fermentation technology

Dr. Harisingh Gour University , Sagar
(2021-2023)
Department Of Pharmaceutical Sciences
Presentation On- Fermentation Technology
Submitted to : Submitted by :
Dr. Nishi Mody Sandeep Kumar Maurya
M. pharma 2nd Sem

Content
1. Fermentation
2. Industrial fermentation
3. General requirements
4. Equipment’s used in fermentation process
5. microbial growth
6. Types of Fermentations and culture systems
7. Industrial fermentation
8. Industrial fermentation of antibiotics
9. Enzymes production
10. Microbial Production of Vitamins
11. Single Cell Protein Production Processes
12. References.

Fermentation
Fermentation is the process of deriving energy from the oxidation of organic
compounds, such as carbohydrates, using an endogenous electron acceptor, which is
usually an organic compound. The most common meaning of fermentation is the
conversion of a sugar into an organic acid or an alcohol. Sugars are the common
substrate of fermentation.
Reaction: The reaction of fermentation differs according to the sugar being used
and the product produced. In the reaction shown below, the sugar will be
glucose(C6H12O6), the simplest sugar, and the product will be ethanol (2C2H5OH).
This is one of the fermentation reactions carried out by yeast, and used in food
production.
Chemical equation:
C6H12O6 + 2 Pi + 2 ADP- → 2 CH3CH2OH + 2 CO2 + 2 ATP (energy released:118 kJ/ mol)
Sugar (glucose or fructose) → alcohol (ethanol) + carbon dioxide + energy (ATP)

General requirements:
Media (Nutrient sources for industrial fermentation): The growth of organisms
involves complex energy-based processes. The rate of growth of micro-organisms is
dependent upon several culture conditions, which should provide for the energy
required for various chemical reactions. The rate of growth of micro-organisms and
hence the synthesis of various chemical compounds under artificial culture, requires
organism specific chemical compounds as the growth (nutrient) medium. The kinds
and relative concentrations of the ingredients of the medium, the pH, temperature,
purity of the cultured organism, etc., influence microbial growth and hence the
production of biomass (the total mass of cells or the organism being cultured), and
the synthesis of various compounds. 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
Carbon source
Glucose: Corn sugar, Starch, Cellulose
Sucrose: Sugarcane, Sugar beet molasses
Lactose: Milk
Fats: Vegetable oils
Hydrocarbons: Petroleum fractions
Nitrogen source
Protein: Soybean meal, Corn steep liquor
Ammonia: Pure ammonia or ammonium salts
Nitrate: Nitrate salts
Nitrogen: Air

Phosphorous source: Phosphate salts
Trace elements: Fe, Zn, Cu, Mn, Mo, Co
Antifoaming agents: Esters, fatty acids, silicones, sulphonates, polypropylene
Buffers: Calcium carbonate, phosphates
Growth factors: e.g. with thiamine, biotin, calcium pentothenate
Precursors: Directly incorporated into the desired product: e.g. Phenyl acetic acid into
Penicillin G.
Inhibitors: To get the specific products: e.g. sodium barbital for rifamycin.
Inducers: enzymes in industry are synthesized in response of inducers: e.g. starch for
amylases, pectin for pectinase.
Chelators: Chelators are the chemicals used to avoid the precipitation of metal ions.
E.g. EDTA, citric acid .

Equipment’s used in fermentation process: fermentation process is mainly carried
out in an equipment known as fermenter or Bioreactor.
Other equipment’s used in the fermentation process are -
i) drawing the culture medium in steady state,
ii) cell separation
iii) collection of cell free supernatant
iv) product purification
v) effluent treatment.
Bioreactors (Fermenters):
A fermenter is the set up to carry out the process of fermentation. The fermenters vary
from laboratory experimental models of one or two litres capacity, to industrial models
of several hundred litres capacity, which refers to the volume of the main fermenting
vessel.

