Unit – I

INTRODUCTION TO INDUSTRIAL BIOPROCESS

1.0 Introduction to Bioprocess technology

1.1 History of Fermentation Technology
1.1.1 Antony Van Leeuwenhoek – contribution
1.1.2 Spontaneous generation
1.1.3 Lizaro Spallanzani – contribution
1.1.4 Robert Koch – Contribution
1.1.5 Eduard Buchner – contribution
1.1.6 Wieldiers – Contribution
1.1.7 Kluyver – Contribution
1.1.8 Alexander Fleming – Contribution
1.2.0 Traditional and Modern Biotechnology
1.3.0 Products relating to Modern Biotechnology
1.4.0 Survey of Microorganism
1.5.0 Fermentation process
1.5.1 Solid state fermentation
1.5.2 Submerged fermentation
1.5.2.1 Batch cultivation
1.5.2.2 Continuous cultivation
1.5.2.3 Fedbatch cultivation
1.6.0 Structure and Anatomy of a Fermenter
1.6.1 Basic structure of a fermenter
1.7.0 Fermentation media
1.7.1 Media composition
1.7.2 Sterilization and Contamination
1.7.3 Inoculum Media
1.7.4 Screening for fermentation media
1.8.0 Isolation and Screening of Microbes
1.8.1 Isolation
1.8.2 Screening
1.8.2.1 Primary screening of Microbes
1.8.2.2 Secondary screening of Microbes
1.9.0 Process flow sheets
1.10.0 Block diagram of Fermentation process

1.0 INTRODUCTION TO MODERN BIOPROCESS TECHNOLOGY

 The modern bioprocess technology is an extension of ancient techniques of
developing useful products from natural biological activities. Alcoholic beverages
are good examples of bioprocessing. The combination of yeast cells and nutrients
from grain forms a fermentation system in which the organisms consume the
nutrients for their own growth and produce alcohol and CO2 that helps to produce
the beverage.
The modern bioprocessing technology is more sophisticated, but its
principle remains the same. Bioprocess is widely used commercially in the
production of enzymes, food products, agricultural products, etc.
With improved techniques and instrumentation, bioprocess may have
applicatioins even in areas where chemical processes are used. The advantages of
bioprocess over chemical methods are,

 They usually require lower temperature, pressure & pH.

 They use renewable raw materials.
 They can produce greater quantities with less energy
consumption.

In most bioprocesses, enzymes are used to catalyse the biochemical

reactions of the microorganisms or their cellular components. The biological
catalyst does not change itself. In the fermentation tanks or fermenters, a series of
reactions take place to change the initial raw materials into the desired product.

Bioprocessing done through lab procedures can produce only small amount
of useful substances. As advances in bioprocess technology are made, especially
separation and purification techniques, commercial firms can produce these
substances in large amounts and thus make them available for use in medical
research, food processing, agriculture, waste management and many other fields of
science.
HUMAN APPLICATION
 Biotechnological methods are now used to produce several pharmaceutical
and other specialized purposes. A harmless strain of E.coli, given a copy of human
insulin gene, can produce insulin which can be purified and used to treat diabetes
in humans. Microbes can also be modified to produce digestive enzyme
deficiency. Products of modern bioprocessing include artificial blood vessel from
collagen tubes coated with a layer of the anticoagulant heparin.

FERMENTATION TECHNOLOGY

Fermentation technology is the oldest of all biotech processes. The term

was derived from Latin ‘fevere’ which means to boil that appears when yeast acts
upon the fruit extracts or malted grain during the production of alcohol.
Fermentation is a process of chemical change caused by organism or thier
products, usually producing effervescence and heat.
In microbiology, the production of a thing by means of mass culture of
microorganisms is considered fermentation. The biochemical metabolism in
fermentation is an energy generating aerobic process in which organic compounds
acts as electron donors.

Louis Pasteur defined this principle as, the anaerobic reactions through
which microorganisms obtained energy for growth in the absence of oxygen.

