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Industrial microbiology involves the use of microorganisms to produce commercially valuable
Industrial microbiology includes many areas, including:

food production,


Biomass utilization


Biotechnology involves the use of an organisms biochemical and metabolic pathways for
industrial production of commercially viable products
Fermentation in food processing typically is the conversion of carbohydrates to alcohols and
carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic
conditions. Fermentation usually implies that the action of microorganisms is desirable, and the
process is used to produce alcoholic beverages such as wine, beer, and cider. Fermentation is
also employed in the leavening of bread, and for preservation techniques to create lactic acid in
sour foods such as sauerkraut, dry sausages,and yogurt, or vinegar (acetic acid) for use in
pickling foods.
Food fermentation has been said to serve five main purposes

Enrichment of the diet through development of a diversity of flavors, aromas, and

textures in food substrates

Preservation of substantial amounts of food through lactic acid, alcohol, acetic acid and
alkaline fermentations

Biological enrichment of food substrates with protein, essential amino acids, essential
fatty acids, and vitamins

Elimination of antinutrients

A decrease in cooking times and fuel requirements


Fermentation is the process of deriving energy from the oxidation of organic compounds, such
as carbohydrates, and using an endogenous electron acceptor, which is usually an organic
compound. In contrast, respiration is where electrons are donated to an exogenous electron
acceptor, such as oxygen, via an electron transport chain. Fermentation is important in anaerobic
conditions when there is no oxidative phosphorylation to maintain the production of ATP
(Adenosine triphosphate) by glycolysis. During fermentation, pyruvate is metabolised to various
different compounds. Homolactic fermentation is the production of lactic acid from pyruvate;
alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide; and
heterolactic fermentation is the production of lactic acid as well as other acids and alcohols.
Sugars are the most common substrate of fermentation, and typical examples of fermentation
products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be
produced by fermentation, such as butyric acid and acetone. Yeast carries out fermentation in the
production of ethanol in beers, wines and other alcoholic drinks, along with the production of
large quantities of carbon dioxide. Fermentation occurs in mammalian muscle during periods of
intense exercise where oxygen supply becomes limited, resulting in the creation of lactic acid.

Ethanol fermentation
Ethanol fermentation (performed by yeast and some types of bacteria) breaks the pyruvate down
into ethanol and carbon dioxide. It is important in bread-making, brewing, and wine-making.
The chemical equation below summarizes the fermentation of glucose, whose chemical formula
is C6H12O6. One glucose molecule is converted into two ethanol molecules and two carbon
dioxide molecules:
C6H12O6 2C2H5OH + 2CO2
C2H5OH is the chemical formula for ethanol.
Before fermentation takes place, one glucose molecule is broken down into two pyruvate
molecules. This is known as glycolysis.

Lactic acid fermentation

Lactic acid fermentation is the simplest type of fermentation. Essentially, it is a redox reaction. In
anaerobic conditions, the cells primary mechanism of ATP production is glycolysis. Glycolysis
reduces transfers electrons to NAD+, forming NADH. However, there is only a limited supply
of NAD+ available in a cell. For glycolysis to continue, NADH must be oxidized have electrons
taken away to regenerate the NAD+. This is usually done through an electron transport chain in
a process called oxidative phosphorylation; however, this mechanism is not available without
Instead, the NADH donates its extra electrons to the pyruvate molecules formed during
glycolysis. Since the NADH has lost electrons, NAD+ regenerates and is again available for
glycolysis. Lactic acid, for which this process is named, is formed by the reduction of pyruvate.

In heterolactic acid fermentation, one molecule of pyruvate is converted to lactate; the other is
converted to ethanol and carbon dioxide. In homolactic acid fermentation, both molecules of
pyruvate are converted to lactate. Homolactic acid fermentation is unique because it is one of the
only respiration processes to not produce a gas as a byproduct.
Homolactic fermentation breaks down the pyruvate into lactate. It occurs in the muscles of
animals when they need energy faster than the blood can supply oxygen. It also occurs in some
kinds of bacteria (such as lactobacilli) and some fungi. It is this type of bacteria that converts
lactose into lactic acid in yogurt, giving it its sour taste. These lactic acid bacteria can be classed
as homofermentative, where the end product is mostly lactate, or heterofermentative, where
some lactate is further metabolized and results in carbon dioxide, acetate or other metabolic
The process of lactic acid fermentation using glucose is summarized below. In homolactic
fermentation, one molecule of glucose is converted to two molecules of lactic acid:
In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose
being converted to one molecule of lactic acid, one molecule of ethanol, and one molecule of
carbon dioxide:
The fermentation process requires the following:

a) a pure culture of the chosen organism, in sufficient quantity and in the correct physiological
b) sterilised, carefully composed medium for growth of the organism;
c) a seed fermenter, a mini-model of production fermenter to develop an inoculum to initiate the
process in the main fermenter;
d) a production fermenter, the functional large model; and
e) equipment for i) drawing the culture medium in steady state, ii) cell separation, iii) collection
of cell free supernatant, iv) product purification, and v) effluent treatment.
Items a) to c) above constitute the upstream and e) constitutes the downstream, of the
fermentation process,
Fermenters/bioreactors are equipped with an aerator to supply oxygen in aerobic processes, a
stirrer to keep the concentration of the medium uniform, and a thermostat to regulate
temperature, a pH detector and similar
The preparation of a population of microorganisms from a dormant stock culture to an active
state of growth that is suitable for inoculation in the final production stage is called inoculum
development. As a first step in inoculum development, inoculum is taken from a working stock
culture to initiate growth in a suitable liquid medium.
Bacterial vegetative cells and spores are suspended, usually, in sterile tap water, which is then
added to the broth. In case of nonsporulating fungi and actinomycetes the hyphae are fragmented
and then transferred to the broth.
Inoculum development is generally done using flask cultures; flasks of 50 ml to 12 litres may be
used and their number can be increased as per need. Where needed, small fermenters may be
Inoculum development is usually done in a stepwise sequence to increase the volume to the
desired level. At each step, inoculum is used at 0.5-5% of the medium volume; this allows a 20200-fold increase in inoculum volume at each step. Typically, the inoculum used for production
stage is about 5% of the medium volume.


Growth media are required for industrial fermentation, since any microbe requires water,
(oxygen), an energy source, a carbon source, a nitrogen source and micronutrients for growth.
Carbon & energy source + nitrogen source + O2 + other requirements Biomass + Product +
byproducts + CO2 + H2O + heat

Raw material


corn sugar, starch, cellulose


sugarcane, sugar beet molasses


milk whey


vegetable oils


petroleum fractions


soybean meal, corn steep liquor, distillers' solubles


pure ammonia or ammonium salts



nitrate salts

Phosphorus source

phosphate salts
Vitamins and growth factors
Yeast, Yeast extract
Wheat germ meal, cotton seed meal
Beef extract
Corn steep liquor

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
Inhibitors: To get the specific products: e.g. sodium barbital
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,olive oil and tween are also used at times.
Chelators: Chelators are the chemicals used to avoid the precipitation of metal ions. Chelators
like EDTA, citric acid, polyphosphates are used in low concentrations.


At about the onset of the stationary phase, the culture is disbanded for the recovery of its biomass
(cells, organism) or the compounds that accumulated in the medium (alcohol, amino acids), and a
new batch is set up. This is batch processing or batch culture.

The best advantage of batch processing is the optimum levels of product recovery. The
disadvantages are the wastage of unused nutrients, the peaked input of labour and the time lost
between batches.
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 reached, but growth is renewed by the addition of the limiting
component. A certain amount of the whole culture medium (aliquot) can also be added
periodically, at the time when steady state sets in. The addition of nutrients will increase the
volume of the medium in the fermentation vessel. 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 steady state sets in; and another aliquot of medium will
reverse the process.
This is continuous culture or processing. Since the growth of the organism is controlled by the
availability of growth limiting chemical component of the medium, this system is called a
chemostat. The rate at which aliquots are added is the dilution rate that is in effect the factor that
dictates the rate of growth.
The events in a continuous culture are:
a) the growth rate of cells will be less than the dilution rate and they will be washed out of the
vessel at a rate greater than they are being produced, resulting in a decrease of biomass
concentration both within the vessel and in the overflow;
b) the substrate concentration in the vessel will rise because fewer cells are left in the vessel to
consume it;
c) the increased substrate concentration in the vessel will result in the cells growing at a rate
greater than the dilution rate and biomass concentration will increase; and
d) the steady state will be re-established.
Hence, a chemostat is a nutrient limited self-balancing culture system, which may be
maintained in a steady state over a wide range of sub-maximum specific growth rates.
The continuous processing offers the most control over the growth of cells.
Commercial adaptation of continuous processing is confined to biomass production, and to a
limited extent to the production of potable and industrial alcohol.
The steady state of continuous processing is advantageous as the system is far easier to control.
During batch processing, heat output, acid or alkali production, and oxygen consumption will
range from very low rates at the start to very high rates during the late exponential phase. The

control of the environmental factors of the system becomes difficult. In the continuous
processing, the rates of consumption of nutrients and those of the output chemicals are
maintainable at optimal levels. Besides, the labour demand is also more uniform.
Continuous processing may suffer from contamination, both from within and outside. The
fermenter design, along with strict operational control, should actually take care of this problem.
The production of growth associated products like ethanol is more efficient in continuous
processing, particularly for industrial use.
Continuous culturing is highly selective and favours the propagation of the best-adapted
organism in culture.
A commercial organism is highly mutated such that it will produce very high amounts of the
desired product. But physiologically such strains are inefficient and give way in culture to
inferior producers--a kind of contamination from within.
In the fed-batch system, 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.
Fed-batch achieved some appreciable degree of process and product control.
A low but constantly replenished medium has the following advantages:
a) maintaining conditions in the culture within the aeration capacity of the fermenter;
b) removing the repressive effects of medium components such as rapidly used carbon and
nitrogen sources and phosphate;
c) avoiding the toxic effects of a medium component; and
d) providing limiting level of a required nutrient for an auxotrophic strain.
Production of baker's yeast is mostly by fed-batch culture, where biomass is the desired product.
Diluting the culture with a batch of fresh medium prevents the production of ethanol, at the
expense of biomass; the moment traces of ethanol were detected in the exhaust gas.

