Microbial Biotech Chapter 3

3.
4 Stages of fermentation process

• The fermentation process is divided into two stages
1. upstream processing and

2. downstream processing
• The upstream process deals with:
I. Screening of industrially important microbes

II. Primary screening, preparation of pure culture and selection
of production strain at lab scale level
III.Secondary screening and optimization of production
parameter of production strain at lab and pilot scale
IV.Genetic
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modification ofFermentation
production strain if required
Technology 1
V. The formulation of inoculum and production media and
optimization of growth parameters at lab scale for product
formation
VI. The sterilization of the inoculum and production medium
VII. Sterilization of fermenter and its auxiliary equipments
VIII. The production of sufficient pure and active microbial
culture for the inoculation in production vessel
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Upstream processing…
• In general, Upstream processing involves all the steps related to:
 inoculum development,
 media development,
improvement of inoculum by genetic engineering and related
processes
 optimization of growth kinetics so that product development
can improve tremendously.

Media development
• Availability of nutrients is necessary to the cell to maintain
metabolic activity
• Formulation of proper medium is an essential requirement for the
success of a fermentation process
• Medium used for industrial fermentations should fulfil the
following criteria:
1. Maximum yield and conc of product or biomass per
unit mass of substrate
2. Minimum production of undesirable metabolites
3. Consistent quality of product
4. Minimal problems during sterilization and fermentation
5. Ease of disposal of wastes
6. Cheap

Inoculum preparation
 If the preserved culture is used it should be initially revived by

growth in Erlenmeyer flask on a biological shaker or on a solid
medium (if spore formation is needed).
 In order to obtain sufficient inoculum for small fermentors, a
second series of shake cultures is usually made in more flasks.
 Out from lyophilized strains the growth of inoculum takes around
4 -10 days, out from frozen cultures the growth of inoculum takes
4-48 hours for bacteria and 1-7 days for fungi.
 Finally, out of refrigerated cultures the growth of inoculum takes
4 -24 hours for bacteria and 1-5 days for fungi.
In most cases, fermentation produces metabolites, not the cells
themselves. However, there are a few exceptions where the cells
are the desired product, such as in the production of single-cell
protein or yeast.
Cells to be used must be actively growing, young and vigorous

and must therefore be in the phase of logarithmic growth.
Since organisms used in most fermentations are aerobes, the

inocula will usually be vigorously aerated in order to encourage
maximum cell development.
 The chemical composition of the medium may differ in the

inoculum and production stages.
Fermentation medium
• Like any other living system, microorganisms also require a

source of energy, carbon, nitrogen, oxygen, iron and other
minerals, micronutrients, and water for growth, and
multiplication.
• All these nutrients that are essential for the growth and
multiplication of microbial organisms are supplied in the form of
nutrient media.
• For laboratory-scale cultivation we may use certain costly media

components, but for industrial purposes they should be
economical
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and readily available. Fermentation Technology
7
• If the nutritive components of the media are not of natural origin,
such nutritive media are known as synthetic media, which we can
synthesize in the laboratory following certain recipes, mixing the
required salts, minerals, and carbon source.
• Many nutrient media available commercially contain both natural

and added components and are semi-synthetic media.
• For example, commercially available nutrient broth, trypticase

soya broth (TSB), brain-heart infusion (BHI) broth, yeast extract,
potato dextrose agar, casein digest, etc., are some examples of
semi-synthetic media.
• For laboratory-scale cultivation of bacteria and other
microorganisms, these synthetic or semi-synthetic media are
preferred.
• For commercial purposes, the recommended media should be

cheap and available year round.

Media design for fermentation processes
Water is at the center of all biotechnological processes and in

most cases will be the dominant component to the media in
which organisms (microorganisms, plant and animal cells) will
grow.
The quality of water is highly relevant
In media production there is usually quality control of the raw

materials.

