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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—
•
•
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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 maxima
capacity of the equipment is variable that may range from small laboratory scale to industrial scale
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Plate evaporators:
The liquid to be concentrated flows over plates
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Falling film evaporators:
In this case, the liquid flows down long tubes which gets distributed as a thin film over the heating
surface
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Forced film evaporators:
The liquid films are mechanically driven and these devices are suitable for producing dry product
concentrates
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In these evaporators, a centrifugal force is used to pass on the liquid
over heated plates or conical surfaces for instantaneous evaporation
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Besides concentration, this technique is also useful for partial purification of
a product
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e
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➢ The process of liquid-liquid extraction may be broadly categorized as extraction of low
molecular weight products and extraction of high molecular weight products
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However, it
is quite difficult to extract hydrophilic compounds
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Physical extraction:
The compound gets itself distributed between two liquid phases based on the physical properties
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Dissociation extraction:
This technique is suitable for the extraction of ionisable compounds
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Reactive extraction:
In this case, the desired product is made to react with a carrier molecule (e
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, phosphorus
compound, aliphatic amine) and extracted into organic solvent
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g
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Supercritical fluid (SCF) extraction:
This technique differs from the above procedures, since the materials used for extraction are
supercritical fluids (SCFs)
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Supercritical CO 2, with a low critical temperature
and pressure is commonly used in the extraction
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Extraction of high molecular weight compounds:
Proteins are the most predominant high molecular weight products produced in fermentation
industries
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They are extracted by using an aqueous two-phase systems or reverse micelles
formation
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g
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Water is the main component in ATPS, but the
two phases are not miscible
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The distribution of the desired product is based on its surface and ionic character and the nature
of phases
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Membrane Filtration:
➢ Membrane filtration has become a common separation technique in industrial biotechnology
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➢ The membrane filtration technique basically involves the use of a semipermeable membrane
that selectively retains the particles/molecules that are bigger than the pore size while the
smaller molecules pass through the membrane pores
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It is rather difficult to sterilize membrane filters
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Membrane adsorbers can bind to proteins and retain them
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Pervaporation:
This is a technique in which volatile products can be separated by a process of permeation through
a membrane coupled with evaporation
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However, this procedure has a limitation since it cannot be
used for large scale separation of volatile products due to cost factor
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Further, precipitation technique can also be
employed for the removal of certain unwanted byproducts e
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nucleic acids, pigments
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In addition to these non-specific protein precipitation
reactions (i
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the nature of the protein is unimportant), there are some protein specific
precipitations e
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, affinity precipitation, ligand precipitation
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➢ Ammonium sulfate increases hydrophobic interactions between protein molecules that result
in their precipitation
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Organic solvents:
Ethanol, acetone and propanol are the commonly used organic solvents for protein precipitation
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Since proteins are denatured by organic solvents, the
precipitation process has to be carried out below 0°C
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It reduces the quantity of water available for protein solvation and precipitates protein
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Ionic polymers:
The charged polymers such as polyacrylic acid and polyethyleneimine are used
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Increase in temperature:
The heat sensitive proteins can be precipitated by increasing the temperature
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Affinity precipitation:
The affinity interaction (e
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, between antigen and antibody) is exploited for precipitation of
proteins
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Adsorption:
The biological products of fermentation can be concentrated by using solid adsorbent particles
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In recent years, cellulosebased adsorbents are employed for protein concentration and for concentration of low molecular
weight compounds (vitamins, antibiotics, peptides) polystyrene, methacrylate and acrylate based
matrices are used
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The desired product, held by the adsorbent, can
be eluted
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Purification by Chromatography:
The biological products of fermentation (proteins, pharmaceuticals, diagnostic compounds and
research materials) are very effectively purified by chromatography
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Chromatography usually consists of a stationary phase and mobile phase
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The compounds are eluted by a mobile phase
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The eluate from the column can be monitored continuously (e
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protein
elution can be monitored by ultraviolet adsorption at 280 nm), and collected in fractions of definite
volumes
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In this technique, the separation of
molecules is based on the size, shape and molecular weight
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A solution mixture
containing molecules of different sizes (e
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different proteins) is applied to the column and eluted
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On the other hand,
the larger molecules cannot pass through the pores and therefore come out first with the mobile
liquid (Fig
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At the industrial scale, gel-filtration is particularly useful to remove salts and low
molecular weight compounds from high molecular weight products
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Ion-exchangers are of two
types (cation- exchangers which have negatively charged groups like carboxymethyl and
sulfonate, and anion- exchangers with positively charged groups like diethylaminoethyl (DEAE)
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In ion-exchange chromatography, the pH of the medium is very crucial, since the net charge varies
with pH
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The ionic bound molecules can be eluted from the matrix by changing the pH
of the eluant or by increasing the concentration of salt solution
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Affinity chromatography:
This is an elegant method for the purification of proteins from a complex mixture
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The ligand can
be a specific antibody, substrate, substrate analogue or an inhibitor
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In Table, some examples of ligands used for the purification of proteins are given
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Hydrophobic interaction chromatography (HIC):
This is based on the principle of weak hydrophobic interactions between the hydrophobic ligands
(alkyl, aryl side chains on matrix) and hydrophobic amino acids of proteins
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The elution
of proteins can be done by lowering the salt concentration, decreasing the polarity of the medium
or reducing the temperature
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Formulation:
Formulation broadly refers to the maintenance of activity and stability of biotechnological
products during storage and distribution
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For certain small molecules, (antibiotics, citric acid), formulation can be done by crystallization
by adding salts

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