Modern (Today) fermentation technology can be defined as both aerobic

and anaerobic metabolic activities of microorganisms in which specific chemical
changes are brought about in an organic substrate.
1.1 HISTORY OF FERMENTATION TECHNOLGY
1.1.1 ANTONY VAN LEEUWENHOEK- CONTRIBUTION
Antony Van of Holland started his hobby of making simple microscopes.
During his lifetime, he made more than 250 microscopes. These consisted of home
ground lenses mounted in brass and silver. Thus, Antony was able to obtain
magnifications upto 160 diameters. This magnification is enough to allow
observation of some of the larger microbial forms. He described beer yeast as
being very small spherical or oval granules. His observation was important,
because it revealed that this yeast mass was actually composed of many minute
spherical forms, but he failed to explain those spherical forms were living entities.
1.1.2 SPONTANEOUS GENERATION
Redi’s experiment in the 17th century demonstrated that the macro concepts
of spontaneous generation were fallacious; mice did not spring forth de novo from
old rags, nor did maggots from meat. These experiments did not explain the
apparent spontaneous generation at the micro level, such as the spontaneous
appearance of microscopically observable microorganisms in solutions of organic
materials, particularly in sugar solutions undergoing alcoholic fermentation.
1.1.3 CONTRIBUTION OF LAZARO SPALLANZANI
In 1799, Lazaro attempted to disprove spontaneous generation by
hermetically sealing infusions of seeds in flasks, followed by heating of the flasks
for varying periods of time in a boiling water bath. In these experiments, he
observed that a short period of heating killed one group of microbes, probably the
bacteria, were more resistant to heating.
Thus his experiment first attempted the difference in heat resistance of
various types of organisms and that prolonged heating may be necessary for
sterilization.
Microbial growth occurred when Lazaro’s flasks were opened to the
atmosphere. Questions were raised by other investigators as to whether this growth
reflected introduction into the medium from the air of new viable organisms or
whether the organisms originally present in the flasks were not killed by the heat
and were just waiting for the introduction of air to resume growth. These
investigators also proposed an alternate hypothesis that supported the spontaneous
generation theory that the heat had killed the microorganisms but that reentry of
atmosphere into the media in the flasks had caused the spontaneous transformation
of insert medium components into living microorganisms.
1.1.4 CONTRIBUTION OF ROBERT KOCH
Robert Koch had been studying the relationship of microorganisms to
disease, but was encountering difficulty in not being able to work with pure
cultures of the disease producing microorganisms, especially since Lister
technique was not feasible for his studies. Koch’s observation that boiled potato
slices on incubation allowed development of colonies of microorganisms and that
these colonies could be transferred to fresh potato slices prompted him to search
for a solidifying agent which could be added to liquid medium so as to provide a
similar type of colonial growth. Potato slices could not be used for his animal
pathogens, since these organisms did not grow on this substrate. He therefore
employed gelatin as a solidifying agent and as a result was able to obtain
individual colonies well separated from each other on the surface of the gelatin
medium.
Furthermore, he realized that colony morphology could be utilized for the
description and identification of microorganisms and that this technique also could
be used for the purification of cultures.
Frau Hesse, the wife of one of Koch’s students, realized that agar provided
a solidifying that demonstrated none of the faults of gelatin or of solidified blood
serum.
While Koch’s plating procedures provided tremendous advantages for the
study of microorganisms, there was still the of a suitable culture vessel.
In 1887, Petri solved this problem by describing what is known as the Petri
plate, a culture dish is still used extensively today.
Further studies on this fermentation, various individuals postulated that,
possibly it was mediated by an enzyme associated with the yeast.

1.1.5 EDUARD BUCHNER (1897)

 Buchner ground up yeast cells and expressed the juice from the broken
cells. He then filtered the juice to obtain a clear solution and mixed the solution
with a concentrated sugar solution. On incubation, CO2 was evolved and he
identified and quantified the ethyl alcohol that was formed.
He considered that the active agent was a soluble proteinecious enzyme,
because filtration that removed all the yeast cell fragments had no effect and
because the activity was not susceptible to chloroform treatment.
He called this enzyme Zymase and considered it probable that zymase,
under natural conditions was extra cellular and operated in the medium outside the
yeast cells. Thus, Buchner was the first to demonstrate enzymatically mediated
fermentation reactions.

1.1.6 WILDIERS (1901)

He demonstrated that Yeast has Vitamin requirement for growth and won
the first demo of a growth factor requirement for microbes. He applied the name
‘bios’ to this growth factor.

