Chapter 10 Bioprocessing and

Biomanufacturing

Students are already aware that living organisms especially 10.1 Historical
microbes and biological processes therein are used for Perspective
making various household products (curd/yoghurt, idli, 10.2 Instrumentation
kinema, etc.) and industrial products (ethanol). We have in Bioprocessing:
Bioreactor and
already learnt in the previous class that living organisms Fermenter Design
are endowed with a variety of metabolic processes which
10.3 Operational Stages
lead to the formation of chemical compounds, called of Bioprocess
metabolites, that are broadly classified into primary
10.4 Bioprocessing and
and secondary metabolites. Primary metabolites are the Biomanufacturing
compounds produced directly out of primary metabolic of Desired Product
pathways associated with essential cellular functions such
as growth and development. On the other hand, secondary
metabolites are intermediates or indirect products,
elaborated by entirely different metabolic pathways called
secondary metabolic pathways. Secondary metabolites take
part in a variety of functions. For example, they are used in
defence for protection against pathogens, phytoplanktons
and herbivores, to improve tolerance to abiotic stresses;
as attractants for insects and animals for fertilisation,
seed dispersal in plants or to contribute in causing the
displeasure to the unwanted feeders.

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 Nowadays, most of these secondary metabolites have
a variety of applications in the form of pharmaceuticals,
dyes, food additives, enzymes, vitamins, etc. In view of the
variety of applications, production of these compounds
(biochemicals) at commercial level requires their production
in bulk quantities, as quantities produced naturally are
not sufficient. These compounds are usually produced at
commercial level in a purified form through a series of steps
covered under bioprocessing. For large scale production,
where large volume (on an average 100–10,000 litres) of
culture can be processed, the development of bioreactors
was required. Thus, bioreactors can be thought of as
vessels in which raw materials are biologically converted
into specific products using microbial, plant or animal
cells or their components. Bioreactors provide optimal
growth conditions (temperature, pH, substrate, salts,
vitamins, oxygen) for achieving the desired product. Thus,
bioprocessing deals with the manufacturing of desired
biochemicals using a living system or their components.
Bioprocess involves the maintenance of sterile (microbial
contamination free) atmosphere or environment to enable
the growth of only the desired microbial or eukaryotic cell
in large quantities for manufacturing biotechnological
products like antibiotics, vaccines, enzymes, organic acids
and so on.

10.1 HISTORICAL PERSPECTIVE

After the breakthrough discovery of Penicillin and its
role in killing bacteria by Alexander Fleming in 1928,
the significance of products from biological systems was
well understood. Now, the challenge before the scientific/
research community was to enhance the production
of penicillin. Production in larger amount for its use in
treatment would obviously require a systematic process
using the culture of biological entity in question i.e.,
Penicillium species. The need of involvement of microbial
physiologist and other life scientists and technologists was
identified. This followed the identification of a number of
products of living organisms and processes especially from
microbes for application in bioprocessing. Many companies

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 and government laboratories, assisted by different
universities and institutions came together to take up this
challenge and efforts were made to increase the production
of penicillin. All this paved a way for the emergence of a
new area of biological applications, which is now known
as bioprocessing. Thus, bioprocessing involves biological
or living systems or their components (e.g., enzymes,
chloroplasts, etc.) and chemical engineering processes to
obtain the desired products at commercial level as depicted
in Fig. 10.4.
At industrial or commercial stage, all bioprocesses are
carried out in vessels called fermenter or bioreactor. We
are also aware that after the advent of rDNA technology,
microbes are extensively employed for the production of a
number of biological material for the welfare of mankind.

Box 1
Discovery of Penicillin
It was in 1928 when Alexander Fleming
at St. Mary’s Hospital in London, while
trying to isolate boils causing bacterium,
Staphylococcus aureus, found that one of the
Petridishes was contaminated inadvertently
with a foreign entity. Instead of discarding
the Petri plate for disinfection, Fleming made
an important observation in the unwashed
contaminated plate that no bacteria grew near the invading entity. The observation surprised
Fleming’s intellect and he soon realised that this chance observation may be a meaningful
arena of interest.
Later this antibacterial foreign entity was identified as a common mould, the Penicillium
notatum, and the metabolite secreted has powerful antibacterial properties called penicillin.
Its full potential as an effective antibiotic was established by Ernest Chain and Howard
Florey. This antibiotic was extensively used to treat the American soldiers wounded in World
War II. Fleming, Chain and Florey were awarded the Nobel Prize in 1945 for this discovery.