The basic nutritional requirements of microorganisms are an
energy or carbon source, an available nitrogen source, inorganic
elements, and for some cell types, specific growth factors.
In most biotechnological processes, carbon and nitrogen sources

are more often derived from relatively complex mixtures of cheap
natural products or by-products.
Availability and type of nutrient can exert strong physiological

control over fermentation reactions and product formation.
 Raw material input to a fermentation will be largely dependent
on the cost of the material at a particular time.
The medium must allow maximum growth potential throughout
the fermentation if biomass is required , whereas for a compound
not growth limited, e.g. organic acids, antibiotics, etc., a medium
must be formulated that after an initial growth phase will become
deficient in one or more nutrients.
Depending on the process, limitation of P, N, carbohydrates or

trace metals can induce product formation.
 In some fermentations high levels of glucose can create

catabolite repression of, for example, enzyme synthesis; this can
be overcome by using other slowly fermentable carbohydrates or
by incremental or continuous feeding of the glucose.
Media components
• Carbon is a major component of cell biomass, since 50% of

the dry weight of cells is carbon.
• Facultative organisms incorporate about 10% of the carbon in

the substrate into cell material under conditions of anaerobic
metabolism.
• In the presence of oxygen, up to 55% of the substrate carbon

is converted into carbon incorporated into the cell biomass.
• CO2 is the source of carbon for autotrophs (e.g., alga) and
autolithotrophs (some types of archae bacteria found at the
bottom of the ocean).
 A simple carbon source, which is simple to use and easily
available, can be used.
 Sugars such as glucose, lactose, sucrose, and complex

polysaccharides such as starch, glycogen cellulose used.
 A mixture of various carbohydrates, and other compounds such

as cereal grain powders, cane molasses, etc., are usually used as
carbon sources in microbial culture media.
 The main purpose of the carbon source is to provide energy and

carbon skeleton for the synthesis of various other biological
compounds.
Nitrogen Source
Nitrogen is about 10% of the weight of most organisms, and is

supplied to microbial cultures through a variety of compounds.
The most direct source of nitrogen is inorganic, and may consist of

ammonia or ammonium salts.
However, this is insufficient if a specific amino acid needed for

growth cannot be made by the organism. The amino acid must then
be supplied in the broth.
Proteins, nucleic acid and vitamins, as well as individual amino acids

are recognized as organic sources of nitrogen.
Complex sources of nitrogen include soybean, peanut, fish
meals, yeast extract, whey, casein, and hydrolyzed proteins.
Byproducts from sugar processing, such as molasses from

sugarcane or corn steep liquor from corn wet milling, contain
such nutrients.
Corn steep liquor or molasses is therefore used as a supplement

or replacement for water in making up a major part of the liquid
fraction of the starting culture media.

The minimum components required in a microbial medium:
Trace elements: Fe, Zn, Cu, Mn, Mo, Co
Antifoaming agents: Esters, fatty acids, fats, silicones, sulphonates,

polypropylene glycol
Buffers: Calcium carbonate, phosphates
Growth factors: Some microorganisms cannot synthesize the required

cell components themselves and need to be supplemented, e.g. with
thiamine, biotin, calcium pentothenate
Precursors: Directly incorporated into the desired product: Phenyl

ethyl amine into Benzyl penicillin, Phenyl acetic acid into Penicillin G

Inhibitors: To get the specific products: e.g. sodium barbital for
rifamycin
Inducers: The majority of the enzymes used in industrial

fermentation are inducible and are synthesized in response of
inducers: e.g. starch for amylases, maltose for pollulanase,
pectin for pectinase.
Chelators: Chelators are the chemicals used to avoid the

precipitation of metal ions. Chelators like EDTA, citric acid,
polyphosphates are used in low concentrations.

