increase in membrane permeability).
The media composition for different stages of production is given below. The
production flow-diagram is given in Fig. 28.1.
Medium A Amount, g/liter (unless specified)

Sugar beet molasses 60
Yeast extract 1
N-Z amine (enzymatic casein hydrolysate) 1
(NH4)2HPO4 2
MgSO47H2O 1
MnSO4.H2O 0.2
ZnSO4.7H2O 0.02
Na2MoO4.2H2O 0.005
Agar 25
Tap water To make 1 liter
pH 7.4
Medium B
Same as Medium A but without agar
392
Medium C Amount, g/liter (unless specified)
Sugar beet molasses 100
Yeast extract 2
(NH4)2HPO4 5
MgSO47H2O 3
MnSO4.H2O 0.2
Co(NO3)2.6H2O 0.188
5,6-dimethyl benzimidazole 0.025
ZnSO4.7H2O 0.02
Na2MoO4.2H2O 0.005
Agar 25
pH 7.4
Pseudomonas denitrificans MB 2436 Inoculum storage: Lyophilized

in dry milk
Agar slant with medium A:

Inoculum cultivation Incubation at 28°C for 96 hrs
Preculture 1 lit Erlenmeyer flask with 150ml medium B

Incubation at 28°C for 72 hrs
Production culture 5-lit fermenter with 3.3 lit medium C; sterilize at

120°C for 75 min
Inoculum: 150ml preculture
Incubation: 29 °C, 90 hrs
Stirring: 420 rpm
Aeration: 1 v/v/min
Fig. 28.1 Flow diagram of vitamin B12 production
28.3 MICROBIAL PRODUCTION OF -CAROTENE
28.3.1 INTRODUCTION
Carotenoids are found in many animal and plant tissues but originate exclusively
from plants or microbes. -carotene (provitamin A) is converted into vitamin A in
the intestinal mucous membrane and is stored in the liver as the palmitate ester.
There is a good demand for β-carotene as provitamin A and as food coloring agent.
Other carotenoids such as lycopene or xanthophylls do not have provitamin A
activity but are used as food coloring agents. Carotenoids are synthesized by
chemical means or by microorganisms but the fermentation process is not
economical. Production processes for several carotenoids is given in Table 28.1.
393
Table 28.1 Production processes for several carotenoids
Carotenoid Organism Medium Time (days) Yield (mg/liter)

β-carotene Blakeslea trispora CSL, distiller’s solubles ~ 8days 3000
Lycopene Streptomyces Starch, soymeal 6 500
chrestomyceticus
Zeaxanthin Flavobacterium sp Glucose, CSL 335
28.3.2 PRODUCTION PROCESS USING BLAKESLEA TRISPORA STRAINS
The production flow diagram using Blakeslea trispora strains NRRL 2456(+) and
NRRL 2457 (-) is given in Fig. 28.2 and the media compositions for the same are
Isoniazid and kerosene are sterilized separately. After 48 hrs, 1 g/liter of β-ionone
and 5 ml kerosene/liter are added. Glucose feeding (total addition of 42 g/liter) is
done until the end of fermentation.
The observation that production occurs during the process of zygospore formation
in this organisms has had an impact on process development. When cultures of both
sexual forms (+) and (-) strains are mixed, a significant increase in carotene
production in the (-) strain is achieved. The production is also increased by trisporic
acid. Another activator of β-carotene synthesis is isoniazid, particularly in
combination with β-ionone. Alone, β-ionone is toxic to the production organism,
but in the presence of plant oils, it promotes carotene production. The addition of
purified kerosene to the medium doubles the yield.
Table 28.2 Medium composition for -carotene production
Medium A Amount, g/lit Medium B Amount, g/lit

Cornsteep liquor 70 Distillers solubles 70
Corn starch 50 Corn starch 60
KH2PO4 0.5 Soybean meal 30
MnSO4.H2O 0.1 Cottonseed oil 30
Thiamin-HCl 0.01 Antioxidant 0.35
Tap water To make 1 liter MnSO4.H2O 0.2
Thiamin-HCl 0.5
Isoniazid 0.6
Kerosene 20ml
pH 6.3
394
Blakeslea trispora Blakeslea trispora Inoculum storage:
NRRL 2456(+) NRRL 2457(-) spores in sterile soil
Culture on agar slant Culture on agar slant Incubation: 168hrs, 27°C
2 lit Erlenmeyer flask with 400ml medium A.

Preculture
Incubation: 48hr, 26 °C, shaker
170 lit fermenter with 120 lit medium A.

Mixed preculture
Inoculum: 40ml of each preculture
Incubation: 40hrs, 26°C
Stirring: 170 rpm
Aeration: 1.1 v/v/min
Production culture 800 lit fermenter with 320 lit medium B

