Fermentor

A fermentor (bioreactor) is a closed vessel with adequate arrangement for aeration, agitation, temperature and pH
control, and drain or overflow vent to remove the waste biomass of cultured microorganisms along-with their
products. A fermentor is used for commercial production in fermentation industries and is a device in which a
substrate of low value is utilized by living cells or enzymes to generate a product of higher value. Fermentors are
extensively used for food processing, fermentation, waste treatment, etc.

De Beeze and Liebmann (1944) used the first large scale (above 20 litre capacity) fermentor for the production of
yeast. But it was during the First World War, a British scientist named Chain Weizmann (1914-1918) developed a
fermentor for the production of acetone. Since importance of aseptic conditions was recognised, hence steps were
taken to design-and construct piping, joints and valves in which sterile conditions could be achieved and manufactured
when required.

Design of typical fermentor

All bioreactors deal with heterogeneous systems dealing with 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. Chemical engineering principles are employed for design and operation of bioreactors.
A bioreactor should provide for the following:
(i) Agitation (for mixing of cells and medium),
(ii) Aeration (aerobic fermentors); for O2 supply,
(iii) Regulation of factors like temperature, pH, pressure, aeration, nutrient feeding, liquid level etc.,
(iv) Sterilization and maintenance of sterility, and
(v) Withdrawal of cells/medium (for continuous fermentors).
Modern fermentors are usually integrated with computers for efficient process monitoring, data acquisition, etc.

Generally, 20-25% of fermentor volume is left unfilled with medium as “head space” to allow for splashing, foaming
and aeration. The fermentor design varies greatly depending on the type and the fermentation for which it is used.
Bioreactors are so designed that they provide the best possible growth and biosynthesis for industrially important
cultures and allow ease of manipulation for all operations.

Size of Fermentors:
The size of fermentors ranges from 1-2 litre laboratory fementors to 5, 00,000 litre or, occasionally, even more,
fermentors of upto 1.2 million litres have been used. The size of the fermentor used depends on the process and how it
is operated.

Construction of Fermentors:
Industrial fermentors can be divided into two major classes, anaerobic and aerobic. Anaerobic fermentors require little
special equipment except for removal of heat generated during the fermentation process, whereas aerobic fermentors
require much more elaborate equipment to ensure that mixing and adequate aeration are achieved.
Since most industrial fermentation processes are aerobic, the construction of a typical aerobic fermentor is as follows:

1. Cooling Jacket:
Large-scale industrial fermentors are almost always constructed of stainless steel. A fermentor is a large cylinder
closed at the top and the bottom and various pipes and valves are fitted into it. The fermentor is fitted externally with a
cooling jacket through which steam (for sterilization) or cooling water (for cooling) is run.
Cooling jacket is necessary because sterilization of the nutrient medium and removal of the heat generated are
obligatory for successful completion of the fermentation in the fermentor. For very large fermentors, insufficient heat
transfer takes place through the jacket and therefore, internal coils are provided through which either steam or cooling
water is run.
2. Aeration System:
Aeration system is one of the most critical part of a fermentor. In a fermentor with a high microbial population
density, there is a tremendous oxygen demand by the culture, but oxygen being poorly soluble in water hardly
transfers rapidly throughout the growth medium.
It is necessary, therefore, that elaborate precautions are taken using a good aeration system to ensure proper aeration
or oxygen availability throughout the culture. However, two separate aeration devices are used to ensure proper
aeration in fermentor. These devices are sparger and impeller.

The sparger is typically just a series of holes in a metal ring or a nozzle through which filter-sterilized air (or oxygen-
enriched air) passes into the fermentor under high pressure. The air enters the fermentor as a series of tiny bubbles
from which the oxygen passes by diffusion into the liquid culture medium.

The impeller (also called agitator) is an agitating device necessary for stirring of the fermenter.

The stirring accomplishes two things:

(i) It mixes the gas bubbles through the liquid culture medium and
(ii) It mixes the microbial cells through the liquid culture medium. In this way, the stirring ensures uniform access of
microbial cells to the nutrients.
Ideally, the impeller should be 1/3 of the fermentors diameter fitted above the base of the fermentor. The number of
impeller may vary from size to size to the fermentor.
3. Baffles:
The baffles are normally incorporated into fermentors of all sizes to prevent a vortex and to improve aeration
efficiency. They are metal strips roughly one-tenth of the fermentors diameter and attached radially to the walls.

4. Controlling Devices for Environmental Factors:

In any microbial fermentation, it is necessary not only to measure growth and product formation but also to control the
process by altering environmental parameters as the process proceeds. For this purpose, various devices are used in a
fermentor. Environmental factors that are frequently controlled includes temperature, oxygen concentration, pH, cells
mass, levels of key nutrients, and product concentration.

