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Fermentation

Fermentation The term fermentation

derives from the Latin verb
'ferver', that means 'to boil'
describe the appearance of
malted grain or fruit juice
functioned upon by yeast,
during the process of
alcohol production.

Term

In context to the microbiology and biotechnology term,

In a general sense, fermentation is the fermentation means any process for the production of
economically useful products by the growth of microorganisms in
conversion of a carbohydrate such as sugar into nutrient medium under either aerobic or anaerobic condition.
an acid or an alcohol by use of yeast or bacteria.
The earliest use of fermentation was to prepare While in context to biochemistry fermentation is an energy-
alcoholic beverages since thousands of years. generating anaerobic process in which organic substances act as
both electron donors and acceptors.
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History of Development of Fermentation Products

Time Fermentation products
History of fermentation Antiquity Alcoholic beverages, Soy sauce, Vinegar
1700s Production of Vinegar
Before 18th century fermentation processes were carried out to preserve
and alters foods without much understanding about the role of 1800s Yeast induced fermentation process
microorganisms. 1929 Discovered Penicillin by Alexander Fleming
1930 Discovery of biotechnological method for production of organic acids
Ethanol was the only product produced on large scale before world war I. 1940s Industrial production of organic acids
1950s Steroidal bioconversion and several antibiotics development.
1960s Production of single cell protein from petroleum or natural gas.
In 1940s manufacturing of organic acids and antibiotics increased the 1970s Production of enzymes
interest of industrial fermentation technology. From 1980s Production of foreign proteins by use of genetically modified
microorganisms. Production of monoclonal antibodies.

Applications or Scope of Fermentation Processes (b) Production of Microbial metabolites

• Mainly two types:

Primary metabolites:
• These are Produced during the log phase or exponential phase or trophophase of
(a) Production of biomass or Microbial cells the growth cycle.
• Baker's Yeast, Lactic acid bacillus, Bacillus sp. And Single cell protein • These are essential for their growth or the by-products of energy-yielding
metabolism
• Examples: Ethanol, Citric acid, Glutamic acid, Lysine, Vitamins, Polysaccharides
etc.

Secondary metabolites:
• These are produced during the stationary phase or idiophase of growth cycle.
• These compounds do not have any obvious role in growth of microorganisms.
• These compounds are produced by the filamentous and spore forming bacteria.
• Examples: Antibiotics, growth promoters and inhibitors.
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(d) Production of Transformation products

(c) Production of Microbial enzymes • Fermentation processes, in which microorganisms are
used for conversion of a compound into a structurally
•Enzymes produce by various microorganisms. similar, but financially more valuable compound. These
processes are termed as transformation or
•Examples: biotransformations or bioconversions.
•Produce by Bacteria: Protease, Glucose • Microorganisms can act as chiral catalyst with
Isomerase, Amylase And Lipase stereospecificity hence, such reactions are specific than
the purely chemical reaction.
•Produce by Fungi:, Protease, Pectinase, • Examples: Phenyl acetyl carbinol, Steroid
Amylase And Amyloglycosidase Biotransformation

(e) Production of Recombinant products Screening of microorganisms

•Microorganisms are capable of

synthesizing foreign proteins upon Screening is defined as set of techniques that allow the detection and
isolation of microorganisms of interest from natural microbial population.
insertion of genes of higher organisms. This procedure allows selection of desired microorganism while discarding
•Examples: Insulin, Human serum the undesired microbes at the same time.

albumin, Interferon, Epidermal growth There are various screening techniques usually can be classified as Primary
and secondary screening techniques.
factor, Clotting factors-VII & IX.
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Selection of the microorganisms should be

Primary screening
based on following characteristics:
Nutritional characteristics (can be grow on cheap medium to bring economy in the These are preliminary tests to detect the presence of the microorganism of
interest.
process).

Ease of cultivation on large scale. It gives basic idea about which microorganisms having an ability to produce a
product but does not give estimation about productivity or yield.
Ease of product recovery.

High productivity. It is a qualitative approach.

Stable in biochemical characteristics.

There are various approaches.
Minimum sensitivity to genetic manipulation.