10.2 INSTRUMENTATION IN BIOPROCESSING:

BIOREACTOR AND FERMENTER DESIGN
Bioreactor is an engineered vessel made up of glass or
steel that supports a biologically active environment,
where cells can be cultivated under aseptic conditions with
appropriate nutritional and environmental requirements.

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 In a bioreactor, the biochemical processes involve
the cultivation of microbial, plant and animal cells
or biochemically active substances derived from such
cell cultures or organisms. Commonly, bioreactors are
cylindrical and vary in size. The design and components of
a typical bioreactor are shown in Fig. 10.1.

Motor
(a)

Temperature Probe
pH Probe
In In Antifoam
HCl NaoH
Inoculation Port

Outlet for air

Water
Outlet Digital controller
Agitator Shaft

Air Sparger
Bafe
Impeller
Water
Bath
Water
Inlet

Exhaust

(b)

Fig. 10.1: (a) Diagrammatic representation of the design and components of a typical bioreactor
(b) Photograph of a laboratory bio-reactor

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 A bioreactor should fulfil the following requirements:
1. a sterile environment, so that a pure culture may be
grown without contamination
2. adequate supply of air for cellular respiration in
culture
3. uniform mixing of nutrients, cells and air throughout
the bioreactor vessel without causing any shear stress
to the cultured cells
4. a system for maintenance of optimum temperature
conducive for the growth and product formation in a
desired culture
5. a system for monitoring the environmental process
parameters, such as pH, dissolved oxygen, etc.
Thus, in order to fulfil these requirements a typical
bioreactor consists of the following:
• Agitator shaft: It helps in mixing contents of the
bioreactor and keeps the cells in perfect homogenous
conditions that provide better transport of nutrient
and oxygen throughout the running bioprocess. An
impeller is fixed at the bottom of an agitator shaft.
• Sparger: It helps in providing an adequate and
continuous supply of sterilised air (oxygen) using
microfilters for growing cells submerged in the liquid
media inside the bioreactor system.
• Baffle: It helps in breaking the vortex formation which
is highly undesirable as it changes the centre of gravity
of the system making it consume extra power to run
the system.
• Jacket: It provides area for the circulation of water
at the given temperature for maintenance of optimum
temperature inside the device required for the growth
of cultivated cells and product formation.
• Sensitivity probe for temperature and pH: These
are the probes to sense temperature and hydrogen ion
concentration of a bioprocess.
• Digital controller for controlling process
parameters: Digital controller is connected to
bioreactor through probes and its one separate unit is
connected to a water bath that pumps water of desired
temperature in and out of the jacket present around
the bioreactor unit for the maintenance of temperature
throughout. It is also connected to pH probes and
bottles containing 1M NaOH and 1N HCl. As the

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 probe senses acidity or alkalinity, digital controller
commands any of the two bottles for addition of either
dilute NaOH or HCl for the maintenance of the desired
pH. All process parameters including temperature,
pH, speed of stirring (rpm), etc., are exhibited over the
display of the controller.

10.2.1 Types of bioreactors

Based on the design or configuration, important types of
bioreactors are discussed below:
• Stirred tank reactors are the most conventional
bioreactors. In these reactors, agitator facilitates the
mixing of nutrients, oxygen and growing cells. The
reactor is characterised by the presence of agitator
shaft. Design, shape and size of the impeller differ for
various bioprocesses [Fig. 10.2(a)].
Motor
Agitator Gas
Headspace
Region

Downcomer
Sparger
Liquid
Media

Bafe Draft Tube

Impeller Air
Grooved Bubble Sparger
Nozzle Bank

(a) Stirred tank (b) Air-lift (c) Bubble column

Fig. 10.2: Various types of bioreactors

• In Air-lift reactors, a motion of air is created using

a draft tube. The mixing of nutrients and oxygen is
maintained by creating the air current that lifts the
fluid broth and cells up and down, inside-out or vice-
versa through a draft tube inside the reactor vessel
[Fig. 10.2(b)].
• In Bubble column reactor, the mixing of nutrients
and oxygen is maintained with the help of air bubbles
produced through sparger jet. These reactors
provide low shear environment, which may be a
critical consideration for some cells and high oxygen
transferred per unit of power input [Fig. 10.2(c)].