A bioreactor is basically a large vessel, generally made of thick stainless-steel body. A
fermenter usually refers to the containment system for the cultivation of prokaryotic
cells (bacteria), while in a bioreactor grows the eukaryotic cells (Mammalian, insect,
plant). Industrial fermenters are designed to provide the best possible growth and
biosynthesis conditions for industrially important microbial cultures, and to allow ease
of manipulation for all operations associated with the use of the fermenters. The main
function of a fermenter is to provide a controlled environment for growth of a
microorganism, or a defined mixture of microorganism, to obtain a desired product. An
ideal fermenter should have the provisions and control over various operations like
temperature, pH, adequate aeration and agitation, less evaporation loss, low power
consumption, proper sampling facilities, use of cheapest material, operated aseptically
for a long period throughout the operation, vessel of similar geometry (small and larger
vessels), vessel with minimal use of labor during operation, harvesting, cleaning,
suitable for variety of reactions etc. Modern fermenters are usually integrated with
computers for efficient process monitoring, data acquisition etc.

Large scale production fermenter design and its various controls
Design of industrial fermentation process: The fermentation process requires the
following:
a) a pure culture of the chosen organism, in sufficient quantity and in the correct
physiological state;
b) sterilized, 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.
Essential features of a bioreactor
(I) The vessel should be strong enough to hold large volumes of culture broth and
pressure that could be generated at times due to gas production. It should be
completely free from leakages, otherwise contaminations will occur.
(ii) Adequate aeration and agitation of the fermenting broth should be provided for
optimum growth.

(iii) It should have a system for monitoring and regulating temperature, pH .
(iv) Allow monitoring and / or control of dissolved oxygen.
(v) The vessel should be connected to inlet pipes to receive culture medium and
inoculum of the microorganism.
(vi) It should have facility for sampling and should require a minimum of labor in
maintenance, cleaning, operating and harvesting operations.
(vii) It should be constructed by cheapest materials and should be adequate service
provisions for individual plants.
(viii) The size and the internal volume of the bioreactor is designed by bio-engineers
with proper knowledge of fluid dynamics, and the microbiologists should maintain the
sterility of the entire system.

Types of Fermenters (Bioreactors): The most widely used bioreactors in industries are
1. Stirred tank bioreactor
2. Tower bioreactors
3. Air lift bioreactors
4. Packed - bed bioreactors
5. Fluidized bed bioreactors
6. Photo bioreactors

1. Stirred tank bioreactor (STRS): It is an upright cylindrical vessel with motor driven
central shaft that supports one or more agitators (impellers) so as to maintain the
homogeneity. The number of impellers is variable and depends on the size of the bioreactor.
These bioreactors are used in a range of sizes from one-liter laboratory unit to production
scale vessels of typically 100-ton capacity. A continuous flow culture provides a continuous
source of cells. At the lateral side, there is an opening through which culture medium is
being pumped into the fermenter. Another opening acts as the outlet for harvesting the
products is present at the base of the fermenter. The volume of the vessel is about 30 to 50%
larger than the required culture volume which allow disengagement of the liquid droplets
from the exhaust gas and room for foaming. A heating coil is used to raise the temperature
inside the vessel.

2. Tower bioreactors: These are huge bioreactors mainly designed for the brewing
industry. In these, the cells tend to aggregate and settle at the bottom of the vessel
inspires of the up flow of the fluid. A high hydrostatic pressure generated at the bottom
of the reactor increases the solubility of oxygen in the medium. Tower fermenters are of
different types based on their design. They are Bubble-up fermenters, Bubble column
tower fermenters, Multistage tower fermenters, Vertical-tower Beer fermenters. The
high density of cells may create anaerobic condition which is advantageous to alcohol
fermentation. Tower bioreactor has high aeration capacities without having moving
parts.

3. Air lift bioreactors: In airlift bioreactors, the medium is circulated around two
nested columns within a long tubular tower. The rate of airflow of the reactor depends
on the volumetric mass transfer co-efficient in the reactor system. The incoming air
forces the medium up the inner column, or riser, and it then descends down the outer
column, or down comer tube. The riser tube may be placed within the down comer
tube or it may be externally located and connected to the latter.