Sterilization
Sterilization - MOs are either killed or removed from the
material/equipment
Sterilization ensures that
 only the desired MO is present to carry out the fermentation
 products are made of predicted quality

 the env`t is protected from undesirable contamination
 deterioration (microbial spoilage) of products is prevented

The various methods of sterilization are:
1. Physical methods
a. Thermal (heat) methods
b. Radiation method
c. Filtration method
2. Chemical method
 Different gases

Physical Methods
1. Heat sterilization
Heat sterilization is the most widely used and reliable method of

sterilization, involving destruction of enzymes and other essential
cell constituents.
This method of sterilization can be applied only to the thermo-

stable products, but it can be used for moisture-sensitive materials.
i) Dry Heat (160-180˚C) sterilization for thermo-stable products
ii) Moist heat (121-1340˚C) sterilization is used for moisture-

resistant materials.
Heat sterilization
The action ofThe efficiency with which heat is able to inactivate MOs
is dependent upon
i) the degree of heat,

ii) the exposure time and
iii) the presence of water.
 heat will be due to induction of lethal chemical events mediated
through the action of water and oxygen.
In the presence of water much lower temperature time exposures are
required to kill microbe than in the absence of water.
Thermal (heat) methods
Thermal methods includes:
i) Dry heat sterilization
1.Incineration
2.Red heat
3.Flaming
4. Hot air oven
ii) Moist heat sterilization
1.Dry saturated steam (autoclaving)
2. Boiling water/steam at atmospheric pressure
3. Hot water below boiling point

Dry heat sterilization
It employs higher temperatures in the range of 160-180˚C and requires
exposures time up to 2 hours, depending upon the temperature
employed.
 The benefit of dry heat includes good penetrability and non-corrosive

nature which makes it applicable for sterilizing glass wares and metal
surgical instruments.
It is also used for sterilizing non-aqueous thermo-stable liquids and

thermo-stable powders.
Dry heat destroys bacterial endotoxins (or pyrogens) which are

difficult to eliminate by other means and this property makes it
applicable for sterilizing glass bottles which are to be filled aseptically.
Moist heat sterilization
Moist heat sterilization involves the use of steam in the range of
121-134˚C.
Steam under pressure is used to generate high temperature needed

for sterilization.
Saturated steam acts as an effective sterilizing agent.

Autoclave
Autoclaves use pressurized steam to destroy MOs, and are the most
dependable systems available for the decontamination of laboratory
waste and the sterilization of laboratory glassware, media, and
reagents.
 For efficient heat transfer, steam must flush the air out of the
autoclave chamber.
Generally the conditions employed are temperature upto121-134˚C for

15-20 min under 15 lbs pressure, based on type of material used.

Radiation sterilization
Many types of radiation are used for sterilization like
electromagnetic radiation (e.g., gamma rays, UV light,etc).
The major target for these radiation is microbial DNA.
Radiation sterilization with high energy gamma rays or

accelerated electrons has proven to be a useful method for
the industrial sterilization of heat sensitive products.
Microwave irradiation, commonly in food industries.

Radiation…
Radiation sterilization is generally applied to articles in the dry state;
including surgical instruments, sutures, prostheses, unit dose
ointments, plastic syringes and dry pharmaceutical products.
UV light, with its much lower energy, and poor penetrability finds uses
in the sterilization of air, for surface sterilization of aseptic work areas.
Fig. Laminar Air Flow Bench

Filtration sterilization
Filtration process does not destroy but removes the MOs.
It is used for both the clarification and sterilization of liquids and gases
as it is capable of preventing the passage of both viable and non-viable
particles.
 The major mechanisms of filtration are sieving, adsorption and

trapping within the matrix of the filter material.

Chemicals used for sterilization or disinfection
Alcohol
Ethylene Oxide
Ozone
Bleach
Sanitizer
Glutaraldehyde and Formaldehyde
Phthalaldehyde
Hydrogen Peroxide
Peracetic Acid
Metals (e.g.,silver)

• Newer techniques
 e.g., high voltage pulses and photo- semiconductor powers
 involve the rupture of the cell membrane by increasing the

transmembrane
 electric field strength beyond a certain threshold

Fermentation process
The culturing of the microbial system can be achieved in different
ways.
The type of culture method sometimes depends on the type of the

microbial system or on the type of the product that we expect.
For example, one can get two entirely different products from the
same organism by changing the nutritional and other parameters or
even culturing vessels.
Batch culture. This is a small-scale laboratory experiment in

which a microbial culture is growing in a small volume flask.