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 10.3 OPERATIONAL STAGES OF BIOPROCESS
A bioprocess is composed mainly of two stages for converting
raw material into the final product namely, upstream and
downstream processing (Fig. 10.3).

Stock Culture Raw Material

Nutrient Media
Shake Flask

Sterilisation
Upstream
Processing

Bioreactor/
Fermentor

Seed for
Inoculation
Controller

Bioprocess/Fermentation

Recovery

Downstream
Processing
Purication

Packaging

Fig. 10.3: Different stages of upstream and downstream processing

The upstream bioprocessing consists of four components

as detailed below:
1. optimisation of nutritional conditions in artificial
media and formulation for culturing the living
organisms, cells or its components
2. sterilisation of media, bioreactor and other additional
tools and equipment
3. production of pure, active and healthy inoculums in
sufficient quantity

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 4. optimisation of environmental conditions for growth
and product formation
On the other hand, downstream processing consists of two
steps as detailed below:
1. extraction, recovery and purification of product
2. the disposal of effluents produced by the process
Upstream and downstream processing are described in
detail in the following sections.

10.3.1 Upstream processing

A typical upstream bioprocess involves raw materials such
as the biomass of microbial, plant or animal cells to be
usually treated and mixed with other ingredients that are
required for the cells to grow well. The raw material to
be used in bioprocessing is first converted to a suitable
fermentable form. Preparation of liquid or solid nutrient
medium, sterilisation, aeration, agitation and shear
sensitivity besides many other preparatory operations and
scaling up for high product formation are described in the
following sections.
Nutrient media or culture media required for maximum
growth and product formation of a particular culture is
formulated using chemicals and nutrients, etc. Different
media formulations are prescribed for microbial, plant and
animal cell culture (Chapter 6–8).
Requirement of medium constituents varies with the
species of an organism for biosynthesis and cell
maintenance. The following equation based on
stoichiometry may be considered for growth and product
formation:
Carbon and energy source + Nitrogen source Cell biomass + products +
+ other requirements Carbon dioxide + Water + heat

The equation is important for economical designing of

the media by minimising wastage of media components. For
example, under aerobic conditions, carbon requirement of
a particular culture may be estimated by determining the
cellular yield coefficient (Y) as:
Quantity of cell dry weight produced by the culture
Y=
Quantity of carbon substrate utilised

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 Fig. 10.4: Bioprocess Development

Similarly, in a bioprocess other media components may

also be determined for their minimal wastage and optimum
productivity of the culture. Thus, media formulation is very
important for a bioprocess.
Inoculum of viable, healthy, fast growing and high
producing living cells, organ or organism is very much
required for good growth and technologically viable and
economically efficient production. Inoculum may be
developed on solid culture or liquid culture. Liquid culture
may usually be developed in shake flask and called
suspension culture. The culture to be used as inoculum
must fulfil the following criteria:
1. inoculum should be healthy and in log or exponential
phase of the growth cycle (i.e., actively dividing).
2. in subsequent transfer, inoculum should not exhibit
long lag phase.
3. it should be available in sufficient amount to provide
an inoculum of optimum size.