Sterilize: 55min, 122°C
Inoculum: 32 lit of mixed preculture
Incubation: 185 hrs, 26°C
Stirring 210 rpm
Aeration 1.3 v/v/min
Fig. 28.2 Flow diagram of vitamin β-carotene production
Because of the low stability of β-carotene within the cells, the addition of an
antioxidant is necessary during the fermentation process. The carotenoid-rich
mycelium can be used directly as a feed additive. To obtain pure β-carotene, the
mycelium is removed, dehydrated (with methanol), extracted with methylene
chloride (75-92% yield) and the crude product is further purified.
395
CHAPTER 29
BIOFERTILIZERS
29.1 INTRODUCTION
The term biofertilizer denotes the ‘nutrient inputs of biological origin for plant
growth’. Here biological origin should be referred to as microbiological process
synthesizing complex compounds and their further release into outer medium, to the
close vicinity of plant roots which are again taken up by plants. Therefore, the
appropriate term for biofertilizer should be ‘microbial inoculant’. In the recent years,
use of microbial inoculants as a source of biofertilizers has become a hope for most
countries, as far as environmental- and economical viewpoints are concerned.
Development and use of biofertilizers is mainly concerned with the exploitation of a
group of nitrogen fixing organisms called diazotrophs for harvesting atmospheric
nitrogen for plant crops.
Nitrogen compounds account for 40-50% of the dry matter of protoplasm of plant
cells. Nitrogen is therefore required in large quantities by growing plants and is
indeed the key to soil fertility. Plant crops obtain nitrogen from fertilizers and
atmospheric nitrogen. Atmospheric nitrogen is in fact the cheapest and ubiquitous
source of nitrogen for the plant kingdom.
The big reservoir of atmospheric nitrogen, however, is not directly available to the
crop plants: plants simply cannot use the atmospheric dinitrogen (molecular
nitrogen). An important intermediary involved here is the heterogeneous group of
microorganisms collectively called diazotrophs. This group of organisms, limited in
type, is able to change the dinitrogen into forms readily assimilable by crop plants,
either by reduction to NH3 or oxidation to NO3¯. This microbial process of
producing the inorganic forms of nitrogen from molecular nitrogen is known as
nitrogen fixation or diazotrophy. Nitrogen fixation is of great economic importance in
agriculture. The soil supports the plant growth indefinitely when it is replenished
with nitrogen taken away (by crop plant year after year) and this task is carried out by
diazotrophs.
The various microorganisms that have realized or potential applications as

biofertilizer are:
1. Bacteria: Rhizobium sp, Azospirillum, Azotobacter

2. Fungi: Mycorrhiza
3. Blue-green algae (cyanobacteria): Anabena, Nostoc
4. Fern: Azolla (containing a symbiont Anabena azollae)
The diazotrophs exhibit two modes of nitrogen fixation, viz., (i) non-symbiotic and
(ii) symbiotic. Those microorganisms that pass independent life and fix atmospheric
nitrogen are known as free-living diazotrophs, notable among which are species of
Azotobacter, Bacillus, Clostridium, and Anabena. By analogy, those microorganisms
which establish symbiotic relationships with plants for fixing nitrogen are called
symbiotic diazotrophs. The plants and the symbiotic diazotroph exhibit mutualism
whereby the plant exchanges carbohydrates (energy source) with the diazotroph for
the nitrogen the latter fixes.
The modes of nitrogen fixation, however, are not confined to any particular group
of microorganisms. In fact, the same microorganism may exhibit both the modes of
diazotrophy. A remarkable characteristic that all diazotrophs share is the presence in
them of an enzyme complex called nitrogenase which helps in the conversion of
atmospheric nitrogen into ammonia. The overall reaction scheme is:
Nitrogenase complex
N 2  6e  12 ATP  H 2 O 
 2NH 4   12 ADP  12 Pi  4 H +
At present, diazotrophs cultured in commercial scale for the biological nitrogen

fixation are mostly based on rhizobial-, cyanobacterial-, and mycorrhizal cultures.
The symbiotic relationship between legumes and rhizobia is the most talked-about
topic as regards symbiotic nitrogen fixation. The relation has been found to be
extremely specific (commonly described by what is called host specificity). Stated
differently, Rhizobium species or strains effective for one group of legume plants are
less effective or ineffective for another group. Even within the species, certain
strains are more effective than other with the given host plant. For the purpose of
inoculation, and commercial preparation of the bacteria, legumes are therefore
classified into seven major categories as given in Table 29.1.
Table 29.1 Species of Rhizobium and cross inoculation groups of hosts
Legume category/group Rhizobium species

Soybeans Rhizobium japonicum
Peas and vetch Rhizobium leguminosarum
Beans Rhizobium phaseoli
Lupines Rhizobium lupini
Cowpeas Rhizobium sp
Alfalfa Rhizobium meliloti
Clover Rhizobium trifoli
Before rhizobia can fix nitrogen, they must establish themselves in the cells of the
root tissue of the host plant. Infection of the root hair system by rhizobia is closely
associated with the formation of ‘infection thread’ that develops into certain root
hairs. The bacteria invade the host plant cells via this infection thread, causing
enlargement and an increased rate of cell division. This event leads to the formation
of abnormal growth (nodule formation) in the root system. Within the nodules the
bacteria convert free nitrogen to nitrates, which the host plant utilizes for its
development. See Fig. 29.1 for an idea.
397
Infection thread
Nodules
Bacteria
II III
Root hair I
IV
Epidermis
Cortical cells Root system of
Cross section of root leguminous plant
Fig 29.1 Different stages (I, II, II, and IV) of root nodule formation in legume plant
Once the inoculant having high nitrogen-fixing ability is introduced into the field, it
promptly enters into ecological competition with indigenous strains already present
in the soil. Sometimes, the introduced strain may not actually populate the roots but
may simply be overwhelmed by the indigenous strains. This event renders the
microbial inoculant ineffective.
29.2 PRODUCTION OF RHIZOBIUM CULTURE
Bacteria to be inoculated in soil as biofertilizer need to be multiplied on artificial