After an organism producing a valuable product is identified, it becomes necessary to increase
the product yield from fermentation to minimise production costs. Product yields can be
increased by
(i) developing a suitable medium for fermentation,


(ii) refining the fermentation process and

(iii) improving the productivity of the strain.
Generally, major improvements arise from the last approach; therefore, all fermentation
enterprises place a considerable emphasis on this activity. The techniques and approaches used to
genetically modify strains, to increase the production of the desired product are called strain
improvement or strain development.
Strain improvement is based on the following three approaches:
(ii)recombination, and
(iii) recombinant DNA technology.


Chief feature

The main approach to strain
A. Mutant Selection :
improvement; produces new alleles
of existing genes
Used in, the initial stages of strain
Occur without any treatment
1. Spontaneous Mutations
improvement; also for maintenance
with a mutagen
of improved strains
Induced by chemical
Mutagenesis followed by selection;
2. Induced Mutations
(mainly) or physical
several cycles employed
Production of 6-demethyl
Affect the pattern of
3. Major Mutations
tetracycline in place of tetracycline
metabolite production
by S. aureofaciens
Small gains in each cycle of
Affect the rate metabolite
4. Minor Mutations
selection; substantial improvement
after several cycles
B. Mutant Selection
Enhanced production of an amino
Defective biosynthesis of a
1. Auxotrophic mutants
acid, e.g., phe mutants accumulate
2. Analogue resistant
Feedback insensitive
Overproduction of metabolites,
e.g., amino acids by C. glutamicus
Some mutants are high producers,
3. Revertants of
e.g., chlortetracycline by S.
nonproducing mutants
4. Revertants of
Some are high produces, e.g.
auxotrophic mutants
chlortetracycline by S. viridifaciens
5. Resistance to the
Increased production, e.g.,
antibiotic produced by the
chlortetracycline by S. aureofaciens


organism itself
C. Recombination
1. Sexual reproduction
2. Heterokaryosis

3. Protoplast fusion

Produces new combinations of

existing alleles
Some bacteria and Actinomycetes;
fungi and yeast

Conjugation; fusion of
Nuclear fusion followed, by
mitotic recombination and Fungi
mitotic reduction
Protoplasts produced by lytic
Bacteria, Actinomycetes, fungi;
enzymes fusion by PEG,
quite; successful
recombinant recovery

When an organic compound is modified by simple chemically defined reactions, catalyzed by
enzymes present in cells, into a product that is recoverable, it is called biotransformation. Both
the substrate and the product are not involved in the primary or secondary metabolism of the
organism employed.
This is in contrast to the various metabolites, e.g., organic acids, amino acids, antibiotics etc.,
produced by the complex pathways of primary or secondary metabolism of the organism.
Biotransformations are far more efficient and economical due to the rapid microbial growth and
high metabolic rates of microorganisms.
Immobilization can be defined as the confinement of a biocatalyst inside a bioreaction system,
with retention of its catalytic activity and stability. Cells can be immobilized
Immobilized Cell Bioreactors - Bioreactors of this type are based on immobilized cells. Cell
immobilization is advantageous when
(i) the enzymes of interest are intracellular,
(ii) extracted enzymes are unstable,
(iii) the cells do not have interfering enzymes or such enzymes are easily inactivated/removed
(iv) the products are low molecular weight compounds released into the medium.


Under these conditions, immobilized cells offer the following advantages over enzyme
(i) enzyme purification is not needed,
(ii) high activity of even unstable enzymes,
(iii) high operational stability,
(iv) lower cost and
(v) possibility of application in multistep enzyme reactions. In addition, immobilization permits
continuous operation of bioreactor, which reduces the reactor volume and, consequently,
pollution problems. Obviously, immobilized cells are used for such biotransformations of
compounds, which require action of a single enzyme.
Cell immobilization may be achieved in one of the following ways.

(1) Cells may be directly bound to water insoluble carriers, e.g., cellulose, dextran, ion exchange
resins, porous glass, brick, sand, etc., by adsorption, ionic bonds or covalent bonds.
(2) They can be cross linked to bi- or multifunctional reagents, e.g., glutaraldehyde, etc.
(3) Polymer matrices may be used for entrapping cells; such matrices are polyacrylamide cell,
-carrageenan (a polysaccharide isolated from a seaweed), calcium alginate (alginate is extracted
from seaweed), poly glycol oligomers, etc.
Out of these approaches, calcium alginate immobilization is the most commonly used since it
can be used for even very sensitive cells. Cell immobilization has been used for commercial
production of amino acids, organic acids, etc.

A bioreactor is a device in which a substrate of low value is utilized by living cells or enzymes
to generate a product of higher value. A bioreactor is a device in which the microorganisms are
cultivated and motivated to form the desired products by maintaining optimum conditions for
growth and metabolic activity. A fermenter refers to the device used for the cultivation of
prokaryotic cells e.g. bacteria, fungi etc. whereas a bioreactor is used for growing eukaryotic


Bioreactors are extensively used for food processing, fermentation, waste treatment, etc. On the
basis of the agent used, bioreactors are grouped into two broad classes:
(i) those based on living cells and,
(ii) those employing enzymes.
But in terms of process requirements, they are of the following types:
(i) aerobic,
(ii) anaerobic,
(iii) solid state, and
(iv) immobilized cell bioreactors.
All bioreactors deal with heterogeneous systems having two or more phases, e.g., liquid, gas,
solid. Therefore, optimal conditions for fermentation necessitate efficient transfer of mass, heat
and momentum from one phase to the other.
A bioreactor should provide for the following:
(i) agitation (for mixing of cells and medium),
(ii) aeration (aerobic fermenters; for O2 supply),
(iii) regulation of factors like temperature, pH, pressure, aeration, nutrient feeding, liquid level,
(iv) sterilization and maintenance of sterility, and
(v) withdrawal of cells/medium (for continuous fermenters). Modem fermenters are usually
integrated with computers for efficient process monitoring, data acquisition, etc.
The size of fermenters ranges from 1-21 laboratory fermenters to 500,000 1 or, occasionally,
even more; fermenters of upto 1.2 million litres have been used. Generally, 20-25% of fermenter
volume is left unfilled with medium as "head space" to allow for splashing, foaming and
aeration. The fermenter design varies greatly depending on the type of fermentation for which it
is used.




A typical conventional bioreactor has cylindrical vessel with domed top and bottom generally
made up of stainless steel. The reaction vessel which is surrounded by a jacket, is provided with
a sparger at the bottom through which air (or other gases such as CO2 and NH3 (for pH
maintenance) can be introduced. The reaction vessel also has side ports for pH, temperature and
dissolved O2 sensors. The agitator shaft is connected to a motor at the bottom. Above the liquid
level of the reaction vessel, connections for acid, alkali, antifoam chemicals and inoculum are
located. The bioreactor is designed to work at very high temperatures (150-1800C), high pressure
(377-412 kPa) and also to withstand vacuum which prevents its collapse while cooling.

Types of Bioreactors
Depending on the design of the reactor, the bioreactors are of following types:
a) Continuous stirred tank bioreactors - These bioreactors have a cylindrical vessel with motor
driven central shaft which gives support to one or more agitators (impellers). The shaft is fitted at
the bottom of the bioreactor. The diameter of the impeller is usually one third of the vessel
diameter. The impellers are available in different designs like- Rustom disc, concave bladded,
marine propeller etc. In stirred tank reactors, the air is added to the culture medium under
pressure through a device called sparger. The sparger along with the impellers (agitators) enables
better and efficient gas distribution through out in the vessel. The advantages of using stirred
tank reactors are: the efficient transfer of gas to growing cells which keeps the growth of cells in
healthy limits, stirring ensures good mixing of the contents, the operating conditions are flexible
and the bioreactors are easily available which makes them commercially viable products.
b) Bubble column bioreactors - In these bioreactors, the gas or air is introduced at the base of
the column through perforated pipes or plates, or metal microporous spargers. The vessel used
for bubble column bioreactors is usually cylindrical with an aspect ratio (height o diameter ratio)
of 4-6. The rate of flow of gas affects the O2 transfer and mixing.
c) Airlift bioreactors - Airlift bioreactors are commonly used for aerobic bioprocessing
technology. In the airlift bioreactors, the medium of the vessel is divided into two interconnected
zones by means of baffle or draft tube. The air/gas is pumped into one of the two zones referred
to as riser and the other zone that receives no gas is known as downcomer. The dispersion
flows up the riser zone while the down flow occurs in the downcomer. Further there are two
types of bioreactors:
1) Internal loop bioreactor - These bioreactors have a single container with a central draft tube
that creates interior liquid circulation channels which keeps the volume and circulation at a fixed
rate for fermentation.


