Continuous stirred tank bioreactors

A continuous stirred tank bioreactor consists of a cylindrical vessel with motor

driven central shaft that supports one or more agitators (impellers). The shaft is
fitted at the bottom of the bioreactor Fig. 19. IA).
The number of impellers is variable and depends on the size of the bioreactor i.e.,
height to diameter ratio, referred to as aspect ratio. The aspect ratio of a stirred
tank bioreactor is usually between 35. However, for animal cell culture
applications, the aspect ratio is less than 2.
The diameter of the impeller is usually of the vessel diameter. The distancebetween two impellers is approximately: 1.2 impeller diameter. Different types of
impellers (Rustom disc, concave bladded, marine propeller etc.) are in use.
In stirred tank bioreactors or in short stirred tank reactors (STRs), the air is added
to the culture medium under pressure through a device called sparger. The sparger
may be a ring with many holes or a tube with a single orifice. The sparger along
with impellers (agitators) enables better gas distribution system throughout the
vessel. The bubbles generated by sparger are broken down to smaller ones by
impellers and dispersed throughout the medium.
This enables the creation of a uniform and homogeneous environment throughout
the bioreactor.
Advantages of STRs There are many advantages of ST Rs over other types. These
include the efficient gas transfer to growing cells, good mixing of the contents and
flexible operating conditions, besides the commercial availability of the
bioreactors.

Bubble column bioreactors

In the bubble column bioreactor, the air or gas is introduced at the base of the
column through perforated pipes or plates, or metal microporous spargers. The
flow rate of the air/gas influences the performance factors 02 transfer, mixing.
The bubble column bioreactors may be fitted with perforated plates to improve
performance. the vessel used for bubble column bioreactors is usually cylindrical
with an aspect ratio of 4-6 (i.e., height to diameter ratio).

Airlift bioreactors
In the airlift bioreactors; the medium' of the vessel is divided into two interconnected
zones by means of a baffle or draft tube.
In one of the two zones referrer to riser, the air/gas is pumped. The other zone that
receives no gas is the downcomer

 The dispersion flows up the riser zone while the down flow occurs in the downcomer.
There are two types of airlift bioreactors.
Internal-loop airlift bioreactor (Fig. 11.10 has a single container with a central draft
tube that creates interior liquid circulation channels. These bioreactors are simple in
design, with volume and circulation at a fixed rate for fermentation.
External loop airlift bioreactor (Fig. 19. ID) possesses an external loop so that
the liquid circulates through separate independent channels. These reactors can be
suitably modified to suit the requirements of different fermentations. In general, the
airlift bioreactors are more efficient than bubble columns, particularly for of
microorganisms, this is mainly because in these bioreactors, the mixing of the contents
is better compared to bubble columns.
Airlift bioreactors are commonly employed for aerobic bioprocessing technology.
They ensure a controlled liquid flow in a recycle system by pumping. Due to high
efficiency, airlift bioreactors are sometimes preferred e.g., methanol production, waste
water treatment, single-cell protein production. In general, the performance of the airlift'
bioreactors is dependent on the pumping (injection) of air and the liquid circulation.
Two-stage airlift bioreactors
Two-stage airlift bioreactors are used for the temperature dependent formation of
products. Growing cells from one bioreactor (maintained at= temperature 300 C) are
pumped into another bioreactor (at temperature 420C. There is a necessity for the
two-stage airlift bioreactor, since it is very difficult to raise the temperature quickly
from 300 C to 420 C in the same vessel. Each one of the bioreactors is fitted with
valves and they are connected by a transfer tube and pump (Fig. 19.2A). The cells
are grown in the first bioreactor and the bioprocess proper takes place in the second
reactor.

Tower bioreactors
A pressure-cycle fermenter with large dimensions constitutes a tower bioreactor
(Fig, 19.2B): A high hydrostatic pressure generated at the bottom of the reactor
increases the solubility of 02 in the medium. At the top of the riser, (with expanded top)
reduces pressure and facilitates expulsion of C02. The medium flows back in the
downcomer and completes the cycle. The advantage with tower bioreactor is that it has
high aeration capacities without having moving parts.