Use of Computer in Fermentor:

Computer technology has produced a remarkable impact in fermentation work in recent years and the computers are
used to model fermentation processes in industrial fermentors. Integration of computers into fermentation systems is
based on the computers capacity for process monitoring, data acquisition, data storage, and error-detection.
Some typical, on-line data analysis functions include the acquisition measurements, verification of data, filtering, unit
conversion, calculations of indirect measurements, and differential integration calculations of estimated variables, data
reduction, and tabulation of results, graphical presentation of results, process stimulation and storage of data.

Fermentation process
Fermentation systems may be liquid, also known as submerged or solid state, also known as surface. Most fermentors
used in industry are of the submerged type, because the submerged fermentor saves space and is more amenable to
engineering control and design.

1. Submerged Liquid Fermentations

Submerged liquid fermentations are traditionally used for the production of microbially derived enzymes. Submerged
fermentation involves submersion of the microorganism in an aqueous solution containing all the nutrients needed for
growth.

Fermentation takes place in large vessels (fermenter) with volumes of up to 1,000 cubic metres. Most industrial
enzymes are secreted by microorganisms into the fermentation medium in order to break down the carbon and
nitrogen sources. Batch-fed and continuous fermentation processes are common. In the batch-fed process, sterilized
nutrients are added to the fermenter during the growth of the biomass. In the continuous process, sterilized liquid
nutrients are fed into the fermenter at the same flow rate as the fermentation broth leaving the system. Parameters like
temperature, pH, oxygen consumption and carbon dioxide formation are measured and controlled to optimize the
fermentation process.

Next in harvesting enzymes from the fermentation medium one must remove insoluble products, e.g. microbial cells.
This is normally done by centrifugation. As most industrial enzymes are extracellular (secreted by cells into the
external environment), they remain in the fermented broth after the biomass has been removed. The enzymes in the
remaining broth are then concentrated by evaporation, membrane filtration or crystallization depending on their
intended application. If pure enzyme preparations are required, they are usually isolated by gel or ion exchange
chromatography.

Several types of submerged type of fermentors are known and they may be grouped in several ways:. The most
commonly used type of fermentor is the Aerated Stirred Tank Batch Fermentor.

Aerated stirred tank batch fermentor

A typical fermentor of this type is an upright closed cylindrical tank fitted with one or more baffles attached to the
side of the wall, a water jacket or coil for heating and/ or cooling, a device for forcible aeration (known as sparger), a
mechanical agitator usually carrying a pair or more impellers, means of introducing organisms and nutrients and of
taking samples, and outlets for exhaust gases. Modern fermentors are highly automated and usually have means of
continuously monitoring, controlling or recording pH, oxidation-reduction potential, dissolved oxygen, effluent O2
and CO2, and chemical components.

Advantages of Submerged fermentation:

• Lower total investment costs;
• Improved process control;
• Reduced fermentation time;
• Reduced floor space requirements;
• Lower labour costs;
• Simpler operations; and
• Easier maintenance of aseptic conditions on an industrial scale.
Disadvantages of submerged fermentation:
 Expenses for equipment are higher
 Consumption of electrical energy is higher;
 The process is very sensitive to short interruptions or breakdowns in aeration and vulnerable to infections,
which result not only in losses of yield, but also in a total breakdown of respective batches

2. Surface fermentation:
In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These
techniques are very complicated and rarely used in industry. A. niger forms a mycelium layer on the liquid surface of
the aluminum or stainless steel trays. These trays are stacked in fermentation rooms supplied with filtered air which
serves both to supply oxygen and to control the temperature of fermentation. Surface fermentation is easy to control
and to implement. It needs no aeration or agitation of the fermentation broth, so it needs no instrumentation for
aeration and agitation. The separation of citric acid from the mycelium is easy because the microorganism is not
dispersing into the medium. Only the temperature and humidity of the fermentation chamber need controlling. It can
be used easily in small plants as well as in third world countries.

With surface fermentation, the fermentation broth is concentrated due to a high evaporation rate during fermentation.
Thus, expenses and losses during recovery and purification are low. However, surface fermentation has the following
disadvantages: Building investment costs are high. Personnel expenses are high in developed industrial countries with
extremely high wages. Fermentation time is long and therefore productivity is low.

3. Solid state fermentation

Solid-state fermentation (SSF) is defined as the fermentation process in which microorganisms grow on solid
materials without the presence of free liquid. The SSF is alternative to submerged fermentation for production of value
added products like antibiotics, single cell protein, Poly unsaturated fatty acids, enzymes, organic acids, biopesticides,
biofuel and aroma production.

The aim of SSF is to bring cultivated fungi or bacteria in tight contact with the insoluble substrate and to achieve the
highest nutrient concentration from the substrate for fermentation.

