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FERMENTATION, TYPES OF FERMENTERS, DESIGN & USES OF FERMENTERS

AND OPTIMZATION OF FERMENTATION PROCESS

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 FERMENTATION, TYPES OF FERMENTERS, DESIGN & USES OF
FERMENTERS AND OPTIMZATION OF FERMENTATION PROCESS.

*1Dr. Chy. Ezemba; 1Ekwegbalu E.A and 2Ezemba A.S

AFFLIATED INSTITUTION:
1
Department of Microbiology, Chukwuemeka Odumegwu Ojukwu University (COOU) P.M.B 02, Uli,
Anambra State, Nigeria.
2
Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University Awka, Anambra State,
Nigeria.
E-MAIL:* constancechinyere790@gmail.com

1. INDUSTRIAL FERMENTATION SYSTEM; MEANING

1. 1. Introduction Concept:
Fermentation comes from the latin verb “fevere”, which means to boil. Ironically, fermentation is possible
without heat. It’s originated from the fact that early at the start of wine fermentation gas bubbles are released
continuously to the surface giving the impression of boiling.
It has 3 different meaning which might be confusing.
A. Usage of the word in Microbial Physiology; Fermentation is defined as the type of metabolism of a
carbon source in which energy is generated by substrate level phosphorylation (one of the 3 mechanism of
phosphorylation atom to generate ATP from ADP) such as:
- Substrate Level
-Oxidative
-and Photophosphorylation.
And in which organic molecules function as the final electron acceptor generated during catabolism.
Note: If inorganic compound such as: (Sulphate or nitrate, and oxygen) is the final acceptor is called
Respiration. Respiration is referred to as aerobic if the final acceptor is oxygen. And anaerobic when it is
some other inorganic compound outside oxygen e.g sulphate or nitrate.
*Final electron acceptor or (as acceptors of the reducing equivalents).

Microorganisms generate ATP using respirations. Also they use carbohydrates (sugar) for energy and a fuel
organic chemical like (ATP) adenosine triphosphate delivers that energy to every part of a cell when needed.

Fermentation is similar to anaerobic respiration; the kind that takes place when there is not enough oxygen
present.
Depending upon environmental conditions, individual cells and microbes have the ability to switch between
the two different modes to energy production.

B. The second usage of the word is in Industrial microbiology which defines fermentation as any process
in which micro-organisms are grown on a large scales, even if the final electron acceptor is not an organic
compound (i.e if the growth is carried out under aerobic conditions) e.g yeast cells (Saccharomyces
cerevisiae) prefer fermentation to aerobic respiration even when oxygen is abundant.

C. The third usage concerns Food microbiology defines fermentation as any metabolic process in which
micro-organism’s activity creates a desirable change in food and beverages, whether it’s increasing flavor,
preserving food stuffs, providing health benefit or more.

1
 Here micro-organisms determine the nature and general character of the food, but micro-organisms form
only a small portion of the finished product by weight. Food such as vinegar, cheese, bread and yoghurt,
are fermented foods.

1.2. Three (3) Distinct Types of Fermentation

i. Lactic Acid Fermentation: yeast strains and bacteria convert starches
or sugars into lactic acids, requiring no heat in preparation. These anaerobic chemical reactions pyruvic acid
uses nicotinamide adenine dinucleotide + hydrogen (NADH) to form lactic acid and NAD+. The method
makes, pickles, yogurt and sourdough bread.
ii. Ethanol / Alcohol Fermentation: yeasts breaks pyruvate molecules further down into alcohol and
carbon dioxide molecules to produce wine and beer.
NOTE: Pyruvate molecules from end product of glycolysis or output of the metabolism of glucose
(C6H12O6).
iii. Acetic Acid Fermentation: starches and sugars from grains and fruit ferment into sour tasting
vinegar and condiments. e.g apple cider vinegar and Kombucha.

1.3 Fermentation Process Stages

- Primary fermentation.
- Secondary fermentation
Depending upon what you are fermenting, the process can have several stages.
- Primary fermentation: in this brief phase, microbes begin rapidly working on raw ingredients such
as fruit, vegetables or dairy. E.g Yeasts or other microbes convert carbohydrates (sugars) into other
substances such as alcohols and acids.
The microbes present or in the surrounding liquid (such as brine for fermented vegetables) prevent
putrefying bacteria from colonizing the food instead.
- Secondary fermentation: In this longer stage of fermentation, which last several days or even weeks,
alcohol levels rise and yeasts and microbes die off and their available food source (the carbohydrates)
becomes scarcer. Wine makers and brewers use secondary fermentation to create their alcoholic
beverages.
The pH of the ferment can differ significantly from when it started out, which affects the chemical
reactions taking place between the microbes and their environment. Once alcohol is between 12-15% it
kills the yeast, preventing further fermentation, distillation is needed to remove water, condensing
alcohol content to create a higher percentage of alcohol.

1.4. INDUSTRIAL FERMENTATION SYSTEM

Industrial fermentation process comprises of biological chemical & physical aspect of fermentation.
It begins with suitable microorganisms, requirement of sterility, cellular reaction and specified conditions.
 Screening for suitable microorganisms
 Development of seeding inoculum for multiplication and support (Good growth for microorganisms)
 Development of production medium which is almost & always complex to maximize yields of the
metabolite.
 Appropriate measures to ensure contamination control.
 Determination of physical condition such as pH, temperature, time.
 Determination of industrial metabolites (isolation and purification of the metabolite after termination of
fermentation) which are very costly and expensive.