It consists of a limited volume of broth culture in a flask
inoculated with the bacterial or microbial inoculum and follows a
normal growth phase.
 It is a closed-culture system because the medium contains a

limited amount of nutrients and will be consumed by the growing
microorganisms.
In batch cultures, the nutrients are not renewed and the
exponential growth of cells is limited to a few generations.

During the log phase the consumption of the nutrients will be the
maximum resulting in the maximum biomass output with the
excretion of the product.
At the stationary phase the rate of growth decreases and becomes
zero. This is because at the stationary phase the cells are exposed to
a changed environment.
Fed-batch culture. The batch culture can be made into a semi-

continuous culture or fed-batch culture by feeding it with fresh
media sequentially at the end of the log phase or in the beginning of
the stationary phase without removing cells.

Because of this the volume of the culture will go on increasing as
fresh media is added.
This method is specially suited for cultures in which a high
concentration of substrate is inhibitory to cell multiplication and
biomass formation.
In such situations the substrate can be fed at low concentrations to
achieve cell growth.
This method can easily produce a high cell density in the culture
medium, which may not be possible in a batch fermentor or shake
flask culture.
This is especially important when the product formation is

intracellular to achieve maximum product output per biomass.

3. Continuous culture. Bacterial cultures can be maintained in a
state of exponential growth over long periods of time using a system
of continuous culture.
Continuous culture, in a device called a chemostat, can be used to

maintain a bacterial population at a constant density.
This is a very convenient method to get continuous cell growth

and product formation over a long period of time.
The nutrient medium including the raw material is supplied at a

rate that is equal to the volume of media with cells and product
displaced or removed from the culture.

The volume removed and the volume added is the same.
 In effect there is no change in the net volume as well as the

chemical environment of the culture.
In a chemostat, the growth chamber is connected to a reservoir of

sterile medium. Once the growth is initiated, fresh medium is
continuously supplied from the reservoir.
The volume of fluid in the growth chamber is maintained at a

constant level by some sort of overflow drain. Fresh medium is
allowed to enter into the growth chamber at a rate that limits the
growth of the bacteria.
The bacterial cells grow (cells are formed) at the same rate at
which bacterial cells and spent medium are removed by the
overflow.
The rate of addition of the fresh medium determines the rate of

growth because the fresh medium always contains a limiting
amount of an essential nutrient.
Thus, the chemostat relieves the insufficiency of nutrients, the

accumulation of toxic substances, and the accumulation of excess
cells in the culture, which are the parameters that initiate the
stationary phase of the growth cycle.

 Since the culture is fed with the fresh medium at specific rate, a
steady state of growth and metabolism is achieved.
 At a steady state, the cell multiplication and substrate

consumption for growth and product formation occur at a fixed
rate. The growth rate is maintained constantly.
 The formation of new biomass is balanced with the removal of

cells from the outlet. Continuous culture is very suitable for the
production of cell biomass and products.
 It is widely used for the production of single-cell protein from

liquid effluents as a byproduct of the waste treatment.

Microbial growth
• Growth is an orderly increase in the quantity of cellular
constituents.
• It depends on the ability of the cell to form new protoplasm from

nutrients available in the environment.
• Four general patterns of cell multiplication:

I. Binary fission- bacteria
II. Budding- yeast
III. Elongation of mycellium and its branching- fungi
IV. In the case of virus, there is no regular pattern for
multiplication, mainly because its multiplication is host
dependent and grow Fermentation
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intracellularly.
Technology 41
When bacteria are grown in a closed system such as a test tube,
the population of cells almost always exhibits these growth
dynamics: cells initially adjust to the medium (lag phase) until
they can start dividing regularly in the exponential phase.
When their growth becomes limited, the cells stop dividing

(stationary phase), until eventually they show loss of viability
(death phase).
Growth is expressed as change in the number of viable cells

versus time or the cell biomass versus time.