(2) External loop airlift bioreactor-These have an external loop to keep the liquid in circulation
through separate independent channels. The modifications can be made in these bioreactors
depending on the requirements of different fermentation processes.
(3) Two stage airlift bioreactors -These bioreactors have two bioreactors which are basically
used for the temperature dependent formation of products. The growing cells from one bioreactor
(maintained at temperature 300C are pumped into another bioreactor (at temperature 420C). This
is done because it is very difficult to increase the temperature quickly from 300C to 420C in the
same vessel. The cells are grown in the first bioreactor and with the help of the fitted valves and
a transfer tube and pump, they are transferred into the second bioreactor, where the actual
bioprocessing takes place.
(4) Tower bioreactors - In this type of bioreactor, a high hydrostatic pressure is generated at the
bottom of the reactor which increases the solubility of O2 in the medium. Since the top is
expanded, the pressure is reduced which helps in the expulsion of CO2. The cycle completes
with the medium flowing back into the downcomer. The advantage with Tower bioreactor is that
it has high aeration capacities without having moving parts.
d) Fluidized bed bioreactors - These bioreactors are mainly suitable to carry out reactions
involving fluid suspended biocatalysts such as immobilized enzymes, immobilized cells,
microbial flocs etc. The design of the bioreactors is such that the top is extended and the reaction
column is narrow which retains the solids in the reactor and allows the liquids to flow out. To
maintain an efficient operation of fluidized beds, gas is sparged to create a suitable gas-liquidsolid fluid bed. The recycling of the liquid ensures continuous contact between the reaction
contents and biocatalysts which increases the efficiency of bioprocessing.
e) Packed bed bioreactors- A packed bed bioreactor consists of a bed of solid particles, with
biocatalysts on or within the matrix of solids, packed in a column. The solids are generally
porous or non-porous gels which maybe compressible or rigid in nature. The nutrient broth
continuously flows over the immobilized biocatalyst and the products are released into the fluid
from where they are removed. However, due to poor mixing, it is difficult to control the pH of
packed bioreactors by the addition of acid or alkali.
f) Photobioreactors - These bioreactors are specialized for fermentation that can be carried out
either by exposing to sunlight or artificial illumination. The photobioreactors are made up of
glass or transparent plastic which are the solar receivers. The cell cultures are circulated through
the solar receivers by using centrifugal pumps or airlift pumps. These bioreactors work in the
temperature which ranges from 25-400C. In these bioreactors, the microorganisms e.g.
microalgae, cyanobacteria etc. grow during the day time while the products (e.g. beta-carotene,
asthaxanthin) are produced during the night.
Solid substrate Fermentation (SSF)
In some biotechnological processes, the growth of the microorganisms is carried out on solid
substrates more or less in the absence of free water. Only approximately 15% of moisture is


present which is essential for solid-substrate fermentation. Cereal grains, wheat bran, sawdust,
wood shavings etc are some of the commonly used solid substrates for SSF. The technique of
SSF is used for the production of edible mushrooms, cheese, soy sauce, and many enzymes and
organic acids. It is carried out as non-aspetic process and therefore saves sterilization costs. The
bioreactors used in this type of fermentation process have simple designs with simple aeration
process and effluent treatment. The yield of the products is very high, at low energy expenditure.
However, in this process only microorganisms, that can tolerate only low moisture content can be
used. It also difficult to monitor O2 and CO2 levels in SSF. The slow growth of microorganisms,
also become a limiting factor for product formation.
Working of a bioreactor
In the operation of a bioreactor, there are a few steps that are followed.
a) Sterilization - The most important requirement is to maintain aseptic or sterile conditions for
aseptic fermentation. In order to achieve this, the air supplied during fermentation, he growth
medium and the bioreactor it self and all its accessories are sterilized. There are two methods of
sterilization that are followed: (1) In situ sterilization- In this, the bioreactor is filled with the
required medium followed by injection of pressurized steam into the jacket or coil surrounding
the reaction vessel. The whole system is heated to about 1200C and maintained at this
temperature for about 20 minutes. However, this method is not energy efficient and prolonged
heating destroys the vitamins and precipitates the components of the medium. (2) Continuous
heat sterilization - In this, the empty bioreactor is first sterilized by injecting pressurized steam
and the medium is rapidly heated to 1400C for a short period again by injecting the pressurized
steam. This is an energy efficient method and also does not precipitate the medium components.
b) Aeration - Oxygen is stored in compressed tanks and is introduced at the bottom of the
bioreactor through a sparger. Aeration of the fermentation medium supplies oxygen to the
production microorganisms and remove carbon dioxide from the bioreactor. The gases released
during the fermentation accumulate in the headspace and then pass out through an air outlet. The
headspace is a vacant space on the upper part of the bioreactor and is generally about 20% of its
volume. The air-lift system of aeration involves sparging of air done at the bottom of the
fermenter with an upward flow of air bubbles. The aeration capacity of the system depends on
the air-flow rate and the internal pressure. The stirred system of aeration involves increasing the
aeration capacity by stirring using impellers driven by motor. The aeration capacity of the
fermenter depends on the rate of stirring, rate of air flow and internal pressure.
c) Inoculation and sampling - The sterilized bioreactors with growth medium are inoculated
with the production organisms. The size of the inoculum is generally 1-10% of the total volume
of the medium. During the fermentation process, the samples are regularly withdrawn to check
contamination and to measure the amount and quantity of product formed.
d) Control systems - Various factors like- pH, temperature, dissolved oxygen, adequate mixing,
concentration of the nutrients, foam formation etc are continuously monitored to maintain
optimal growth environment in the bioreactor. Very sensitive sensors are available which carry
out automated monitoring of these variables. The ideal pH range for optimal growth of


microorganisms is between 5.5- 8.5. The pH changes due to the release of metabolites into the
medium by the growing microorganisms. The required pH level is maintained by adding acid or
alkali followed by thorough mixing of the medium components. The optimal temperature is
maintained by using the heating and cooling systems fitted in the bioreactor. Continuous
monitoring of dissolved oxygen concentration is also a must for the optimal bioreactions. The
oxygen is sparingly soluble in water (0.0084g/l at 250C) and is introduced into the bioreactor as
the air bubbles.
The concentration of the nutrients is also important because the limiting concentrations of
nutrients helps in the optimal product formation and the high nutrient concentrations have
inhibitory effect on the microbial growth. Another important factor to control is the foam
formation. The protein rich media is used in industrial fermentation which leads to froth or
foam formation on agitation during aeration. Some antifoam chemicals lower the surface
tension of the medium and causes the foam bubbles to collapse. Mineral oils with silicone or
vegetable oils are also used as antifoam agents. The bioreactors can also be fitted with
mechanical foam control devices which break the foam bubbles and throw back into the
fermentation medium. The proper and continuous mixing of the microbial culture is very
important to maintain optimal levels of oxygen in the nutrient medium and to prevent the
accumulation of toxic metabolic products.
e) Cleaning - After the completion of the fermentation process, the products are harvested
(removal of contents for processing) and the bioreactor is prepared for the next round of
fermentation after cleaning technically referred to as turn around. The cleaning of the
bioreactors is carried out by using high-pressure water jets from the nozzles fitted into the
reaction vessel. In order to maintain the cost effectiveness of the bioreactor, the time taken for
turn around which is known as down time, is kept as short as possible.