4. Packed - bed bioreactors: it is a vessel filled about 2/3rd with solid particles.
These fermenters are usually installed with a forced aeration device. A column
which is filled with a solid matrix, traps microorganisms within it. The solid matrix
may be gels absorbed with the biocatalyst (enzyme/cells), or more rigid particles.
The temperature adjustment between the top and bottom of the substrate depends on
the thickness of the bed and the aeration rate. A nutrient broth is continuously
poured over the immobilized biocatalyst. The products obtained in the bioreactor are
released into the fluid and removed. The concentration of the nutrients can be
increased by increasing the flow rate of the nutrient broth. The depth of the packed
bed is limited by several factors like pH gradient formation, nutrient concentration
and oxygen requirement.

Fluidized bed bioreactors: In the fluidized bed bioreactor, the solid substrate is
fluidized by upward flow. In this type of bioreactor, the solids are retained in the rector
while the liquid flows out. A fluidized bed has a biological film developed on particles
which are suspended in an upward flow of liquid in which they are then free to circulate.
Fluidized beds are designed using a biocatalyst such as immobilized enzyme or cells
adsorbed to particles. The support particles can be solids such as sand or glass beads or
porous such as plastic or stainless-steel mesh. The solid particles carrying the
biocatalyst are suspended in the liquid substrate. For an efficient operation of fluidized
beds, gas is sparged to create a suitable gas-liquid-solid fluid bed. The up flowing
stream of nutrient (substrate) is used to fluidize the solid particles which get dispersed in
the liquid. The top of the reactor is kept broad and the bottom narrow so that the
particles concentrate more on the lower narrow region. These bioreactors are mostly
used in conjunction with immobilized cells or enzyme system and are operated
continuously.

Photo bioreactors: The photosynthetic
organisms (Cyanobacteria, Microalgae)
require light for the production of
important products (e.g. βCarotene,
single cell protein and astaxanthin) and
bioreactors designed for them are called
photo bioreactors. In addition to light,
the cells require CO2 which can be
provided by dissolving bicarbonate.
These bioreactors comprise an array of
transparent tubes (glass or clear plastic
tubes), that are placed horizontally or
vertically. The array of tubes or flat
panels constitute light receiving system.
The culture is circulated through the
light receiving system solar receivers by
centrifugal pumps or airlift pumps. The
light penetration depends on the density
of the biomass and the temperature is
maintained at 22-370C.

Phases of microbial growth: When an organism is introduced into a growth medium
(inoculation process). Growth of the inoculum does not occur immediately, but takes a
little while. This is the period of adaptation, called the lag phase. Following the lag phase,
the rate of growth of the organism steadily increases, for a certain period--this period is the
log or exponential phase. After a certain time of exponential phase, the rate of growth
slows down, due to the continuously falling concentrations of nutrients and/or a
continuously increasing (accumulating) concentrations of toxic substances. This phase,
where the increase of the rate of growth is checked, is the deceleration phase. After the
deceleration phase, growth ceases and the culture enter a stationary phase or steady state.
The Bacterial Growth Curve: In the laboratory, under favorable conditions, a growing
bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2,
4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of generations). This is called
exponential growth. In reality, exponential growth is only part of the bacterial life cycle,
and not representative of the normal pattern of growth of bacteria in Nature. When a fresh
medium is inoculated with a given number of cells, and the population growth is
monitored over a period of time, plotting the data will yield a typical bacterial growth
curve .

1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the
population remains temporarily unchanged. Although there is no apparent cell division
occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins,
RNA, etc., and increasing in metabolic activity. The length of the lag phase depends on a
wide variety of factors including the size of the inoculum; time necessary to recover from
physical damage or shock in the transfer; time required for synthesis of essential
coenzymes or division factors; and time required for synthesis of new (inducible)
enzymes that are necessary to metabolize the substrates present in the medium.