1.1.7 KLUYVER (1924)

Kluyver (1924) publication entitled “Unity and diversity in the metabolism
of Microorganisms” discussed the striking diversity and a yet basic similarity of
metabolic pathways and of thermodynamic considerations among microorganisms
as well as higher forms of life.

1.1.8 ALEXANDER FLEMING (1929)

The discovery of Penicillin altered research workers to the possibility that
other microorganisms might synthesize antibiotics. Thus, with intensive research
effort during the Second World War and thereafter, Streptomycin,
Chloromphenicol, Tetracycline and a series of other antibiotics of great
commercial and medicinal value were discovered.
 It is apparent that many of these studies do have a strong bearing on the
technology and thinking of modern day industrial microbiology as well as on the
technology of the times in which the studies were carried out. Also, we see that
many of the techniques and the concepts developed are still valid today.
 TABLE 1.1 The chronological development of the fermentation industry may be represented as five overlapping stages

STAGE MAIN PRODUCTS REACTOR NATURE PROCESS CULTURE QUALITY STRAIN

CONTROL METHOD OF THE SELECTION
PRODUCT
Wooden, 1500 barallel Thermometer, Batch fermentation Pure cultures
I Alcohol capacity,Copper used in Hydrometer and Medium of Yeast
Pre 1900s later brewaries. heat exchanger
Vinegar Barrels, Shallow trays, “ “ “ “ Pure cultures
trickle filters “ “ of Yeast
Baker’s yeast, Steel vessel of 200m3 pH electrode with Batch and Fedbatch Pure cultures
II Glycerol, Citric acid, capacity for acetone / offline control. fermentation
Lactic acis and butanol. Air spargers & Temperature “ “
1900-1940 acetone / Butanol mechanical stirrers were control
introduced.
Penicillin, Mechanically aerated Sterilizable pH & Batch, Fedbatch and Mutatiuon
Streptomycin, vessels,aseptic operation, oxygen electrodes Continuous and selection
III Gibberelin, true fermenters with fermenattion
Aminoacids, computerized High
1940- date Nucleotides, controlloops
transformation,
enzymes
IV Sigle cell protein Pressure cycle and Computer linked Continuous culture Genetic Engg’
production using pressure jet vessels to controlloops with medium recycle on strains
1964- date hydrocarbons overcome problems with High attempted
gas & heat exchange
Heterologous proteins Feremnters developed in Control and Batch, fedbatch & Introduction
by microbial and stage 3 & 4. Animal cell sensors developed Continuous of foreign
V animal cells. reactors developed in stages 3 & 4 fermantations. High genes through
Monoclonal antibodies Perfusion methods rDNA
1979- date produced by animal are developed for technology
cells animal cell processes for strain
improvement.
1.2.0 TRADITIONAL & MODERN BIOTECHNOLOGY
Many authors prefer to use the term old or traditional biotechnology to the
natural process that have been in use for many centuries to produce beer, wine,
curd, cheese and many other foods.
The new or modern biotechnology embraces all the genetic manipulation,
cell fusion techniques and the improvements made in the old biotechnological
processes.
We have to accept that the present day biotechnology is not something new,
but it represents a series of technologies. Some of them dating back to thousands
of years of production of foods, beverages, modification of plants and animals
with desired tracts. It is only in recent years that these traditional practices are
being subjected to scientific scruting, understood and improved atleast in some
instances.
The development of any fermentation process requires constant
experimental work and patience. No book can provide complete details about
fermenttation processes, since such details are trade secrets. Hence,
experimentation is a must in establishing new fermentation plants.