Medium containing pH indicative dye for Crowded plate method

detection of organic acid or amine producers. Approach used for the detection of antibiotic producing
microorganism.
pH indicating dye such as neutral red, bromo thymol blue
incorporated into the poorly buffered agar medium. Preparation of different dilutions of soil or natural
microbial population.

After inoculation and incubation, production of organics

acids or amines can be detected as change in the colour Spreading of dilutions on nutrient agar plate.
of the dye in the vicinity of the colony.
Selection of agar plate having colonies in range of 300 –
These colonies are then subcultured to purified but 400, as it is useful in locating the desired colonies.
further testing is required as many microorganisms are
produced inorganic acid or base as a metabolic products.
zone of inhibition surrounding colonies indicates
presence of Antibiotic producer.
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Limitations of crowded plate method Improvement in the crowded plate method

• Further confirmatory tests are required to perform as zone of
inhibition surrounding the colonies may be due to other causes like Incorporation of test microorganism.
depletion of nutrient in the medium, change in the pH.
• This method gives idea about the activity of compound produce Use of dilution that gives isolated colonies in range of 30-300.
against unknown microorganisms while researchers are interested in
activity against specific microorganisms.
After incubation test microorganism to be sprayed and plates are further
incubated to allow growth of test microorganism.

Antibiotic producer can be detected as zone of inhibition surrounding colonies

which can measure in terms of mm.
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Secondary screening must confirm or reveal the following information about

Secondary Screening: the product produced/synthesized by newly detected microorganisms

Novelty of the product. [Link]

These methods are useful in finding out microorganisms Chemical form/s of the product.
with a potential application or real inherent value in the
Chemical stability of the product in production medium.
fermentation processes or industries and discarding the
valueless microorganisms at the same time to minimize Destruction or modification of the product by the producer in the culture broth.
the cost of research. It may be a qualitative (the spectrum Yield of the product.
of microorganisms sensitive to new product) or
Economy of the process and scope of improvement w.r.t. overall process and cost.
quantitative approach (product yield).
Critical requirements w.r.t. growth of microorganism and product formation.

Method of Estimation of the product.

Improvement of culture
screening design Numbers of product form.

is to carry out Highest yield possible.

Naturally isolated microorganism generally gives very less yield of industrially
interested compound.
series of
experiments in Presence of contamination.
Thus, it is required to increase the yield of the product that can be achieved by
liquid media to Process conditions like pH, aeration, agitation
optimization of the parameters related to fermentation process.
collect the
details. Such as: Chemical stability w.r.t . time, temperature and pH. However, using this approach it is not possible to bring the economy in the
production up to desirable level as that gives increase the yield up to certain
level only.
Solubility of product in various solvent.
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Approaches for Improvement of the culture Sources of culture

• Due to various novel product manufactured by the fermentation
technology, importance of microorganisms to industry, research and
Selection of a high yielding strain i.e. natural variant with different genetic makeup.
educational institute increased that necessitate culture collections.
• These culture collections are serving as authentic source for the
Genetic improvement by induction of mutation (by chemical or physical distribution of microbial strains and repositories for strains as part of
methods). patent deposits.
• Microbial Type Culture Collection & Gene Bank (MTCC) (India)
Genetic recombinant techniques to design high yielding strain (by transformation, • National Collection for Industrial Microorganism, India
transduction, conjugation, protoplast fusion and parasexuality in fungi).

American Type Culture Collection (ATCC), USA

Preservation of culture
Australian Collection of Antarctic Microorganisms (ACAM) (Australia)

Japan Collection of Microorganisms (Japan) Microorganisms give high yield or new fermentation product are
required to store for future use.
National Collection of Type Cultures (NCTC), UK
Microbial cultures are preserved to maintain the cultures:
National Collection of Yeast Cultures (NCYC), UK
• Viable and genetically stable and free from contamination.
The National Collection of Industrial, food and Marine Bacteria (NCIMB) • Physical and physiological properties.

Hence, stock cultures of interested strains are maintained in as

Collection Nationale De Cultures De Microorganismes (CNCM), France
primary stock culture and working stock culture.
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Periodic transfer on the fresh medium

Most simple method.