2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced
growth wherein all the cells are dividing regularly by binary fission, and are growing by
geometric progression. The cells divide at a constant rate depending upon the composition
of the growth medium and the conditions of incubation. The rate of exponential growth of
a bacterial culture is expressed as generation time, also the doubling time of the bacterial
population.
Generation time (G) is defined as the time (t) per generation (n = number of generations).
Hence, G=t/n is the equation from which calculations of generation time (below) derive.
3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture.
Population growth is limited by one of three factors:
1. exhaustion of available nutrients;
2. accumulation of inhibitory metabolites or end products;
3. exhaustion of space, in this case called a lack of "biological space".

During the stationary phase, if viable cells are being counted, it cannot be determined
whether some cells are dying and an equal number of cells are dividing, or the
population of cells has simply stopped growing and dividing. The stationary phase, like
the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary
metabolites, such as antibiotics, do so during the stationary phase of the growth cycle
(Secondary metabolites are defined as metabolites produced after the active stage of
growth). It is during the stationary phase that spore-forming bacteria have to induce or
unmask the activity of dozens of genes that may be involved in sporulation process.
4. Death Phase. If incubation continues after the population reaches stationary phase, a
death phase follows, in which the viable cell population declines. (Note, if counting by
turbidimetric measurements or microscopic counts, the death phase cannot be
observed.). During the death phase, the number of viable cells decreases geometrically
(exponentially), essentially the reverse of growth during the log phase.

Types of Fermentations and culture systems
1. Solid substrate (solid state) fermentation (SSF): It is a microbial process in which a solid
material is used as the substrate or the inert support of microorganisms growing on it. Although
SSF was developed for the manufacturing of traditional foods and alcoholic beverages, its
application has been extended to the pharmaceutical and biochemical industries. The most
commonly used solid substrates for SSF are cereal grains, wheat bran, saw dust, wood shavings
and several other plant and animal materials. Fungi and actinomycetes are the best suited for SSF,
because of their larger biomass and reach by means of hyphae. SSF techniques are also being used
for the production of high-value products such as enzymes and toxins. Most enzymes from fungi
are now being cheaply produced through SSF. More recently, this approach has been used for the
production of extracellular enzymes, certain valuable chemicals, fungal toxins, and fungal spores.

In solid substrate fermentation, the microbial distribution occurs on the solid surface, and
microbial growth and product formation also occur mainly on the surface. The moisture
content required for SSF is normally low, depending on the physical or chemical
characteristics of the substrate. Heat derived from the metabolism and growth of the
microorganism raises the temperature of the solid substrate bed and causes the loss of
moisture. The air supply and temperature of the solid substrate bed is controlled by forced
aeration, in which the large surface area of the solid substrate promotes heat transfer and
gas exchange of oxygen and carbon dioxide. The bioreactors used in SSF are tray
fermenters without any agitation, drum fermenters with continuous or staggered slow
agitation, and column fermenters with forced aeration. The tray type fermenters consist of
wooden, metallic (aluminum, iron), or plastic trays with perforated bottom. The trays are
sterilized and filled with a layer of substrate mixed with inoculum. The trays are stacked
one above the other to a convenient height. A humid atmosphere is created inside the
chamber, and the temperature is controlled by cool or warm air. After fermentation
process is completed, the trays are removed and the fermented mash is pooled for
downstream processing for product recovery. he column fermenter is a glass or plastic
column, with a jacket for water circulation and usually aerated through forced air and it is
more expensive.

2. Submerged fermentation: this is of following type
A. Batch processing or culture: 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.
B. Continuous processing or culture: 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.

C. Fed-batch culture or processing: 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. The
production of penicillin, a secondary metabolite, is also by fed-batch method. Penicillin process
has two stages: an initial growth phase followed by the production phase called the 'idiophase'.
The culture is maintained at low levels of biomass and phenyl acetic acid, the precursor of
penicillin, is fed into the fermenter continuously, but at a low rate, as the precursor is toxic to the
organism at higher concentrations.