1.3.0 PRODUCTS RELATING TO MODERN BIOTECHNOLOGY

Microorganisms were exploited for useful purposes long before anything

was known about their existence or their characteristics. As early as 6000 B.C, the
Babylonians and Sumerians used to make alcohol. History reveals many other
applications of microbial processes that resulted in the production of desirable
materials,particularly foods and beverages. However, as we have mentioned
earlier, it was not until the studies of Pasteur in the second half of the 19 th century
that the role of microrganisms in these processes was understood.
Microorganisms can be considered chemical factories in nature. They have
the capacity to convert a raw material into end product. If the end products have
value for human use, then it becomes attractive to exploit the microbiological
process, which is to produce the end products on commercial value.
Industrial biotechnology has experienced two explosions during the last few
decades. In the 1940s, the discovery of antibiotics, led by Penicillin, initiated a
major new industry built upon the products of microorganisms. More recently, as a
result of the great advances in our knowledge of microbial genetics, it is possible
to manipulate microorganisms genetically to produce new products. This process
is known as recombinant DNA Technology. The major modern biotechnology
products were tabulated below.
 TABLE 1.2 Some Industrial Products produced by Bacteria
PRODUCT MICROORGANISM USES
Acetone / Butanol Clostridium acetobutylicum Solvents; Chemical manufacturing
2,3 Butanediol Bacillus polymyxa, Enterbacter Sp. Solvent; humectants; chemical intermediate
Dihydroxy acetone phosphate Glucanobacter suboxydans Fine chemical
2-keto gluconic acid Pseudomonas Spp. Intermediate for D- ascorbic acid production
5- keto gluconic acid Glucanobacter suboxydans Intermediate for Tartaric acid
Lactic acid Lactobacillus bulgaricus food products; textile & Laundry; chemicals;
deliming hides
Bacterial amylase Bacillus subtilis Modified starches; sizing paper; desizing textiles
Bacterial protease Bacillus subtilis Bating hides; desizing fibres; spot remover;
tenderizing meat
Cobalamin ( Vit.B12) Streptomyces olivaceus, Treatment of pernicious anaemia; food and feed
Propionibacteria supplements
Glutamic acid Brevibacterium Spp. Food additive
Lysine Micrococcus glutamicus Animal feed additive
Insulin, Interferon, Somatostatin rDNA varities of E.coli Human therapy
Bioinsecticiedes Bacillus thuringiensis, Control of Insects
Bacillus popillae
Sorbose Glucanobacter suboxydans Manufacture of ascorbic acid.
 TABLE 1.3 SOME COMMERCIAL PRODUCTS OF YEAST
PRODUCT MICROORGANISM USES
Baker’s yeast, Beer, Wine & Saccharomyces cerevisiae Baking industry; Brewing
Bread. Industry
Soy sauce Saccharomyces rouxii Food condiment
Sour french bread Candida milleri Baking
Commercial alcohol Saccharomyces cerevisiae, Fuel; Solvent
(Ethanol) Kluyveromyces fragilis
Riboflavin Eremothecium ashbyi Vitamin supplement
Candida utilis Animal feed (SCP) from
Microbial protein paper waste.
(Single cell protein) Saccharomyces lipolytica Microbial protein from
petroleum products.

1.4.0 SURVEY OF MICROORGANISMS

The major group of microorganisms are briefly described below. Although
viruses are not protists or cellular organisms, they are included for two reasons,

 The techniques used to study viruses are microbiological in

nature.
 Viruses are causative agents of diseases, hence, digestive
procedures for their identification are employed in the clinical
microbiological lab as well as the plant pathology laboratory.
ALGAE:
These are relatively simple organisms. The most primitive types are
unicellular. Others are aggregation of similar cells with little or no differentiation
in structure or function. Still other algae, such as the large brown kelp, have a
complex structure with cell types specialized for particular functions. Regardless
of size or complexity, all algal cells contain chlorophyll and are capable of
photosynthesis. Algae are found most commonly in aquatic environments or in
damp soil.

VIRUSES:

These are very small non cellular parasites or pathogens pf plants, animals
and bacteria as well as other microorganisms. They are so small that they can be
visualized only by the electron microscope. Viruses can be cultivated only in
living cells.
BACTERIA:
These are unicellular prokaryotic organisms or simple associations of
similar cells. Cell multiplication is usually by binary fission.
PROTOZOA:
These are unicellular eukaryotic organisms. They are differentiated on the
basis of morphological, nutritional and physiological characteristics. Their role in
nature is varied but the best known protozoa are the few that cause disease in
human beings and animals.
FUNGI:
Fungi are eukaryotic lower plants devoid of chlorophyll. They are usually
multi cellular but are not differentiated into root, stems and leaves. They range in
size and shape from single celled microscopic yeasts to giant multi cellular
mushrooms and puffballs. We are particularly interested in those organisms
commonly called molds, the mildews, the yeasts and the plant pathogens known as
rusts. True fungi are composed of filaments and masses of cells which make up the
body of the organisms, known as mycelium. Fungi reproduce by fission, by
budding or by means of spores borne on fruiting structures that are quite
distinctive for certain species.