2
1.4.1 Types of Microbial fermentation processes
Microbial culture process can be carried out in different ways. Such as:
1. Batch fermentation
2. Fed-batch fermentation
3. Continuous fermentation

-Batch fermentation:
Batch fermentation, is a closed system in which a large volume of nutrient medium is inoculated to proceed
for the harvest and recovery of the product. This ends the batch fermentation as the vessel is cleaned and re-
sterilized for the subsequent batches. This is a close system in which all the nutrients are initially added to
the vessel and inoculated.
Subsequent treatments include maintaining adequate aeration by providing stirrers and pH control by the
addition of an acid or alkali. In aerated stems, antifoam agents such as palm oil or soybean oil are added.
Large scale growth of microorganisms tends to generate heat in the system. Temperature control is
maintained by providing water circulation system around the vessel for heat exchange.
In batch fermentation, the growth of microorganisms follows the characteristics growth curve with a lag
phase followed by a log phase, finally reaching the stationary phase due to limitation of nutrients and other
factors. A diauxy growth curve can be observed when complex nutrient solutions are used, two lag phases
frequently occurs separated by a second log phase, this is due to one of the substrates utilized preferentially.
The presence of one substrates represses the breakdown of other substrate.
In summary, microorganisms goes through all the stages of growth (Lag phase, transient acceleration phase,
exponential phase, deceleration, stationary phase), prior to the collection of product.

-Fed-Batch Fermentation:
In Fed-batch, substrate is added in increments as the fermentation progresses (small concentrations and
small doses) because during the formation of many secondary metabolites is subject to catabolite repression
by high concentration of glucose, other carbohydrates, or nitrogen compounds. Because critical elements of
the nutrient solution are added in small concentration in the beginning of the fermentation and these
substances (substrates) continue to be added in small doses during the production phase.
It is difficult to measure the substrate concentration directly and continuously during the fermentation.
Indirect parameters, which are correlated with the metabolism of the critical substrates, have to be measured
in order to control the feeding process. For example, in the process of organic acids, the pH is to be used to
determine the rate of glucose feeding. Sometimes, dissolved O2 or the CO2 content in the exhaust air are
monitored. Here microorganism is maintained at a peak rate of growth (exponential phase).

-Continuous Fermentation:
During continuous fermentation, some part of the components (include media and inoculums) of upstream
process are withdrawn intermittently and replacement of the withdrawn substances are made by adding the
fresh medium or nutrients.
The withdrawn part from the fermenter is used for recovery of the products. During continuous
fermentation, the equipment is always in use and secondly inoculum is not required in subsequent addition
of the nutrients.

This industrial fermentation is being carried out in bioreactors, which include:

1. Fermentors or Reactors or Bioreactors.
2. Other vessels like test tubes, flask and petridishes

3
1.5 Fermenters (Bioreactors)
A fermenter is an enclosed and sterilized vessel that maintains optimal conditions for the growth of a
microorganism. The microorganisms undergo fermentation to produce large quantities of a desired
metabolite (products) for commercial use. This product can be collected from a fermenter after a fixed
amount of time (batch cultivation) or ongoing (continuous cultivation).
Fermenters also called Bioreactor are special vessels equipped with control devices for broth mixing,
aeration and control of recation (culture) condition. Eg. Heat exchanger for temperature control.
A bioreactors can be as small 1 -20 litres in laboratory scale, but 100,000 – 500,000 liters for production in
a fermentors
Fermenter size is measured by the total volume only about 75% of the volume is usually utilized for actual
fermentation, the rest being left for foam and exhaust gasses.

1.6 Main function of a Fermenter (Bioreactor)

1. To provide a conducive and controlled environment for the biological agent (microorganisms) such as
pH, temperature and pressure.
2. To serve as a defined mixture of microorganism before adequate mixing for nutrient and product
transfer.
3. Adequate sparging for aeration and degassing of generated gasses.
4. To obtain a desired product after fermentation.
5. To obtain a homogenous culture with mineral power input.
6. To enable to maintain optimal conditions for cell growth and product formation.
7. A vessel that is robust, strong enough to withstand the various treatments required high heat, pressure
sterilization.
8. Inoculation point for aseptic transfer in inoculums and sampling valve for withdrawing a sample for
different tests.

1.7 Components of Bioreactor

1. Probes and sensors (pH, O2, heat, temp, cell mass, pressure, antifoam probes). Control devices to also
check the levels of key nutrients and product concentration.
2. External water jacket
3. Head plate
4. Sampling port / product outlet
5. Exhaust outlet
6. Acid / base inlet
7. Nutrient inlet
8. Shaft
9. Motor
10. String paddles
11. Impeller
12. Air sparger / aeration system
13. Baffles
14. Sealing

A flow chart of major component of a bioreactor are shown and listed below in figure 1.

4
 Here is flow chart
that show major
parts of fermenter.

Figure 1: Flow chart that show major part of a fermenter.