There are various methods practiced for Periodic transfer to freshly prepared slant before exhaustion of nutrients.

the preservation or maintenance of the Storage at refrigerator condition.

microbial cultures and the particular Period for transfer may be from week to few years depending upon the
technique is selected on the basis of time microorganism and media used for storage.

and type of culture to be preserved. Development of variant and mutant may occur due to changes in the
characteristics of the strain.
Time of preservation can be extended by covering the growth with sterile mineral
oils.

Preservation in sterile soil Storage at very low temperature

In this method microorganisms are preserved as dense culture in the presence

Widely use for the spore forming microorganisms. of cryoprotectant (Glycerol or Dimethyl sulfoxide).

Spores or active culture transferred to soil (previously sterilized).

Cryoprotectant are added to prevent the damage to delicate cell structure.
Then soil is dried under reduced atmospheric pressure.
Dense culture is stored at -1500C or less using liquid nitrogen in a sealed
Culture in soil then preserved at room temperature. ampoules or vials.

This method is used for preservation of many microorganisms which cannot

Non-sporulated microorganisms may died over a period of time. withstand the lyophilization method.
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Lyophilization or freeze drying Microbial Growth cycle

Many microorganisms can be preserved for several years effectively by this

method that killed by other methods of preservation.

Dense culture in vials frozen at temperature -600C to -780C is then connected

to a high vacuum to freeze dried.

This process makes the culture dehydrated with minimum damage.

Finally, ampoules or vials are stored at refrigerated condition.

Lag Phase: Log Phase or Exponential Phase or Trophophase

Growth rate increase as cells begin to divide.

Initial, no growth phase and number of cell changes very less.
During this phase growth rate is reciprocal of generation time.

Period of adaptation of microorganisms to its new Microbial cells are nearly uniform and clearly in define condition.
environment.
Although nutrients are continuously consumes, growth rate remain constant.
In this phase cells are increase in size and synthesizing various
enzymes and molecules depending upon surrounding medium. Products produced during this phase are essential for their growth and are known as primary
metabolites (proteins, lipids and carbohydrates).
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Stationary phase:
Death or Decline phase:

Nutrients become depleted and growth rate decrease.

At this stage, rate of cell death increase over new
Exhaustion of nutrient, accumulation of toxic metabolite and pH shift lead to cell formation
death of the microorganisms

Numbers of new cell formation and cell death are in equilibrium. Exhaustion of key nutrients and accumulation of
inhibitory substances resulting in a death of
Products produced during this phase are known as secondary metabolites
(antibiotics). microbial cells.

Types of Fermentation processes Batch culture

• Closed system.
Batch culture • Limited quantity of nutrients in the fermentor.
• The culture passes through the lag, log, stationary, and then death
phase.
Continuous culture • Products are accumulated in the fermentor until the maximum yield
obtained and collected at the end of the process.
• Before and after process fermentor is cleaned and sterilized.
Fed-Batch culture • Use to produce biomass, primary and secondary metabolites.
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Conditions that give the high productivity

during batch fermentation process are Continuous culture
summarized as under:
Nutrients/medium supply at constant rate and also, continuous removal of
Type of Product Set culture condition that culture/product.

Formation of microbial cell in the process is balanced by loss of the cell due to
Biomass gives maximum growth rate and cell concentration culture/product removal from the fermentor.

Primary metabolites extend the lag phase Steady state process.

Secondary metabolites Shorten the lag phase and extended stationary phase.
Growth of the culture should be substrate limited.

Chemostat:
In this approach, culture growth is regulated by availability of a single
growth limiting nutrient.