Some morphological and characteristic features of these various microbial

groups are shown in the given below table 1.4
 TABLE 1.4 CHARACTERISTICS OF MAJOR MICROORGANISMS

GROUP SIZE IMPORTANT FEATURES PRACTICAL SIGNIFICANCE

Bacteria 0.2 – Prokaryotic; unicellular; simple internal Some cause disease; having important role in cycling
100µm structure; grow on artificial lab media; of elements for soil fertility; used in manufacture of
asexual reproduction by simple cell important components; some spoil foods and some
division make foods
Viruses 0.015 – Do not grow on artificial lab media; Cause disease in humans; other animals and plants;
0.2 µm require living cells within which they are also infect microorganisms.
reproduced; all are obligate parasites; can
view only by electron microscope.
Fungi – 5.0 - 10 Eukaryotic; unicellular; lab cultivation Production of alcoholic beverages; also used as food
Yeasts µm much like that of bacteria; asexual supplement; some cause disease.
reproduction by cell division, budding and
sexual reproduction.
Fungi – 2.0 – Eukaryotic; multi cellular; with many Responsible for decomposition of many materials;
Molds several distinctive structural features; cultivated in useful for industrial production of many chemicals
mm laboratory much like bacteria; reproduction including Penicillin; cause diseases of humans,other
by sexual and asexual process. animals and plants.
2.0 - 200 Eukaryotic; unicellular; some cultivated in Food foe aquatic animals; some cause disease.
µm laboratory much like bacteria; some are
Protozoa intracellular parasites; reproduction by
asexual and sexual processes.
Algae 1.0 µm to Eukaryotic; unicellular and multi ceelular; Important to the production of food in aquatic
many most occur in aquatic environments; environments; used as food supplement and in
feet contain chlorophyll and are photosynthetic; pharmaceutical preparations; source of agar for
sexual and asexual reproduction microbiological media; some produce toxic
substances.
1.5.0 FERMENTATION PROCESS

The anaerobic or aerobic, energy generating catabolic reactions of organic

substances by means of mass cultivation of particular microorganisms is called as
fermentation process.
This principle can be broadly classified into two major types. They are,
 Solid state fermentation
 Submerged fermentation
This classification is based on the nature of substrate what we use.

1.5.1 SOLID STATE FERMENTATION

In Solid state fermentation solid or semi solid medium is used to carry out
fermentation. This fermentation principle is preferable for cultivation of
filamentous fungi because the fungi can cover the solid substrate with mycelium
and utilize the nutrients completely. But, some of the fungi can also be cultivated
by submerged fermentation with certain conditions.
A solid porous matrix which can be biodegradable or not but with a large
surface area per unit volume in the range of 10 3 – 106 m2 / cm3, is used for
microbial growth on the solid / gas interface.
The matrix should absorb water several times its dry weight with a
relatively high water activity on the solid/gas interface in order to allow high rates
of biochemical processes.
The solid/gas interface should be a good habitat for the fast development of
specific cultures of moulds, yeasts or bacteria, either in pure or mixed cultures.
The mechanical properties of the solid matrix should withstand
compression or gentle stirring, as required for this fermentation process. This
requires small granular or fibrous particles which do break or stick to each other.
 The solid matrix should not be contaminated by inhibitors of microbial
activities and should be able to absorb or contain available microbial foodstuff
such as Carbohydrates, nitrogen sources and mineral salts.