Source: (Kumaresan and Jyeshtharaj, 2006 )

5
1.8 Major parts of the fermenter:
-Probes and sensors: Are used to monitor the condition within the fermenter in order to maintain optimal
level of microbial growth example:
(1) Antifoam probes to prevent contamination in fermentation process.
(2) Acid / base probes a flow to add acid and bases during fermentation process.
-External water jacket: It can be used to absorb excess heat and maintain a constant viable86 temperature.
-Head plate: To cover the top of the vessel of bioreactors.
-Sampling pot: A valve to get the sample of the fermentation
-Exhaust outlets / nutrient inlet: Allow for the introduction of sugar of the removal of the metabolic
wastes.
-Acid/base inlet: Allows for the regulation of pH level within the chamber (formation of product may alter
the pH).
-Shaft: Not allow medium to escape or microorganisms to enter the edges.
-Motorized string paddles: Function to distribute heat and materials evenly within the reaction chamber.
-Impeller: To diminish the size of air bubbles to give a bigger interfacial area for O2 transplant and to
decrease diffusion paths. This also maintain uniform environment throughout the vessel content.
-Air sparger: To ensure better dispersal of air for microorganisms in the fermenter example; porous
sparger, orifice sparger and nozzle sparger.
-Baffles: To prevent a vortex and improve aeration efficiency. Also to prevent a whirl pool effect that could
impede proper mixing per mixing.
-An aerator: Can be used to introduce compress air into the chamber while a defoamer can hindered the
formation of foam.
-Sealing: Between top plate & vessel to maintain airtight condition, aseptic and containment.

1.9. 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.
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 (Gueguim et al ., 2005).

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.

6
Since most industrial fermentation process are aerobic, the construction of a typical
aerobic fermentor (Fig. 2) are show below:

Figure 2: A Typical Aerobic fermenter

Source: (Gueguim et al ., 2005)

7
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 an 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 (Gueguimet
al ., 2005).

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.

The size and position of the impeller in the fermentor depends upon the size of the fermentor. In tall
fermentors, more than one impeller is needed if adequate aeration and agitation is to be obtained. 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 (Gueguim et al ., 2005).
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 (Gueguim et al ., 2005).

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 (Gueguimet al ., 2005).

8
1.10Types of Fermentors

There are various types of fermentors that are described below-

1. Stirred Tank (Continuous) Bioreactor (Used for making antibiotics).

2. Airlift Bioreactor (Yeast production)
3. Packed tower (Bed) Bioreactor (in vinegar production)
4. Fluidized Bed Bioreactor (for enzymes production)
5. Photobioreactor (Spirulina production)
6. Membrane Bioreactor
7. Bubble Column Bioreactor
8. Nathan fermentor (used in brewing industry)
9. Pulsed column bioreactor

-Continuous Stirred Tank Bioreactor

The continuous stirred-tank bioreactor consists of a vessel, pipes, valves, pumps, agitator, shaft, impeller
and a motor. Which is shown in figure 3. Below.
Sparger is mostly used to add air to the culture medium under pressure and the impellers serve as a gas
distributor throughout the fermentor and also break down the larger bubbles for a uniform distribution.
The motor power the bioreactor which helps in mixing cultures and also there are sensors that can detect
temperature, PH, dissolved oxygen, glucose, lactic acid, ammonia, ammonium ion, and other parameters
in the culture medium.
The main target of desired products enlists the cells or primary metabolite which mostly acquire
microorganisms like yeast or bacteria.

Figure 3: A Continuous Stirred Fermentor.

9
 Advantages of continuous stirred tank reactor
 It is a continuous operation
 It has a good capture over temperature
 It provides a homogeneous environment for cell growth and proliferation
 Easily adapts to two-phase runs
 It is quite cost-efficient in both investment and operation
Disadvantages of continuous stirred tank reactor
 Their mechanic agitation produces shear stress which may harm the cultured cells. but this can be
overcome by changing the shape and diameter of the impeller blade or adding bovine serum albumin or
dextran
 Foaming can also cause a problem but again can be solved by antifoaming agents.

-Airlift Bioreactor
The medium of the vessel consists of a baffle or a draft tube through which air is pumped. This might
create a bubble in the medium which ultimately helps in rising up through the baffle tube and drags the
surrounding fluid as well. The diagram is shown in figure 4. Below.
This, in the process, stirs up the contents by air.
There are two types of airlift bioreactors;
Internal loop type,
External loop type
 Internal-loop airlift bioreactor has a single container with a draft carrying out the fermentation process.
 External loop airlift bioreactor has an external loop to separate samples or liquids in different channels
carrying out fermentation.

Figure 4: Airlift Bioreactor

10
Airlift Bioreactor Advantages
 It produces very little shear stress, less friction
 It requires fewer efforts to construct the bioreactor.
 It is cost-efficient
 Less energy is required
Airlift Bioreactor Disadvantages
 High Pressure is required in this system
 No shaft is present which helps as a foam breaker which creates a major drawback.

-Packed Bed Bioreactor

Packed-bed bioreactors are tubular reactors which are stuffed with an immobilized enzyme or microbial
cells as biocatalysts as immobilization enhances the stability of the enzyme and these enzymes can be
utilized multiple times. The diagram is shown in figure 5.below.
The substrate is then allowed to flow through the packed-bed bioreactor and it is generally operated in a
single pass.