Downstream processing (DSP)

The extraction and purification of a biotechnological product from fermentation is referred to as
downstream processing. The methods adopted for downstream processing depends on the nature
of the end products, its concentration, stability, and the degree of purification required. Both
intracellular metabolites as well as extracellular metabolites are isolated by DSP. The
intracellular metabolites are products located within the cells e.g. vitamins, enzymes etc. The
extracellular metabolites are the products present outside the cells e.g. most antibiotics, amino
acids, alcohol, citric acid, enzymes like amylases, proteases etc. some products like vitamin B12,
flavomycin etc are present both as intracellular as well as extracellular products.
Downstream processing involves a number of steps which are as follows:
a) Solid-liquid separation - The first step is to separate whole cells and other insoluble
substances from the culture broth. This is done by using several methods:


1) Flotation- The process of aeration involves the bubbling of gas in to the liquid broth. The
cells and other solid particles get adsorbed on gas bubbles which form a foamy layer which is
collected and removed.
2) Flocculation - The cells or cellular debris form large aggregates and settle down which can be
easily removed. Some flocculating agents like inorganic salt, organic poly-electrolyte, mineral
hydrocolloid etc are often used to achieve appropriate flocculation.
3) Filtration - this is the most commonly used technique to separate the biomass and culture
filtrate. The rate of filtration depends on many factors such as the size of the organism, presence
of other organisms, viscosity of the medium, and temperature. Several filters like depth filters,
absolute filters, rotary drum vacuum filters, membrane filters etc. are used. There are three major
types of filtration processes used depending on the size of the particles- microfiltration,
ultrafiltration, and reverse osmosis.
4) Centrifugation - The centrifugation is mostly used for separating solid particles from liquid
phase. The technique of centrifugation is based on the principle of density differences between
the particles to be separated and the medium. In the bioreactors, the continuous flow industrial
centrifuges are used where there is a continuous feeding of the slurry and collection of clarified
fluid. The solid deposits are intermittently removed. Various models of centrifuges used are:
Tubular bowl centrifuge, Disc centrifuge, Multichamber centrifuge, Scroll centrifuge or decanter
b) Release of intracellular products - The biotechnological products that are located with in the
cells like vitamins, enzymes, etc are released in an active form for their further processing and
isolation. The microorganisms or cells can de disintegrated or disrupted by physical, chemical, or
enzymatic methods depending on the nature of the cells.
1) Physical methods of cell disruption are (i) Ultrasonication, (ii) Osmotic shock (used for
releasing hydrolytic enzymes and binding proteins from gram-negative bacteria), (iii) Heat
Shock treatment, (iv) High pressure homogenization, (v) Impingement which involves
hitting a stationary surface or a second stream of suspended particles with a stream of suspended
cells at high velocity and pressure. Microfluidizer is a device developed on the basis of the
principle of impingement. (vi) Grinding with glass beads where the cells mixed with glass
beads are subjected to a very high speed in a reaction vessel. The cells break as they are forced
against the wall of the vessel by the beads
2) Chemical methods- Treatment with alkalies, organic solvents, and detergents lyse the cells to
release the content. Alkali treatment is used for the extraction of some bacterial proteins. The
organic solvents like methanol, ethanol, isopropanol, butanol etc also disrupt the cells. The
organic solvent, toluene, which is commonly used, dissolves membrane phospholipids and
creates the membrane pores to release intracellular contents. The ionic detergents denature the
membrane proteins and lyse the cells e.g. cationic-cetyl trimethyl ammonium bromide or
anionic-sodium lauryl sulfate. Non-ionic detergents are less reactive and also affect the
purification steps.
3) Enzymatic methods - Lysozyme is the most commonly used enzyme which hydrolyses beta1,4-glycosidic bonds of the mucopepide in bacterial cell walls e.g. gram positive bacteria. This
enzyme is commercially available produced from hen egg white. For gram-negative bacteria,
lysozymes are in use with EDTA to break the cells. When the cell wall gets digested by


lysozyme, the periplasmic membrane breaks due to osmotic pressure, which releases the
intracellular contents. Glucanase and mannanase along with proteases lyse the cell wall of yeast.
c) Concentration - The biological products are concentrated by getting rid of water which is
present in the filtrate. Depending on the nature of the desired products and the cost effectiveness,
the techniques used to concentrate the biotechnological products are evaporation, liquid-liquid
extraction, membrane filtration, precipitation, and adsorption.
(i) Evaporation - The water from the broth is removed by the process of evaporation using
evaporators. The evaporators use a heating device which supplies the steam. There is a unit for
the separation of concentrated product and vapour and a condenser for condensing vapour. The
commonly used evaporators are Plate evaporator, Falling film evaporator, Forced film
evaporator, Centrifugal forced film evaporator.
(ii) Liquid-Liquid extraction - In liquid-liquid extraction, the biological products are
concentrated by transferring the desired product from one liquid phase to another liquid phase.
The process of liquid-liquid extraction is categorized as extraction of low molecular weight
products and extraction of high molecular weight products.
(iii) Membrane filtration - This technique involves the use of a semipermeable membrane that
selectively retains the particles/molecules that are bigger than the pore size while the smaller
molecules pass through the membrane pores. The membranes are made up of polymeric
materials such as polyethersulfone and polyvinyl difluoride. Now a days, microfilters and
ultrafilters made up of ceramics and steel are being used which are easy to clean and sterilize.
Pervaporation is a technique in which the volatile products are separated by a process of
permeation through a membrane coupled with evaporation and is used to extract and concentrate
volatile products. Perstraction- This technique is used to recover and concentrate hydrophobic
compoundsvand is based on the principle of membrane filtration coupled with solvent extraction.
(iv) Precipitation - This is the most common method used to concentrate proteins and
polysaccharides in industrial processes. The alteration in temperature and in pH, neutral salts,
organic solvents, high molecular weight polymers (ionic and non ionic) etc are used in
precipitation. Neutral salts like commonly used ammonium sulphate increases the hydrophobic
interactions between protein molecules which leads to their precipitation. Ethanol, acetone and
propanol are the commonly used organic solvents for protein precipitation which reduce the
dielectric constant of the medium and increase the electrostatic interaction between the protein
molecules. The precipitation process is carried out below 00C because the proteins get denatured
by organic solvents. Polyethylene glycol (PEG) is a high weight non-ionic polymer that also
precipitates the proteins by reducing the quantity of water available for protein salvation. In the
category of ionic polymers e.g. polyacrylic acid, polyethyleneimine are used which form
complexes with oppositely charged protein molecules and neutralize the charges. This leads to
the precipitation of proteins. Besides this, the physical factors like increase in temperature,
increase in pH, etc also leads to the precipitation of proteins. Besides these, the affinity
precipitation (affinity interaction e.g. between antigen and antibody)and precipitation using
ligands is also used.
d) Purification The products of fermentation are purified by using the technique of
chromatography. Chromatography consists of a stationary phase and a mobile phase. The porous
solid matrix packed in a column constitutes the stationary phase and the mixture of the


compound to be separated is loaded on this. The compounds are eluted by a mobile phase. A
large number of matrices are commercially available for the purification of proteins e.g. agarose,
cellulose, porous silica, cross linked dextran etc. Some of the commonly used chromatography
techniques used are: Gel-filtration chromatography- In this technique, the separation of
molecules is based on the size, shape and molecular weight using sponge-like gel beads with
pores serving as molecular sieves for separation of smaller and bigger molecules. The Ionexchange chromatography involves the separation of molecules based on their surface charges. It
is useful for the purification of antibiotics, besides the purification of proteins. In ion exchange
chromatography, the pH of the medium is very crucial because the net charge varies with pH.
The ionic bound molecules are eluted from the matrix by changing the pH of the eluant or by
increasing the salt concentration. The ion-exchangers are of two types- Cation exchangershaving negatively charged groups like carboxymethyl and sulfonate e.g. Dowex, HCR, Amberlite
IR etc. Anion exchangers have positively charged groups like DEAE (diethylaminoethyl) e.g.
Dowex SAR, Amberlite IRA etc. The Affinity chromatography is based on an interaction of a
protein with an immobilized ligand. The ligand can be a specific antibody, substrate, substrate
analogue or an inhibitor. The protein bound to the ligand is eluted out by changing the pH of the
buffer, altering the ionic strength or by using another free ligand molecule.
e) Formulation- The maintenance of activity and stability of biotechnological products during
storage and distribution is known as formulation. As proteins are highly sensitive and lose their
biological activity, hence their formulations requires special care. In order to prolong their shelf
life, certain stabilizing additives are added e.g. sugars (sucrose, lactose), salts (sodium chloride,
ammonium sulphate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol).
Proteins are formulated in the form of solutions, suspensions or dry powder. For some small
molecules (antibiotics, citric acid), formulation is done by crystallization using salts. Drying is
an important component of product formulation which involves the transfer of heat to a wet
product for removal of moisture. The commercially available dryers in use are- contact,
convection and radiation dryers. Spray drying is used for drying large volumes of liquids where
small droplets of liquid containing the product are passed through a nozzle directing over a
stream of hot gas. After the evaporation of water, the solid particles are left behind.
Freeze drying or lyophilization is the most preferred method for drying and formulation of a
wide range of products- pharmaceuticals, food stuff, bacteria, viruses etc. Freeze drying does not
cause loss of biological activity of the desired product. Lyophilization is caused due to the
sublimation of a liquid from a frozen state. The product with the liquid is frozen and then freeze
dried under vacuum. After releasing the vacuum, the product containing vials are sealed
A biosensor is an analytical device for the detection of an analyte that combines a biological
component with a physicochemical detector component.
It consists of 3 parts:


the sensitive biological element (biological material (e.g. tissue, microorganisms,

organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.), a biologically derived
material or biomimic) The sensitive elements can be created by biological engineering.

the transducer or the detector element (works in a physicochemical way; optical,

piezoelectric, electrochemical, etc.) that transforms the signal resulting from the
interaction of the analyte with the biological element into another signal (i.e.,
transducers) that can be more easily measured and quantified;

associated electronics or signal processors that are primarily responsible for the display of
the results in a user-friendly way. This sometimes accounts for the most expensive part of
the sensor device, however it is possible to generate a user friendly display that includes
transducer and sensitive element.