media to harvest on a large scale so that they can be supplied to farmers.
Strains of Rhizobium are grown in Yeast Extract Mannitol (YEM) broth, the
composition of which is: 1 g Yeast extract; 10 g Mannitol; 0.5 g K2HPO4; 0.2 g
MgSO4.7H2O; 0.1 g NaCl; 1000 ml Distilled water. The pH is maintained at 6.5-7.0.
The principal steps for mass cultivation are:
1. Sterilize the growth medium and inoculate with broth of mother culture
prepared in advance
2. Incubate for 3-4 days at 30-32°C
3. Test the culture for its purity and transfer to a large fermentor equipped
with temperature control and aeration device. Allow aerobic fermentation
for 4-9 days. There should be profuse growth of bacteria
4. Check the quality of the broth
5. Blend the broth with sterile carrier, e.g., peat, lignite, farmyard manure and
charcoal powder. The carrier should contain (1-4) × 109 rhizobial cells/g
6. Pack the culture in polyethylene bags and store at 4°C or supply to the
farmer
The increase in yield of legumes by using rhizobial culture ranges from 2.4% (Vigna
munga) to 16.4% (arahar: Cajanus cajan).
29.2.1 APPLICATION OF RHIZOBIAL CULTURE AT THE FARM LEVEL
There are variations in the method of application of rhizobial culture at the farm
level. One very successful method entails seed inoculation with aqueous suspension
398
of carrier culture during sowing (Fig. 29.2). The method of preparing seed inoculant
is as follows:
1. Prepare 10% sugar or jaggery solution by boiling in water and then cool
2. Add Gum Arabic (10%) to help rhizobial cells stick to the seed
3. Add the carrier-based rhizobial culture to the solution and mix well. For one
hectare, 400 g of charcoal-based culture would be sufficient
4. Add seeds in the slurry and again mix well. The number of rhizobial cells
per seed should be between 105 and 106
5. Spread the seeds in shade for drying
6. Store the seeds at 4°C or use them in the farm
Water in a container
50g sugar or jaggery
Boil for 15 min
Gum arabic (200g)
Cool it
Sticker solution Rhizobial culture
Mix properly
Inoculum slurry Seeds
Mix properly
Dry seeds in shade, keep them covered
Seeds coated with rhizobial cells
Sow in the field
Fig 29.2 Procedure for seed inoculation with rhizobial culture
29.3 PRODUCTION OF BLUE-GREEN ALGAE
In water logging conditions, cyanobacteria multiply, fix atmospheric nitrogen, and

release it into surroundings in the form of amino acids, proteins and other growth
promoting substances. The process of application of blue-green algal culture in field
as biofertilizer is also called algalization. Algalization has been reported to increase
yield in paddy by around 1200 kg/ha.
The biofertilizer is beneficial for paddy. The mass cultivation of cyanobacterial

fertilizers is done in various ways, viz.,
1. Cemented tank method

2. Shallow metal trough method
3. Polyethylene lined pit method
4. Field method
399
The polyethylene-lined pit method is most suitable for small and marginal farmers.
In this method, small pits are prepared in the field and lined with thick polyethylene
sheets. Mass cultivation of cyanobacteria is done by using any of the four methods
under the following steps:
1. Prepare the cemented tanks, shallow trays of iron sheets, or polyethylene-

lined pits in an open area. Width of tanks or pits should not be more than 1.5
m. This will facilitate the proper handling of culture
2. Transfer 2-3 kg soil (collected from open place for 1 m2 area of tank) and
add 100 g of super-phosphate. Water the pit to about 10 cm height. Mix
lime to adjust to pH 7.0. Add 2 ml of insecticide, e.g., malathion to protect
the culture from mosquitoes. Mix well and allow soil particles to settle down
3. When the water becomes clear, sprinkle 100 g of starter inoculum on the
surface of water
4. When temperature remains between 35 and 40°C during the summer,
optimum growth of cyanobacteria is achieved. Always maintain the water
level to about 10 cm during this period
5. After drying, the algal mat will get separated from the soil and form flakes.
During summer, about 1 kg pure algal mat/m2 area is produced. These are
collected, powdered, kept in sealed polyethylene bags, and supplied to the
farmers
The algal flakes can be used as starter inoculum if the same process is repeated.
400
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INDEX
A Amylase, 2, 116, 121, 125, 126, 127,