Figure 5: A packed Bed Bioreactor

Advantages of Packed Bed Bioreactor

 It’s quite easy to operate
 It provides better quality
 The products can be controlled.
Disadvantages of Packed Bed Bioreactor
 As the substrate and product molecules are carried out both in and out of the carrier matrix during the
reaction there is a problem of external mass transfer resistance.
11
Fluidized Bed Bioreactor
A fluidized bed bioreactor is an immobilized cell reactor which is a combination of stirred tank and
packed bed continuous flow reactors.
It can be explained as beds of regular molecules that are suspended in a flowing liquid stream. Below is
the diagram of Fluidized Bed Bioreactor in figure 6.
This can be used for particles such as immobilized enzymes, immobilized cells, and microbial flocs.
Immobilized-cell particles are retained in the bed by gravity critical parameter here
is the settling velocity
It involves cell as biocatalysts including 3 phases; gas-liquid-solid.

Figure 6: Fluidized Bed Bioreactor

Advantages of Fluidized Bed Bioreactor

 It is used for wastewater treatment and hydrogen production.

Disadvantages of Fluidized-Bed Bioreactor

 It requires more energy in order to achieve fluidization in the bioreactor.

-Photobioreactor
 These are carried out either by exposing to sunlight or artificial illumination.
 These are majorly used for the cultivation of algae. The diagram of Photobioreactor is shown below in
figure 7.
0
 The temperature range used is between 25-40 C

Photobioreactor Advantages
 Contamination is lower.
 It can be space-saving as it can be placed vertically, horizontally or at an angle, indoors or outdoors.

12
 Photobioreactor Disadvantages
 The control of PH and temperature is quite difficult
 It is somewhere susceptible to contamination.

Figure 7: A Photobioreactor

-Membrane Bioreactor
It consists of a biological reactor with suspended biomass and solids removal by ultra- and microfiltration
membranes.
It is used for alcoholic fermentation, solvents, organic acid production, wastewater treatment.
It involves high-quality effluent through the membranes and eliminates the sedimentation and filtration
processes. Below is the Diagram of Membrane Bioreactor shown in figure 8.
The membrane materials mostly used are polysulfonte, polyamide and cellulose acetate.

Figure 8: Membrane Bioreactor

13
Advantages of Membrane Bioreactor
 The loss of the enzyme is minimized.
 Effluent quality is high
 The effluent is properly disinfected from all the pathogenic microbes
Disadvantages of Membrane Bioreactor
 Quite costly and energy-consuming
 The aeration is limited
 Membrane pollution is also a drawback

-Bubble Column Bioreactors

The bioreactor involves agitation by density-driven
density driven fluid motions and sparger is used to provide air into
the continuous liquid phase at the bottom of the reactors
reactors. Below is the
he diagram of Bubble Column
Bioreactors shown in figure 9.
The fluid mixed intensively at high gas flow rates when convective flows become turbulent.

Figure 9: Bubble Column Bioreactors

Advantages of Bubble Column Bioreactors

 It acquires good heat and mass transfer
 Easy to operate
 Low maintenance
Disadvantages of Bubble Column Bioreactors
 Back mixing is a major drawback which adversely affects product conversion.

The type used extensively in industries are:

 The stirred tank fermentor
 Airlift fermentor
 Bubble column fermentor

The most widely used bioreactors is the continuous stirred type while the most commonly used bioreactor
is aerated stirred tank batch fermentor because most industrial fermentation process are aerobic.

Several types of fermenter are further grouped base on different concept of function and designs:
1. Shape or configuration
2. Liquid (submerged) or solid state (surface)
3. Aerated or aerobic.
4. Batch or continuous production

Based on configuration (mixing devices) mechanically and non-mechanically (pneumatically) agitated

bioreactors.
 Mechanically agitated bioreactors (impellers propeller). They are very popular and most studied.
 Non mechanically (pneumatically); these are bioreactor which are mixed without mechanical
agitators either by air bubbling or pneumatically.
-Bubble coiumn
-Airlift bioreactors: split airlift, Airlift with interneral draft tube, external loop airlift.
They have a lot of advantages than mechanical.
1. Cheaper, simpler and easier to construct.
2. The energy required for mixing is lower.
3. The hydrodynamic stress is usually very low.

1.11 Designs and construction of Bioreactors

There are two types of materials which can be used in construction a fermenter:
(i) Stainless steel; according to American iron and Steel Institute (AISI), if a steel contains 4%
chromium, it is called stainless. The long and continuous use of stainless steel sometimes shows
pitting it is also important to consider the material used for aseptic seal.
(ii) Glass; It can be made between glass and glass and metal, or using metal for joints between a vessel
and detachable top or base plate (glass)
On pilot scale, any material to be used will have to be assessed in their ability to withstand pressure,
sterilization, corrosion and their potential toxicity and cost.

1.12 Major Requirement for Bioreactor Construction

1. The construction materials 0.4% chromium stainless steel or glass
2. The Agitator (Impeller): The size and position of the impeller in the vessel depends upon the size
of the fermenter. In all vessels, more than one impeller is needed if adequate aeration-agitation is
to be obtained. Ideally, the impeller should be 1/3/ or 1/2 of the vessel diameter (D) above the
base of the vessel. The number of impeller may vary size to size of the vessel.