Various approaches are reported to In a chemostat, growth rate is determined by the supply of a single
control the microbial activity that limiting nutrient. It is a nutrient regulated self balancing culture.
Steady state condition may be maintained at sub-maximal growth
maintains the steady state condition rate for prolong period.

in continuous fermentation process. Most widely used approach as minimum wastage of nutrient as
residue in the harvested culture and less complex system.
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Turbidostat Biostat

Here, total number of the microbial cells is to be kept constant by monitoring

the turbidity of the culture using the device like photoelectric cell in narrow
limit. Here, biomass concentration is to
Ultimately, based on the turbidity rate of nutrients/medium supply and be monitor such as concentration
culture/product removal is regulated.
of CO2 or pH to maintain the
So, if turbidity exceeds the set level, pump is switched on to supply the
medium and switch off when turbidity fall below the set level.
steady state condition.

Advantages of continuous culture: Disadvantages of continuous culture

Productivity is higher than the batch culture.

If process is not properly optimized than large

Growth rate and concentration of limiting nutrient may be controlled. quantity of the residual unused nutrient loss in the
harvesting culture.
System as steady state may be operated for infinite time.

It is sensitive to contamination.
Nonproductive time associated w.r.t. operation such as cleaning,
sterilization, filling of fermentor and lag time is much lower in the case of
continuous culture compared to batch culture.
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Fed-Batch culture In this process, initially fermentor partially filled with the medium and process behaves as a batch
culture.

Then, fresh medium is to be added just before the culture is likely to enter in the decline phase and
Fed batch culture is an intermediate between the dilution rate should be adjusted such that the biomass concentration remains constant throughout
the process below the specific maximum growth rate.
batch and continuous culture.

If culture is nutrient limited, it provides a means of System is similar to the chemostat and the growth rate approximately equal to the dilution rate
until the fermentor is completely filled with medium.
controlling the growth rate of the microorganisms.
The process ultimately stopped to recover the desired product

Salient features of fermentor

Minimum power
Operated Prevent the escape Minimal
requirement and
aseptically. of microbes. evaporation losses.
cheap.
Design of Fermenter
Provision for Provision for
Smooth internal Suitable for a range aeration, agitation controlling of
surfaces. of processes. and sampling of temperature and
the medium. pH of the medium.

Simple operation,
harvesting, Adequate service
cleaning and provisions.
maintenance.
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Fermentor design:
Aeration:
Sparger or diffuser:
• This is a most widely used approach in stirred fermenter. Device consists of pipe
Vessel: or plate with minute holes which is used to introduce pressurized air into the
liquid medium in the fermentor in the form of small bubbles. It is arranged at the
base of the fermentor. Bubbling of air through sparger provides both agitation and
Major part and common requirement for any kind of fermentation aeration.
process irrespective of size and utility. Vessels are constructed of
stainless steel and/or borosilicate glass. The selection of the material Impeller:
is based on the several parameters. Visual observation of the process • Air is introduced to the hollow drive shaft of the impeller and pressurized air
is the greatest advantage of the glass fermentor, while less introduce in to the liquid medium in the fermentor through the holes in the shaft.
contamination problem with fermentor made-up of stainless steel. Air lift or Gas lift fermentor:
• In this type of fermentor air or gas is used to circulate the content of the
fermentor.

Agitation
The agitating device consists of shaft to which
Impellers: impeller is fitted. An impeller consists of disc
fitted with various types of bolt. Baffles
Forced In this type pump is used to circulate the medium
convection: in fermentor.
Baffles are metal strips in fermentor that prevent
Sparger or air Introduction of air by means of sparger or air lift vortexing. Usually four baffles are present and
lift device: fermentor provides agitation along with aeration. attached radially to the wall of fermentor.
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Parameters to control and measure during the Types of fermentor:

fermentation process Mechanically stirred
Stirred tank fermentor
fermentor
• Using hot and cold water circulation in the jacket surround the
Temperature control:
fermentor and thermocouples are used to measure the tempe.
Submerge fermentor:
• By the addition of acid or alkali through an auto-titer connected Microorganisms are Fermentor with forced
pH control: Airlift fermentors
to a pH electrode a pH measurement system. dispersed and growing convection of liquid media
in the liquid medium.
• Using tachometer fitted to the drive motor and determining the
Speed control:
power supply by the speed controller to control the speed. These are further
categorized as:
Air or gas flow • using the pressure controlled valve connected to the rotameter.
control: Rotameter is used to measure the airflow. Fluidized bed fermentor
Dissolved oxygen • level of oxygen control by air or speed or both in combination.
control: Polarographic electrode is used to measure the dissolved oxygen. Surface fermentors:
Microorganisms are Pneumatically operated
• Using conductance probe that detect the foam in head space growing on the surface of fermentor Packed bed fermentor
Antifoam control:
connected with pump delivering antifoam reagent. solid medium.