In a microbial culture under a steady state the substrate is the
nutrients and the product is the cell biomass.
In such a reaction the rate of growth will be proportional to the

cell biomass present in the culture.
The cell culture behaves like an autocatalytic reaction.
When the microbial culture is under a steady state the rate of

increase in cell biomass is equal to the product of specific growth
and cell concentration (biomass concentration).

Agitation in a Fermentor
Agitators with their attached impellers serve a number of ends.

They help to distribute the incoming air as fine bubbles, mix
organisms uniformly, create local turbulence, as well as ensure a
uniform temperature.
Since the viscosity of the broth may alter as fermentation

proceeds, a satisfactory compromise of size, shape, and number of
impellers must be worked out.
In un baffled fermentors a vortex or inverted pyramid of liquid

forms and liquid is thrown up on the side of the fermentor.

The result is that heavier particles sediment and thorough
agitation is not achieved.
 The insertion of baffles helps eliminate the formation of a

vortex and interferes with the upward throw of liquid against the
side of the fermentor.
The use of baffles thus ensures not only a more thorough

mixing of the nutrient and air but also the breakup of the air
bubbles.

Aeration in a Fermentor
Oxygen is essential for growth and yield of metabolites in aerobic

organisms.
In those fermentations where aerobic organisms are used, the

supply of oxygen is therefore critical.
For the oxygen to be absorbed by MO. it must be dissolved in

aqueous solution along with the nutrients.
At 20°C for example, water holds only about nine parts per million
of oxygen. Furthermore, the higher the temperature the less oxygen
and other gases water can hold.
For some highly aerobic fermentations such as the growth of yeast
or production of citric acid, oxygen is so critical that even if the
broth were entirely saturated with oxygen it would contain only a
15 second supply for the organisms.
The oxygen control in industrial fermentations is as important as

pH, temperature and other environmental controls.

The air used in fermentation, whether, sterile or not, is forced
under pressure into the bottom of the fermentor just below the
lowest impeller the air enters through a sparger which is a pipe
with fine holes.
The smaller the holes the finer the bubbles and the more
effective the supply of oxygen to the microorganisms.
However, if the holes are too small, then a greater pressure will
be required to force the air through, with consequent higher
consumption of energy and therefore of costs.

Process Control in a Fermentor
 Process control is concerned with making adjustments to the

process consequent upon the measurement of one or more of the
variables that change as a result of the action of the process.
 The course of a fermentation may be followed by monitoring

various operational parameters within the fermentor e.g. pH, air
input, effluent gases, temperature; factors such as cell yield, or the
output of metabolites may also be followed.
• The degree of accuracy of the monitoring depends of course on
the instruments being used for the purpose.
pH measurement and control
 In some industrial fermentations, good yield depends on
accurate control of the pH of the fermentation broth.
 Sterilizable pH probes are inserted in the fermentor or in a
suitable projection therefrom in which the broth bathes the
electrode.
 With these electrodes it is now possible to use an arrangement
which will monitor pH changes and automatically induce the
introduction into the medium of either acid or alkali.

Carbon dioxide measurement
The measurement of CO2 helps determine the course of the

fermentation as well as the carbon balance.
 Three principles are employed in for CO2 determination.