15
 There are four classes namely: Disc turbine, Vaned disc, open turbin of variable pitch and marine
impeller.
3. Stirrer Gland and Bearing: Four basic types assembly have been used: the packed gland seal, the
simple bush seal, the mechanical seal and the magnetic drive.
4. Baffles: There are metal strips used for checking or preventing the vortex formation around the
walls of the vessel which results to foaming. The baffles are normally incorporated into agitated
vessel of all sizes to prevent a vortex and to improve aeration efficiency. They are attached
radically to the walls for every roughly one-tenth (1/10th )of the vessel diameter. The gap between
wall& baffle facilicites the scouring action around the vessel. This movement also minimized
growth on baffles and fermentation walls.
5. Sparger: A sparger may be defined as a device for introducing air into the liquid in a fermenter. It
is important to know whether sparger is to be used on its own or with mechanical agitation as it
can influence initial bubbles size. The efficiency of sparging is a function of the sparging rate and
the extent of gas distribution/dispersion by spargers ie (number of pores, the diameter of the pores
and the distribution of the pores in the sparger). The types of gas used for sparging depends on the
aim.
e.g *Air, oxygen or mixture of two is used for anerobic process where aeration is the main
objective of sparging.
*Inert gasses such as nitrogen are often used for anerobic processes where mixing and de-gassing
are the main objectives.
*A mixture of air and carbon dioxide are used in some plant & animal cells and also
photobioreactor.
Three basic types of sparger have been used and may be described as the porous sparger, the
orifice sparger and the nozzle sparger. They also have one called sparger agitator.
Addition of O2 to the culture (aeration) to remove unwanted gasses from the culture (de-gas) or to
keep the cells in suspension the method is called sparging.
6. Sealing: Sealing between top plate and vessel is an important criteria to maintain airtight
condition, aseptic and containment. There are 3 types of sealing.
a. Gasket.
b. Lipseal.
c. “O” ring: It has simple sealing and double sealing.
Note: 2 way sealing in “O” ring with steam between two seals.
Sealing have to be done between 3 types of surfaces viz;
- Glass- glass.
- Glass-metal
- Metal – metal.
Using materials such as fabric-nitryl or butyl rubbers.

Others are:
 Dissolved oxygen / carbon dioxide concentrations
 Temperature control
 pH control
 Pressure control
 Foam control

16
1.13 Designs
Some of the bioreactors were designed for specific processes while others are more robust and can be used
for various processes.
Since the optima conditions for cell growth and product formation varies depending on the cell strain and
the desired product, it must be decided if the bioreactor will be used for a specific cell / product or whether it
will be used for a variety of cells / products before designing a bioreactors.
The basic points of consideration while designing a fermenter includes:
- Productivity and yielding.
- Fermenter operability and reliability and contamination free.
- Product purification.
- Waste management.
- Waste treatment.
- Energy requirements.
- Material for construction either glass or stainless steel 4% chromium.
The aim or objectives of bioreactor designs:
- When the bioreactor size is increased, to be able to maintain optima conditions for cell growth and
product formation.
- To construct a bioreactor which is cheap and simple to operate and maintain.
- To be able to change one process parameter while the other parameters are held constant.
- To obtain a homogeneous culture with mineral power input.
- To maintain they hydrodynamic stress as low as possible.

Development of Bio-Processes
This is usually done in stages which include;
A. The basic experiments at laboratory/ bench scale.
B. Elucidation of the optima conditions at pilot scale.
C. Establishment of the industrial scale process where the optima conditions established at lower seals
are maintained at industrial scale.
1. This involves controlling the culture conditions such as mass transfer.
- Mixing
- Hydrodynamic stress.
- Constant power consumption per unit volume.
2. As well as medium composition and concentration. Lastly screening of micro-organisms.
Microorganisms are not left out in this process as they are the micro-reactants in the fermenter
(bioreactor). Various microorganisms have been reported to produce an array of primary and
secondary metabolites, but in a very low quantity.

2. OPTIMIZATION OF FERMENTATION PROCESS

2.1 Concepts: Fermentation technology is widely used for the production of various economically important
compounds which have application in the energy production, pharmaceutical, chemical and food industry.
For any fermentation based product, the most important thing is the availability of fermented product equal
to that of the market demand.
Optimization literally means the design and operation of a system or process to make it as good as possible
in some defined sense.

17
 Generally optimizations of fermentation process are those method, activities or practices and
parameters applied during fermentation to ensure optimum performance of the fermenter and production of
quality and products in optimum quantity. It is another approach to medium design and is used to determine
the limiting concentration of each media component.
Optimization of fermentation process aims at:
- Identifying and determining the limiting concentration of each media component.
- Identifying and to know the right nutrient to choose for growth, multiplication and their metabolic
activities.
- Adjusting fermentation conditions such as pH, temperature, agitation speed, fermentation time.
- Increasing the yield, activity of the desired product.
- Maximize the profits from fermentation process i.e minimize the product cost and undesired product
otherwise known as by-products.
Some process assessment Index (PA1) parameters consider during optimization of fermentation media
are;
1. Volume of the inoculum/volume of the inoculate.
2. Volume of the fermenter vessel/the capacity of the fermenter.
3. Carbon/nitrogen sources/its concentration.
4. Nutrient availability (nutritive and non nutritive components).
i. Buffer
ii. Agar
iii. Surfactants (antifoaming agent (fatty acid and derivatives)
iv. Growth factors
v. Phosphates e.t.c.
Standardize the
5. pH
6. Temperature
physical
7. Agitation speed parameters
8. Fermentation time
9. Aeration requirements.