Stirred tank fermentor

Most widely used.

It is an upright cylindrical vessel.

A shaft in centre supports the various types of impeller for stirring the content and baffles
to prevent vortexing and efficient aeration are incorporated in to fermentor.

Liquid medium is used to provide growth to microorganisms

A head space is provided above the liquid medium to allow aeration and splashing so
working volume is less than the volume of fermentor.

Adequate provision for delivery of medium, culture, air and reagents.

Adequate control system for pH control, temperature control and speed control.
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Key points

Hydrodynamic shear is low, so this fermentor is widely used

for animal and plant cultures, where the cells are delicate.
Air lift fermentor
Good ability to transfer O2 and heat.

This fermentor do not handle the highly viscous media.

Flow of medium is adjusted to

Fluidized bed fermentor balance the gravitational force
in such a way that:
• This is a cylindrical vessel with expanded top section. • The trapped cells are remained in
• Fermentor equipped with sparger at the bottom and the pressurized fluidized or suspended form.
air or gas provides agitation. • Allow the trapped cells to convert the
medium constituent (substrate) in to
• Microorganisms are trapped in a polymer structure and held in the product without washed out along
fermentor using mesh. with the product.
• Fresh or re-circulated liquid medium is continuously pumped to the
As the velocity of the medium
bottom of the vessel.
reduced in expanded upper
part of the fermentor that also,
prevent the loss of microbial
cells in harvested culture.
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Packed bed fermentor In this type of fermentor microorganisms

are fixed instead of fluidized or Hollow fiber fermenter In this type of fermentor, microorganisms
suspended state as in the case of are embedded to hollow fibre that having a
fluidized bed fermentor. very small diameter.

Liquid medium is flow through the Anchorage-dependent cell lines can be

packed bed or column can be upward or cultured in bundles of fibers held together
downward direction depending upon the in the cartridge and medium circulate out
design of fermentor. sides of the fibers.

The product is released along with the So, this type of fermentor has very large
consumed medium. surface to volume ratio.

There is no danger of microorganism This fermentor widely used in production

being washout. of monoclonal antibody, gonadotropin, and
antigen.

Steps of a Typical Fermentation process

• Formulation of media for production and

Upstream processes: inoculums development
Processes before • Sterilization of the medium, fermentor and
fermentation auxiliary instruments
• Inoculums preparation

• Growth of microorganisms under optimum

Fermentation: conditions in fermentor

Downstream processes: • Product recovery and purification

Processes after fermentation • Effluent or waste disposal
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Formulation of media for production and There are two types of fermentation medium
inoculums development Synthetic or chemically defined
Complex or crude media.
medium
Microorganisms can be maintain their metabolic activity and synthesize desired
products in the presence of nutrients. • Synthetic media contains • Crude media on other hand are
defined, known and pure cheap hence, wide spread use in
Nutrients are provided by means of fermentation media. components, the large scale production.
• Their composition is known. • Crude media contain crude or
• Synthetic media are undefined nutrients.
reproducible but expensive and • Problem with this type of media
give low yield are unpredictable growth rate
during fermentation process,
One of the most critical steps in the fermentation process is to design a and relatively difficult recovery
suitable medium. Generally it is to be selected by the trial or error method or or purification process.
by systemic optimization study.