3. Anaerobic Fermentation: Anaerobic microorganisms do not utilize molecular oxygen
in biosynthesis and are not capable of using oxygen as a terminal electron acceptor.
Instead, they use diverse array of organic and inorganic electron donors and acceptors in
their energy metabolism. In anaerobic fermentations, the production of biomass is less
than aerobic organisms. With less biomass production, more carbon can be converted to
end-products and the product yield is higher. Anaerobes can use a variety of substrates
including polysaccharides, molasses, sugars and other complex substrates which may be
obtained from agricultural waste streams and reduce the overall cost of the fermentation
process. Many anaerobes grow at high temperatures at which oxygen is poorly soluble
and growth under these conditions can contribute to efficient product recovery. Anaerobic
organisms of industrial importance include a wide range of genera in the domains of
bacteria and archaea. The genus Clostridium is important medically and for the
production of metabolites including enzymes and solvents.

4. Aerobic Fermentation: If the growth of the fermentation microorganism is to occur
aerobically, then provision must be made for rapid incorporation of sterile air into the
medium in such a manner that the oxygen of this air is dissolved in the medium and,
therefore, readily available to the microorganism, and the carbon dioxide resulting from
microbial metabolism is largely flushed from the medium. Usually, the sterile air is
supplied to the fermenter. The fermenters mostly employed in aerobic fermentations
include stirred tank type and air lift type. The fermenter should provide aseptic means for
the withdrawal of culture samples during the fermentation as well as for the introduction
of inoculum at the initiation of the fermentation.

Industrial fermentation: industrial fermentation is considered as the breakdown of
organic substances and reassembly into other substances. Fermenter culture in
industrial capacity often requires highly oxygenated and aerobic growth conditions,
whereas fermentation in the biochemical context is a strictly anaerobic process.
There are 5 major groups of commercially important fermentation:
1. Microbial cells or biomass as the product, e.g. baker’s yeast, lactobacillus, etc.
2. Microbial enzymes: catalase, amylase, protease, pectinase, glucose isomerase,
cellulase, lipase, lactase, etc.
3. Microbial metabolites:
A. Primary metabolites – ethanol, citric acid, glutamic acid, lysine, vitamins,
polysaccharides etc.
B. Secondary metabolites: all antibiotic fermentation.
4. Recombinant products: insulin, HBV, interferon, streptokinase.
5. Biotransformation’s: phenyl acetyl carbinol, steroid biotransformation, etc.

Industrial fermentation of antibiotics
Downstream Processing
• Products in a fermenter are impure and dilute, so need to be purified by
downstream processing.
• This usually involves filtration to separate the microbial cells from the liquid
medium, followed by chemical purification and concentration of the product.
• Downstream processing can account for 50% of the cost of a process.
• Antibiotics are antimicrobial agents produced naturally by other microbes (usually
fungi or bacteria).
• The first antibiotic was discovered in 1896 by Ernest Duchesne and
"rediscovered" by Alexander Flemming in 1928 from the filamentous fungus
Penicilium notatum.
• Penicillin was the first important commercial product produced by an aerobic,
submerged fermentation

• When penicillin was first made at the end of the second world war using the
fungus Penicilium notatum, the process made 1 mg dm3.
• Today, using a different species (P. chrysogenum) and a better extraction
procedures the yield is 50 g dm3. Penicillin is produced by the fungus Penicillium
chrysogenum which requires lactose, other sugars, and a source of nitrogen (in this
case a yeast extract) in the medium to grow well.
• There is a constant search to improve the yield.
• Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the
stationary phase.
fermenter require
It requires a batch fermenter, and
a fed batch process is normally
used to prolong the stationary
period and so increase production.

Downstream processing is relatively easy since penicillin is secreted into the
medium (to kill other cells), so there is no need to break open the fungal cells.
However, the product needs to be very pure, since it being used as a therapeutic
medical drug, so it is dissolved and then precipitated as a potassium salt to separate
it from other substances in the medium.

The resulting penicillin (called
penicillin G) can be chemically
and enzymatically modified to
make a variety of penicillin's
with slightly different properties.
These semi-synthetic penicillin's
include penicillin V, penicillin O,
ampicillin and amoxicillin.