Some chemical and biological variables including the physical parameters.

1) The optimum medium even for a single industrial process may differ depending on the stage.
Inoculum (seed culture)/ fermentation medium. The microorganisms require different medium i.e for
the cell growth phase and the product formation phase).
2) Both the choice of the nutrient sources and their concentrations affects the amount of undesired
products (by-products) formed.
3) E.g Antifoaming agent (fatty acid and derivatives, polyglycoles, higher alcohols and silicones) are
also determined since some of these agents are either toxic or impart some undesirable characteristics
to the culture if added in excess.

2.2 Media Optimization Strategies/Methods:

There are various strategies/methods used during the course of medium design/medium fermentation
process optimization they includes:
- Borrowing.
- Component replacing.
- Biological Mimicry
- One-factor-at-a-time.
- Factorial Design.

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 - Placket and Burmar’s Design.
- Central composite Design.
- Surface methodology.
- Evolutionary operation.
- Evolutionary operation factorial design.
- Artificial neural network.
- Fuzzy logic.
- Genetic Algorithms.

But the most frequently used and historically is one-factor-at-a-time (OFAT) followed by full factorial
technique and response surface methodology. But placekett and burmar’s design and component
replacing can be useful for screening medium components.
In bioprocess industry it is often needs to conduct optimization experiments because new mutants and
strains are continuously being introduced. In medium fermentation process optimization, different
combinations and sequence of process conditions and medium components are needs to be investigated to
determine the growth condition that produce the biomass with the physiological state best constituted for
product formation.

1.Borrowing
This is an open-ended system for process optimization. The medium components and process conditions are
obtained from the literatures and what other workers were used to grow the same genus, species or strains
are analyzed. The problem with this method is that there are too many options for a given fermentation
process. Therefore short listing is necessary and advantage of this method is that it is simple, easy and
requires no mathematical skill (Kennedy and Krouse, 1999).

2.Component Replacing
This is an open-ended system for process optimization and only used to compare the component of one type
in a fermentation medium (Nandi and Mukherjee, 1988). In this method, one of component of the medium
was replaced by a new one at same incorporation level. However this method does not consider the
components interactions. But this method can useful for screening different carbon, nitrogen and other
source for improving the medium utilization (Kennedy and Krouse, 1999; Jatinder et al., 2006; Tavares et
al., 2005). Screening of suitable carbon source for mevastatin and citric acid production by solid-state
fermentation was carried out by component replacing techniques (Ahamad et al., 2006; Kumar et al., 2003).

3.Biological Mimicry
Biological mimicry is a close-ended system for fermentation process optimization. This method is useful for
optimization of various components of fermentation media and based on concept that cell grow well in a
medium that contains every things it needs in right proportion (mass balance strategy). The medium is
optimized based on elemental composition of microorganisms and growth yield. The limitation of this
method is measuring elemental composition of microorganisms is expensive, laborious and time consuming
moreover it does not consider the component interaction however this method gives an idea about different
micro and macro elements level require in the media for optimal growth of microorganisms (Kennedy and
Krouse, 1999).

4.One-factor-at-a-time
One-factor-at-a-time is a close-ended system for fermentation process optimization. This method can be
applied for optimization of medium components as well as for process condition and it is based on the
classical method of changing one independent variable while fixing all other at a certain level (Ahamad et
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al., 2006; Alexeeva et al., 2002; Patidar et al., 2005). This strategy has the advantage that it is simple, easy
and the individual effects of medium components and process condition can be seen on graphs (Kar et al.,
1999; Kumar et al., 2003) but the limitations of this method are interaction between the components are
ignored, extremely time consuming, expensive for large number of variable as it involves a relatively large
number of experiments. Because of its easy and convenience one-factor-at-a-time method has been the most
popular method for improving the fermentation medium and process condition.

5. Factorial Design
Factorial design is a close-ended system for process optimization. In this method, level of factors/parameters
are independently varied, each factor at two or more levels. This effects that can be attributed to the factors
and their interactions are assessed with maximum efficiency in factorial design more over it allow for the
estimation of the effects of each factor and interaction.

The optimization procedure is facilitated by construction of an equation that describes the experimental
results as a function of the factor level. A polynomial equation can be constructed in the case of a factorial
design where the co-efficient in the equation are related to the effects and interactions of the factors. In a full
factorial (complete factorial) design every combination of factor level was tested. Typical factors are
microbial strain, medium components, temperature, humidity, initial pH and inoculum volume. The most
commonly used full factorials in medium improvement experiments are two factorial designs (denoted by
2n when there are n factors). These designs are the smallest capable of providing detailed information on
factor interaction (i.e., antagonistic or synergistic effects) (Xie et al., 2003).

A partial factorial design provides a compromise when the number of runs required in full factorials is
impracticable. These are usually two-level factorial design. Two level fractional factorial are denoted by 2n-
k, where n is the number of factors and ½ k is the fraction of the complete factorial used. This notation gives
an immediate idea of the number of runs required. For example, 26-1 is a half fraction of complete factorial
25 and requires 32 (i.e., 25) runs per replicate (Rajendhran et al., 2002). In most case factorial design were
in combined with other different optimization techniques such as central composite design (Park et al.,
2005) and evolutionary operation (Tunga et al., 1999) to optimize fermentation process.