1.5.2 SUBMERGED FERMENTATION

The fermentation process that is carried out using broth or liquid medium is
known as submerged fermentation. Basically, three types of cultivation methods
are carried out such as,
1. Batch Cultivation
2. Fed batch Cultivation
3. Continuous Cultivation
1.5.2.1BATCH CULTIVATION

The batch fermentation is a technique for large scale production of

microbes or microbial products in which, at a given time, the fermenter is stopped
and the culture is worked up. Besides, the culture is allowed to cross all the phases
of its growth and there is no addition of fresh medium or recovering of culture till
the end of process. At about the onset of the stationary phase, the culture is
disbanded for the recovery of its biomass or the compounds that accumulated in
the medium and a new batch is set up.
The advantage of this method is that optimum levels of product can be
recovered, it can be used for different reactions everyday and it can be properly
sterilized. Little risk of infection or strain mutation and complete conversion of
substrate are possible.
The disadvantages are the wastage of unused nutrients, the peaked input of
labour and the time lost between batches much idle time for sterilization, growth
of inoculums, cleaning after the fermentation and the safety problems when filling,
emptying and cleaning.
1.5.2.2CONTINUOUS CULTIVATION

The culture medium may be designed such that growth is limited by the
availability of one or two components of the medium. When the initial quantity of
this component is exhausted, growth ceases and a steady state is required, but
growth is renewed by the addition of the fresh limiting component. A certain
amount of whole culture medium can also be added periodically at the time of
steady state sets in. The addition nutrients will increase the volume of the medium
in the fermenter. It is so arranged that the increased volume will drain off as an
overflow, which is collected and used for recovery of products. At each step of
addition of the medium, the medium becomes dilute both in terms of the
concentration of the biomass and the products. New growth, stimulated by the
added medium will increase the biomass and the products till another log state sets
in; and another aliquot of medium will reverse the process. This is continuous
cultivation.
[

Since the growth of the organism is controlled by the availability of growth

limiting chemical component of the medium, this system is called a chemostst.
The rate at which aliquots are added is the dilution rate that is in effect the factor
that dictates the rate of growth.
The continuous processing offers the most control over the growth of cells.

1.5.2.3 FED BATCH CULTIVATION

In the fedbatch cultivation, a fresh aliquot of the medium is continuously or
periodically added, without the removal of the culture fluid. The fermenter is
designed to accommodate the increasing volumes. The system is always at a quasi
steady state.
1.6.0 STRUCTURE AND ANATOMY OF A FERMENTER

The fermenter is a device used to carry out submerged fermentation. Some

times, fermenters are also called as Bioreactors. There is a quite difference
between fermenters and bioreactors based on the nature of cultivation that is
carried out using the device.

If fermentation is carried out for the purpose of metabolites

(Ex. Antibiotics & Enzymes)
the device is called as fermenter. If fermentation is carried out for the purpose of
cell biomass (Ex. Cultivation for SCP), the device is called as bioreactor.

The shape and provisions present in the fermenter differ with the nature of
the desired product. Fed batch reactors are commonly used to produce biological
products. Batch reactors are the second commonly used. Continuous reactors are
rarely used for large scale production of biochemicals, used in waste treatment
processes.

1.6.1 BASIC STRUCTURE OF A FERMENTER

The fermenter comprises several components, big and small. We shall

discuss selective components that make fermentation feasible on a large scale,
which include the following,
 Stirrers
 Spargers
 Baffles
 Components
 Heating and cooling system
 Sensors
 Steaming unit
STIRRERS
The stirrer is used to agitate the medium constantly and allow even
distribution of air throughout the medium. The stirrer comprises two parts, namely
centre shaft and impellers. The impellers are fixed to the electric motor which
rotates the impellers.

IMPELLERS

Two types of impellers are used in industrial fermentation.

(a) The propeller agitator resembles marine propellers, the difference being
that the vessel remains stationary while the fluid moves.

(b) Axial impellers are similar to the radial ones, except that the blades are
pitched usually at 45 degree angle, which causes flow to move downward, parallel
to the shaft and then upward along the wall of the tank. The impellers are
generally installed vertically in the tank so as to allow the fluid to circulate in one
direction along the axis while flowing in the opposite direction along the walls.

SPARGERS
Spargers are a type of equipment used to introduce smallest bubbles into a
fermenter. The smallest sparger is the single orifice sparger. As the size of the
fermenter increases, the gas requirements increase. To achieve this, the diameter
of the orifice can be increased.
The aim is to keep bubbles as small as possible. For better aeration needs,
the ring sparger is incorporated. The ring sparger has many holes to introduce the
maximum expected air into the fermenter. The sparger is located under the disc of
the lower impeller so that air steam is directed through the impellers. The combine
spargers are a combination of agitator with sparger. During agitation, sterile air is
passed through the pores of the sparger which also aerate the medium. Thus
aeration and agitation are carried out in a single device.
BAFFLES
In a fermenter, there is a need for agitation in both aerobic and anaerobic
operations. Agitators which are centrally mounted in the fermentation tanks
exhibit poor mixing characteristics if baffles are absent. Baffles reduce the
impellers to deliver power to the fluid by preventing it from swirling. The baffled
reactor shows that the lesser agitator speed and power consumption is required to
achieve the better agitation of the medium than unbaffled reactor.