Fluidized bed bioreactors

Fluidized bed bioreactor is comparable to bubble column bioreactor except the top
position is expanded to reduce the velocity of the fluid. The design of the fluidized
bioreactors (expanded top and narrow reaction column) is such that the solids are
retained in the reactor while the liquid flows out (Fig. 19.3A). These bioreactors are
suitable for use to carry out reactions involving fluid suspended biocatalysts such as
immobilized enzymes, immobilized cells, and microbial flocs.
For an efficient operation of fluidized beds, gas is sparged to create a suitable gas-liquidsolid fluid bed. It is also necessary to ensure that the suspended solid particles are not
too light or too dense (too light ones may float. whereas dense ones may 'settle at the
bottom), and they are in a good suspended state. Recycling of the liquid is important to
maintain continuous contact between the reaction contents and biocatalysts. This enable
good efficiency of bioprocessing
Packed bed bioreactors
A bed of solid particles, with biocatalysts on or within the matrix of solids, packed
in a column constitutes a packed bed bioreactor (Fig. 19.3B). The solids used may be
porous or non-porous gels, and they may be compressible or rigid in nature. A nutrient
broth flows continuously over the immobilized biocatalyst. The products obtained in the
packed bed bioreactor are released into the fluid and removed. While the flow of the fluid
can be upward or downward, downflow under gravity is preferred. The concentration of
the nutrients (and therefore the products formed) can be increased by increasing the
flow rate of the nutrient broth.

Because of poor mixing, it is rather difficult to control the pH of packed bed

bioreactors by the addition of acid or alkali. However, these bioreactors are preferred for
bioprocessing technology involving product-inhibited reactions. The packed bed
bioreactors do not allow accumulation of the products to any significant extent.
Photobioreactors

These are the bioreactors specialized for fermentation that can be carried out
either by exposing to sunlight or artificial illumination. Since artificial illumination is
expensive, only the outdoor photobioreactors are preferred. Certain important
compounds are produced by employing photobioreactors e.g., -carotene, asthaxanthin.
The different types of photobioreactors are depicted in Fig. 19.4. They are made up of
glass or more commonly transparent plastic. The array of tubes or flat panels constitute
light receiving systems (solar receivers). The culture can be circulated through the solar
receivers by methods such as using centrifugal pumps or airlift pumps. It is essential that
the cells are in continuous circulation without forming sediments. Further adequate
penetration of sunlight should be maintained. The tubes should also be cooled to prevent
rise in temperature.
Photobioreactors are usually operated in a continuous mode at a temperature in the
range of 25-40 C. microalgae and cyanobacteria are normally used. The organisms grow
daylight while the products are produced during night.
Features of a conventional bioreactors;

Conventional bioreactors are cylindrical vessels with domed top and bottom. The
reaction vessel, surrounded by a jacket, is provided with a sparger at the bottom
through which air (or other gases such as CO2 and NH3 for pH maintenance) can be
introduced.

 The agitator shaft is connected to a motor at the bottom. The reaction vessel has
side ports for pH, temperature and dissolved O2 sensors.
Above the liquid level of the reaction vessel, connections for acid, alkali, antifoam
chemicals and inoculum are located.
The bioreactor is usually designed to work at higher temperature (150-180 0 C),
higher pressure (377-412 kPa).
The reaction vessel is also designed to withstand vacuum, or else it may collapse
while cooling. The materials used for the construction of bioreactor must be nontoxic and must withstand the repeated sterilization with high pressure stream.
The bioreactor vessel is usually made up of stainless steel. It should be free from
crevices and stagnant areas so that no solids/liquids accumulate.
Easy to clean channels and wedded joints (instead of couplings) are preferred.
Transparent materials shield be wherever, possible, since it is advantageous to
inspect the medium and culture frequently.