6.Plackett and Burman’s Design

Plackett and Burman’ s design may be useful to find out the important variable in a system this design is
suitable when more then five independent variables are to be investigated. Plackett and Burman’ s design are
useful to screen out important factor, which influence the fermentation process (Naveena et al., 2005).
Which are optimized by response surface methodology in further studies (Sayyad et al., 2006; Singh and
Satyanarayana, 2006). This technique allows for evaluation of n variables by n+1 experiments. n+1 must be
multiple of 4 e.g., 8, 12, 16, 24, etc. therefore the number of independent variables which can be investigated
by this method are 7, 11, 15, 19, 23, etc. Any factors not assigned to a variable can be designated as a
dummy variable. The incorporation of dummy variable into an experiment makes it possible to estimate the
variance of effects (Plackett and Burman, 1946).

7.Central Composite Design

Central composite design (CCD) was introduced by Box And Wilson; CCDs are formed from two level
factorials by addition of just enough points to estimate curvature and interaction effects. The design can be
viewed as partial factorials with factors at five levels. The number of runs in CCD increases exponentially
with number of factors. Optimization of media components for compaction production in complex and
chemically defined production medium using CCD has been reported (Kennedy and Krouse, 1999).

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CCD can be combined with response surface methodology, in which experiments were designed by CCD
and thereafter optimized by response surface methodology (Chakravarti and Sahai, 2002; Dahiya et al.,
2005).

8.Response Surface Methodology

Statistical experiment design is a powerful method for accumulating information about a process rapidly and
efficiently from a small number of experiments, thereby minimizing experimental costs. Box and Wilson
introduced Response Surface Methodology (RSM). RSM seeks to identify and optimize significant factors
with the purpose of determining what levels of factors maximize the response (Sayyad et al., 2006; Singh
and Satyanarayana, 2006). RSM uses statistical experimental design such as Central Composite Design
(Chakravarti and Sahai, 2002; Dahiya et al., 2005), Box-Behnken Design (Sayyad et al., 2006) etc. in order
to develop empirical models that relate a response and mathematically describes the relationships existing
between the independent and dependent variables of the process under consideration.

The contours of a response surface optimization plot show lines of identical response. Response means the
results of an experiment carried out at particular values of the variables being investigated. The axes are the
contour plots are the experimental variable and the area within the axes is termed the response surface. To
construct a contour plot, the results (response) of a series of experiments employing different combination of
variable are inserted on the surface of the plot at the points delineated by the experimental conditions, points
giving the same results (equal response) are joined together to make a contour line (Kumar et al., 2004).

The purpose of response surface methodology was to obtain a predicted model and this model can be useful
for optimizing the fermentation media formulation or for optimization of fermentation process condition, to
carry out simulation with model equation and for better understanding the fermentation process.

9.Evolutionary Operation
Evolutionary operation employs factorial design sequentially to improve yield. The changes made to
variable from one cycle to the next are restricted and can only be made when the estimated improvements
are greater then the estimated experimental error. Optimization of production of protease
by Rhizopusoryzae using Evolutionary operation has been reported (Banerjee and Bhattachaaryya, 1993).

10.Evolutionary Operation Factorial Design

The evolutionary operation (EVOP) factorial design methodology was a hybrid of evolutionary operation
and factorial design technique here, experiments are designed based on factorial technique and results are
analyzed by EVOP. This methodology is considered to be a multi variable sequential search technique, in
which the effects of n variable factors are studied and response analyzed statistically. The decision-making
procedure is easy and clear-cut it directs the change of variable to wards the objective maximum or
minimum values. Evolutionary operation factorial design technique combines the advantage of factorial
technique for designing experiments with n parameters and that of evolutionary operation methodology for
systematic analysis of experimental results and facilitate the selection of optimum condition or direct the
change desired for individual parameters for design of subsequent experiments. For a study of a five variable
system, the total number of new experiments to be conducted is 25, apart from two control experiments
(search level regions). The parameters for the above experiments are arranged in both higher level (+) and
lower level (-) compared to search level regions (0), the parameters and the total number of experiments are
represented in a [5 X (25+2)] matrix Which has been divided in two blocks i.e., overall negative blocks and
overall positive blocks. All experiments were replicated for two cycles. The magnitude of effects, change in
mean effects, standard deviation and error limits (of average, of effects and of change in mean effects),
analyzed as per the decision making procedure of Evop to arrive at the optimum. When the experimental
21
results of the first set did not satisfy the optimum conditions, a second set of experiments was planed
selecting the best condition of the first set as the new search level for second set. This procedure was
repeated till the optimum condition was obtained (Tunga et al., 1999; Panda, 2001).
Optimization of protease enzyme production under solid-state fermentation by Rhizopusoryzae and
optimization of gallic acid production under solid-state fermentation using evolutionary operation and
factorial design technique has been reported (Tunga et al., 1999; Kar et al., 2002; Mukherjee and Banerjee,
2004).

11.Artificial Neural Network

Artificial neural network is the model and trained on a given set of data and then used to predict new data
point and provide a mathematical alternative to quadratic polynomial for representing data derived from
statistically designed experiments. Artificial neural network’s strong points are that they work well with
large amount of data and handles them easily without requiring no mechanistic description of system, this
make artificial neural network particularly well suited to medium optimization (Kennedy and Krouse, 1999).