Enzymes production
Microbial enzymes are widely used in different industries mainly because of vast
availability of sources. Microbial enzymes could be genetically modified and are
considered as economical in comparison to plant and animal enzymes. Production of
microbial enzymes by application of fermentation procedures involves microbial
propagation like bacteria, mold and yeast to get desired product. The improvement
in concentration, purity and percentage of recovery of enzymes can be achieved
based on standard principles which are microbial sources, improvement of strain
and application of membrane augmented downstream processing method to improve
specific activity of enzyme. There are two methods of fermentation used to produce
enzymes. These are submerged fermentation and solid-state fermentation. Carbon
containing compounds in or on the substrate are broken down by the
microorganisms, which produce the enzymes either intracellular or extracellular.
Industries that use enzymes generated by fermentation are the brewing, wine
making, baking, cheese making, dairy, milling, beverages, and cereals.

1. Solid substrate cultivation : Adopted for the extraction of enzymes from fungi,
such as Penicillum, aspergillus sp.,.
• This is also called Tray cultivation or thin layer culture or Koji fermentation.
• Microbes are culture in a tray which are 2x 40 cm in size.
• Trays are made up of wood or metal.
• Trays are filled with raw materials/ nutrients (1/4th of the depth).
• Organisms are inoculated on the solid medium and incubated on the solid medium.
Incubated at an air-conditioned room or in incubator for few days.
• Dense mat of microbes is formed over the surface of solid medium.
• Mat is then separated and used for the extraction of enzymes.
2. Deep-Bad cultivation: microbes are cultivated in Rectangular culture vessels, with
the size of 18x200’’.
• Wheat bran, rice bran, potato flakes etc., are used as substrate.
• Any one substrate is added into vessel upto 2’’ hight.
• Microbial inoculum poured over medium

Type of medium would you use to stimulate a microbe to synthesize a protease
I. A medium with proteins but no amino acids is used.
Microbes are encouraged into the log phase initially with a medium with a lot of protein
This encourages rapid increase in the number of cells, but not much protease is produced.
Cells are then introduced into the fermentation vessel and allowed to grow for a further 1-8
days.
Micro-organisms are suitable for use in the large scale production of enzymes in fermenters
because:
•They have rapid growth rates and are able to produce larger numbers of enzyme molecules
per body mass than many other organisms
• Micro-organisms can be genetically engineered to improve the strain and enhance yields
• Micro-organisms are found in a wide variety of different habitats such that their enzymes
are able to function across a range of temperatures and pH
• Micro-organisms have simple growth requirements and these can be precisely controlled
within the fermenter
• Micro-organisms can utilise waste products such as agricultural waste as substrates

The Biotechnological Process of Enzyme Production

Down stream processing
The remaining mixture contains enzymes, waste materials, nutrients and cells . The
enzyme is extracted by downstream processing.
The nature of the downstream processing depends on two considerations:
Whether enzyme is intracellular or extracellular.
How pure the final product needs to be.
Industrial enzymes can be quite crude, but medicinal enzymes must be extremely pure.
The purer the enzyme, the more complex the downstream processing, and the more
expensive it is.

Microbial Production of Vitamins
• Vitamins are organic compounds that perform specific biological functions for normal
maintenance and optimal growth of an organism. These vitamins cannot be synthesized
by the higher organisms, including man, and therefore they have to be supplied in small
amounts in the diet.
• Microorganisms are capable of synthesizing the vitamins. In fact, the bacteria in the gut
of humans can produce some of the vitamins, which if appropriately absorbed can
partially meet the body’s requirements. It is an accepted fact that after administration of
strong antibiotics to humans (which kill bacteria in gut), additional consumption of
vitamins is recommended.
• Microorganisms can be successfully used for the commercial production of many of the
vitamins e.g. thiamine, riboflavin, pyridoxine, folic acid, pantothenic acid, biotin,
vitamin B12, ascorbic acid, P-carotene (pro-vitamin A), ergosterol (pro-vitamin D).
However, from economic point of view, it is feasible to produce vitamin B12, riboflavin,
ascorbic acid and p-carotene by microorganisms.