169, 170, 179, 375
Abnormal bonding, 54 Anaerobic growth factors, 149
Abzymes, 96 Analog resistance, 17
Acclimatization, 240 Analyzer, 221
Acetobacter, 2, 118, 252, 264, 265, 267, Antibiosis, 272
271 Antibiotics, 1, 2, 9, 29, 33, 40, 42, 44,
Acetobactor suboxidans, 391 49, 132, 191, 272-274, 277, 280,
Acetyl-ScoA, 311, 330 281, 289, 386, 389
Acid proteases, 127 Anticodon, 54, 56
Acidification power, 191 Antigen removal, 280
Active dry yeast, 145, 158 Antigen-antibody reaction, 34, 35
Acylase, 135 Antisense, 58
Adjuncts, 173 Apoenzymes, 96
Adsorption, 130 Appetizer, 248
After-fermentation, 241 Appetizer wine, 236
Aging, 180, 199, 209, 244, 247, 248, Architectural genes, 72
249, 367, 368 Armagnac, 254
Alcohol content, 235 Artificial vinegars, 271
Alcohol-free, 162 Ascomycotina, 139
Ales, 161 Aseptic sampling, 82, 83
Aleurone, 168 Ashbya gossypii, 315
Algalization, 399 Aspergillus, 2, 41, 116, 118, 125, 127,
Alginate, 131, 303, 304, 305 128, 139, 319, 320, 321, 323, 326,
Alkaline phosphatase, 30 349, 363, 365, 367, 369, 371
Alkaline proteases, 127 Asymmetric transcription, 58
Alkaline serine protease, 127 Attenuation, 77, 78, 196, 210
Alkylating agents, 14 Autoxidation, 377
Allosteric activator, 104, 105 Auxanogram, 5
Allosteric enzymes, 104, 114 Auxanography, 5, 282
Allosteric inhibitor, 105, 106 Auxotrophic mutants, 17, 18
Allosteric site, 104, 105, 115 Azeotropic, 225, 226
Allosteric transition, 104, 105 Azotobacter, 303, 304, 396, 397
Amelioration, 240
Amidase, 135 B
Amino acids, 1, 18, 44, 51-56, 65, 68,
69, 107, 120, 130, 148, 149, 162, Bacterial α-amylase, 126
169, 196, 220, 243, 276, 328, 340, Bacteriophage, 268
360, 361, 364, 386, 387, 390, 399 Baffles, 89, 92
Aminoadipate pathway, 339 Bakers yeast, 1, 2, 143-155, 157, 158,
Ammonia, 148, 151, 317, 324, 331, 160, 293, 343
332, 338, 354, 355, 364, 371, 377, Baking process, 248
397 Barley, 129, 161, 165, 167- 170, 173,
Ammonium persulfate, 191 179, 261, 365, 366, 370
Ammonium salts, 129, 148, 151, 153, Base analogs, 15
326, 354, 355, 375 Basidiomycotina, 139
Amoco, 353 Beer, 42, 80, 86, 125, 141, 143, 161-
Ampicillin, 32, 33, 281 163, 165, 166, 170, 171, 173, 174,
178-180, 183, 184, 190, 191, 194, Categorization of yeasts, 141
195, 197-212, 216, 218, 219, 221, cDNA library, 23
280, 291, 316, 320, 321, 383 Cell wall permeability, 17, 321, 330
Bentonite, 199, 209, 244, 249, 270 Cellulosic materials, 216
Beta carotene, 309 Central Dogma, 55
Bifunctional enzymes, 114 Chaetomium cellulolyticum, 359, 360
Binning, 244 Champagne process, 250
Biofertilizer, 396, 398, 399 Chaptalization, 240
Biological weapons, 45 Cheese, 296, 300, 346
Biomass, 1, 133, 146, 153, 180, 266, Chemotherapeutic agents, 272
279, 310, 343, 352, 356-360, 362, Chillproofing, 209
382 Chlorella pyrenoidosa, 306, 310
Bioprocess technology, 44 Chorismic acid pathway, 335
Bioreactor, 80, 88, 120 Chromatography, 122, 123
Biosensors, 137 Chromosomes, 47, 48
Biotechnology, 21, 41, 43, 45, 401, 403 Cider, 234, 253, 264, 364
Blackstrap molasses, 216 Citric acid, 1, 2, 41, 160, 294, 319, 321-
Blakeslea trispora, 306, 394 324
BOD, 147, 157, 356, 360 Classification and nomenclature of enzymes,
Bordeaux, 237, 245 97
Botrytized, 247 Clones, 11, 21, 22, 35, 282
Bottom yeast, 142 Cloning vector, 21, 26, 28, 30
Bouquet, 236, 237, 244, 247-249, 254 Clostridium, 41, 296, 297, 315, 397
Branching enzymes, 305 Codon, 23, 51-54, 56, 66-71
Brandy, 253 Cognac, 253
Brevibacterium, 329, 335 Cold trub, 190
Brew kettle, 182, 188, 189 Colony hybridization, 35
Brewers yeast, 144, 145, 238, 293 Color extraction, 240
Bright Beer Tank, 201 Common mutagens, 14
Bub vat, 217 Competitive inhibition, 101, 103
Budding, 140, 141 Composition of beer, 162
Burton Upon Trent, 179 Compound lipids, 306
Butylated Hydroxy Anisole, 159 Compressed-yeast, 151
Butyric acid, 296 Constitutive genes, 74
Contamination, 36, 37, 40, 81-83, 85,
C 87, 88, 93, 120, 155, 164, 191, 216,
Candida, 2, 139, 143, 145, 146, 231, 219, 229, 286, 300, 355, 372, 375,
237, 310, 315, 319, 335, 337, 346, 376
349, 360, 374 Cooperative binding, 104
CAP site, 76 Corepression, 76, 77
Carbon dioxide, 80, 213, 227 Corn steep liquor, 118
Carbonation, 201, 251 Corn stover, 359
Carboxymethyl cellulose, 130 Corrosion, 169, 355
Carlsberg, 127, 128, 163 Corynebacterium, 2, 310, 319, 329, 335-
Carlsberg flask, 194, 195 337
Cascade, 85, 219, 232 Cosmids, 22, 28, 29, 32
Catabolite repression, 18, 76, 115, 120, Covalent bonding, 131
126, 164, 291, 344 Crabtree effect, 154, 219
Catalase, 170 Cracking, 52
Catalytic power, 97, 99 Crowded plate, 7
406
Cryptococcus terricolus, 310 Entrapment, 131
CSTBR, 120 Environmental technology, 44
Culture yeast, 141 Enzyme engineering, 42, 116, 137, 138
Cup-plate method, 388 Enzyme kinetics, 108