The operation of a bioreactor basically involves the following steps.

1. Sterilization
2. Inoculation and sampling
3. Aeration
4. Control systems
5. Cleaning.

STERILIZATION
Aseptic conditions are the basic requirements for successful fermentation. That is
the bioreactor and its accessories, the growth medium and the air supplied during
fermentation must be sterile.
In situ sterilization
The bioreactor filled with the required medium is injected with pressurized steam
into the jacket or coil surrounding the reaction vessel. The whole system is heated to
about 1200 C and held at this temperature of or about 20 minutes. In situ sterilization has
certain limitations. It is not energy efficient (i.e., energy is wasted) since the bioreactor
has to be heated for a long period to rise the temperature of the whole system to 1200 C.
Prolonged heating may destroy vitamins, besides precipitating the medium components
Continuous heat sterilization
In this technique, empty bioreactor is first sterilized by injecting pressurized steam.
The Medium is rapidly heated to 1400 C for a short period by injecting the pressurized
steam. Alternately, the medium can be sterilized by passing through a heat exchanger
heated by pressurized steam. Subjecting the medium to high temperature for a short
period does not precipitate medium components. Further, there is no energy Wastage in
continuous heat sterilization
INOCULATION AND SAMPLING

The bioreactor with the growth medium under aseptic conditions is ready for
inoculation with the production organism. The size of the inoculum is generally 110%
of the total volume of the medium.
A high yielding productions train of the organism taken from a stock culture (lyophilized
and stored in a deep freezer or in liquid nitrogen) is used. During the course of
fermentation, samples are regularly drawn from the bioreactor. This is required to check
the contamination (if any) and measurement of the product formed.
AERATION
Aeration of the fermentation medium is required to supply 02 to the production
organisms and remove C02 from the bioreactor. The aeration system is designed for good
exchange of gases. Oxygen (stored in tanks in a compressed form) is introduced at the
bottom of the bioreactor through a sparger. The small bubbles of the air pass through the
medium and rise to the surface. The bioreactor usually has about 20% of its volume as
vacant space on the upper part which is referred to as head space. The bioreactor has
about 80% working volume. The gases released during fermentation accumulate in the
headspace which pass out through an air outlet.
Air-lift system of aeration
In this type of aeration, sparing of air is done at the bottom of the fermenter. This
allows an upward flow of air bubbles. The more is the aeration capacity of the fermenter,
the more is the dissolved 02 in the medium. Further, the aeration capacity of the air-lift
system is directly proportional to the airflow rate and the internal pressure. Oxygen
demand refers to the rate at which the growing culture requires 02. For all the aerobic
organisms, the aeration capacity should be more than the oxygen demand or else the
growth of the organisms will be inhibited due to oxygen depletion (starvation).
Stirred system of aeration
The aeration capacity of the medium can be enhanced by stirring. This can be done by
using impellers driven by a motor. The aeration capacity of the stirred fermenter is
proportional to the stirring speed, rate of air flow and the internal pressure Stirred
fermenters are better suited than air-lift fermenters to produce better aeration
capacities.
CONTROL SYSTEMS
It is essential to maintain optimal growth environment in the reaction vessel for
maximum product formation. Maximal efficiency of the fermentation can be achieved by
continuously monitoring the variables such as the pH, temperature, dissolved oxygen,
adequate mixing, nutrient concentration and foam formation. Improved sensors are now
available for continuous and automated monitoring of these variables (i.e. on line
measurement of pH).
Most of the microorganisms employed in fermentation grow optimally between pH
5.5 and
8.5. In the bioreactor, as the microorganisms grow, they release metabolites into the
medium which change pH. Therefore, the pH of the medium should be continuously
monitored and maintained at the optimal level. This can be done by the addition of acid
or alkali base (as needed) and a thorough mixing of the fermentation contents.
Sometimes, an acid or alkaline medium component can be used to correct pH, besides
providing nutrients to the growing microorganisms.
Temperature