A common example of a commercial biosensor is the glucose biosensor, which uses the enzyme
glucose oxidase to break blood glucose down. In doing so it first oxidizes glucose and uses two
electrons to reduce the FAD (a component of the enzyme) to FADH2. This in turn is oxidized by
the electrode (accepting two electrons from the electrode) in a number of steps. The resulting
current is a measure of the concentration of glucose. In this case, the electrode is the transducer
and the enzyme is the biologically active component.
There are many potential applications of biosensors of various types. The main requirements for
a biosensor approach to be valuable in terms of research and commercial applications are the
identification of a target molecule, availability of a suitable biological recognition element, and
the potential for disposable portable detection systems to be preferred to sensitive laboratorybased techniques in some situations.


Since olden times people are using fermented products in their daily food consumption. There
are many advantages of fermented foods such as they easily digestible and have improved
flavour, texture and nutritive value. Some of the most commonly used fermented products are
cheese, bread, yoghurt, sausages, soy sauce etc. With the advances made in microbiology and
biotechnology, food and beverage fermentation and production has become a major industry. The
food biotechnology has helped in improving the quality, nutrition value, safety and preservation
of foods which in turn has helped in making these foods available through out the year.

Two species of bacteria Lactobacillus bulgaricus
and Lactococcus thermophilus in approximately equal proportions, are used to make yoghurt.
Commercial producers pasteurize and homogenize the milk before adding the starter. After
stirring, the mixture is then incubated for 3-6 hours at 40-450C. At this temperature the two
bacteria have a mutually stimulating effect on one another. Proteolytic enzymes from L.
bulgaricus break down milk proteins into peptides. These stimulate the growth of L.
thermophilus which, in turn, produce formic acid and carbon dioxide, growth stimulants for L.
bulgaricus. As the incubation proceeds, L. bulgaricus converts the lactose to lactic acid and the
pH falls to 4.2-4.4 which leads to the coagulation of proteins by lactic acid and the thickening of
the yoghurt. Further processing involves the addition of flavour, colour, fruit pulp and heat
treatment to kill off any bacteria. Typical products
(a) Sweetened and flavored yoghurt
To offset its natural sourness, yoghurt can be sold sweetened, flavored or in
containers with fruit or fruit jam
(b)Strained yoghurts
Strained yoghurts are types of yoghurt which are strained through a paper or
cloth filter, traditionally made of muslin, to remove the whey, giving a much
thicker consistency and a distinctive, slightly tangy taste.


Metabolic products of bacteria used in yoghurt making


Stages in the manufacture of processed yoghurt




The process of fermentation in wine is the catalyst function that turns grape juice into an
alcoholic beverage. During fermentation yeast interact with sugars in the juice to create ethanol,
commonly known as ethyl alcohol, and carbon dioxide (as a by-product. Fermentation may be
done in stainless steel tanks, which is common with many white wines, in an open wooden vat,
inside a wine barrel and inside the wine bottle itself as in the production of many sparkling
The most common species of wine grape is Vitis vinifera, which includes nearly all varieties of
European origin. Red wine is made from the must (pulp) of red or black grapes that undergo
fermentation together with the grape skins. White wine is made by fermenting juice which is
made by pressing crushed grapes to extract a juice; the skins are removed and play no further
role. To start primary fermentation yeast is added to the must for red wine or juice for white
wine. Cultured yeasts most commonly used in winemaking belong to the Saccharomyces
cerevisiae (also known as "sugar yeast") species. Within this species are several hundred


different strains of yeast that can be used during fermentation to affect the heat or vigor of the
process and enhance or suppress certain flavor characteristics of the varietal. During this
fermentation, which often takes between one and two weeks, the yeast converts most of the
sugars in the grape juice into ethanol (alcohol) and carbon dioxide. . Upon the introduction of
active yeasts to the grape must, phosphates are attached to the sugar and the six-carbon sugar
molecules begin to be split into three-carbon pieces and go through a series of rearrangement
reactions. During this process the carboxylic carbon atom is released in the form of carbon
dioxide with the remaining components becoming acetaldehyde. The absence of oxygen in this
anaerobic process allows the acetaldehyde to be eventually converted, by reduction, to ethanol.
During the conversion of acetaldehyde a small amount is converted, by oxidation, to acetic acid
which, in excess, can contribute to the wine fault known as volatile acidity (vinegar taint). After
the yeast has exhausted its life cycle, they fall to the bottom of the fermentation tank as sediment.
The carbon dioxide is lost to the atmosphere.
After the primary fermentation of red grapes the free run wine is pumped off into tanks and the
skins are pressed to extract the remaining juice and wine, the press wine blended with the free
run wine at the wine makers discretion. The wine is kept warm and the remaining sugars are
converted into alcohol and carbon dioxide. The next process in the making of red wine is
secondary fermentation. This is a bacterial fermentation which converts malic acid to lactic acid.
This process decreases the acid in the wine and softens the taste of the wine. Red wine is
sometimes transferred to oak barrels to mature for a period of weeks or months, this practice
imparts oak aromas to the wine. The wine must be settled or clarified and adjustments made prior
to filtration and bottling.
During fermentation there are several factors that winemakers take into consideration. The most
notable is that of the internal temperature of the must. Typically white wine is fermented between
18-20 C though a wine maker may choose to use a higher temperature to bring out some of the
complexity of the wine. Red wine is typically fermented at higher temperatures up to 29 C. For
every gram of sugar that is converted, about half a gram of alcohol is produced, so to achieve a
12% alcohol concentration, the must should contain about 24% sugars. Sulfur dioxide has two
primary actions, firstly it is an anti microbial agent and secondly an anti oxidant. In the making
of white wine it can be added prior to fermentation and immediately after alcoholic fermentation
is complete. If added after alcoholic ferment it will have the effect of preventing or stopping
malolactic fermentation, bacterial spoilage and help protect against the damaging effects of
The clarification and stabilization of wine in winemaking involves removing insoluble and
suspended materials that may cause a wine to become cloudy, gassy, form unwanted sediment
deposit or tartaric crystals, deteriorate quicker or develop assorted wine faults due to physical,
chemical or microbiological instability. These processes may include fining, filtration,


centrifugation, flotation, refrigeration, barrel maturation, pasteurization and racking. Most of

these processes will occur after the primary fermentation and before the wine is bottled. The
exception is for white wine production which will usually have the must separated from some of
the grape skins and particles prior to fermentation so as to avoid any unwanted maceration. Some
of the materials that are removed from the must during this stage of winemaking include dead
yeast cells (lees), bacteria, tartrates, proteins, pectins, various tannins and other phenolic
compounds, and pieces of grape skins, pulp, stems and gums.
Tartaric acid is the most prominent acid in wine with the majority of the concentration present as
potassium acid salt. The crystallization of these tartrates can happen at unpredictable times if the
wine is exposed to low temperature. To prevent this from happening after the wine has been
bottled, winemakers stabilize the wine by putting it through a cold stabilization process where it
is exposed to temperatures below freezing to encourage the tartrates to crystallize and precipitate
out of the wine. During the cold stabilizing process after fermentation, the temperature of the
wine is dropped to close to freezing for 12 weeks. This will cause the crystals to separate from
the wine and stick to the sides of the holding vessel. When the wine is drained from the vessels,
the tartrates are left behind
Filtration in winemaking is used to accomplish two objectives, clarification and microbial
stabilization. While fining clarifies wine by binding to suspended particles and precipitating out
as larger particles, filtration works by passing the wine through a filter medium that captures
particles that are larger than the hole size of the medium. Most filtration in a winery can be
classified as either depth filtration or surface filtration.
Depth filtration is often the first type of filtration a wine sees after fermentation when the wine is
pushed through a thick layer of pads made from cellulose fibers, diatomaceous earth or perlite
which traps the particles and can be removed from the wine. Surface filtration involves running
the wine along a thin film of polymer material filled with holes tinier than the particles that are
being filtered out.
In winemaking, fining is the process where a substance (fining agent) is added to the wine to
create an adsorbent, enzymatic or ionic bond with the suspended particles, making them a larger
molecule that can precipitate out of the wine easier and quicker. The most common organic
compounds used include egg whites, casein derived from milk, gelatin and isinglass obtained
from the bladders of fish. Pulverized minerals and solid materials can also be used as fining
agents with bentonite clay being one of the most common fining agent used due to its
effectiveness in absorbing proteins and some bacteria. Activated carbon derived from charcoal is
used to remove some phenols that contribute to browning colors as well as some particles that
produce "off-odors" in the wine.[5] In a process known as blue fining, potassium ferrocyanide is


used to remove copper and iron particles that may have entered the wine through the use of metal
winery and vineyard equipment, vineyard sprays such as the bordeaux mixture, and the use of
bentonite as a fining agent
A final dose of sulfite is added to help preserve the wine and prevent unwanted fermentation in
the bottle. The wine bottles then are traditionally sealed with a cork, although alternative wine
closures such as synthetic corks and screwcaps, which are less subject to cork taint, are
becoming increasingly popular. The final step is adding a capsule to the top of the bottle which is
then heated for a tight seal.
There are four main methods of sparkling wine production. The first is simple injection of
carbon dioxide (CO2), the process used in soft drinks, but this produces big bubbles that dissipate
quickly in the glass. The second is the Metodo Italiano - Charmat process, in which the wine
undergoes a secondary fermentation in bulk tanks, and is bottled under pressure. The third
method is the traditional method or mthode champenoise. With this method the bubbles for
more complex wines are produced by secondary fermentation in the bottle. As the name
suggests, this is used for the production of Champagne and other quality sparkling wines, but is
slightly more expensive than the Charmat process. The fourth method is the "transfer method".
This method will take the cuvee to bottle for secondary fermentation, which allows for the
additional complexity, but then will transfer the wine out of the individual bottles into a larger
tank after it has spent the desired amount of time on yeast.