Cyanogen bromide, 136 Enzyme technology, 44, 80, 96
Cyclic AMP, 76 Enzyme unit, 99
Cyclodextrin, 97 Enzyme-substrate complex, 101, 108-110
Cycloheximide, 40, 164 Episomes, 48
Dahi, 363 Eremothecium ashbyii, 315
Decline phase, 86 Ergosterol, 306, 309, 149, 190, 309
Decoction, 183 Esterases, 348
Deep bed process, 120 Ethanol, 1, 40, 44, 103, 124, 154, 156,
Degeneracy, 24, 53, 55 163, 208, 215, 216, 219, 220, 221,
Desiccation or drying, 39 224, 225-227, 231, 232, 241, 264-
Dessert wine, 236 269, 311, 324, 325, 338, 340, 347,
Determination of Km value, 112 353, 355, 364, 372, 383
Deuteromycotina, 139 Exoamylases, 125
Dextran, 301, 303 Exon, 23
Diacetyl, 198, 206, 293, 294 Exonucleases, 26, 60
Diacetyl level, 206 Extremozymes, 96, 97
Diaminopimelate pathway, 339 Extrusion, 157
Diatomaceous earth, 121, 144, 199, 244,
270, 303 F
Diazotrophs, 396, 397 Factor A, 277, 279
Digestive enzymes, 125 Fed-batch fermentation, 84, 87, 286, 332
Disgorging, 249, 250 Feed yeast, 2, 145, 146, 360
DL-α-aminocaprolactam, 341 fermentation, 1, 2, 3, 5, 13, 18, 41, 80-
DNA, 13, 15, 16, 21-33, 35, 42, 45, 87, 89, 91, 93-95, 115, 117-121,
47-51, 53, 55, 56, 58, 61-64, 68, 71, 125-129, 142, 143, 145, 146, 148,
73, 75, 97, 118, 275, 276, 360 150-156, 161-163, 179, 190, 191,
Dosage, 247, 250, 251, 288, 289 195-199, 206, 207, 210, 212, 215,
Down time, 86, 87, 219 216-220, 227, 229-243, 246-253,
Downstream processing, 86, 117, 118, 256, 262, 264-266, 269, 272, 277,
120, 133, 334, 343 279-286, 291, 293, 295, 296-307,
Draft beer, 205 312, 315, 316, 319,-321, 324, 326,
Drawing off, 241 327, 331-343, 347, 350, 353- 355,
Dried yeast, 143 358-383, 391-395, 398
Drop-plate, 388 Fermented foods, 362-364, 374
Dry milling, 171 Fermenter, 44, 80-83, 85, 87, 88-91, 93,
95, 146, 151-153, 155, 191, 196,
E 213, 217-219, 240, 242, 269, 279,
Ebullition, 188 285, 286, 291, 296, 302, 308, 326,
Effervescence, 235, 236 332, 333, 338, 340, 350, 355, 365,
Endoamylases, 125 372
Endonucleases, 26, 27 Fermenter configuration, 88
Endosperm, 167, 169 Fermenter design, 87
Endosymbionts, 50 Fermosin, 311
Enriched pellets, 176 Fill Cold, 244
Entomopthoraceae, 311 Filtration, 81, 120, 121, 136, 144, 148,
Entrainer, 226 157, 169, 171, 172, 180, 184, 186,
407
198- 207, 209, 212, 245, 246, 250, Glutamic acid, 148, 328-334, 370, 374
251, 256, 261, 280, 287, 288, 297, Glutaraldehyde, 131, 134
299, 312, 320, 321, 322, 325, 334, Glutathione, 160
342, 348, 358, 359, 369, 372 Gluten, 160, 285, 331
Fine chemicals, 117 Glycogen, 125, 190, 191
Fining agents, 199, 243, 244 Glyoxylate cycle, 323, 324, 329
Flavors and fragrance, 293 Grapes, 238
Flocculation, 142, 210, 215 Growth factor, 149
Fluidized-bed, 159 Gundruk, 363, 378, 381, 382
Foam, 179, 196, 197, 208, 249, 269,
354, 355, 372 H
Fodder yeast, 145, 146, 360 Hardness, 150, 179
Food yeast, 143 HART, 35
Fortified and sweet wines, 247 Haze, 180, 206, 209, 210
Fring’s generator process, 267 Head, 208
Fumaric acid, 2, 102, 324, 325 Headiness, 220
Fumigation, 40 Heads, 31, 222, 254
Functional proteins, 357 Health care, 45
Fungal alkaline protease, 128 Hemocytometer, 191, 192, 193
Fungal α-amylase, 125 Heterofermentative bacteria, 379
Fusel oils, 220, 224, 254 Heterotropic, 105
High fructose corn syrup, 134
G High gravity brewing, 206
Gallization, 240 Higher alcohols, 162, 212, 218, 220, 237,
Gel permeation, 122 246, 248, 253
GEMS, 42, 45, 46 High-test molasses, 216
Gene bank, 22, 23 Holoenzyme, 61-63, 96
Gene cloning, 21, 26 Homeostasis, 72
Gene machine, 24 Homofermentative, 379
Gene manipulation, 21, 45 Hop extracts, 176
General Electic Process, 231 Hop extracts, 176, 177
General methods of isolation, 10 Hops, 161, 173-176, 188, 202, 208
Gereneral process of enzyme recovery, 120 Hordein, 167, 169
General types of immobilization, 130 Host specificity, 397
Generation time, 118, 155 Hot Bottling, 244
Genetic engineering, 21, 26, 42, 45 Hot break, 188, 189
Genetic material, 47 HTST, 148, 241, 244, 247
Genome, 22, 26, 29, 45, 47, 50, 345 Hydrolase, 70, 341
Genomic DNA, 22 Hypheal tip method, 11
Genomic Libraries, 22
Geometric ratios, 90 I
German process, 267 IADY, 160
Germination, 167-170, 320, 386 Idiophase, 277, 284, 286
Germination index, 166 Immobilized enzymes, 130, 133, 137, 289
Gesammte konzentration, 266 Impeller, 89, 92, 93
GK, 266, 267 Indispensable amino acid, 335
Gluconic acid, 325, 327 Induced-fit, 108
Gluconobacter, 252, 264, 265 Inducer, 73-76, 114, 116, 120, 277, 341
Glucono-δ-lactone, 326 Inducible genes, 74
Glucose-repressible, 345 Industrial enzymes, 117
408
Infusion, 161, 181-183 Lysine, 6, 7, 17, 27, 52, 107, 131, 164,
Inoculum, 5, 7, 36, 81-83, 85, 151- 153, 283, 338-342, 361
179, 190, 217, 218, 266, 279, 285, Lysine auxotrophs, 283
290, 291, 300, 302, 321, 324, 326,
332, 347, 373, 374, 376, 377, 382, M