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 4. it should be available in a suitable morphological form.
5. it should be free of contamination.
6. it should retain its product forming capability.
Aeration is required to fulfil oxygen requirement
of submerged suspension culture in shake flask and
bioreactors. Ideally, maximum amount of dissolved oxygen
in pure water is approximately 8 g/L, which is available for
growing submerged culture in liquid media. In bioreactor,
pure sterilised air is sent through sparger to maintain the
oxygen levels in the media for growth of the culture.
A mild agitation is required for uniform distribution of
oxygen and other nutrients. Some cells such as animal cells
are more shear sensitive than plant cells. Plant cells are
more sensitive to shear than microbial cells. Thus, in shake
flask, shear stress or sensitivity of cells to shear can be
controlled by increasing or decreasing the speed of shaker
i.e., revolution per minute (rpm) of the shaking condition.
The same is maintained in bioreactors by increasing or
decreasing the speed of agitator.
Temperature required for maximum growth and
production has to be maintained. Temperature requirement
for different cultures is different. There may be an optimum
temperature for the formation of a desired product. The
optimum temperature may be similar or different for
culture growth and product formation.
Hydrogen ion concentration (pH) is another
parameter that affects growth and product formation.
Optimum pH for growth and product formation has to be
maintained for an efficient bioprocess.
Thus, before a bioprocess is run, all processing
parameters, such as nutrient media formulations,
temperature, hydrogen ion concentration, etc., are
optimised separately.
Sterilisation is an essential requirement for a successful
bioprocess. It requires sterilisation of tools, glassware,
media, air or in-site sterilisation of bioreactor, etc., and even
the maintenance of aseptic conditions to perform processes
and scale up in the bioreactor. Thus, contamination in a
bioprocess may be avoided by the following:
1. sterilisation of nutrient media
2. sterilising the bioreactor vessel

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 3. sterilising all the materials to be added to the
bioreactor vessel during the process.
4. maintenance of aseptic conditions during the
fermentation.
5. using a pure culture as inoculum.
• A typical bio process development stage are summarised
in fig. 10.4

Modes of Bioprocess Operation

One of the important decisions in the development of a
bioprocess is that the mode of bioprocess operations is
to be applied to a particular bioprocess. Mainly there are
three different modes under which a bioprocess may be
operated:
1. Batch
2. Fed-batch
3. Continuous
1. Batch mode: It is a closed culture system with an
initial fixed amount of nutrients. An inoculated batch
culture passes through a number of phases of growth
cycle. When cells are grown in a nutrient medium, cells
start growing in number and size to some extent. In a
suitable nutrient medium, the cells utilise nutrients
from the medium for growth and energy production and
convert the material to be bioprocessed (fermented) into
[X] [X]
[S]

[QS] [X]

[QS] [S] [QS]

[S]

Time Time Time

(a) Batch mode (b) Fed-batch mode (c) Continuous culture mode

S = Substrate conc, X = Microbial cells, QS = Rate of utilisation of substrate

Fig. 10.5: Graphical representation of growth of cell culture in (a) Batch mode (b) Fed batch mode and
(c) Continous culture mode.
the product. Fig. 10.5(a) is showing cell biomass [X], rate of
substrate consumption [Qs] and substrate concentration
[S] in a batch culture.
2. Fed-batch mode: In a batch culture if the growth of

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 cells become limited due to the concentration of one or more
substrate components, the same are fed intermittently or
continuously to the growing culture as per the requirement
from time to time. In such culture, volume in the fermenter
increases due to extra feed added and no removal of any
volume of culture. This is known as fed-batch mode
of culture, which is advantageous for maintaining the
residual substrate concentration at very low levels and
thereby avoiding their toxic effects. In this culture system,
substrate concentration‘s’ and rate of substrate utilisation
[Qs] remains constant and cell biomass/product keeps on
increasing Fig. 10.5(b).
3. Continuous mode: In this mode, the design of the
bioprocess (reactor) is such that the fresh lot of nutrient
media is added and the used media is removed in such a
way so that continous supply of the desired product is to
be ensured. Similarly, a fresh lot of inoculum could also
be added. Thus, in the continuous mode, a steady state is
maintained that is, the formation of new biomass by the
culture is balanced by the loss of cells from the reactors
vessel Fig 10.5(c). Thus, during steady state rate of growth
and product formation, substrate concentration and rate
of substrate utilisation remains constant.