Solid substrates generally provide a good dwelling environment to the microbial flora comprising bacteria, yeast and
fungi. Among these, filamentous fungi are the best studied for SSF due to their hyphal growth, which have the
capability to not only grow on the surface of the substrate particles but also penetrate through them. Several agro crops
such as cassava, barley, etc. and agro-industrial residues such as wheat bran, rice bran, sugarcane bagasse, cassava
bagasse, various oil cakes (e.g. coconut oil cake, palm kernel cake, soybean cake, ground nut oil cake, etc), fruit pulps
(e.g. apple pomace), corn cobs, saw dust, seeds (e.g. tamarind, jack fruit), coffee husk and coffee pulp, tea waste,
spent brewing grains, etc are the most often and commonly used substrates for SSF processes.

Solid substrate (matrix), must contain enough moisture. Depending upon the nature of the substrate, the amount of
water absorbed could be one or several times more than its dry weight, which leads relatively high water activity (aw)
on the solid/gas interface in order to allow higher rate of biochemical process. Low diffusion of nutrients and
metabolites takes place in lower water activity conditions whereas compaction of substrate occurs at higher water
activity. Hence, maintenance of adequate moisture level in the solid matrix along with suitable water activity is
essential elements for SSF processes.

Solid substrates should have generally large surface area per unit volume. Smaller substrate particles provide larger
surface area for microbial attack but pose difficulty in aeration/respiration due to limitation in inter-particle space
availability. Larger particles provide better aeration/respiration opportunities but provide lesser surface area.

The majority are aerobic fermentations, requiring aeration and occasional or continuous agitation. Bioreactors
commonly used include the following.
1. Rotating drum fermenters, comprising a cylindrical vessel of around 100 L capacity mounted on its side onto
rollers that both support and rotate the vessel. These fermenters are used in enzyme and microbial biomass
production. Their main disadvantage is that the drum is filled to only 30% capacity, otherwise mixing is
inefficient.

2. Tray fermenters, which are used extensively for the production of fermented oriental foods and enzymes.
Their substrates are spread onto each tray to a depth of only a few centimetres and then stacked in a chamber
through which humidified air is circulated. These systems require numerous trays and large volume incubation
chambers of up to 150m3 capacity.
After the required time of incubation in khoji, product recovery is done either by drying the product in dryer
and used whole product as such, or it can be further solvent extracted for purified enzymes.

3. Bed systems, as used in commercial koji production, consisting of a bed of substrate up to 1m deep, through
which humidified air is continuously forced from below. And the product recovery is done similar way as
explained above.
Advantages of Solid State Fermentation
 Usually simpler with lower energy requirements and higher volumetric productivity
 Provides the natural habitat of some fungi and bacteria
 Might be easier to meet aeration requirements
 Easier downstream processing
 The fungal hyphae are bathed in a liquid medium and do not run the risk of desiccation;
 Temperature control is typically not overly difficult, such that the organism is exposed to a constant
temperature throughout its growth cycle;
 The availability of O2 to the biomass can be controlled reasonably well at a particular level of saturation of
the medium
 The availability of the nutrients to the organism can be controlled within relatively narrow limits if desired,
through the feeding of nutrient solutions.
 pH control is relatively easy to provide.
TYPES OF FERMENTATION PROCESSES
There are three types of fermentation processes and they are
1. Batch fermentation
2. Fed batch fermentation
3. Continuous fermentation
1. Batch Fermentation:
Batch fermentation is a closed culture system, because initial and limited amount of sterilized nutrient medium is
introduced into the fermenter. The medium is inoculated with a suitable microorganism and incubated for a definite
period for fermentation to proceed under optimal physiological conditions. Oxygen in the form of air, an antifoam
agent and acid or base, to control the pH, are being added during the course of fermentation process.

During the course of incubation, the cells of the microorganism undergo multiplication and pass through different
phases of growth and metabolism due to which there will be change in the composition of culture medium, the
biomass and metabolites. The fermentation is run for a definite period or until the nutrients are exhausted. The culture
broth is harvested and the product is separated.

Batch fermentation may be used to produce biomass, primary metabolites and secondary metabolites under cultural
conditions supporting the fastest growth rate and maximum growth would be used for biomass production. The
exponential phase of growth should be prolonged to get optimum yield of primary metabolite, while it should be
reduced to get optimum yield of secondary metabolites.

The used medium along with cells of microorganism and the product is drawn out from the fermenter. When the
desired product is formed in optimum quantities, the product is separated from the microorganism and purified later
on.

It has both advantages and disadvantages which are detailed below:

Merits:
(a) The possibility of contamination and mutation is very less.
(b) Simplicity of operation and reduced risk of contamination.