400 Maceration carbonique, 241
Inoculum build-up, 81, 290 Maillard, 170
Intercalating agents, 15 Malo-lactic fermentation, 242, 243, 252
Intron, 23 Malt, 151, 163, 165, 171, 181, 255,
Invertase, 1, 98, 107, 143, 324, 343-345 363
Ionizing radiations, 16 Malting, 167
Iotech Process, 232 Mame miso, 366
Isoacceptors, 54 Marker genes, 28, 282
Isoprenoid, 309 Mash filter, 184, 185
IUPAC, 41, 143 Mashing, 161, 171, 179, 181-183, 365,
366
J Maturation, 37, 142, 151, 197- 199, 244
Jand, 80, 85, 363, 382, 383, 384 Mead, 363
Meat analogs, 360
K Medium formulation, 81
Kanegafuchi, 353 MEL genes, 142
Kieselguhr, 180, 199, 200, 201, 204 Melibiose, 142
Kilning, 170 Melle-Bionot Process, 218
Kinema, 85, 364, 373-375 Membrane confinement, 132
Kinetics of enzyme biosynthesis, 115 Membrane filter, 204
Koji, 229, 319, 320, 365, 366, 369- 372 Messenger RNA, 56
Krausen, 195, 196, 199 Metabolite, 3, 4, 17, 81, 95, 150, 219,
272, 283, 284
L Metal activators, 106
Lactobacillus brevis, 380 Metalloenzymes, 106
Lactobacillus casei, 386 Michaelis-Menten, 100, 101, 104, 108,
Lactobacillus plantarum, 380, 381 110, 112
Lactoflavin, 314 Microbial genetics, 47
Lactones, 294 Microbial production of fats, 306
lacZ, 33 MIcrobial production of α-amylase, 125
Lag phase, 86 Microbiological assay, 1, 386- 389
Lagering, 180 Micromanipulator technique, 10, 11
Lagers, 161 Miso, 365, 370
Lauter tub, 187 Mixed codon, 51
Leaky cells, 116, 330 Modification, 13, 61, 71, 72, 99, 104,
Leuconostoc mesenteroides, 301, 302, 379- 106, 120, 132, 137, 169, 170, 314
381 Modules, 48
Ligase, 22, 26, 27, 29, 30, 118 Molasses, 118, 147-151, 153, 156, 157,
Lineweaver-Burk plot, 104, 112, 113 216, 217, 311, 320, 330, 331, 332,
Linkers, 27, 30 336, 340, 341, 392, 393
Lipase, 1, 97, 143, 343, 349, 375 Molds, 2, 9, 11, 118, 126, 127, 135,
Lipases, 348 139, 158, 215, 216, 220, 270, 271,
Log phase, 83, 86, 87 280, 281, 306, 311, 319, 349, 358,
Lyophilization, 37, 38, 40, 216 363, 365, 375, 382
Moniliella acetoabutens, 270
409
Monomeric, 106, 113 PEG-induced protoplast fusion, 20
Moromi, 369, 371, 372 Pekilo process, 358
Mother of vinegar, 267 Penicillin, 40, 273, 274, 281-283, 285,
mRNA, 22, 23, 50, 51, 52, 55, 56, 58, 287, 288, 331
59, 62, 64- 67, 68, 69, 70-72, 75, 77- Penicillin G, 135, 281, 282, 288
79, 115, 276, 345 Penicillin V, 135, 282
Mucor, 2, 116, 127, 128, 139, 215, 349, Penicillinase, 281
375 Penicillium, 2, 13, 19, 41, 128, 135, 139,
Multienzyme complex, 108, 113, 114, 133 273, 280, 281, 283
Murcha, 38, 363, 382, 383 PEP carboxylase, 330
Must treatment, 240 Periodic transfer, 37
Mutation, 5, 13, 16, 17, 36, 49, 54, 116, Perlite, 199
276, 278, 336, 345 Perry, 253, 363
Mute, 248 Phage, 22, 29, 31, 32, 33, 268
Mycoproteins, 358 Phagemids, 22, 28
Phenylenediamine, 314
N Pitching, 153, 190, 217
Natick Process, 231 Plasmid, 22, 28, 31, 33, 48, 49
Natto, 85, 363, 364, 370, 373, 374, 375 Plate and frame filter, 244
Negative regulation, 73, 74, 75 Plate count, 157
Niacin, 387 Podbielniak, 288
Nigar, 383 Poliovirus, 46
Nitrogen fixing, 396 Polyethylene glycol, 124
Nitrous acid, 14 Polygalacturonase, 350, 351
Non-renewable, 353 Polymerase, 22-24, 55, 58, 61-64, 71, 75,
Nonsense codons, 51, 70 77, 78, 97, 118
Nucleic acids, 16, 55, 117, 124, 276, Polymerase Chain Reaction, 24
343, 357, 360, 361 Polymixin, 191
Polyphenolase, 170
O Polysaccharides, 1, 44, 125, 145, 169,
Oil overlay method, 37 209, 216, 298, 363
Oleaginous, 306, 310, 313 Pomace, 241
Oligomeric enzymes, 113, 114 Positive regulation, 73
Operon, 72, 74, 75, 76, 77, 78 Post-translation, 72, 114
Organic acids, 319 Potential hazards of biotechnology, 45
Orleans process, 266 Pour-plate technique, 10
Overoxidation, 265, 266, 268 Precipitation, 123
OverProof, 225 Premasher, 182
Prenyl mercaptan, 202
P Press wine, 241
Paecilomyces variotii, 358 Primary metabolites, 1
Parasexual cycle, 19 Primary screening, 4
Passenger DNA, 29 Primary stock cultures, 36
Pasteur effect, 154 Primary transcript, 22, 55, 71
Pasteurization, 180, 203, 244, 369 Primer, 23, 25, 58, 62, 303
Pasteurization unit, 203 Proanthocyanidin-free barley, 166
PBR, 134, 136 Proenzymes, 106
PCR, 24, 25 Promoter, 28, 61-64, 73, 74, 76, 77
Peatiness, 261 Proof, 225
Pectin esterase, 1, 220
410
Propagation, 81, 83, 85, 194, 217, 247,
256, 257, 291 S
Propionibacterium freudenreichii, 391 Saccharomyces, 2, 49, 139, 147, 215, 231,
Protease, 2, 97, 121, 127, 128, 129, 375 237, 248, 306, 345, 350, 351, 360,
Protein-rest period, 183 363, 366, 369, 387
Protoplast fusion, 19 Sake, 41, 80, 85, 125, 363, 364, 371,
Pruteen, 353, 355 372, 383
Pseudomonas cocovenenans, 378 Salmonellosis, 289
Puncheons, 247 Sauerkraut, 85, 363, 364, 378-380
Purdue Process, 231 Sauterne, 238, 248
Pure culture techniques, 10, 238 scale-up, 95
Schizosaccharomyces, 139, 215