The first method, which is the most widely used, depends on the
ability of CO2 to absorb infrared rays. A sensitive sensor translates
this absorption to a gauge or record, from which it can be read off.
 In the second method the effluent gas emerging from the broth is
bubbled through a dilute solution of NaOH containing phenol red.
The change in color of the phenol red is reflected in a photocell
and the amount of CO2 may be calculated from a standard curve.
The third method depends on the thermal conductivities of the
various gases in a mixture.
Oxygen determination and control
• Two principles:
1. The polarographic-In the polarographic method, a negative electric

current 0.6-0.8 in voltage is passed through an electrode immersed in an
electrolyte made of neutral potassium chloride. This negative electrode
(cathode) is made of a noble metal such as platinum or gold. The
electric current generated in the system is proportional to the quantity of
oxygen reacting at the cathode
2. The galvanic method- In the galvanic method, no external source of

electricity is applied. Instead the electricity generated between a base
metal anode (zinc, lead, or cadmium) and noble metal cathode (silver or
gold) is sufficient to cause the reduction of oxygen at the cathode.
Pressure
It is important to know the pressure of gases in order to ensure that

a positive pressure is maintained.
A positive pressure helps eliminate contamination and

contributes to the maintenance of proper aeration.
Pressure may be determined with aid of a manometer.

Downstream processing
Downstream processing refers to the isolation and purification of a
biotechnologically formed product to a state suitable for the intended use.
 In most, but not all, biotechnology processes the desired product(s) will
be in dilute aqueous solution and the ultimate level of downstream
processing will mirror the type of product and required degree of purity.
The range of products is considerable and varied in form and can include
whole cells amino acids, vitamins, organic acids, solvents, enzymes,
vaccines, therapeutic proteins and monoclonal antibodies.
Within these products there will be considerable variation in molecular

size and chemical complexity, and a wide range of separation methods
will be required for recovery and purification.
Fig. 3.2 Examples of unit downstream
processing.
 Improvements in downstream processing will benefit the
overall efficiency and costs of the processes.
 Downstream processing will primarily be concerned with initial

separation of the bioreactor broth into a liquid phase and a solid
phase, and subsequent concentration and purification of the
product.
 Downstream processing is a multistage operation. It will

depend largely on the chemical and physical properties of the
products.

Initial pre-treatment or conditioning of the broth can involve
flocculation of cells or solids, or the breakdown of cells to
release intracellular products such as enzymes.
Separation of components in the liquid phase of the broth can

involve settling, filtration, centrifugation or electrokinetic
separation, with the final degree of processing dependent on the
final use of the products.
 Final product isolation will utilize established chemical and

biochemical technologies.

Molecular size or centrifugation will determine the extraction processes
that will be most suitable, and will be influenced by product quality and
overall economics. Final product purity will reflect the type of product
and the standard of purity required.
Methods currently used include evaporation and distillation,

precipitation, membrane filtration, adsorption, affinity, ion-exchange and
gel-filtration chromatographies.
Final products of the downstream purification stages should have some

degree of stability for commercial distribution.
Stability is best achieved for most products by using some form of

drying. In practice this is achieved by spray-drying, and freeze-drying.

Products sold in the dry form include organic acids, amino acids,
antibiotics, polysaccharides, enzymes, single cell protein and
many others.
Many products cannot be supplied easily in a dried form and must

be sold in liquid preparations. Care must be taken to avoid
microbial contamination and deterioration and, when the product
is proteinaceous, to avoid denaturation.
Purity and stability are the hallmarks of most high-value

biotechnological products.

Steps needed
1. Removal of insoluble
2. Isolation or extraction of product
3. Product purification
4. Concentration of the product
5. Product polishing (packaging of product)

Removal of insolubles
• Capture of the product as a solute in a particulate-free liquid
• Example separation of cells, cell debris or other particulate
matter from fermentation broth containing a production e.g., an
antibiotic.
• Types of operation
• Filtration =is technically defined as the process of separating suspended solid
matter from a liquid, by causing the latter to pass through the pores of a membrane
• Centrifugation =is a method of separating molecules having different densities by
spinning them in solution around an axis (in a centrifuge rotor) at high speed
• Flocculation = is a process by which a chemical coagulant added to the water acts
to facilitate bonding between particles, creating larger aggregates which are easier
to separate
Product isolation
• Reducing the volume of material to be handled and
concentrating the product.
• The unit operations involved
• solvent extraction
• ultra filtration
• precipitation
• adsorption