Desired Characteristics of the fermentation medium

Support rapid growth
Components of the media for fermentation
Reproducible Provides maximum
of microorganisms or
performance. yield.
desired product.
Water :
Minimum yield of
Stable during Readily available •Major component of fermentation media.
formulation and throughout the year
undesired products.
sterilization process. in large quantity. •Also, used for auxillary process like cooling,
heating and cleaning.
Not interfere with
fermentation process with Free from toxic effect •Dissolved salt, effluent contamination and pH
respect to aeration,
agitation, product
Cheap. on microorganisms or
products
is required to control.
extraction and
purification.
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Source of carbon: Cost: • selected on the basis of price, yield and availability in
different locations

• Concentration of carbon in the media governs the growth rate of Purity: • it may affect the yield as in the case of citric acid production
(carbon source must be free of metallic impurities)
microorganisms.
• Low yield of secondary metabolites may results from the rapid • During sterilization at high temperature sugar may react
metabolism of the high concentration of the sugar. Method of with the ammonium ions or amino acids present in the
medium to form black nitrogen containing compound.
sterilization of Hence, it is advisable to sterilize sugars separately in heat
sterilization method.
media: • When starch is present as a carbon source, upon heating it
will produce high viscosity of the medium.

Government laws: • About the use of particular substrate.

Carbohydrates
Molasses By-product of the cane and beet sugar industries. Cane molasses is rich in biotin,
pantothenic acid, thiamine, phosphorus and sulphur. Beet molasses contain biotin,
pyridoxine, thiamine, pantothenic acid and inositol.
Vegetable oils Oils obtained from vegetable seeds like olive,
It is cheap and widely used but contains many impurities.
maize, cotton seed, linseed, soybean etc.
Fruit juices Contains soluble sugars like juice of grapes
Starch Wheat, rice, maize cereals, potatoes etc Hydrocarbons Methane, natural gas, paraffin etc.
These are sources of starch that requires pretreatment to convert in to Paraffin and gas oil widely used in production of
fermentable sugars single cell protein
Cellulose material Sulphite waste liquor is a by-product of paper pulp production
Rice straw is a by-product in agriculture.
Corn steep liquor and Used as source of nitrogen but also contains carbon source
soyabean meal
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Minerals:
Source of nitrogen • For the growth of microorganisms and metabolism certain minerals
Inorganic Ammonia (used to control pH), ammonium salts (produce are required to incorporate in to the medium. Following minerals are
essential components.
acid condition) or nitrates (produce alkaline pH).
Organic Amino acid, protein or urea Minerals added Calcium, Phosphorous, Magnesium, sulphur, potassium
Other proteinaceous material serving as a source of amino
acids are in the medium and chlorine
Corn steep liquor: concentrated by-product from corn steeping
to produce starch, gluten and other products.
Soyabean meal: material after removing oil from soyabean Minerals present Copper, ion, manganese, cobalt, molybdenum, zinc and
seeds. as impurities nickel
Cotton seed meal (pharmamedia)
Peanut meal
Distillers soluble

Growth factor Used for production of

Growth factors Biotin Glutamic acid

Precursors
Calcium pantothenate Vinegar
these are the compounds when present in the media, directly
incorporated in to product without any major changes.
Certain preformed compounds are required to incorporate in the
fermentation media as many microorganisms cannot be able to produce a These compounds increase the yield of product.
full complement to cell components.

Precursor Increase the synthesis of product

Vitamins, amino acids, fatty acids or sterols are the examples of the growth
factors. Usually natural source of carbon and nitrogen contain various Phenyl ethylamine Penicillin G
growth factors. The following are some examples of the growth factors Phenyl acetic acid
used in the fermentation process.
Cyanide Vitamin B12
Chloride Chlortetracycline
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Inducers Inhibitors
Many enzymes are inducible. Inducers are the compounds when These are the compound that enhance the desired product
present in the medium induce the synthesis of the enzymes. formation while at the same time retard the formation of
Inducer Induce the synthesis of enzyme
undesired related products.
Starch Amylase Inhibitors Desired product Undesired product

Cellulose Cellulose (enhance formation) (Inhibit formation)

Bromide Tetracycline Chlortetracycline
Pectin Pectinase
Alkali metal or phosphate and Citric acid Oxalic acid
Fatty acids Lipase
pH less than 2
Sodium bisulphate Glycerol acetyldehyde