Temperature control is absolutely essential for a good fermentation process. Lower

temperature causes reduced product formation while higher temperature adversely
affects the growth of microorganisms. The bioreactors are normally equipped with
heating and cooling systems that can be used as per the requirement, to maintain the
reaction vessel at optimal temperature.
Dissolved oxygen
Oxygen is sparingly soluble in water (0.0084 8/1 at 25 0C). Continuous supply of
oxygen in the form of sterilized air is done to the culture medium. This is carried out by
introducing air into the bioreactor in the form of bubbles. Continuous monitoring of
dissolved oxygen concentration is done in the bioreactor for optimal product formation.
Adequate mixing
Continuous and adequate mixing of the microbial culture ensures optimal supply of
nutrients and 02, besides preventing accumulation of toxic metabolic byproducts (if any).
Good mixing (by agitation) (if favourable environment for optimal and homogeneous
growth environment product formation. However, excessive may damage microbial cells
and increase temperature of the medium, besides increased foam
Nutrient concentration
The nutrient concentration in a bioreactor limited so that its wastage is prevented.
In addition limiting concentrations of nutrients may advantageous for optimal product
formation, since high nutrient concentrations are often associated with inhibitory effect
on microbial growth. It is now possible to do on-line monitoring of the nutrient
concentration, and suitably modify as per the requirements.
Foam formation
The media used in industrial fermentation is generally rich in proteins. When
agitated during aeration, it invariably results in froth or foam formation that builds in
head space of the bioreactor. Antifoam chemicals are used to lower surface tension of
the medium, besides causing foam bubbles to collapse. Mineral oils based on silicone or
vegetable oils are commonly used as antifoam agents. Mechanical foam control devices,
referred to as mechanical foam breakers, can also be used. Such devices, fitted at the
top of the bioreactor break the foam bubbles and the throw back into the fermentation
medium.
CLEANING
As the fermentation is complete, the bioreactor is harvested i.e. the contents are
removed for processing. The bioreactor is then prepared for the next round of
fermentation after cleaning (technically called turn round). The time taken for turn round,
referred to as down time, should be as short as possible (since it is non-productive).
Due to large size of the bioreactors, it is not possible to clean manually. The cleaning of
the bioreactors is carried out by using high-pressure-water jets from the nozzles fitted
into the reaction vessel.
SOLID SUBSTRATE (SOLID STATE) FERMENTATION
There are certain fermentation processes that do not involve liquid medium. For
these biotechnological processes, the growth of the microorganisms is carried out on
solid substrates in the complete absence or almost complete absence of free water. The
presence of some moisture (about 1 5%) is necessary for solid substrate (or solid state)

fermentation (SSF). The most commonly used solid substrates for SSF are cereal grains,
wheat bran, sawdust, wood shavings and several other plant and animal materials.
These solid substrates are polymeric in nature, insoluble or sparingly soluble in water,
and contain concentrated source of nutrients for the growth of microorganisms. SSF is a
very old traditional technique carried out in many countries. It is used for the production
of edible mushrooms, cheese, soy sauce and many other fermented products (including
enzymes and organic acids). A selected list of solid state fermentations is given Table
19.2. Composting is a good example of SSE Solid substrate fermentation has been very
popular for the production of fermented foods (idli, dosa, dhokla, bread, beverages,
fermented fish, meat, yogurt, cheese, pickles). Fermentation often makes the food more
nutritious, easily digestible and better in flavour.
Table 19.2
product

Substrate

Edible mushrooms
Cheese
Soy sauce
Sauerkraut
Enzymes
Organic acids
Leaching of metals
Composting

Straw manure
Milk, curd
Soy beans, wheat
Cabbage
Wheat bran
Cane sugar, molasses
Low grade ores
Mixed organic material

Sewage treatment

Sewage compounds

Microorganisms
involved
Agaricus bisporus
Lentinula edodes
Penicillium roquefortii
Lactic acid bacteria
Aspergillus niger
Aspergillus niger
Thiobacillus
Fungi, bacteria,
actinomycetes
Bacteria, fungi, protozoa