Demerits:
(a) For every fermentation process, the fermenter and other equipment are to be cleaned and sterilized.
(b) Only fraction of each batch fermentation cycle is productive.
(c) It is useful in fermentation with high yield per unit substratum and cultures that can tolerate initial high substrate
concentration.
(d) It can be run in repeated mode with small portion of the previous batch left in the fermenter for inoculum.
(e) Use of fermenter is increased by eliminating turn round time or down time.
(f) Running costs are greater for preparing and maintaining stock cultures.
(g) Increased, frequency of sterilization may also cause greater stress on instrumentation and probes.
(h) Fresh sterilized medium and pure culture are to be made for every fermentation process.
(i) Yield of the desired product may also vary.
(j) There will be a non-productive period of shutdown between one batch productive fermentation to the other,
(k) More personal are required.

2. Continuous Fermentation:
It is a closed system of fermentation, run for indefinite period. In this method, fresh nutrient medium is added
continuously or intermittently to the fermenter and equivalent amount of used medium with microorganisms is
withdrawn continuously or intermittently for the recovery of cells or fermentation products.

As a result, volume of the medium and concentration of nutrients at optimum level are being maintained. This has
been operated in an automatic manner. The continuous fermenter has their maximum uses that take long time to reach
high productivity, reduces down time and lowers the operating costs.

In continuous mode, starting medium and inoculum are added to the fermenter. After the culture is grown the
fermenter is fed with nutrients and broth is withdrawn at the same rate maintaining a constant volume of broth in the
fermenter. In continuous mode with cell cycle, the cell mass is returned to the fermenter using micro filtrations with
bacteria or screens with fungal mycelium.

A continuous fermentation is generally carried out in the following ways:

(a) Single stage fermentation
(b) Recycle fermentation
(c) Multiple stage fermentation

(a) Single Stage Fermentation: In this process, a single fermenter is inoculated and the nutrient medium and culture
are kept in continuous operation by balancing the input and output of nutrient medium and harvested culture,
respectively.

(b) Recycle Fermentation: In this method, a portion of the medium is withdrawn and added to the culture vessel.
Thus, the culture is recycled to the fermentation vessel. This method is generally adopted in the hydrocarbon
fermentation process. The recycling of cells provides a higher population of cells in the fermenter which results in
greater productivity of the desired product.

(c) Multiple Stage Fermentation: In this process, two or more fermenters are employed simultaneously and the
fermentation is operated in a sequence. Different phases of fermentation process like growth phase and synthetic phase
are carried out in different fermenters. Generally, growth phase is allowed in the first fermenter, synthetic phase in the
second and subsequent fermenters.
This process is adapted particularly to those fermentations in which growth and synthetic activities of the
microorganisms are not simultaneous. Synthesis is not growth related but occurs when cell multiplication rate has
slowed down.
The process of continuous fermentation is monitored either by microbial growth activity or by product formation and
these methods are called:

(i) Turbidostat method, and

(ii) Chemostat method.

(i) Turbidostat Method: In this method the total cell content is kept constant by measuring the culture turbidity at a
regular interval of fermentation process. By turbidity measurement it is possible to the fermenter to regulate both the
nutrient feed rate and the culture withdrawal rate.
Fermentation, in which this method is employed, must be carried out at a low maximum cell population which leads to
the usage of less amount of substrate and wastage of greater amount of substrate as unused and residual medium,
which is removed from the fermenter along with the harvested culture.

(ii) Chemostat Method: In this method nutrient feed rate and harvest culture withdrawal rate are maintained at
constant value. This is achieved by controlling the growth rate of the microorganism by adjusting the concentration of
any one of the chemicals of the medium, like carbon source, nitrogen source, salts, O2 etc. which acts as a growth
limiting factor.

Apart from the above chemicals, sometimes the concentration of the toxic product generated in the fermentation
process, the pH values and even temperature also act as growth limiting factors. This method is employed more often
than turbidostat method because of fewer mechanical problems and presence of less amount of unused medium in the
harvested culture
 However, continuous fermentations have certain advantages and limitations which are as follows:
Merits:
1. The fermenter is continuously used with little or no shutdown time.
2. Only little quantity of initial inoculum is needed and there is no need of additional inoculum.
3. It facilitates maximum and continuous production of the desired product.
4. There is optimum utilization of even slow utilizable substances like hydrocarbons.

Demerits:
1. Possibility of contamination and mutation because of prolonged incubation and continuous fermentation, are more.
2. Possibility of wastage of nutrient medium because of continuous withdrawal for product isolation.
3. The process becomes more complex and difficult to accomplish when the desired products are antibiotics rather
than a microbial cells.
4. Lack of knowledge of dynamic aspects of growth and synthesis of product by microorganism used in fermentation.