Antifoams

Certain components in the medium like corn steep liquor or soybean

meal and condition like agitation produced foaming during the Problem of • Designing the suitable medium and
fermentation process which is not desirable.
the fermentation conditions like
agitation and aeration
Problems associated with foaming are:
foaming • Using mechanical foam breaker
can be • Using antifoams. (Various alcohols,
ester, oils, sulphonates and silicones)
avoided by
• Cells may entrapped in the foam and removed from medium that results in autolysis
of microorganisms.
• Further autolysis of the microorganisms stabilizes the foam.
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Sterilization of the medium, fermenter and

auxiliary instruments
Buffers • Foreign microorganisms may utilize the medium
During the that result in loss of productivity.
fermentation • Foreign microorganisms may contaminate the
Chelators processes, product
foreign
• Extraction and purification of the desired product
microorganisms
Oxygen availability may enter in the
may be difficult due to product of foreign
microorganism
• Presence of a rapidly metabolized carbohydrate in the medium. process which
may lead to • Foreign microorganisms may bring the degradation
• Viscosity of the medium following of product.
• Presence of surface active agent as antifoam in the medium, which problem. • Virus e.g. bacteriophage if enter as foreign
reduce diffusion of oxygen. microorganisms may bring lysis of the cells.

Sterilization of the fermentation medium:

• Using pure culture

Contamination • Sterilizing the medium and other material
to the added to fermentor
fermentation
process may be • Sterilizing the fermentor and auxiliary
prevented by instrument used for fermentation process.
• Maintaining aseptic condition.
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Cleaning and sterilization of fermentor Inoculum preparation

Cleaning-In-Place (CIP) and Sterilization- Desired • Microorganisms should be growing rapidly to

In-Place (SIP) are systems designed and characteristics minimize the lag phase or incubation period.
• Microorganisms should be in desired
of inoculum to morphological form
validated for automatic cleaning and be used for • Inoculum should be in a pure form i.e. free
sterilizing the fermentor without any fermentation
from contamination
• Microorganisms should preserve its
major disassembly and assembly work. processes characteristics to synthesize the product

Reduction fermentation process in the length can

be achieved by
Reducing the By growing the microorganisms on the medium containing the
fermentation same nutrients as the production medium, consequently,
process eliminating the need for a change in the enzyme profiles. Limitations of this series of transfer to inoculum
length of lag
phase during
Using large quantity of inoculum (0.5 - 10% by volume of the preparation
medium) to inoculate the fermentation medium.
the
fermentation To shorten the time for preparation of inoculum for the •Microbial strain may undergo mutation,
process. production medium, series of steps are to be designed in such a
way that inoculum is transferred to next medium with increasing
•Contamination may occur so it is advisable to
the volume at each step. check the contamination at each step of
These subsequent inoculums are called seed cultures. The final inoculum preparation.
seed culture serves as the inoculum for the production
fermentor.
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Product recovery and purification

Success of any fermentation process of valuable products lies on their designed,

separation and purification steps and stability of the product during this process. Various criteria to select the approach for the
product recovery and purification includes
Fermentation products produced during the process are present in less amount
along with the consumed medium • Product formation by microorganisms i.e. intra or extracellular
hence, required to separate it from the exhausted media component and • Physicochemical properties of the product
subsequently purify for further use. • Type and level of impurities
Various approaches are used to recover and purify the product and particular • Price of the product
approach to be selected on the basis of the various criteria. • Biohazard of the product and culture.

Steps and objective Methods

Filtration
Separation of crude product
Centrifugation Effluent or waste disposal
Sedimentation
(if product is intracellular then first microbial cells • Consumed media,
are ruptured by different techniques like application Effluent or waste from • Unconsumed inorganic and organic compounds,
of shear, osmotic shock, enzyme treatment or fermentation
• Microorganisms or cell residues and
sonication) processes contains
• Water.
Extraction of the product / Ion exchange
Ultrafilteration
Concentration Solvent extraction It should not be
Crystallization
discarded directly to
Purification the sewage or nearby • Content of organic matter and microorganisms.
Precipitation
Chromatography water reservoir
because of their
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The organic matter present in the that result in the decreased dissolved oxygen
waste may be oxidized by the
microorganisms
level and bring undesirable changes in the types Fermentation process and recovery of some products
of macro organisms like fish.
• Hence, waste is treated with microorganisms that are actively decompose the
organic matter to carbon dioxide and water.
In terms of
Unconsumed organic matters Biochemical oxygen demand (BOD) or
in waste are determined Chemical oxygen demand (COD).