For solid substrate fermentation, single pure

cultures, mixed cultures or mixed organisms may be used. Pretreatment of substrate raw
materials is sometimes done to facilitate the availability of nutrients.
Solid substrate fermentation is normally carried out as a non-aseptic process. This saves
sterilization costs. It is important that the substrates used in SSF have adequate spaces
in between to allow good air circulation. This facilitates adequate exchange of gases,
besides promoting heat elimination. Forced air circulation may be done to maintain
optimal conditions in SSE
Bioreactors for SSF
Bioreactors designed for solid state fermentation are much simpler' compared to
liquid-state fermentation. In the Fig 19.6, tower reactor, drum reactor and forced aeration
reactor, used in SSF are depicted.

Advantages of SSF
Solid substrate fermentation employs simple natural solids as the media.
Low technology, low energy expenditure and requires less capital investment.
No need for sterilization, less microbial contamination and easy downstream
processing.
Yield of the products is reasonably high
Bioreactor design, aeration process, and effluent treatment are quite simple.
Many domestic, industrial and agricultural wastes can be fruitfully used in SSE
Limitations of SSF
The microorganisms that tolerate only low moisture content can be used.
Precise monitoring of SSF (e.g., O2 and CO2 levels, moisture content) is not possible.
The organisms grow slowly and consequently there is a limitation in product
formation
Heat production creates problems and it is very difficult to regulate growth.
MEDIA (SUBSTRATES) FOR INDUSTRIAL FERMENTATION
The media used for the growth microorganisms in industrial fermentation must
contain all the elements in a suitable form for the synthesis of cellular substances as well
as the metabolic products. While designing a medium several factors must be taken into
consideration. The most important among them is the ultimate product desired in the
fermentation. For growth-linked products (primary metabolites e.g. ethanol, citric acid),
the product formations is directly dependent on the growth of the organisms hence the
medium should be such that it supports good growth. On the other hand, for products
which are not directly linked to the growth (secondary metabolites e.g. antibiotics,
alkaloids, gibberellins), the substrate requirements for product formation must also be
considered.
In the laboratory, pure defined chemicals may be used for culturing
microorganisms. Howeverf,o r industrial fermentations, undefined and complex
substrates are frequently used for economic reasons. Cheaper substrates are
advantageous since they minimize the production cost of the fermented products.
Wastes from agriculture, and byproducts of other industries are generally preferred,
although they are highly variable in composition. Raw materials used in fermentation
largely depend on their cost at a particular time, since there are seasonal variations.
The choice of the medium is very critical for successful product formation. For
industrial fermentation, the microorganisms, in general, utilize a luxury metabolism.
Therefore, good production yields are expected with an abundant supply of carbon and
nitrogen sources, beside requisite growth factors. The media used in fermentation
processes may be synthetic or crude.
Synthetic media
Media with all the requisite constituents in a pure form in the desired proportion
represents synesthetic media. Use of this type of media in fermentations is not
practicable.
Crude media