Downstream processing
In the process, the desired product is recovered in an

Discard

Cell as products Cell debris

(Intracellular
product)
Cell biomass Cell rupture Recovery Purification

Solid liquid
Separation

Fermenter Supernatant Recovery Purification

(for extracellular product)

Fig. 10.6: Major steps in downstream processing

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 efficient way which involves efficient separation and
purification technique. Thus, the product of bioprocess
may be cell biomass, extracellular component of liquid
media (broth) or intracellular product of cell. The major
process of downstream processing is shown in Fig. 10.6.
The extraction and purification of product in the culture
fluid may be difficult and costly. High quality efficient
recovery requires the following considerations:
1. the process chosen must be quick.
2. the chosen process must have minimum investment
and operated at minimum cost.
The downstream processing mainly consists of physical
separation as well as purification operations, which
includes the separation of particulate, dialysis, reverse
osmosis, solid-liquid separations, adsorption, liquid-liquid
extraction, distillation, drying, etc.
Solid-Liquid Separation: The first step in product
recovery is separation of solids, such as biomass, insoluble
particles and macromolecules from culture fluid or
fermentation broth. In some cases, the culture broth or
fermentation fluid needs pre-treatment, such as heating or
pH adjustment or treating with coagulating and flocculating
agents for the separation of insoluble products from the
fluid or broth. Major methods used for the separation of
cell biomass are filtration or centrifugation.
Filtration is the most common cost-effective method to
be used for separation of large particles and cell biomass
from the culture fluid. The conventional filtration involves
the separation of large particles (pore diameter dp>10
mm) by using canvas, synthetic fabrics or glass fibre as
filter medium. Continuous rotary filters are most widely
used filters in the industry. Ultra-filtration or microporous
filtration is also used for the separation of cell biomass.
Centrifugation is used for the separation of particle
size between 100 µ — and 0.1 µ — from liquid in centrifuge
and ultracentrifuge.
Cell disruption: if the product is intracellular, it may
be recovered by cell rupture. Cell rupture techniques may
be powerful but mild enough so that it should not damage
the desired product. Disruption of cell may be achieved by
physical, chemical and biological methods.

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 Physical methods include the mechanical means
of cell disruption by milling in high-speed bead mills,
homogenisation by creating very high shear rates using
high pressure homogeniser and ultrasonic vibrations
through sound waves in ultrasonicator. Cell disruption
using ultrasonicator is very effective with most of the cell
suspension culture.
Non-mechanical methods include the treatment of cells
with either chemicals, such as surfactants, alkalis, organic
solvents, or by osmotic shocks besides biological methods
such as enzymatic degradation of the cell wall.

Recovery
After solid and liquid are separated, a dilute aqueous
solution is obtained from which the product has to be
recovered and purified. Extraction and adsorption are the
processes that are exclusively categorised as techniques
for recovery of the product.
Choice of recovery process is based on the following criteria:
1. intracellular or extracellular location of the product.
2. concentration of product in the culture fluid.
3. physical and chemical properties of the desired
product.
4. minimal acceptable standard of purity.
5. impurities in the culture fluid.
The separation of a component from a liquid mixture by
treatment with a solvent in which the desired component
is preferentially soluble is known as liquid-liquid
extraction. The specific requirement is obtaining a high
percentage extraction of product but concentrated in a
smaller volume of solvent. Efficient extraction requires
choosing a suitable solvent for extraction and optimising
conditions of temperature, pH, light, etc. After complete
extraction, the solute rich phase is called the extract and
the residual liquid from which solute has been removed is
called raffinate.
Purification: Purification techniques include
precipitation, chromatography, electrophoresis, membrane
separation, dialysis, reverse osmosis, ultra-filtration, etc.,
some of these technique are also used for the recovery
of products. In Class XI, you have already learnt about
chromatography and electrophoresis. Some of the other
purification techniques are discussed below:
• Precipitation is a technique widely used for

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 the recovery of proteins and antibiotics. It can be
induced by the addition of salts, organic solvents and
ultrafiltration.
Membrane Separation process can be classified
into three categories: microfiltration, ultrafiltration and
reverse osmosis. These are all pressure driven membrane
separation processes in which separation is achieved
through very small pore sizes. In microfiltration, the pore
size is 0.1 to 10 µm, while in ultrafiltration, it ranges from
0.01 — 0.1 µm.
Table 10.1: Different types of filtration processes