Penicillin
• BOD and COD are amount of oxygen consumed in decomposition of organic
matters by microorganisms and hot acidified dichromate.
• Both this parameters give indication of level of organic matter present in the waste.

The fermentation waste may contain the microorganisms that may be pathogenic or
it may be taken out by competitor.

• Hence, it is desirable to sterilize the waste before disposal.

• Penicillium chrysogenum (high yielding strain) Medium Various media are reported by the researcher and used
components: by the different industries for the production of penicillin.
• The production strain of penicillium
Culture: chrysogenum maintained by sporulated agar
slant maintain in frozen state, lyophilisation of Inoculum medium generally not contain the lactose and
spores or preserving spores in soil at low precursor as at this stage goal is to increase the growth of
temperature. inoculum and not the production of antibiotic.
• Surface culture method Media composition has significant effect on the yield of
Method: • Submerged culture method (widely used product; hence, the media are optimized before using for
method) the routine production.
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• lactose, glucose or sucrose.

• Glucose in the medium supports the rapid growth of microorganisms while
Source of carbon: • lactose is converted slowly to glucose and galactose and produces starvation
condition that is required for the production of penicillin.
• If glucose is used as a source of carbon, then it is added gradually in the
fermentor to produce starvation condition. pH of the medium rise during the fermentation process due to utilization of
corn steep liquor that contains lactic acid (depleted during the process) and
amino acid (which upon deamination convert to ammonia).
Source of nitrogen: • corn steep liquor, ammonium sulphate, ammonium acetate.

At the end of the process pH is about 8 due to exhaustion of lactose which

Minerals: • the various ions required are Cu++, Mg++,Zn+, K+, PO4-2., SO4-2 resulting autolysis of mycelium. Generally penicillin fermentation is harvested
before autolysis of the cells occur.
Precursors: • phenyl acetic acid for penicillin G

Sterilization of the medium by steam sterilization method:

Medium pH (optimum): • 5.5 -6.

Steps Methods
separation Filtration usually rotary filter
-To remove microbial cells and mycelium from broth
Extraction of the Solvent extraction
Fermentation Aerobic process.
process product -First extracted with organic solvent after acidification and
conditions Agitation generally provided by impeller. then back extract with aqueous solution of potassium
are:
hydroxide, calcium hydroxide or sodium hydroxide.
Temperature: 22 -25 0C.
Repeat this extraction and back extraction after
Fermentation time: 5 – 6 days. acidification, which also help in purification of product.

pH: 6 – 7.5. Purification Carbon treatment to remove pigment and/or pyrogen.

Finally it is subjected to crystallization
Yield: 200-800 units/ml Different salt of penicillin can be prepared by minor
modification in the method.
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Citric Acid

• Aspergillus niger(widely used), Penicillium luteum,

Culture: P. Citrinum, Yeast

• The production strain of Aspergillus niger
maintained in the form of dry spores.

• (i) Surface culture method (Old method yet in

Method: practice)
• (ii) Submerged culture method

Steps Methods / Purpose

separation -To remove microbial cells and mycelium from broth. (First
mycelium is pressed to obtain intracellular citric acid)
Precipitation Treatment of broth with milk of lime in hot neutral condition to

• Aerobic process. precipitate citric acid as calcium citrate. Then precipitates are
Fermentation • Agitation required. subjected to filtration and washing.
process • Temperature: 26 -30 0C. Treatment with Precipitates of calcium citrate are treated with sulphuric acid

conditions • Fermentation time: 5 – 8 days. Sulphuric acid. that release the citric acid and to remove calcium as sulphate

are: • Yield: 60 to 80 grams of citric acid precipitates.

per 100 grams of added sugar. Treatment with Charcoal Decolourization using activated charcoal.
Purification Finally citric acid obtained from the solution by evaporation
and crystallization.
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