Single Cell Protein Production Processes
The production of single cell protein takes place in fermentation. This is done by
selected strains of microorganisms which are multiplied on suitable raw materials in
technical cultivation process directed to the growth of the culture and the cell mass
followed by separation processes.
Process development begins with microbial screening, in which suitable production
strains are obtained from samples of soil, water, air or from swabs of inorganic or
biological materials and are subsequently optimized by selection, mutation, or other
genetic methods. Then the technical conditions of cultivation for the optimized
strains are done and all metabolic pathways and cell structures will be determined.
Besides, process engineering and apparatus technology adapt the technical
performance of the process in order to make the production ready for use on the
large technical scale. Here is where the economic factors (energy, cost) come into
play. Safety demands and environmental protection is also considered in the
production of SCP in relation both to the process and to the product. Finally, safety
and the protection of innovation throw up legal and controlled aspects, namely
operating licenses, product authorizations for particular applications and the legal
protection of new process and strains of microorganisms.

Submerged fermentation
In submerged process , the substrate used for fermentation is always in liquid state which
contains the nutrients needed for growth.
The fermentor which contains the substrate is operated continuously and the product
biomass is continuously harvested from the fermentor by using different techniques then
the product is filtered or centrifuged and then dried.
Aeration is an important operation in the cultivation, heat is generated during cultivation
and it is removed by using a cooling device. The microbial biomass can be harvested by
various methods. Single cell organisms like yeast and bacteria are recovered by
centrifugation while filamentous fungi are recovered by filtration. It is important to
recover as much water as possible prior to final drying done under clean and hygienic
conditions.

Semisolid fermentation
In semisolid fermentation, the preparation of the substrate is not cleared and it is also
more used in solid state e.g. cassava waste.
Submerged culture fermentations require more capital investment and have high
operating cost.
The cultivation involves many operations which include stirring and mixing of a
multiphase system, transport of oxygen from the gas bubbles through the liquid phase to
the microorganisms and the process of heat transfers from liquid phase to the
surroundings. A special bioreactor is designed for identifying mass and energy
transportation phenomena, called U-loop fermentor.
Production of single cell protein involves basic steps of preparation of suitable
medium with suitable carbon source, prevention of the contamination of medium and the
fermentor, production of microorganisms with desired properties and separation of
synthesized biomass and its processing. Carbon source used can be n-alkenes, gaseous
hydrocarbons, methanol and ethanol, renewable sources like carbon oxide molasses,
polysaccharides, effluents of breweries and other solid substances.

Solid state fermentation
Solid state fermentation (SSF) has been extensively studied for the production of various
value added products like SCP, feeds, enzymes, ethanol, organic acids, Bcomplex
vitamins, pigments, flavours.
This process consists of depositing a solid culture substrate, such as rice or wheat bran,
on flatbeds after seeding it with microorganisms; the substrate is then left in a
temperature-controlled room for several days.
Accurately managing the synthesis of the desired metabolites requires regulating
temperature, soluble oxygen, ionic strength and pH and control nutrients

The main steps involved are: medium preparation, fermentation, separation and
downstream processing.
A simple common flow diagram of SCP production by fermentation has been shown in
the Figure.

References
1. Amann, R.I., Ludwig, W. & Schleifer, K.H. Phylogenetic identification and in situ
detection of individual microbial cells without cultivation. Microbial,
(1995);Torsvik, V., Goksoyr, J. & Daae, F.L. High diversity in DNA of soil
bacteria. Appl. Environ. Microbiol, (1990).
2. Robinson, P.K., Enzymes: principles and biotechnological applications. Essays in
biochemistry, 2015. 59: p. 1-41.
3. Fermentation Microbiology and Biotechnology By EMT Mansi et al.
4. Industrial Microbiology : An Introduction By MJ Waites.
5. Industrial Microbiology By HS Patel.
6. Food and Industrial Microbiology By Raveendra Reddy.

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