Molecular Pressure
Process Size Cutoff Material Retained
Wt. Cutoff Drop (psi)

Suspended material
Microfiltration 0.01–10 µm <1000,000 Da 10
(bacteria, etc.)
300-300,000 Biological, colloids,
Ultrafiltration 0.01–0.1 µm 10–100
Da macromolecules

Reverse 0.001 µm All suspended and

<300 Da 100–800
osmosis or <1µm dissolved materials

Protein products are under the range of molecular

cut-off for ultrafiltration.
If saline water is separated from pure water by a
semipermeable membrane, osmosis occurs, i.e., water
molecules move from pure water phase to saline water

p
p p

Reverse
Osmosis
Osmotic
Pressure

Pure Saline Pure Saline

Pure (a) Saline water (b) water water (c) water
water water

Fig. 10.7: Reverse Osmosis: Pressure driven membrane separation processes

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 phase [Fig. 10.7(a)]. As the water moves to saline phase,
its pressure increases. This pressure is called osmotic
pressure [Fig. 10.7(b)]. In reverse osmosis (RO), pressure
is applied onto a salt containing phase, which drives water
molecules in reverse direction, that is, from salt containing
phase to pure water phase [Fig. 10.7(c)]. The pressure
required for the movement of water in reverse direction
is slightly larger than the osmotic pressure as the solvent
flux takes place in the direction against the concentration
gradient [Fig. 10.7(c)].
Dialysis is an operation used for the removal of
low-molecular weight (MW) solutes, such as organic acids
(MW = 100–500 Da) and inorganic ions (molecular weight =
10–100 Da) from a solution through
a selectively permeable membrane.
A well-known example is the use of
dialysis membranes to remove urea
(MW = 60) from urine in artificial
kidney (dialysis) devices.
As depicted in Fig. 10.8, the
dialysis membrane separates two
phases containing low and high
molecular weight solutions. Since,
the cut-off size of pores of a dialysis
membrane is very small, only low
Semi-permeable Membrane
molecular weight molecules move
Fig. 10.8: Dialysis from a high to low concentration
region. At equilibrium, the chemical
potentials of diffusing compounds on
both sides of a membrane are equal.

10.4 BIOPROCESSING AND BIOMANUFACTURING

OF DESIRED PRODUCTS
These days bioprocessing industries have successfully
provided a number of valuable products from primary
(amino acids and organic acids) as well as secondary
metabolites (antibiotics) using microorganisms, animal
and plant cells as well as their constituents. Some of
the examples are: production of alcohols, antibiotics,
amino acids, organic acids, enzymes, vitamins, vaccines,
recombinant proteins, pigments, plant alkaloids, etc.,
(Table 10.2). These products have now become an integral
part of our day-to-day life. Some of these are described in
Table 10.3.

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 Since time immemorial, yeasts have been used for
the production of ethanol. A common species of yeast
Saccharomyces cerevisiae, commonly called brewer’s or
baker’s yeast, is used in the fermentation of malted cereals
and fruit juices for ethanol production. Depending upon
the type of raw material and the processes, a variety of
alcoholic drinks are obtained. Wine and beer are produced
without distillation, whereas whisky, brandy and rum are
produced by distillation of the fermented broth.
Table 10.2: Examples of variety of bioprocessing products

Types Products

Cell biomass Baker’s yeast, single cell protein

Extracellular Alcohols, organic acids, amino acids, enzymes, antibiotics

Intracellular Recombinant DNA protein

Table 10.3: Some of the major products of bioprocesses at commercial level

Microorganisms or Plants or
Products Category
Animals cells used
Ethanol Alcohol Saccharomyces cerevisiae

L-glutamic acid Amino acids Corynebacterium glutamicum

Lactic acid Organic acid Lactobacillus delbrueckii

Proteases Enzymes Bacillus spp.