3. Fed Batch Fermentation:

It is a modification to the batch fermentation. In this process substrate is added periodically in instalments as the
fermentation progresses, due to which the substratum is always at an optimal concentration. This is essential as some
secondary metabolites are subjected to catabolite repression by high concentration of either glucose, or other
carbohydrate or nitrogen compounds present in the medium.

For this reason, the critical elements of the nutrient medium are added in low amount in the beginning of the
fermentation and these substrates continue to be added in small doses during the production phase. This method is
generally employed for the production of substances such as penicillin. Yoshida (1973) introduced this term for the
first time for feeding the substrates to the medium as the nutrients are exhausted, so as to maintain the nutrients at an
optimum level.

The fed-batch fermentation may be of three types:

(i) Variable Volume Fed Batch Culture: The same medium is added resulting in an increase in volume.

(ii) Fixed Volume Fed Batch Culture: A very concentrated solution of the limiting substrate is added at a very little
amount resulting in an insignificant increase in the volume of medium.

(iii) Cyclic Fed Batch Culture: As it is not possible to measure the substrate concentration by following direct
methods during fermentation, which is necessary for controlling the feeding process, generally indirect methods are
employed. For example – in the production of organic acids, the pH value may be used to determine the rate of
glucose utilization.

Advantages:
1. Production of high cell densities due to extension of working time (particularly growth associated products).
2. Controlled conditions in the provision of substrates during fermentation, particularly regarding the concentration of
specific substrates for e.g. the carbon source.
3. Control over the production of, by products or catabolite repression, effects due to limited provision of substrates
solely required for product formation.
4. The mode of operation can overcome and control deviations in the organism’s growth pattern as found in batch
fermentation.
5. Allows the replacement of water loss, by evaporation.
6. Alternative mode of operation for fermentations dealing with toxic substances or low solubility compounds.
7. Increase of antibiotic marked plasmid stability by producing the correspondent antibiotic during the time span of the
fermentation.
8. No additional special piece of equipment is required as compared with the batch fermentation.
9. It is an effective method for the production of certain chemicals, which are produced at optimum level when the
medium is exhausted like penicillin.
Disadvantages:
1. It is not possible to measure the concentration of feeding substrate by following direct methods like
chromatography.
2. It requires precious analysis of the microorganism. Its requirements and the under-standing of its physiology with
productivity is essential.
3. It requires a substantial amount of operator skill for the set-up of fermentation and development of the process.
4. In a cyclic fed batch culture, care should be taken in the design of the process to ensure that toxins do not
accumulate to inhibitory levels and that nutrients other than those incorporated into the fed medium become limited
also, if many cycles are run. The accumulation of non-producing or low producing variants may result.
5. The quantities of components to control must be above the detection limits of the available measuring equipment.
Fed-batch with recycle of cells can also be used for specific purpose such as ethanol fermentation and waste water
treatment.
Downstream processing
Downstream processing is an essential part of bioprocess technology in that the desired product needs to be
isolated, purified and for different end uses. A variety of microorganisms including genetically engineered
species are used for the production of desired products. The products formed may be secreted into the broth
or may be retained within the cell introducing complexity in the recovery of the product.

In view of the complexity, downstream processing involves various techniques and methodologies.
Bi-products differ greatly in their nature hence different separation principles and mechanisms depending on
molecular mass, charge distribution, hydrophobicity, distribution coefficient, structure and
immunogenic structure and specific affinity towards other biomolecules becomes necessary for their
isolation and purification. The choice of the separation methodology depends to a large extent on the nature
of the product, its quantity and the extent of purity required.

Stages in downstream processing

Precipitation:
Precipitation is the most commonly used technique in industry for the concentration of macromolecules such as
proteins and polysaccharides. Further, precipitation technique can also be employed for the removal of certain
unwanted by-products e.g. nucleic acids, pigments.

Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic), besides alteration in temperature
and pH are used in precipitation. In addition to these non-specific protein precipitation reactions (i.e. the nature of the
protein is unimportant), there are some protein specific precipitations e.g., affinity precipitation, ligand precipitation.
Neutral salts:
The most commonly used salt is ammonium sulfate, since it is highly soluble, non¬toxic to proteins and low-priced.
Ammonium sulfate increases hydrophobic interactions between protein molecules that result in their precipitation. The
precipitation of proteins is dependent on several factors such as protein concentration, pH and temperature.

Organic solvents:
Ethanol, acetone and propanol are the commonly used organic solvents for protein precipitation. They reduce the
dielectric constant of the medium and enhance electrostatic interaction between protein molecules that lead to
precipitation. Since proteins are denatured by organic solvents, the precipitation process has to be carried out below
0°C.

Non-ionic polymers:
Polyethylene glycol (PEG) is a high molecular weight non-ionic polymer that can precipitate proteins. It reduces the
quantity of water available for protein solvation and precipitates protein. PEG does not denature proteins, besides
being non-toxic.