Oxidative fermentation
Vinegar is produced by a second fermentation of beer, cider, or wine. The second fermentation is
aerobic and dependent on a mixed culture of Acetobacter , including A. scheutzenbachii, A.
curium, and A. orleanse. In the presence of oxygen, these bacteria convert alcohol to acetic acid
by the following route:
Alcohol (ethanol)------- acetaldehyde------ acetic acid
Given sufficient oxygen, these bacteria can produce vinegar from a variety of alcoholic
foodstuffs. Commonly used feeds include apple cider, wine, and fermented grain, malt, rice, or
potato mashes. The overall chemical reaction facilitated by these bacteria is:
One of the first modern commercial processes was the "fast method" or "German method", first
practised in Germany in 1823. In this process, fermentation takes place in a tower packed with
wood shavings or charcoal. The alcohol-containing feed is trickled into the top of the tower, and
fresh air supplied from the bottom by either natural or forced convection. The improved air
supply in this process cut the time to prepare vinegar from months to weeks.

Nowadays, most vinegar is made in submerged tank culture, first described in 1949 by Otto
Hromatka and Heinrich Ebner. In this method, alcohol is fermented to vinegar in a continuously
stirred tank, and oxygen is supplied by bubbling air through the solution. Using modern
applications of this method, vinegar of 15% acetic acid can be prepared in only 24 hours in batch
process, even 20% in 60-hour fed-batch process.
Anaerobic fermentation


Species of anaerobic bacteria, including members of the genus Clostridium, can convert sugars
to acetic acid directly, without using ethanol as an intermediate. The overall chemical reaction
conducted by these bacteria may be represented as:
It is interesting to note that, from the point of view of an industrial chemist, these acetogenic
bacteria can produce acetic acid from one-carbon compounds, including methanol, carbon
monoxide, or a mixture of carbon dioxide and hydrogen:
2 CO2 + 4 H2 CH3COOH + 2 H2O
This ability of Clostridium to utilize sugars directly, or to produce acetic acid from less costly
inputs, means that these bacteria could potentially produce acetic acid more efficiently than
ethanol-oxidizers like Acetobacter. However, Clostridium bacteria are less acid-tolerant than
Acetobacter. Even the most acid-tolerant Clostridium strains can produce vinegar of only a few
per cent acetic acid, compared to Acetobacter strains that can produce vinegar of up to 20%
acetic acid. At present, it remains more cost-effective to produce vinegar using Acetobacter than
to produce it using Clostridium and then concentrate it
In the form of vinegar, acetic acid solutions (typically 4% to 18% acetic acid, with the
percentage usually calculated by mass) are used directly as a condiment( A condiment is sauce,
or seasoning added to food to impart a particular flavor or to complement the dish. Often
pungent in flavour and therefore added in fairly small quantities), and also in the pickling of
vegetables and other foods. Table vinegar tends to be more diluted (4% to 8% acetic acid), while
commercial food pickling, in general, employs more concentrated solutions. The amount of
acetic acid used as vinegar on a worldwide scale is not large, but is by far the oldest and bestknown application.
The basic ingredients of beer are water; a starch source, such as malted barley, able to be
fermented (converted into alcohol); a brewer's yeast to produce the fermentation; and a
flavouring such as hops. A mixture of starch sources may be used, with a secondary starch
source, such as maize (corn), rice or sugar, often being termed an adjunct, especially when used
as a lower-cost substitute for malted barley. Less widely used starch sources include millet,
sorghum and cassava root


The purpose of brewing is to convert the starch source into a sugary liquid called wort and to
convert the wort into the alcoholic beverage known as beer in a fermentation process effected by



All beers are brewed using a process based on a simple formula. Key to the process is malted
grain mainly barley, though other cereals, such as wheat or rice, may be added. Malt is made
by allowing a grain to germinate, after which it is then dried in a kiln and sometimes roasted. The
germination process creates a number of enzymes, notably -amylase and -amylase, which
convert the starch in the grain into sugar. Depending on the amount of roasting, the malt will take
on a dark colour and strongly influence the colour and flavour of the beer. The malt is crushed to
break apart the grain kernels, expose the cotyledon which contains the majority of the
carbohydrates and sugars, increase their surface area, and separate the smaller pieces from the
Malting is the process where the barley grain is made ready for brewing. Malting is broken down
into three steps, which help to release the starches in the barley. First, during steeping, the grain
is added to a vat with water and allowed to soak for approximately 40 hours. During
germination, the grain is spread out on the floor of the germination room for around 5 days. The
goal of germination is to allow the starches in the barley grain to breakdown into shorter lengths.
When this step is complete, the grain is referred to as green malt. The final part of malting is
kilning. Here, the green malt goes through a very high temperature drying in a kiln. The
temperature change is gradual so as not to disturb or damage the enzymes in the grain. When
kilning is complete, there is a finished malt as a product.
The next step in the brewing process is milling. This is when the grains that are going to be used
in a batch of beer are cracked. Milling the grains makes it easier for them to absorb the water that


they are mixed with and which extracts sugars from the malt. Milling can also influence the
general characteristics of a beer.
Mashing is the next step in the process. This process converts the starches released during the
malting stage, into sugars that can be fermented. The milled grain is dropped into hot water in a
large vessel known as a mash tun. In this vessel, the grain and water are mixed together to create
a cereal mash. The leftover sugar rich water is then strained through the bottom of the mash in a
process known as Prior to lautering, the mash temperature may be raised to about 75 C (known
as a mashout) to deactivate enzymes. Additional water may be sprinkled on the grains to extract
additional sugars (a process known as sparging).
At this point the liquid is known as wort. The wort is moved into a large tank known as a
"copper" or kettle where it is boiled with hops and sometimes other ingredients such as herbs or
sugars. The boiling process serves to terminate enzymatic processes, precipitate proteins,
isomerize hop resins, concentrate and sterilize the wort.
Hops are added during boiling as a source of bitterness, flavour and aroma. The flower of the
hop vine is used as a flavouring and preservative agent in nearly all beer made today. The
flowers themselves are often called "hops". Hops may be added at more than one point during
the boil. The longer the hops are boiled, the more bitterness they contribute, but the less hop
flavour and aroma remains in the beer.
After boiling, the hopped wort is now cooled, ready for the yeast. In some breweries, the hopped
wort may pass through a hopback, which is a small vat filled with hops, to add aromatic hop
flavouring and to act as a filter; but usually the hopped wort is simply cooled for the fermenter,
where the yeast is added . A type of yeast is selected and added, or "pitched", to the fermentation
tank. Yeast metabolises the sugars extracted from grains, which produces alcohol and carbon
dioxide, and thereby turns wort into beer. In addition to fermenting the beer, yeast influences the
character and flavour. The dominant types of yeast used to make beer are ale yeast
(Saccharomyces cerevisiae) top fermenting yeast and lager yeast (Saccharomyces uvarum) An
example of bottom cropping yeast is Saccharomyces pastorianus, formerly known as
Saccharomyces carlsbergensis. The most common method of categorising beer is by the
behaviour of the yeast used in the fermentation process. Beers using a fast acting warm
fermentation which leaves behind residual sugars are termed "ales", while beers using a sloweracting cool fermentation, with a yeast which removes most of the sugars, producing a clean, dry
beer, are termed "lagers". Ale is typically fermented at temperatures between 15 and 24C. Lager
yeast is a cool bottom-fermenting yeast (Saccharomyces pastorianus) and typically undergoes
primary fermentation at 712 C (the fermentation phase), and then is given a long secondary
fermentation at 04 C (the lagering phase). During the secondary stage, the lager clears and
mellows. The cooler conditions also inhibit the natural production of esters and other byproducts,
resulting in a "cleaner"-tasting beer.


The second to last stage in the brewing process is called racking. This is when the brewer racks
the beer into a new tank, called a conditioning tank. Conditioning of the beer is the process in
which the beer ages, the flavour becomes smoother, and flavours that are unwanted dissipate.
Fermentation is sometimes carried out in two stages, primary and secondary. Once most of the
alcohol has been produced during primary fermentation, the beer is transferred to a new vessel
and allowed a period of secondary fermentation. Secondary fermentation is used when the beer
requires long storage before packaging or greater clarity.
After one to three weeks, the fresh (or "green") beer is run off into conditioning tanks. After
conditioning for a week to several months, the beer enters the finishing stage. Here, beers that
require filtration are filtered, and given their natural polish and colour. Filtration also helps to
stabilize the flavour of the beer. Filtering the beer stabilizes the flavour, and gives beer its
polished shine and brilliance. Some brewers add one or more clarifying agents to beer, which
typically precipitate (collect as a solid) out of the beer along with protein solids and are found
only in trace amounts in the finished product. This process makes the beer appear bright and
clean. Examples of clarifying agents include isinglass, obtained from swimbladders of fish; Irish
moss, a seaweed; kappa carrageenan, from the seaweed Kappaphycus cottonii; Polyclar
(artificial); and gelatin. After the beer is filtered, it undergoes carbonation, and is then moved to a
holding tank until bottling.