Pectinase Enzymes Aspergillus niger

Penicillin Antibiotics Penicillium chrysogenum

Propionibacterium shermaniior,
B12 Vitamins
Pseudomonas denitrificans

Diphtheria vaccine Vaccines Corynebacterium diphtheriae

Recombinant
Insulin Recombinant Escherichia coli
Proteins

Pigments (quinone
Shikonin derivatives or Lithospermum erythrorhizon
naphthaquinone)

Taxol Plant alkaloids Taxus brevifolia

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 Antibiotics production is another significant
contribution of bioprocessing and biomanufacturing
towards the welfare of human society. It has already been
discussed earlier in this chapter as to how the penicillin,
the first antibiotic, was discovered which in fact was a
chance discovery. After penicillin, other antibiotics were
also purified from other microbes.
Amino acids, such as lysine and glutamic acid, are useful
in food industry as nutritional supplements and flavour
enhancing compounds, respectively. Production of amino
acids is typically carried out by mutants that have reduced
the capability to synthesise a specific amino acid or a key
intermediate. Mutants of Corynebacterium glutamicum are
used for the commercial production of glutamic acid and
lysine. Certain chemicals, such as organic acids, enzymes
and several other bioactive molecules, are produced
commercially in industry through bioprocessing. Several
species of microorganisms are used in the bioprocessing
of a number of organic acids. A fungal species Aspergillus
niger was used for citric acid production, and species of
bacteria Acetobacter aceti was used for the production of
acetic acid; Clostridium butylicum for the production of
butyric acid and Lactobacillus sp. for the production of
lactic acid.
Enzymes are also produced commercially through
bioprocessing. Lipases are used in detergent formulations
and are helpful in removing oily stains from the laundry.
You must have noticed that bottled fruit juices bought
from the market are clearer as compared to home-made
ones. This is because the bottled juices are clarified by the
use of pectinases and proteases. Proteases are also used
in leather industry. Bioprocessing of these enzymes at
commercial level uses a variety of fungal genera including
the species of Aspergillus, Bacillus, Mucor, Trichoderma, etc.
Streptokinase, produced using Streptococcus species and
modified by genetic engineering, is used as a ‘clot buster’
for removing clots from the blood vessels of patients who
have undergone myocardial infarction leading to heart
attack. Another bioactive molecule cyclosporin A, used as
an immunosuppressive agent in organ-transplant patients,
is produced using the fungus Trichoderma polysporum.

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 Statins produced by the yeast Monascus purpureus have
been commercialised as blood-cholesterol lowering agents.
It acts by competitively inhibiting the enzyme responsible
for the synthesis of cholesterol.
Several vitamins specifically B12 and riboflavin
are commercially produced by fermentation using
microorganisms. Vitamin B12 was first obtained as a
by-product in the production of various antibiotics—
streptomycin, chloramphenicol, or neomycin, using the
fermentation of bacterial genera Streptomyces. Later,
high-yielding strains of Propionibacterium freudenreichii,
Pseudomonas denitrificans, Bacillus megaterium and
Streptomyces olivaceus were developed for the production
of vitamin B12.
Riboflavin is commercially produced through
biotransformation as well as fermentation. In
biotransformation, glucose is first converted to D-ribose
by mutant strains of Bacillus pumilus. The D-ribose so
produced is converted to riboflavin by chemical reactions.
In acetone-butanol fermentation using Clostridium
acetobutylicum and Clostridium butylicum, riboflavin is
formed as a byproduct. Commercial production of riboflavin
is predominantly carried out by direct fermentation using
the ascomycetes. High-yielding strains of Ashbya
gossypii is preferred due to its high producing capability
of riboflavin.
Vaccines, the single most important health weapon,
have also been produced through bioprocess using various
cell or microbial cultures. Corynebacterium diphtheria
is used in the process for the production of diphtheria
toxin, which in turn is processed as diphtheria toxoid
and then to diphtheria toxoid vaccine. The cell-based
vaccine manufacturing process uses cells from mammals
to culture the influenza virus for vaccine production.
Various pharmaceutical companies use different sources
of mammalian cell cultures for the vaccine manufacturing
process.
Plant cell and tissue culture have long been used in
bioprocess for commercial production of a variety of
biochemicals, such as pigments, quinone derivatives, plant
alkaloids, etc., which have been used in a variety of tasks,