Product purification
• To separate contaminants that resemble the product very
closely in physical and chemical properties
• Expensive & require sensitive and sophisticated
equipments
• After a product is recovered or isolated, it may need to be
purified further
• The purification can be accomplished by numerous
methods:
• precipitation
• chromatography
• electrophoresis
• ultrafiltration
Product polishing
• Final processing steps which end with packaging of the
product in a form that is stable, easily transportable and
convenient
• Typical unit operations in product polishing include:
• Crystallization,
• Desiccation,
• Lyophilization and
• spray drying

Factors Affecting Choice of Recovery Process
 Intracellular or Extracellular Location
Impacts whether cell disruption is required.
 Concentration of Product in Broth
Determines the efficiency and scale of recovery methods.
 Physical & Chemical Properties
Guide the selection of purification techniques.
 Use of the Product
Influences the required level of refinement and purity.
 Minimal Acceptable Standard of Purity
Dictates the extent of purification needed.
 Biohazard of the Product
Safety measures may affect the recovery process.
 Impurities in the Broth
Additional purification steps may be necessary.

Downstream processing operations:
1. Separation
a) Filtration
b) Centrifugation
c) Disruption
A) Filtration -The rotary vacuum filter:
One of the most commonly used filters in industry is the rotary

vacuum filter which is available in several forms.

 Essentially the filter consists of a hollow rotating cylinder divided
into four partitions and covered with a metal or cloth gauze.
A vacuum is created inside the rotating cylinder, which is designed

to enhance the filtration process. As the cylinder turns, the vacuum
generates a pressure difference that effectively pulls liquid from the
slurry surrounding it
B) Centrifugation- In the enzyme isolation industry, centrifugation

is preferred to filtration, probably because unwanted cell debris are
quite efficiently removed by this method. A large number of
centrifuges are available.
C) Cell disruption
A lot of biological molecules are inside the cell, and they must
be released from it. This is achieved by cell disruption (lysis).
Cell disruption is a sensitive process because of the cell wall’s

resistance to the high osmotic pressure inside them.
Furthermore, difficulties arise from a non-controlled cell

disruption, that results from an unhindered release of all
intracellular products (proteins, nucleic acids, cell debris) as well
as the requirements for cell disruption without the desired
product’s denaturation. There are mechanical and non-
mechanical cell disruption methods.
2. Concentration:
Extraction – Liquid –Liquid extraction and dissociation extraction
Liquid–liquid Extraction
Also known as solvent extraction widely used in industry.
 it is a partitioning, is a method to separate compounds or metal

complexes, based on their relative solubilities in two different
immiscible liquids, usually water and an organic solvent.

Dissociation Extraction
Many fermentation products are either weak bases or acids.
When solvent extraction is employed the pH is so selected that

the material to be isolated is unionized since the ionized form is
soluble in the aqueous phase and the unionized form is soluble in
the solvent phase.
Weak bases are therefore extracted under high pH conditions and

weak acids under low pH conditions.

Precipitation
The insolubility of many salts is used in the selective isolation of

some industrial products. It is particularly useful in the
elimination of proteinaceous impurities or in the isolation of
enzymes.
Salts are precipitated by one of several methods:
adding inorganic salts and reducing the solubility with the addition
of organic solvents such as alcohol in the case of enzymes.
Lactate and oxalate salts of erythromycin have been so isolated

and citric acid has been isolated with its calcium salt.
3. Purification
The methods used in this section are finer and further eliminate
the impurities thus leaving the desired product purer.
A. Chromatography
 is based on the principle where molecules in mixture applied onto

the surface or into the solid, and fluid stationary phase (stable
phase) is separating from each other while moving with the aid of
a mobile phase
In chromatography, the components of a mixture of solutes

migrate at different rates on a solid because of varying solubilities
of the solutes in a particular solvent.
B. Crystallization
Crystallization is the final purification method for those materials
which can stand heat.
The solution is concentrated by heating and evaporation at
atmospheric pressure to produce a super saturated solution.
The initial water removal is made by heating at reduced pressure or

by lowering the temperature to form crystals which can be
centrifuged off leaving a concentrated liquor.
 It yields compounds which are highly potent more stable and free
from colored impurities.