Some beers undergo a fermentation in the bottle, giving natural carbonation. When the beer has
fermented, it is packaged either into casks for cask ale or kegs, aluminium cans, or bottles for
other sorts of beer

Cheese Production

Cheese is made from the casein of milk that is produced after separating the whey the liquid
portion of the milk. The bacteria used in cheese making are either gas producers or acid


producers. Gas producers release carbon dioxide, while the acid producers form lactic acid from
lactose. It is the gas producers that determine the texture of a cheese and the acid producers
determine the flavour. The cheese production involves the following steps:
a) Acidification of milk
b) Coagulum formation
c) Separation of curd from whey
d) Ripening of cheese.
Cheddar cheese is made from milk sterilized at 720C for 15 secs. A starter consisting of
Streptococcus lactis is added and the milk is left to ripen for an hour. During the ripening the
lactic acid content rises after which the milk is subjected to renneting. Rennet is a mixture of
chymosin and pepsin from the stomach of a calf which coagulates the casein, the principal milk
protein. There are several sources of rennet for cheese production. These include calves, adult
cows, pigs, and fungal sources. Using genetic engineering, some workers have cloned the genes
of animal chymosin and transfer the same into microorganisms.
After renneting, a semi solid mass or coagulum is formed consisting of water, fat and solutes
trapped in a casein matrix.
The coagulum is cut into pea-sized pieces to separate it into small, creamy particles of curd
suspended in a watery whey. Scalding the mixture at 30-390C for 45 minutes is done to expel
more whey and to change the texture of the curd. After the scalding, the curd is allowed to settle
under gravity or pitch and the whey is run off. After the formation of blocks of curd, the blocks
are cut, stacked, drained and turned in a process called cheddaring. Following the cheddaring,
the pH falls to 5.2 and the curd is milled in to small pieces. In the final stages of preparation,
salt is added which helps to preserve the finished cheese and bring out its flavour. Ripening
consists of storing the cheese under appropriate conditions so that bacteria and other
microorganisms can cause chemical changes in the curd, improving and enhancing its flavour.


An outline of various stages in the manufacture of Cheddar cheese


Yeasts can grow in the presence or absence of air. Anaerobic growth, growth in the absence of
oxygen, is quite slow and inefficient. For instance, in bread dough, yeast grow very little.
Instead, the sugar that can sustain either fermentation or growth is used mainly to produce
alcohol and carbon dioxide. Only a small portion of the sugar is used for cell maintenance and
growth. In contrast, under aerobic conditions, in the presence of a sufficient quantity of dissolved
oxygen, yeast grow by using most of the available sugar for growth and producing only
negligible quantities of alcohol. This means that the baker who is interested in the leavening
action of carbon dioxide works under conditions that minimize the presence of dissolved oxygen.
On the other hand, a yeast manufacturer that wants to produce more yeast cell mass, works under
aerobic conditions by bubbling air through the solution in which the yeast is grown.
The problem posed to the yeast manufacturer, however, is not as simple as just adding air during
the fermentation process. If the concentration of sugar in the fermentation growth media is
greater than a very small amount, the yeast will produce some alcohol even if the supply of
oxygen is adequate or even in abundance. This problem can be solved by adding the sugar
solution slowly to the yeast throughout the fermentation process. The rate of addition of the
sugar solution must be such that the yeast uses the sugar fast enough so that the sugar
concentration at any one time is practically zero. This type of fermentation is referred to as a fedbatch fermentation.
The bakers yeast production process flow chart can be divide into four basic steps. In order
these steps are, molasses and other raw material preparation, culture or seed yeast preparation,
The bakers yeast production process flow chart can be fermentation and harvesting and filtration
and packaging. The process takes approximately five days from start to finish.

The basic carbon and energy source for yeast growth are sugars. Starch can not be used because
yeast does not contain the appropriate enzymes to hydrolyze this substrate to fermentable sugars.
Beet and cane molasses are commonly used as raw material because the sugars present in
molasses, a mixture of sucrose, fructose and glucose, are readily fermentable. In addition to
sugar, yeast also require certain minerals, vitamins and salts for growth. Some of these can be
added to the blend of beet and cane molasses prior to flash sterilization while others are fed
separately to the fermentation. Alternatively, a separate nutrient feed tank can be used to mix and
deliver some of the necessary vitamins and minerals. Required nitrogen is supplied in the form of


ammonia and phosphate is supplied in the form of phosphoric acid. Each of these nutrients is fed
separately to the fermentation to permit better pH control of the process. The sterilized molasses,
commonly referred to as mash or wort, is stored in a separate stainless steel tank. The mash
stored in this tank is then used to feed sugar and other nutrients to the appropriate fermentation

Bakers yeast production starts with a pure culture tube or frozen vial of the appropriate yeast
strain. This yeast serves as the inoculum for the pre-pure culture tank, a small pressure vessel
where seed is grown in medium under strict sterile conditions. Following growth, the contents of
this vessel are transferred to a larger pure culture fermentor where propagation is carried out with
some aeration, again under sterile conditions. These early stages are conducted as set-batch
fermentations. In a set-batch fermentation all the growth media and nutrients are introduced to
the tank prior to inoculation.
From the pure culture vessel, the grown cells are transferred to a series of progressively larger
seed and semi-seed fermentors. These later stages are conducted as fed-batch fermentations.


During a fed-batch fermentation, molasses, phosphoric acid, ammonia and minerals are fed to the
yeast at a controlled rate. This rate is designed to feed just enough sugar and nutrients to the
yeast to maximize multiplication and prevent the production of alcohol. In addition, these fedbatch fermentations are not completely sterile. It is not economical to use pressurized tanks to
guarantee sterility of the large volumes of air required in these fermentors or to achieve sterile
conditions during all the transfers through the many pipes, pumps and centrifuges. Extensive
cleaning of the equipment, steaming of pipes and tanks and filtering of the air is practiced to
insure as aseptic conditions as possible.
At the end of the semi-seed fermentation, the contents of the vessel are pumped to a series of
separators that separate the yeast from the spent molasses. The yeast is then washed with cold
water and pumped to a semi-seed yeast storage tank where the yeast cream is held at 34 degrees
Fahrenheit until it is used to inoculate the commercial fermentation tanks. These commercial
fermentors are the final step in the fermentation process and are often referred to as the final or
trade fermentation. Commercial fermentations are carried out in large fermentors with working
volumes up to 50,000 gallons. To start the commercial fermentation, a volume of water, referred
to as set water, is pumped into the fermentor. Next, in a process referred to as pitching, semi-seed
yeast from the storage tank is transferred into the fermentor. Following addition of the seed
yeast, aeration, cooling and nutrient additions are started to begin the 15-20 hour fermentation.
At the start of the fermentation, the liquid seed yeast and additional water may occupy only
about one-third to one-half of the fermentor volume. Constant additions of nutrients during the
course of fermentation bring the fermentor to its final volume. The rate of nutrient addition
increases throughout the fermentation because more nutrients have to be supplied to support
growth of the increasing cell population. The number of yeast cells increase about five- to eightfold during this fermentation.
Air is provided to the fermentor through a series of perforated tubes located at the bottom of the
vessel. The rate of airflow is about one volume of air per fermentor volume per minute. A large
amount of heat is generated during yeast growth and cooling is accomplished by internal cooling
coils or by pumping the fermentation liquid, also known as broth, through an external heat
exchanger. The addition of nutrients and regulation of pH, temperature and airflow are carefully
monitored and controlled by computer systems during the entire production process. Throughout
the fermentation, the temperature is kept at approximately 86 degrees Fahrenheit and the pH in
the range of 4.5-5.5.
At the end of fermentation, the fermentor broth is separated by nozzle-type
centrifuges, washed with water and re-centrifuged to yield a yeast cream with a
solids concentration of approximately 18%. The yeast cream is cooled to about 45
degrees Fahrenheit and stored in a separate, refrigerated stainless steel cream
tank. Cream yeast can be loaded directly into tanker trucks and delivered to
customers equipped with an appropriate cream yeast handling system.

Alternatively, the yeast cream can be pumped to a plate and frame filter press and
dewatered to a cake-like consistency with a 30-32% yeast solids content. This press
cake yeast is crumbled into pieces and packed into 50-pound bags that are stacked
on a pallet. The yeast heats up during the pressing and packaging operations and
the bags of crumbled yeast must be cooled in a refrigerator for a period of time with
adequate ventilation and placement of pallets to permit free access to the cooling
air. Palletized bags of crumbled yeast are then distributed to customers in
refrigerated trucks.