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 such as dying, clothes, food additives, pharmaceutics,
etc. The bioprocess for the production of dye shikonin was
commercially developed for the first time using cell culture
of plant species Lithospermum erythrorhizon. Successful
commercial production of berberine, ginseng, saponin and
taxol are the examples of bioprocessing that uses plant cell
and tissue culture of Coptis japonica, Panax ginseng and
Taxus brevifolia.
After having cloned the gene of interest and having
optimised the conditions to induce the expression of the
target protein, one has to consider producing it on a large
scale. A protein encoding gene expressed in a heterologous
host leads to the synthesis of desired biological product
which is a recombinant protein. Cells harbouring
cloned genes of interest may be grown on a small scale
in a laboratory. The culture may be used for extracting
the desired protein and then purifying it by using different
separation techniques. The recombinant DNA method is
used for large scale production of human insulin. Two-
phase cultivation process is followed for the production
of human insulin; a glycerol batch and a continuous
methanol fed-batch.
This way in industrial applications, a bioprocess is run
with optimised process parameters for high production
of desired compounds. Many compounds are in pipeline
and the researches are going on all over the world for
commercial production of the desired bioactive compounds
from living organisms.

SUMMARY
• There are various metabolic processes in living systems
which are responsible for the synthesis of many metabolites
which can be classified into primary and secondary.
• Primary metabolites are essential for the growth and
development of living organisms whereas, secondary

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 metabolites have diverse functions in defense system,
tolerance to abiotic stress, etc.
• Secondary metabolites are used in many industries, such
as pharmaceuticals, cosmetics, drugs, food additives, etc.
However, these compounds are synthesized in very small
amounts in the natural system. Therefore, efforts are
being made to scale up the production of these beneficial
metabolites using bioprocess engineering.
• Bioreactor or fermenter is an engineered vessel which may
provide optimum conditions for the product formation and
based on the requirement , different types of bioreactors may
be configured in bioprocessing.
• Bioprocessing can be operated through two stages: upstream
processing and downstream processing.
• In upstream processing, formulation and sterilisation of the
media and equipment take place along with the production
of pure, healthy and active culture for inoculation.
• The growth of organisms under optimum conditions for
desired product formation takes place in a bioreactor or a
fermenter.
• There are three modes of operations in bioprocessing:
(i) batch (closed vessel system), (ii) (fed-batch) (growth
limiting substrate is fed intermittently or continuously) and
(iii) continuous (growth limiting substrate is fed
continuously).
• In downstream processing, the product is recovered and
purified using various techniques, such as reverse osmosis,
distillation, drying, etc.
• Many desired products of animals, plants and microbial
origin have been commercialised till date.

EXERCISES
1. Differentiate between primary and secondary metabolites
on the basis of their functions with example.
2. Explain the challenges encountered during the
development of a bioprocess.
3. Describe briefly the design and components of a typical
bioreactor and their applications.

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 4. Explain the basic operational stages of a bioprocess using
concept map.
5. Describe briefly the following:
(a) upstream processing
(b) downstream processing
6. Explain the recovery and purification process of an
intracellular product with the help of a flow diagram.
7. Write short notes on the following:
(a) reverse osmosis
(b) dialysis
8. Match the following:
(a) Agitator (i) Breaking the vortex formation
(b) Sparger (ii) Provides area for circulation of
water of desired temperature
(c) Baffle (iii) Helps in mixing the contents
(d) Jacket (iv) Provides adequate and
continuous supply of air
9. A culture in a closed vessel to which no additional
medium is added is called ___________________ culture.
(a) Continuous
(b) Batch
(c) Fed-batch
(d) Semi continuous
10. Assertion: Secondary metabolites are used in defense
against pathogens, phytoplanktons, improving tolerance
to abiotic, etc.
Reason: Secondary metabolites are intermediate or
indirect products.
(a) Both assertion and reason are true and the reason is
the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is
not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.

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