4. Product isolation
• Product isolation refers to the final steps in the purification process,
ensuring that the desired compound is obtained in a usable form
The final isolation of the product is done in one of the two following
ways:
(i) processing of crystalline products.
(ii) drying of products direct from solution (removing the solvent

directly from a solution containing the desired product, resulting
in a concentrated or dry form of the compound)

Drying
 Drying is the process of removing liquid from wet crystals or
solids, including those isolated from solutions or biological cells.
Types of Drying:
I. Liquid Phase Moisture Removal:
• Involves the removal of moisture that is present in the liquid
phase of a solution.
• Typically used for crystallizing materials to obtain dry crystals
from a saturated solution.
II. Solid-Phase Moisture Removal:
• Refers to the extraction of moisture that is bound within the solid
matrix of materials.
• Common techniques include oven drying, freeze drying, and air
drying, aimed at achieving the desired dryness in solid products.

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3.

4 Stages of fermentation process

1. upstream processing and

• The upstream process deals with:

I. Screening of industrially important microbes

11/7/2024 Fermentation Technology 2

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 If the preserved culture is used it should be initially revived by

Cells to be used must be actively growing, young and vigorous

Since organisms used in most fermentations are aerobes, the

 The chemical composition of the medium may differ in the

• Like any other living system, microorganisms also require a

• For laboratory-scale cultivation we may use certain costly media

• Many nutrient media available commercially contain both natural

• For example, commercially available nutrient broth, trypticase

• For commercial purposes, the recommended media should be

11/7/2024 Fermentation Technology 9

Water is at the center of all biotechnological processes and in

The quality of water is highly relevant

In media production there is usually quality control of the raw

11/7/2024 Fermentation Technology 10

In most biotechnological processes, carbon and nitrogen sources

Availability and type of nutrient can exert strong physiological

Depending on the process, limitation of P, N, carbohydrates or

 In some fermentations high levels of glucose can create

• Carbon is a major component of cell biomass, since 50% of

• Facultative organisms incorporate about 10% of the carbon in

• In the presence of oxygen, up to 55% of the substrate carbon

 Sugars such as glucose, lactose, sucrose, and complex

 A mixture of various carbohydrates, and other compounds such

 The main purpose of the carbon source is to provide energy and

Nitrogen is about 10% of the weight of most organisms, and is

The most direct source of nitrogen is inorganic, and may consist of

However, this is insufﬁcient if a speciﬁc amino acid needed for

Proteins, nucleic acid and vitamins, as well as individual amino acids

Byproducts from sugar processing, such as molasses from

Corn steep liquor or molasses is therefore used as a supplement

11/7/2024 Fermentation Technology 16

Antifoaming agents: Esters, fatty acids, fats, silicones, sulphonates,

Buffers: Calcium carbonate, phosphates

Growth factors: Some microorganisms cannot synthesize the required

Precursors: Directly incorporated into the desired product: Phenyl

11/7/2024 Fermentation Technology 17

Inducers: The majority of the enzymes used in industrial

Chelators: Chelators are the chemicals used to avoid the

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 products are made of predicted quality

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a. Thermal (heat) methods

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Heat sterilization is the most widely used and reliable method of

This method of sterilization can be applied only to the thermo-

i) Dry Heat (160-180˚C) sterilization for thermo-stable products

ii) Moist heat (121-1340˚C) sterilization is used for moisture-

i) the degree of heat,

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 The benefit of dry heat includes good penetrability and non-corrosive

It is also used for sterilizing non-aqueous thermo-stable liquids and