COMPRESSORS

Compressors are necessary in industrial fermentation. They supply the

fermentation tanks with the air necessary to run the process. Most tank require a
compressors capable of delivering 300 – 400 h.p. A fermentation plant may use
one or several compressors.

SINGLE COMPRESSOR PLANTS: In single compressor plants, the

cost of operation is minimal. The only disadvantage is, if the compressor breaks
down, the production gets disturbed until the compressor is fixed.

MULTIPLE COMPRESSOR PLANT: A compressor for each unit solves

the problems associated with single compressor. The cost of maintenance will be
much higher than that of single compressor plants.

COOLING JACKETS
Cooling jackets are present around the fermenter vessel. There are two
types of cooling jackets. They are,
 Vessel Type
 Tube Type
In vessel type, the jacket is like a vessel which is attached to the fermenter
vessel and connected to the inlet and outlet.
 In tube type, pipe is attached either in a spiral manner to the inner
peripheral or outer peripheral region of fermenter vessel.
Based on temperature, either hot or cold water will be passed through the
jackets. In large fermenters, heating coils and cooling compressors will be present
along with the pipes.

STEAMING UNIT
The steaming unit in the fermenter is a modified autoclave. Steam produced
here is used to sterilize the unit before running the fermenters and after culture
recovery. The steaming unit is automatically controlled when the maximum
limitation of steam is produced and constant pressure of steam is passed through
the heating jackets. Usually steam passing is controlled by the valve system either
controlled manually or automatically.

Picture 1.1 Overall structure of a fermenter

SENSORS
Sensors are devices that can detect and sent he signals to the controlling
units of a feremnter. These sensors are used to measure temperature, pH, dissolved
oxygen (DO), etc.

pH PROBE: It is mainly used to detect the pH of the medium. The pH

electrode is always immersed inside the medium.

DO PROBE: It is an electrode used to detect the dissolved oxygen present

in the medium. It is should be kept immersed in the fermentation medium.
 1.7.0 FERMENTATION MEDIA

Media is the one which supplies the essential nutrients and miscellaneous
growth factors for the successful growth of microorganisms. This media posses the
following characteristic features,

1.7.1 MEDIA COMPOSITION

The choice of a good medium is virtually as important to the success of an

industrial fermentation as is the selection of an organism to carry out the
fermentation. The medium supplies nutrients for growth, energy, etc.

1.7.2 STERILIZATION & CONTAMINATION

Media for industrial fermentations usually are sterilized. In certain
instances, the economics of the fermentation do not allow a sterilization expense,
but the fermentation can still be conducted on an industrial scale. Such
fermentations employ low pH values and possibly other contamination inhibitors
to held in check the levels of contaminating organisms.
1.7.3 INOCULUM MEDIA
Inoculum media usually differ in composition from production media.
Inoculum media are composed to quickly yield large numbers of microbial cells in
their proper physiological and morphological states but without sacrificing genetic
stability of the cells.

1.7.4 SCREENING FOR FERMENTATION MEDIA

All of the previous points in relation to fermentation media must be
considered when we attempt to compound a high yielding medium for an existing
or a new fermentation.

After the above methods the next significant step in the fermentation
process is screening of microorganisms and isolation of for culture.
 1.8.0 ISOLATION & SCREENING OF MICROBES

1.8.1 ISOLATION
Isolation is the important step in bioprocessing techniques, which involves
detecting microorganisms of interest from the whole microbial population. The
isolation of organisms may be done with conventional microbiological methods or
dorect sampling methods. The selection of sample is based on the desirable end
products. When an organism is decided for use in production, it can be isolated
from the related environment of the product.

1.8.2 SCREENING

Screening is the next important step in bioprocessing where the isolates are
investigated for producing the desired product. Screening is basically of two types,
namely primary and secondary screening.