R Screening, 1, 4, 6, 14, 231
Racemase, 341 Secondary metabolites, 1
Racemic mixtures, 328 Secondary Screening, 4
Racking, 243 Seed yeast, 152, 153
Radiation, 16 Self-priming, 269
Raksi, 384 Semi-synthetic antibiotics, 134
Reaction catalyzed, 98, 105, 114 Semi-synthetic penicillins, 1, 135, 274,
Recombinant DNA technology, 21, 42 282, 289
Rectifier, 221 Sense strand, 58, 62, 63
Red table wine, 236, 239 Sensory analysis, 207
Reducing end, 125 Serine proteases, 127
Refinery molasses, 216 Sexual recombination, 19
Regulation of gene expression, 72 Sherry, 248
Regulatory genes, 72 Shigellosis, 289
Relative sweetness, 234, 235 Shotgun approach, 22
Remuage, 250 Shoyu koji, 369
Renewable resources technology, 44 Simple lipids, 306
Repressor, 75, 76, 77 Single cell protein, 1, 352
Respiratory Deficient Mutants, 164 Sinki, 363, 382
Respiratory Quotient, 155 Skimming, 180, 191, 199
Resting cells, 334, 341 Slack, 171
Restriction enzymes, 26, 31 Slaw, 378, 379
Reverse osmosis, 122 Solera process, 248, 249
Reverse transcriptase, 23, 55, 118 Solid state fermentation, 85, 118, 126
Reverse transcription, 22 Solvent extraction, 287, 291, 321
Reverse translation, 23 Soy sauce, 367, 370
Rhizobium, 1, 396, 397, 398 Sparger, 93, 267
Rhizopus, 2, 128, 139, 324, 349, 363, Sparkling wine, 236, 249
375-378 Spread-plate technique, 10, 11
Riboflavin, 2, 143, 149, 314-318, 377, Staphylococcus aureus, 284
391 Stationary phase, 37, 39, 86, 115
Ribosomal RNA, 56, 60 Steady state, 108, 109
Ribozymes, 97 Steeping, 167, 168
Riddling, 250 Sterile filtration, 203, 204
RNA genetic material, 50 Sterilization, 82, 88, 89, 245, 285, 315,
Rotameter, 94 316, 319, 320, 357
rRNA, 50, 55-57, 60-62, 67, 72, 276 Stock culture, 36, 38, 81, 83, 191, 194,
Rum, 363 216
411
Stop codons, 51, 69 Tunnel pasteurization, 204
Streak-plate technique, 10 Turnover number, 99
Streptococcus diacetylactis, 293 Two out of three condition, 54
Streptococcus fecalis, 158, 366, 387, 389
Streptomyces, 2, 19, 118, 134, 273, 275, U
278, 289-391, 394 Ultrafiltration, 122
Streptomycin, 2, 275-277, 279, 280, 391 Underproof, 225
Structural genes, 72 Universal Beer Agar, 164
Stuck fermentation, 196, 210, 241 Universal codon, 23
Subculturing, 11, 14, 35-38, 40, 82, 151 Unmixed family, 51
Submerged cultivation, 118 Unsaturated fatty acids, 149, 190
Submerged fermentation, 120, 126, 248, Urinary tract infection, 273, 275
249 UV-rays, 16
Substrate analog, 101
Sucrase, 98, 302, 303, 343 V
Sulfamides, 102 Vaccine, 46
Sunstruck, 178, 202 Vicinal diketone, 198
Svedberg unit, 56, 57, 276 Vinegar, 2, 242, 252, 264-271, 364, 378
Symba, 353 Vinification, 237, 246
Symbiotic diazotrophs, 397 Vitamins, 1, 96, 120, 144, 238, 360,
Symmetrical process, 58 364, 386, 390, 391
Synonymous, 53 Vitis vinifera, 238, 239
Synzymes, 97
W
T Waste technology, 44
Teminism, 55 Waterloo process, 353, 358, 359
Tempeh, 2, 363, 364, 373-375, 377, 378 Wax, 151, 309
Template, 23, 55, 56, 58, 61, 62, 63, 64 Wet milling, 171, 172
Temporal genes, 72 Whirlpool, 179, 189, 190
Tetracycline, 32, 81, 289-291, 402 Whiskey, 253, 255, 261
The genetic code, 51, 52 White miso, 366
The IUB system, 98 White table wine, 236, 246
The thin layer process, 119 Wide spectrum, 272
Top yeast, 142 Wild yeast, 141, 165
Toxoflavin, 378 Wine, 42, 80, 82, 143, 234-254, 263,
Transcription, 23, 55, 58, 61-65, 70-73, 264, 266, 371, 372, 378, 383, 401,
75-78, 114, 115, 345 402
Transfer RNA, 50, 54-56, 58, 61, 62, Wine diamonds, 244
65, 66, 68, 69-72, 276 Wine yeasts, 237, 238
Transgenic animal, 46 Wobble hypothesis, 54
Translation, 51--56, 60, 66, 69-72, 78, Woolftees plot, 112
114, 345 Working stock cultures, 36, 216
Tributyl phosphate, 322 Wort, 18, 151, 169, 171, 173, 176, 178,
Trickling process, 267 179, 188-190, 212
trp operon, 77 Wort oxygenation, 180, 190
Tryptophan, 53, 76-78, 114, 335-338,
375 X
Tryptophan hydroxamate, 336 Xanthan gum, 2, 298, 299, 300, 301
TTC overlay method, 164 Xanthomonas campestris, 2, 299
Tuberculosis, 275, 310 X-gal, 33
412
346, 358, 360, 362, 363, 365,
Y 366,375, 379, 382, 386
YAC, 28
Yeast autolysates, 1, 144 Z
Yeast machine, 217 Zero order, 111
Yeast purity, 165 Zymurgy, 161
Yeast sexuality, 139, 140 α-acid, 175, 176, 178, 188, 203, 206
Yeasts, 2, 5, 9, 18, 19, 40, 50, 135, 139- α-amylase, 170
143, 148-150, 158, 163, 164, 179, α-ketoglutarate dehydrogenase, 330
181, 183, 191, 210, 212, 215, 221, β-galactosidase, 22, 33, 74, 76, 115, 346
231, 237- 243, 245, 247, 248, 250, β-glucans, 169
270, 271, 310, 311, 315, 319, 343, β-lactam, 280
γ lactones, 325
413
Essentials of Industrial Microbiology
Basanta Kumar Rai is currently an Assoc.
Professor of Food Technology at Central
Campus of Technology, Tribhuvan
University, Nepal.
Essentials of Industrial Microbiology is basically a compilation