Ionic polymers:
The charged polymers such as polyacrylic acid and polyethyleneimine are used. They form complexes with oppositely
charged protein molecules that cause charge neutralisation and precipitation.

Increase in temperature:
The heat sensitive proteins can be precipitated by increasing the temperature.

Change in pH:
Alterations in pH can also lead to protein precipitation.

Affinity precipitation:
The affinity interaction (e.g., between antigen and antibody) is exploited for precipitation of proteins.

Precipitation by ligands:
Ligands with specific binding sites for proteins have been successfully used for selective precipitation.

Adsorption:
The biological products of fermentation can be concentrated by using solid adsorbent particles. In the early days,
activated charcoal was used as the adsorbent material. In recent years, cellulose-based adsorbents are employed for
protein concentration.
And for concentration of low molecular weight compounds (vitamins, antibiotics, peptides) polystyrene, methacrylate
and acrylate based matrices are used. The process of adsorption can be carried out by making a bed of adsorbent
column and passing the culture broth through it. The desired product, held by the adsorbent, can be eluted.

FILTRATION
Filtration is defined as the separation of solid in a slurry consisting of the solid and fluid by passing the
slurry through a septum called filter medium. Filtration is the most commonly used technique for separating
the biomass and culture filtrate. The efficiency of filtration depends on many factors— the size of the
organism, presence of other organisms, viscosity of the medium, and temperature. Several filters such as
depth filters, absolute filters, rotary drum vacuum filters and membrane filters are in use.

Types of filters
(i) Plate and frame filters: In this plates and frames are arranged alternately assembled on a horizontal
framework. Plates are covered with filter clothes and held together by hand screw to prevent leakage
between frames. The slurry is fed through the continuous channel by the holes in the corners of the plates
and frames. The filtrate passes through the filter cloth or pad, runs down grooves in the filter plates and is
then discharged through outlet taps to a channel.

(ii) Rotary vacuum filtration:

These filters are frequently used for separation of broth containing 10-40% solids (by volume) and particles
in the size of 0.5-10µm. Rotary drum vacuum filters have been successfully used for filtration of yeast cells
and filamentous fungi. The equipment is simple with low power consumption and is easy to operate.
The filtration unit consists of a rotating drum partially immersed in a tank of broth. Drum covered with
diatomaceous earth matter and allowed to rotate under vacuum with half immersed in the slurry tank. Small
amount of coagulation agent added to broth and pumped into the slurry tank. As the drum rotates, it picks up
the biomass which gets deposited as a cake on the drum surface. This filter cake can be easily removed.

As the cake portion in the drum comes to the upper region which is not immersed in the liquid it is washed
with water and dewatered immediately by blowing air over it. Then before the dried portion is again
immersed into the liquid it is cut off from drum by knife. The mechanism of cake discharge is achieved by
three ways.

(iii) Micro or Ultra Filtration: In this type of filtration, membranes with specific pore sizes can be used.
However, clogging of filters is a major limitation. Filtration of suspended particles can be achieved by either
dead end (static) filtration or cross flow filtration.
(a) Dead end (static) filtration – Solution poured over the membrane and the filtrate is collected at the
bottom. On prolonged filtration pores become blocked which reduce the filtering capacity.
(b) Cross flow filtration – To prevent the blockage the solution is passed over the membrane. Cell
suspension enters laterally and flows over the membrane. The filtrate gets collected at the bottom whereas
the cells are pushed to the opposite end by the continuous flow of suspension which is sent out via an outlet
at the opposite end. The liquid is again passed through a tube which recycles back to the flow. Since the
cells do not block the pores the filtration process can be performed continuously.

There are 3 major types of filtrations based on the particle sizes and other characters. These are
1. Microfiltration,- Micro filters are available in materials such as ceramic and steel that can be aggressively cleaned
and sterilised.
2. Ultrafiltration - membranes are made of ceramic and steel,
3. Reverse osmosis.

Centrifugation:
Centrifugation is a common method used to separate cells from cultured broth. It employs centrifugal force to promote
accelerated settling of particles in a solid-liquid mixture. Separation is achieved by means of accelerated gravitational
force by rapid rotation.

Microorganisms and other cells from the fermented slurry can be removed by using centrifuge when filtration is not a
satisfactory separation method. The particle size that can be separated range from 0.1μm to 100 μm. Unlike the
centrifugation that is conveniently carried out in the laboratory scale, there are certain limitations for large scale
industrial centrifugation.