Types of baker's yeast

Baker's yeast is available in a number of different forms, the main differences being the moisture
contents. Though each version has certain advantages over the others, the choice of which form
to use is largely a question of the requirements of the recipe at hand and the training of the cook
preparing it. Dry yeast forms are good choices for longer-term storage, often lasting several
months at room temperatures without significant loss of viability.With occasional allowances for
liquid content and temperature, the different forms of commercial yeast are generally considered

Cream yeast is essentially a suspension of yeast cells in liquid, siphoned off from the
growth medium. Its primary use is in industrial bakeries with special high-volume
dispensing and mixing equipment, and it is not readily available to small bakeries or
home cooks.


Compressed yeast is essentially cream yeast with most of the liquid removed. It is a soft
solid, beige in color, and arguably best known in the consumer form as small, foilwrapped cubes of cake yeast. It is also available in larger-block form for bulk usage. It is
highly perishable; though formerly widely available for the consumer market, it has
become less common in supermarkets in some countries due to its poor keeping
properties, having been superseded in some such markets by active dry and instant yeast.
It is still widely available for commercial use, and is somewhat more tolerant of low
temperatures than other forms of commercial yeast; however, even there, instant yeast has
made significant market inroads

Active dry yeast is the form of yeast most commonly available to noncommercial
bakers. It consists of coarse oblong granules of yeast, with live yeast cells encapsulated in
a thick jacket of dry, dead cells with some growth medium. Under most conditions, active
dry yeast must first be proofed or rehydrated. It can be stored at room temperature for a
year, or frozen for more than a decade, which means that it has better keeping qualities
than other forms, but it is generally considered more sensitive than other forms to thermal
shock when actually used in recipes.

Instant yeast appears similar to active dry yeast, but has smaller granules with
substantially higher percentages of live cells per comparable unit volumes. It is more
perishable than active dry yeast, but also does not require rehydration, and can usually be
added directly to all but the driest doughs. Instant yeast generally has a small amount of
ascorbic acid added as a preservative.

Rapid-rise yeast is a variety of dried yeast (usually a form of instant yeast) that is of a
smaller granular size, thus it dissolves faster in dough, and it provides greater carbon
dioxide output to allow faster rising


Citric acid is the product of fermentation of Aspegillus niger. The acid is one of the
principal organic acids produced in the citric acid cycle. During the production of CA, the
activity of the condensing enzyme (operating in the condensation of acetyl CoA and
oxaloacetic acid to citric acid) is increased, while the activities of the isocitrate
dehydrogense and acotinase disappear. The enzyme acotinase is responsible for the
control of biosynthesis of isocitric acid from citric acid and in turn isocitrate
dehydrogenase mediates in the hydrogen removal which yield axalosuccinic acid from
isocitric acid. Inactivity of these enzymes is the reason for CA accumulation.


Culture conditions which also lead to accumulation of CA are:

1. Sugar type and concentration- Carbon source: molasses or sugar solution : only
sugars which are rapidly catabolised by the fungus such as sucrose, maltose or
glucose, allow high yields as well as high rates of acid accumulation
2. Trace metal ions- while all usual trace metal ions are essential for A. niger, some
of them particularly Mn2+, Fe2+, Zn2+ have to be present in the medium at growth
limiting concentrations to give high citric acid yields. Na-ferrocyanide is added to
reduce Iron (1.3 ppm) and manganese (<0.1ppm).
4. pH<2citric acid accumulation has been to accumulate in significant amounts
only when the pH is below 2.5. pKa for CA is 1.8


5. Dissolved oxygen tension higher than that required for vegetative growth is
essential and even sparging with pure oxygen can be useful
6. Nitrogen- nitrogen sources used in medium for Ca production include ammonium
salts, nitrates and the potential ammonia source, urea under special conditions. In
continuous culture the N must be limiting for attaining highest CA yields
7. Phosphates- not very critical but appropriate balance is essential
8. Temperature - 30 oC.
There are two different types of fermentations carried out for the production of citric acid:
1. The surface process
2. The submerged process
1.The surface process
The fermentation is carried out in aluminum trays, filled with nutrient medium to a depth of
between 50-200 cm. Spores are distributed over the surface of the trays and sterile air is passed
over them. The mycelium grows over a period of 7-15 days.
2.Submerged method
A niger may be cultured as submerged mycelium in aerated vats i.e. CSTR. The mycelium
germinates to form stubby. Forked and bulbous hyphae, which aggregate to small pellets, which
have a firm, smooth surface and sediment quickly when harvested.



Beet molasses have been widely used in the pan techniques, although pure sucrose and syrups
have been use in submerged cultures in vats. Metallic cations can be removed from the
carbohydrates using cation-exchange process. The carbohydrate is diluted to a concentration of
about 20% to 25%. This high sugar concentration inhibits formation of acids other than citric
acid. The manufacture of citric acids needs a low pH and HCl as added to reduce the medium to
range of pH 2-5 when fungal spore in the inoculum germinate. In the vat, pH is kept below 3.5.
The prepared medium is added with spores from stock cultures. Highly aerobic conditions are
needed and submerged cultures are aerated with sterile air and agitated.


The temperature must be maintained in the range of 20-30oC during the incubation period (710d). Lime is added during harvesting, to the culture medium to precipitate any oxalic acid
which formed and the mycelium and calcium citrate are filtered off. The recovery of CA from the
broth is accompanied by three steps
1. Precipitation- a slurry of calcium citrate, which precipitates out is filtered and the treated
with sulphuric acid which precipitates the calcium
2. Extraction- solvent extraction, which makes the use of various aliphatic alcohols,
ketones, amines, etc
3. Ion exchange- the dilute CA is purified by treatment with Carbon and is demineralised by
successive passage through cation and anion exchange resins
This purified CA is the evaporated; crystallization is performed in the crystallizers. Citric acid
monohydrate (CAM) is formed below 36oC and citric acid anhydride is formed at higher
The fermentation method is a production process in which a specific amino acid is synthesized in
large amounts by a specially selected microorganism in culture. The selected microorganism is
cultured with carbohydrates and ammonia and releases the l-form of the amino acid into the
culture medium. The cell produces glutamate from 2-oxo-glutarate (2-oxo-pentanedioic acid) by
reductive ammonia fixation that uses the enzyme glutamate dehydrogenase, a normal cellular
constituent. Many bacteria useful in glutamate production have been isolated, including
Corynebacterium glutamicum, Brevibacterium lactofermentum, and Brevibacterium flavum.
These glutamate-producing bacteria are all coryneform bacteria, which are gram positive, nonspore-forming, and nonmotile and require biotin for growth. Glutamate accumulation in the
medium occurs only under biotin-limiting conditions. The requirement for biotin limitation
prevented the use of standard raw materials such as sugar molasses because they contained
biotin. Significant efforts were thus expended to overcome this difficulty. Ultimately, methods
were discovered, such as

the addition of a surfactant or of penicillin

the use of microorganisms auxotrophic for glycerol or oleate, that allowed the
bacteria to produce large amounts of glutamate without biotin limitation.
Addition of local anasthetics


The industrial production of MSG using fermentation technology continues to improve in terms
of conversion yield from sugar to glutamate and in production speed of the fermentation.
Fermentation allows the isolation of l-glutamate to be a simple process because the cells produce
the l-isomer. To improve MSG purity, a new method for purifying l-glutamic acid crystals was
developed, which uses recrystallization of the -form and subsequent conversion to MSG. The
mother liquor of the crystallization process is then concentrated and used as a liquid fertilizer
(after pH adjustment with ammonia). The invention of the fermentation method has dramatically
improved glutamate production methods and allowed production to keep up with demand for the



Mushrooms are fungi (class- basidiomycetes and ascomycetes) which can be cultivated on large
scale for human consumption. Out of 4000 species, 200 are edible out of which only 12 species
are cultivated on large scale. Some of the common edible mushroom varieties are- Agaricus
bisporus (button mushroom), Pleurotus ostreatus (Oyster mushroom), Volvariella volvacea
(Chinese mushroom). The edible mushrooms contain 35-45% protein, 7-10% fats and free fatty
acids, 5-15% carbohydrates and minerals in good concentration. Therefore, edible mushrooms
are also called vegetable meat. Edible mushroom cultivation has become one of the examples
where microbial culture is being used for direct human consumption. Cultivation of edible
mushrooms has become one of the most important and profitable biotechnological industry all
around the world. Mushrooms have a short life 8-12 hours and should be therefore stored at low
temperatures of 2-50C. Some of the mushrooms are cultivated in the summer and rainy season
such as Volirariella sp. Agaricus bisporus and Pleurotus sp grow well in winters.
Process of mushroom production


The production of mushroom involves solid-substrate fermentation which is basically carried out
by using substrates like straw, sawdust, compost, wooden logs etc. The compost with desired
formulation is prepared and sterilized and then spread into trays which are then transferred to
production room. This is followed by inoculation with spawn. Spawn is the term used for the
mushroom inoculums containing spores and/or small pieces of fruiting body. The culture is
maintained at optimal growth conditions of temperature 150C and pH of 7.0. The humidity is
maintained at 70-80% by watering the trays regularly. A single crop of mushroom is ready in 710 days. It is possible to have 3-4 crops before terminating the production process.
The outline of edible mushroom production