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ROLE OF DOWNSTREAM PROCESSING IN BIOTECHNOLOGY
Unit operations in Downstream processing
The operation involved in isolation and purification of a product from a suitable organism to
develop useful product is collectively known as down-stream processing (Figure 1)
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No specialized process is required to collect secreted product but several
methods have been developed to released the product through cell disruption
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Figure 1: An over-view of different steps in down-stream processing

This article throws light upon the five stages in downstream processing
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In Fig
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1
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Several methods are
in use for solid-liquid separation
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Flotation:
➢ When a gas is introduced into the liquid broth, it forms bubbles
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These bubbles rise to the foam layer which can be
collected and removed
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g
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Flocculation:
➢ In flocculation, the cells (or cell debris) form large aggregates to settle down for easy removal
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➢ Addition of flocculating agents (inorganic salt, organic polyelectrolyte, mineral hydrocolloid)
is often necessary to achieve appropriate flocculation
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The
efficiency of filtration depends on many factors—
•
•
•

the size of the organism,
presence of other organisms,
viscosity of the medium, and temperature
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Depth Filters:
They are composed of a filamentous matrix such as glass wool, asbestos or filter paper
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Filamentous fungi can be removed
by using depth filters
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Bacteria
from culture medium can be removed by absolute filters
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5-10µm
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The equipment is simple with low power
consumption and is easy to operate
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3)
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This filter cake can be easily removed
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However, clogging of
filters is a major limitation
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4)
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This reduces the clogging process and hence better than the static
filtration
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These are microfiltration, ultrafiltration and reverse osmosis
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Thus, centrifugation is mostly used for separating solid
particles from liquid phase (fluid/particle separation)
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The different types of centrifuges are depicted in Fig
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Tubular bowl centrifuge (Fig
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Tubular bowl centrifuge
can be operated at a high centrifugal speed, and can be run in both batch or continuous mode
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Disc centrifuge (Fig
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The feed/slurry is fed through
a central tube
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Multi-chamber centrifuge (Fig
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It consists of several chambers
connected in such a way that the feed flows in a zigzag fashion
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The force is much higher in the periphery chambers, as a
result smallest particles settle down in the outermost chamber
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5D):
It is composed of a rotating horizontal bowl tapered at one end
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The solids are deposited
on the wall of the bowl which can be scrapped and removed from the narrow end
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Release of Intracellular Products:

As already stated, there are several biotechnological products (vitamins, enzymes) which are
located within the cells
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The microorganisms or other cells can be
disintegrated or disrupted by physical, chemical or enzymatic methods
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6
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For instance, Gram-negative bacteria and
filamentous fungi can be more easily broken compared to Gram-positive bacteria arid yeasts
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Ultra sonication:
Ultrasonic disintegration is widely employed in the laboratory
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Osmotic shock:
This method involves the suspension of cells (free from growth medium) in 20% buffered sucrose
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Osmotic shock is used for the release of
hydrolytic enzymes and binding proteins from Gram-negative bacteria
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But this technique can
be used only for a very few heat-stable intracellular products
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This sudden release of high pressure creates a liquid shear
that can break the cells
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The cells
break as they are forced against the wall of the vessel by the beads
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Under optimal conditions, one can expect a maximal breakage of about 80% of the
cells
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7
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To this shaft are
fitted radial agitators
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The cell suspension is added through
the inlet and the disrupted cells come out through the outlet
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Mechanical and non-mechanical methods:
Among the physical methods of cell disruption described above, ultra sonication, high-pressure
homogenization, impingement and grinding with glass beads are mechanical while osmotic shock
and heat shock are non-mechanical
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Chemical methods of cell disruption:
Treatment with alkalies, organic solvents and detergents can lyse the cells to release the contents
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However, the alkali
stability of the desired product is very crucial for the success of this method e
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, recombinant
growth hormone can be efficiently released from E
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Organic solvents:
Several water miscible organic solvents can be used to disrupt the cells e
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, methanol, ethanol,
isopropanol, butanol
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The organic solvent toluene is frequently used
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Detergents:
Detergents that are ionic in nature, cationic-cetyl trimethyl ammonium bromide or anionic-sodium
lauryl sulfate can denature membrane proteins and lyse the cells
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g
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The
problem with the use of detergents is that they affect purification steps, particularly the salt
precipitation
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Enzymatic methods of cell disruption:
➢ Cell disruption by enzymatic methods has certain advantages i
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, lysis of cells occurs under
mild conditions in a selective manner
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➢ Lysozyme is the most frequently used enzyme and is commercially available (produced from
hen egg white)
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The Gram- positive bacteria (with high content of cell wall mucopeptides) are more susceptible
for the action of lysozyme
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As the
cell wall gets digested by lysozyme, the osmotic effects break the periplasmic membrane to
release the intracellular contents
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For the lysis
of yeast cell walls, glucanase and mannanase in combination with proteases are used
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Stage #3
...
) usually contains 80-98%
of water
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The water has to be removed to achieve
the product concentration
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The
actual procedure adopted depends on the nature of the desired product (quality and quantity to be
retained as far as possible) and the cost factor
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The evaporators, in
general, have a heating device for supply of steam, and unit for the separation of concentrated
product and vapour, a condenser for condensing vapour, accessories and control equipment
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Some of the important types of evaporators in common use are briefly described
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As the steam is supplied, the liquid gets
concentrated and becomes viscous
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Falling film evaporators are suitable for removing water from viscous products of
fermentation
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Centrifugal forced film evaporators:
These equipment evaporate the liquid very quickly (in seconds), hence suitable for concentrating
even heat-labile substances
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Liquid-Liquid Extraction:
➢ The concentration of biological products can be achieved by transferring the desired product
(solute) from one liquid phase to another liquid phase, a phenomenon referred to as liquidliquid extraction
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➢ The efficiency of extraction is dependent on the partition coefficient i
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the relative
distribution of a substance between the two liquid phases
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Extraction of low molecular weight products:
By using organic solvents, the lipophilic compounds can be conveniently extracted
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Extraction of lipophilic products can be done
by the following techniques
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This technique is used for extraction of non-ionising compounds
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Certain antibiotics can be
extracted by this procedure
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g
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Reactive extraction procedure is
quite useful for the extraction of certain compounds that are highly soluble in water (aqueous
phase) e
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, organic acids
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SCFs are intermediates between gases and liquids and exist as fluids
above their critical temperature and pressure
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Supercritical fluid extraction is rather expensive,
hence not widely used (SCF has been used for the extraction of caffeine from coffee beans, and
pigments and flavor ingredients from biological materials)
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Organic solvents cannot be used for protein extraction, as they lose their biological
activities
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Aqueous two-phase systems (ATPS):
➢ They can be prepared by mixing a polymer (e
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, polyethylene glycol) and a salt solution
(ammonium sulfate) or two different polymers
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➢ Cells and other solids remain in one phase while the proteins are transferred to other phase
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The separation takes much longer time by ATPS
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It can be conveniently used for the separation of biomolecules and particles, and for the
concentration of fluids
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➢ Membranes used in filtration are made up of polymeric materials such as polyethersulfone and
polyvinyl di-fluoride
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Membrane adsorbers:
They are micro- or macro porous membranes with ion exchange groups and/or affinity ligands
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Such proteins can be eluted by
employing solutions in chromatography
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Pervaporation is quite useful for the extraction, recovery
and concentration of volatile products
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Precipitation:
Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides
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g
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Neutral
salts, organic solvents, high molecular weight polymers (ionic or non-ionic), besides alteration in
temperature and pH are used in precipitation
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e
...
g
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Neutral salts:
➢ The most commonly used salt is ammonium sulfate, since it is highly soluble, nontoxic to
proteins and low-priced
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➢ The precipitation of proteins is dependent on several factors such as protein concentration, pH
and temperature
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They reduce the dielectric constant of the medium and enhance electrostatic interaction between
protein molecules that lead to precipitation
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Non-ionic polymers:
Polyethylene glycol (PEG) is a high molecular weight non-ionic polymer that can precipitate
proteins
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PEG does not denature proteins, besides being non-toxic
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They form
complexes with oppositely charged protein molecules that causes charge neutralisation and
precepitation
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Change in pH:
Alterations in pH can also lead to protein precipitation
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g
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Precipitation by ligands:
Ligands with specific binding sites for proteins have been successfully used for selective
precipitation
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In
the early days, activated charcoal was used as the adsorbent material
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The process of adsorption can be carried out by making a bed of adsorbent
column and passing the culture broth through it
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Stage # 4
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It is basically an analytical
technique dealing with the separation of closely related compounds from a mixture
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The stationary phase is the porous
solid matrix packed in a column (equilibrated with a suitable solvent) on to which the mixture of
compounds to be separated is loaded
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A single mobile
phase may be used continuously or it may be changed appropriately to facilitate the release of
desired compounds
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g
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The different types of chromatography techniques
Gel-filtration chromatography:
This is also referred to as size-exclusion chromatography
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The sponge-like gel beads with pores
serve as molecular sieves for separation of smaller and bigger molecules
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g
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The smaller molecules enter the gel beads through their pores and get trapped
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8)
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Ion-exchange chromatography:
It involves the separation of molecules based on their surface charges
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The most commonly used cation-exchangers are Dowex HCR and Amberlite IR, the anionexchangers are Dowex SAR and Amberlite IRA
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In other words, the pH determines the effective charge on both the target molecule and
the ion-exchanger
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Ion-exchange chromatography is
useful for the purification of antibiotics, besides the purification of proteins
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Affinity
chromatography is based on an interaction of a protein with an immobilized ligand
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The immobilized ligand on a
solid matrix can be effectively used to fish out complementary structures
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The protein
bound to the ligand can be eluted by reducing their interaction
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The differences in the
composition of hydrophobic amino acids in proteins can be used for their separation
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Stage # 5
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The formulation of low molecular weight products
(solvents, organic acids) can be achieved by concentrating them with removal of most of the water
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Proteins are highly susceptible for loss of biological activity; hence their
formulation requires special care
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It basically involves the transfer of
heat to a wet product for removal of moisture
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➢ Based on the method of heat transfer, drying devices may be categorized as contact,
convection, radiation dryers
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Spray drying:
➢ Spray drying is used for drying large volumes of liquids
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➢ The water evaporates and the solid particles are left behind
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This is
mainly because freeze-drying usually does not cause loss of biological activity of the desired
product
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In the
actual technique, the liquid containing the product is frozen and then dried in a freeze-dryer
under vacuum
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g
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Economics of DSP in Biotechnology
Bio-products include a wide range of chemicals, which may be broadly classified into three major
categories on the basis of market volume, market price and requirement of purity, these include
•

•

•

Very high value and low volume products such as therapeutic proteins and enzymes, factor
VIII, interferon, urokinase etc
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High value, low volume, high purity products such as diagnostic enzymes, human growth
hormones, tissue plasminogen activator, monoclonal antibodies and insulin produced in
tens or hundreds of kilograms
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Characteristic features of bioproducts
Characteristics
feature

Categories
I (Very high value
II (High value,
III
(Bulk
and
low
volume low volume products) industrial products)
products)
3

5

6

9

Market volume

0
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Type of microorganisms and their morphological features (size

and shape)

2
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Physical and rheological characteristics
Morphology of cells
➢ The cells and cell agglomerates exhibit a variety of shapes
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➢ Certain strains of bacteria form a slimy mass, which is difficult to separate from the liquid
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The slimy mass
may clog and foul the experiment
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The concentrations of biomass as
well as that of the products vary widely as shown below
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➢ The clarified broth after the removal of biomass is almost water like in its flow
characteristics for most fermentation
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➢ The viscosity of a broth is strongly influenced by the cell concentration as well as the cell
shape and to a minor extent by the changes in the concentration of nutrients and excreted
metabolites

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