Primary screening is checking the quality of microbes. Most cases of

primary screening are done on agar plates. Secondary screening is checking of
isolates for their quantitative production in media.

1.8.2.1 PRIMARY SCREENING OF MICRO ORGANISMS

Primary screening for enzyme is done by cultivating the isolates in agar

plates incorporated with specific substrate. The production of a specific enzyme
can be detected in two ways; by observing the substrate utilization zone around the
colonies of isolates or by adding reagents to the end product of the reaction, which
shows the utilization zone.
Screening of microorganisms that produce organic acids from various
carbon substrates is often done in a medium incorporated with pH indicator dyes
like neutral red and bromothymol blue. The production of acids will turn the
colour of the dye. An alternative method for screening of organic acids producing
strains is culturing the strain in calcium carbonate incorporated agar media. The
acid produced will dissolve calcium carbonate and form a clear zone.

The simplest screening technique for antibiotic producers is the crowded

plate technique procedure. In this procedure, the soil or other source of
microorganisms is diluted only to a cell concentration such that agar plates
prepared from these dilutions will be crowded with individual colonies on the
surface of the agar; that is 300 – 400 or more colonies per plate. Colonies
producing antibiotic activity are indicated by an area of agar around the colony
that is free of growth of their colonies. Such a colony is sub cultured to a similar
medium and purified by streaking before making stock cultures.

Antibiotic screening is improved further by incorporation into the

procedure of a test organism, which is an organism used as an indicator for the
presence of specific antibiotic activity. Dilutions of soil or of other microbial
sources are applied to the surfaces of agar plates so that well isolated colonies will
develop roughly 30 – 200 colonies per plate. The plates are incubated until the
colonies are a few mm in diameters and so that antibiotic production will have
occurred for those organisms having this potential. A suspension of the test
organisms is then sprayed or applied in some manner to the surface of the agar and
the plates are further incubated to allow growth of the test organism.

Antibiotic activity is indicated by zone of inhibited growth of the organism

around antibiotic producing colonies. In addition, a rough approximation of the
relative amounts of antibiotic produced by various colonies can be gained by
measuring in mm the diameters of the zones of inhibited test organism growth.
Antibiotic producing colonies again must be isolated and purified before further
testing.
1.8.2.2 SECONDARY SCREENING

Secondary screening is used to test further the capabilities of and gain

information about microorganism.

Secondary screening is conducted on agar plates, in flasks or small

fermenters containing liquid media or as a combination of these approaches.

Secondary screening can be qualitative or quantitative. The qualitative

approach, for example, tells us the spectrum or range of microorganisms which is
sensitive to a newly discovered antibiotic. The quantitative approach tells us the
yields of antibiotic which can be expected when the microorganism is grown in
various differing media.

Secondary screening should determine whether the microorganism are

actually producing new chemical compounds not previously described or
alternatively for fermentation process that are already known, secondary screening
should determine whether more economical process is possible. To achieve this
aim, can utilize paper, TLC and other chromatographic techniques.

Secondary screening should also reveal the problems associated with pH,
aeration or other critical requirements for the growth of the organisms and for the
formation of chemical products.
This screening should also detect gross genetic instability in microbial
cultures. Thus, a microorganism is of little value if it tends to mutate or change in
some manner, so that it losses its ability to accumulate high yields of product.
It should determine whether the product has a simple, complex or even a
macro molecular structure, if this information is not available. It should show
whether the product possesses physical properties such as UV absorption or
Fluorescence or Chemical properties that can be employed to detect the compound
during the use of paper chromatography or other analytical methods in the
structure of the compound.
Secondary screening should reveal whether a product resulting from a
microbial fermentation occurs in the culture broth in more than one form and
whether it is an optically or biologically active material.
It should tell whether microorganisms are able o chemically alter or even
destroy their own fermentation products. The microorganism may because of high
accumulation of product in the culture broth, produce adaptive enzymes that
destroy the usefulness of the product.

The proceeding discussion emphasizes the fact that secondary screening

can provide a broad range of information which helps in deciding which of various
microbial isolates possess possible usefulness as an industrial organism.
1.9.0 PROCESS FLOW SHEETS