A. Tubular centrifuges: This is used to separate particle size of 0.1 - 200 μm. This is simple machine made of a
tube rotating between bearings at each end. The suspension enters at the bottom of the centrifuge and high
centrifugal forces act to separate the solids and liquids. The bulk of solids will adhere to the walls of the bowl,
while the liquids exit at the top of the centrifuge.
B. Disc centrifuge: This is a simplest design of disc stack separator in a closed bowl, containing the disc stack,
with an arrangement to collect residual solids at the outer part of the bowl, from where they have to be
 removed manually after stopping rotation. The solids are discharged from the bowl through nozzles, which are
always open.
C. Chamber bowl centrifuge or Multi-chamber centrifuge: This is basically a modification of tubular bowl
type of centrifuge. It consists of several chambers connected in such a way that the feed flows in a zigzag
fashion. There is a variation in the centrifugal force in different chambers. The force is much higher in the
periphery chambers, as a result smallest particles settle down in the outermost chamber.
D. Decanter centrifuge: This used for continuous handling of slurry. This sedimentation centrifuge is designed to
handle significant solid concentration in feed suspension. It consists of horizontal cylindrical bowl rotating at a
high speed, with a helical extraction screw placed co-axially. The screw perfectly fits the internal contour of
the bowl, only allowing clearance between bowl and scroll. The differential speed between the screw and scroll
provides the conveying motion to collect and remove solids that accumulate at the bowl wall.

Drying:
Drying is an essential component of product formulation. It basically involves the transfer of heat to a wet product for
removal of moisture. Most of the biological products of fermentation are sensitive to heat, and therefore require gentle
drying methods. Based on the method of heat transfer, drying devices may be categorized as contact, convection,
radiation dryers. These three types of dryers are commercially available. Parameters affecting drying are the physical
properties of the solid–liquid system, intrinsic properties of the solute, and conditions of the drying environment and
heat transfer parameters. Heat transfer may be by direct contact, convection or radiation.

a. Rotary drum driers removes water by heat conduction. A thin film of solution is applied to the steam heated
surface of the drum, which is scraped with a knife to recover the dried product.

b. In vacuum tray driers the material to be dried is placed on heated shelves within a chamber to which a
vacuum is applied. This allows lower temperatures to be used due to the lower boiling point of water at
reduced pressure. The method is suitable for small batches of expensive materials, such as some
pharmaceuticals.

c. Spray drying involves atomization and spraying of product solution into a heated chamber, and resultant
dried particles are separated from gases using cyclones. Pneumatic conveyor driers use hot air that suspends
and transports particles.

d. Freeze-drying: Freeze-drying or lyophilization is the most preferred method for drying and formulation of a
wide-range of products—pharmaceuticals, foodstuffs, diagnostics, bacteria, viruses. This is mainly because
freeze-drying usually does not cause loss of biological activity of the desired product. Lyophilization is based
 on the principle of sublimation of a liquid from a frozen state. In the actual technique, the liquid containing the
product is frozen and then dried in a freeze-dryer under vacuum. The vacuum can now be released and the
product containing vials can be sealed e.g., penicillin can be freeze dried directly in ampules.

Cell Disruption:
Physical methods of cell disruption:
The microorganisms or cells can be disrupted by certain physical methods to release the intracellular products.
a. Ultra sonication: This method of cell lysis is achieved with high frequency sound that is produced
electronically and transported through a metallic tip to an appropriately concentrated cellular suspension.
Ultrasonic disintegration is widely employed in the laboratory. However, due to high cost, it is not suitable for
large-scale use in industries.

b. Osmotic shock: This method involves the suspension of cells (free from growth medium) in 20% buffered
sucrose. The cells are then transferred to water at about 4°C. Osmotic shock is used for the release of
hydrolytic enzymes and binding proteins from Gram-negative bacteria.

c. Heat shock (thermolysis): Breakage of cells by subjecting them to heat is relatively easy and cheap. But this
technique can be used only for a very few heat-stable intracellular products.

d. High pressure homogenization: This technique involves forcing of cell suspension at high pressure through
a very narrow orifice to come out to atmospheric pressure. This sudden release of high pressure creates a
liquid shear that can break the cells.

e. Impingement: In this procedure, a stream of suspended cells at high velocity and pressure are forced to hit
either a stationary surface or a second stream of suspended cells (impinge literally means to strike or hit). The
cells are disrupted by the forces created at the point of contact. Micro fluidizer is a device developed based on
the principle of impingement. It has been successfully used for breaking E. coli cells. The advantage with
impingement technique is that it can be effectively used for disrupting cells even at a low concentration.

f. Grinding with glass beads: 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. Several factors influence
the cell breakage-size and quantity of the glass beads, concentration and age of cells, temperature and agitator
speed. Under optimal conditions, one can expect a maximal breakage of about 80% of the cells.

Mechanical and non-mechanical methods:

Among the physical methods of cell disruption described above, ultra sonication, high-pressure homogenization,
impingement and grinding with glass beads are mechanical while osmotic shock and heat shock are non-mechanical.
The chemical and enzymatic methods (described below) are non- mechanical in nature.