of a series of lectures on INDUSTRIAL MICROBIOLOGY and
MICROBIAL BIOCHEMISTRY I have delivered over the years
to B. Tech (Food Technology) and B. Sc. (Microbiology)
respectively at Central Campus of Technology, Dharan, Nepal.
The chapters included herein more than cover the current

syllabus of Industrial Microbiology for B. Tech (III year). Within
the scope and limitation of the syllabus, I have tried to put
together information as meticulously as possible. Some
descriptions have become outdated, genetic engineering in
particular. However, the basic concept is still useful. The book
contains a large number of cross-referenced diagrams, tables and
index to assist the students/readers.
Thanks are due to those authors whose books I have freely

consulted. As an acknowledgement, I have appended a short
bibliography, which I hope will be helpful to the students in
finding out additional reading materials.
ISBN 978-1-300-13701-6
90000
Basanta Rai
9 781300 137016

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Essentials of Industrial Microbiology

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Essentials of

The present book is basically a compilation of a series of lectures on INDUSTRIAL

Dharan, Mar 2012 Basanta Kumar Rai

THE SCOPE OF INDUSTRIAL MICROBIOLOGY

Industrial microbiology is one of the most important areas of applied microbiology.

From industrial microbiology standpoint, microorganisms can be considered chemical

1. Microbial cell (biomass)

The following is a brief treatment of some of the important groups of industrially

They are unicellular prokaryotes that reproduce predominantly by binary fission.

 Streptomyces sp.— for streptomycin production

 Penicillium chrysogenum — for penicillins

 Saccharomyces sp. — for alcoholic fermentation, bakers yeast

spiral coccoid elongate

(a) Bacteria (b) Yeast (d) Mold

Fig. 1.1 Typical morphology of bacteria, yeast, and mold

1.3 DESIRABLE PROPERTIES OF INDUSTRIAL MICROORGANISMS

It is difficult to generalize but the most important desirable properties an industrially

(i) Rapid growth in relatively cheap and readily available substrate

In practice, however, no single microorganism has all the above-listed properties.

Table 2.1 Simplified examples of screening of microorganisms

Amino acids Auxanography

 Classification and identification

2. For Product Yield

 Comparison with yield from known commercial strains

 Identification of the compound

 Shake-flask culture (behavior)

Indeed, a host of techniques must be used to gather all of the above-mentioned

2.2 EXAMPLES OF SCREENING

2.2.1 SCREENING OF AMINO ACID PRODUCERS

Preparation of first plate

Preparation of second plate

Top view Side view

paper Petri plate with

Fig. 2.1 Auxanographic screening of lysine producers

2.2.2 SCREENING OF ANTIBIOTIC PRODUCERS FROM SOIL

1. Take 10 g soil and prepare 10-1 dilution

Dally rod Zone of inhibition

Cross-streaking of Diffusion of Streaking of

a = Bacillus subtilis; b = Staphylococcus aureus; c = Escherichia coli; d = Saccharomyces cerevisiae

Streak culture  Purification

Agar plates/broth/small fermenter

INDUSTRIALLY IMPORTANT MICROORGANISMS

Fig. 2.3 Screening of antibiotic producer from soil

GENERAL TECHNIQUES OF SELECTION OF MICROORGANISMS

3.1.1 CHEMICAL METHODS

3.1.2 PHYSICAL METHODS

 Heat treatment: spore-formers can be selected by heating the sample to 80°C

3.1.3 BIOLOGICAL METHOD

Nature also exerts a selective force on microorganisms. Sometimes, animals can

Selective methods can be employed to obtain a large proportion of the

3.2.1 GENERAL METHODS OF ISOLATION

 Streak-plate technique (radiant, continuous, discontinuous, etc.)

3.2.1.1 Streak-plate technique

Fig. 3.1 Streak-plate isolation

3.2.1.2 Pour-plate technique

3.2.1.3 Spread-plate technique

Fig. 3.2 Spread-plate isolation

3.2.1.4 Hypheal tip method

3.2.1.5 Micromanipulator technique

Fig. 3.3 Isolation by micromanipulator