First data generated by conducting a series of experiments and a network is constructed and getting the
network to learn on these data set, once trained, the network is given new data points (media composition or
fermentation process condition) and the output (microbial performance or product formation) predicted.
Artificial neural networks are well suitable for predicting the outcome from the fermentation process thereby
saving time and efforts (Patnaik, 2005).

However artificial neural networks are simply a modeling tool and does not work properly when input data
sequence are missing neural network s confused when different data are generated for same set of
experiments but averaging the data can solve the problems.

12.Fuzzy Logic
Fuzzy logic utilizes and executes a series of rules using Fuzzy membership functions. At first the Fuzzy
memberships are defined. This defines what should be the level of the components in a fermentation media
whether it is in low or high. Then next sets of experiments are defined based on results obtained from the
first set of experiment (Ul-haq and Mukhtar, 2006). When a new medium composition is entered in Fuzzy
logic programme, it predicts the result or the out come (microbial performance or product formation)
(Anderson and Jayaraman, 2005; Kennedy and Krouse, 1999).

13.Genetic Algorithms
In recent years non-statistical optimization techniques such as genetic algorithms are used in fermentation
technology. This is a powerful stochastic search and optimization technique, this technique can be used to
optimize fermentation process without need of statistical designs and empirical models and based on the
principle that after a continuous process of mutation only best individual exist. These individuals strive for
survivals. After some number of generations only the best individual hopefully represents the optimum
solution. In fermentation media or fermentation process optimization rules of genetic algorithms can be
applied successfully where the set of one experiment i.e. medium composition are coded in one chromosome
and each medium constituent level represents one gene after completing the first generation of experiments
chromosome with highest productivity are selected and replicated proportionally to the productivity then
crossover of chromosome and mutation of some randomly chosen genes are performed. In such a way, new
generations of experiments are obtained. But main disadvantage of genetic algorithms is that it does not
store the information generated at each stage of the optimization process (Zuzek et al., 1996). A hybrid of
genetic algorithms and artificial neural network approach was realized to optimize fermentation process.
This technique based on principle that after a satisfactory neural network model and input space which is
22
generated over the range of independent parameters, can be optimized using genetic algorithms the
advantage of this technique is that neural network provide better fits to experimental data then quadratic
polynomial equation and model optimized by genetic algorithms approach which provide a better alternative
to the conventional RSM approach to optimize fermentation process (Nagata and Chu, 2003).

Conclusions
Designing a fermentation media or optimization of fermentation process can be never ending task and every
optimization techniques have their own advantages and disadvantages (Table 1). Historically one-factor-at-
a-time used mostly fallowed by full factorial technique and response surface methodology but Plackett and
Burman’s design and component replacing can be useful for screening medium components. Recently nural
networks fuzzy logics, genetic algorithms and different hybrid techniques such as CCD-RSM, Plackett and
Burman-RSM, factorial design-RSM, evolutionary operation-factorial design and genetic algorithms-
artificial neural network techniques used efficiently to optimize fermentation medium and fermentation
process parameters.

2.3 Frequently used Media Optimization strategies:

There are various strategies which are frequently used during the course of medium design and optimization.
These strategies are geared towards improving the efficiency of the production medium.
-Classical Medium Optimization Methods
a.One-Factor-at-a-Time (OFAT)
In the classical medium optimization techniques, one-factor-at-a-time (OFAT) experiments, only one factor
or variable is varied at a time while keeping other were changed over a desired range. This method due to its
ease and convenience, has found application during the initial stages of medium formulation for the
production of new metabolic or known compound from new source.
OFAT is further sub-grouped into:
i.Removal experiments: A situation whereby all the medium components are removed from the production
medium one-by-one, and after proper incubation period, their effects on the production of secondary
metabolite or the product of interest is observed in terms of suitable parameters. According to Singh et al.,
(2008), removal of Soybean meal or glycerol or NaCl from the fermentation medium during the production
of antifungal compound from Streptomyces capoamus, decreased the yield by 20-40%.
ii. Supplementation experiments: These are experiments carried out to evaluate the effects of various
Carbon and nitrogen supplements on metabolite production. For example, 70-90-% enhancement in the yield
of antifungal product from Streptomyces violaceusniger was observed by supplementing xylose, sorbitol and
hydroxyl proline in the production medium.
ii. Replacement experiments: Here, carbon / nitrogen sources showing enhancement effects on the desired
metabolite production in supplementation experiments are generally tried to be used as a whole carbon /
nitrogen source.
iii. Physical parameters Standardization: In addition to chemical and biological variables, several
researchers used OFAT experiments to standardize the physical parameters such as pH, temperature,
agitation and aeration requirements of the fermentation process.

Like any other technique, OFAT method of medium optimization has its own advantages and disadvantages.
The major advantage of OFAT is its simplicity by which a series of experiments can be carried out and
results can be analyzed by using simple graphs without the aid of high and statistical analysis. The major
drawback of OFAT is the difficulty in estimating the “interactions” from the experiments as it is a hit-and-
miss scattershot sequence of the experiment. Another disadvantage of OFAT techniques is time
consumption and cost as a large number of variables are analyzed, one at a time.

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