Chemical methods of cell disruption:

Treatment with alkalies, organic solvents and detergents can lyse the cells to release the contents.
(i) Alkalies: Alkali treatment has been used for the extraction of some bacterial proteins. However, the alkali
stability of the desired product is very crucial for the success of this method. Alkali such as NaOH
addition alters the pH and affects the integrity of the cell membrane. It is carried out at pH range of 11 to
12 for about 20 to 30 min. e.g., recombinant growth hormone can be efficiently released from E. coli by
treatment with sodium hydroxide at pH 11.
(ii) Organic solvents: Cell wall absorbs the solvent resulting in swelling and rupture of cell wall. At low
concentration the cell wall is not ruptured but the permeability is increased. Product stability should be
considered when choosing the solvent. Several water miscible organic solvents can be used to disrupt the
cells e.g., methanol, ethanol, isopropanol, butanol. These compounds are inflammable; hence require
specialised equipment for fire safety. The organic solvent toluene is frequently used. It is believed that
 toluene dissolves membrane phospholipids and creates membrane pores for release of intracellular
contents.
(iii) Detergents: Detergents permeabilize cells by solubilising cell membranes. They are amphipathic capable
of interacting with both water and lipids and solubilise the cell wall Detergents that are ionic in nature,
cationic-cetyl trimethyl ammonium bromide or anionic-sodium lauryl sulfate can denature membrane
proteins and lyse the cells. Non-ionic detergents (although less reactive than ionic ones) are also used to
some extent e.g., Triton X-100 or Tween. The problem with the use of detergents is that they affect
purification steps, particularly the salt precipitation. This limitation can be overcome by using
ultrafiltration or ion-exchange chromato¬graphy for purification.

Enzymatic methods of cell disruption:

Cell disruption by enzymatic methods has certain advantages i.e., lysis of cells occurs under mild conditions in a
selective manner. This is quite advantageous for product recovery. Lysozyme is the most frequently used enzyme and
is commercially available (produced from hen egg white). It hydrolyses β-1, 4-glycosidic bonds of the mucopeptide in
bacterial cell walls. The Gram- positive bacteria (with high content of cell wall mucopeptides) are more susceptible
for the action of lysozyme.
For Gram-negative bacteria, lysozyme in association with EDTA can break the cells. As the cell wall gets digested by
lysozyme, the osmotic effects break the periplasmic membrane to release the intracellular contents. Certain other
enzymes are also used, although less frequently, for cell disruption. For the lysis of yeast cell walls, glucanase and
mannanase in combination with proteases are used.

Distillation:
Distillation is used to recover fuel alcohol, acetone and other solvents from fermentation media and for the production
of potable spirits. Batch distillation in pot stills continues to be used for the production of some whiskies but for most
other purposes continuous distillation is the method of choice. With ethanol, the continuous process produces a
product with maximum ethanol concentration of 96.5% (v/v). This azeotophic mixture is the highest concentration that
can be achieved from aqueous ethanol, unless a dehydration step is introduced using a water entrainer such as benzene
or cyclohexane.

Some continuous stills may be in the form of four or five separate columns but the Coffey type still comprises two
columns – the rectifier and analyser, each containing a stack of 30-32 perforated plates. Incoming fermentation broth
is heated as it passes down a coiled pipe within the rectifier column by the ascending hot vapour produced by the
analyser column. The now hot broth is released into a trough at the top of the analyser column and as it falls down the
column it is heated by steam. Hot vapours generated are then conveyed from the top of the analyser column to the
bottom of the rectifier column. As it passes upwards it is condensed on the coils carrying incoming broth. There is a
temperature gradient in the rectifier column and each volatile compound condenses at its appropriate level, from
where the fraction is collected.
Crystallization:
Product crystallization may be achieved by evaporation, low temperature treatment, or the addition of a chemical
reactive with the solute. The product’s solubility cannot be reduced by adding solvents, salts, polymers, e.g., non-ionic
PEG and polyelectrolytes or by altering the pH.

Crystallization is typical downstream processing method for high quality products with high purity requirements. The
method is widely used for example in organic acid production. One of the most common organic acids is citric acid
which is used in several industries such as pharmaceutical, food and beverage industries.

Production of citric acid by crystallization is one of the best examples. After fermentation the broth is filtered and
precipitated with Ca(OH)2 at pH of 7.2 and temperature of 70-90 °C. Calcium citrate crystals are formed in the
reaction. After filtration the calcium citrate is reacted with sulfuric acid to precipitate the calcium in the form of
calcium sulfate. Anhydrous citric acid is released (when reaction occurs above 40 °C) and then clarified with active
carbon and crystallized with evaporation. The resulted product has very high purity level due to crystallization and
therefore is suitable for foodstuffs or pharmaceuticals.