The non-synthetic media with naturally available sources are better suited for
fermentations.
In practice, crude media with an addition of requisite synthetic constitutes is ideal for
good product yield in fermentation.
The most frequently used substrates for industrial fermentation with special
reembrace to the supply of carbon and nitrogen sources and growth factors are briefly
described below.
SUBSTRATES USED AS CARBON SOURCES
Carbohydrate constitutes the most predominant source of energy in fermentation.
Industry refined and pure carbohydrates such as glucose or sucrose are rarely used for
economic reasons.
Molasses
Molasses is a byproduct of sugar industry and is one of the cheapest sources of
carbohydrates. Sugar cane molasses (sucrose around 48%) and sugar beet molasses
(sucrose around 33%) are commonly used. Besides being rich in sugar, molasses also
contain nitrogenous substances, vitamins and trace elements. There occurs variation in
their composition of the molasses which mostly depend on the climatic conditions and
production Process. Hydro molasses, a byproduct in glucose production from corn, is
also used as a fermentation substrate
Malt extract
Malt extract, an aqueous extraction of malted barley contains about 80% of
carbohydrates (glucose, fructose, sucrose and maltose). Nitrogen compounds constitute
around 4.5% (proteins, amino acids, purines and pyrimidines).
Starch, dextrin and cellulose
The polysaccharides such as starch, dextrin and cellulose can be metabolized by
microorganisms. They are frequently used for the industrial production of alcohol. Due to
its wide availability and low cost, the use of cellulose for alcohol production is extensively
studied.
Whey
Whey is a byproduct of dairy industry and is produced worldwide. Most of it is
consumed by humans and animals. Whey is a reasonably good source of carbon for the
production of alcohol, single-cell protein, vitamin B12 lactic acid and gibberellic acid.
Storage of whey is a limiting factor for its widespread usage in fermentation industry.
Methanol and ethanol
Some of the microorganisms are capable of utilizing methanol and/or ethanol as
carbon source.
Methanol is the cheapest substrate for fermentation. However, it can be utilized by only
a few bacteria and yeasts. Methanol is commonly used for the production of single-cell
protein. Ethanol is rather expensive. However, at present it is used for the production of
acetic acid.
SUBSTRATES USED AS NITROGEN SOURCES

The nitrogen supply to the fermentation microorganisms may come from inorganic
or organic sources.
Inorganic nitrogen sources
Ammonium salts and free ammonia are cheap inorganic nitrogen sources,
particularly in industrialized countries. However, not all the microorganisms are capable
of utilizing them, hence their use is limited.
Organic nitrogen sources
Urea is fairly a good source of nitrogen. However, other forms of nitrogen sources
are preferred.
Corn steep liquor: This is formed during starch production from corn. Corn steep
liquor is rich in nitrogen (about 4%) and is very efficiently utilized by
microorganisms. It is rich in several amino acids (alanine, valine, methionine,
arginine, threonine, glutamate).
Yeast extracts: They contain about 8% nitrogen and are rich in amino acids,
peptides and vitamins, Glucose formed from glycogen and trehalose during yeast
extraction is a good carbon source. Yeast extracts are produced from baker's yeast
through autolvsls (at 50-530 C) or through plasmolysis (high concentration of NaCI).
Yeast extracts are very good sources for many industrially important
microorganisms.
Soy meal: After extracting the soy bean oil from the soy bean seeds, the left out
residue is soy meal. It is rich in proteins (about 50%) as well as carbohydrates
(about 300/0) contents. Soy meal is often used in antibiotic production.
Peptones The protein hydrolysates are collectively referred to as peptones, and
they are good sources for many microorganisms. The sources of peptones include
meat, soy meal, peanut seeds, cotton seeds and sunflower seeds. The proteins
namely casein, gelatin and keratin can also be hydrolyzed to yield peptones. In
general, peptones derived from animal sources have more nitrogen content while
those from plant sources have more carbohydrate content. Peptones are relatively
more expensive, hence not widely used in industries

Sources of growth factors

Some of the microorganisms are not capable of synthesizing one or more growth
factors such as vitamins. These growth factors are very expensive in pure form, hence
crude sources are preferred. Yeast extract is a rich source of almost all growth factors.
Generally, the substrates derived from plant or animal sources in a crude form are
reasonably rich in mineral content. Sometimes, however mineral (phosphate, sulfate)
supplementation may be required.

STERILIZATION OF CULTURE MEDIA AND GASES

For successful fermentation, it is absolutely essential to ensure.
Sterility of the media containing the nutrients.
Sterility of incoming and outgoing air
Sterility of the bioreactor.
Prevention of contamination during fermentation.

A brief account on the sterilization of has been already described. A bioreactor can also
be sterilized by destroying the organisms by heat/chemicals/ radiation or sometimes by
physical procedures such as filtration .