Down Stream Processing:

Downstream processing refers to the recovery and purification of biosynthetic products,

Filtration:
Filtration is the most commonly used technique for separating the biomass and culture filtrate.
The efficiency of filtration depends on many factors— the size of the organism, presence of
other organisms, viscosity of the medium, and temperature. Several filters such as depth filters,
absolute filters, rotary drum vacuum filters and membrane filters are in use.
Depth Filters:
They are composed of a filamentous matrix such as glass wool, asbestos or filter paper. The
particles are trapped within the matrix and the fluid passes out. Filamentous fungi can be
removed by using depth filters.
Absolute Filters:
These filters are with specific pore sizes that are smaller than the particles to be removed.
Bacteria from culture medium can be removed by absolute filters.

Membrane Filters:
In this type of filtration, membranes with specific pore sizes can be used. However, clogging of
filters is a major limitation. There are two types of membrane filtrations—static filtration and
cross-flow filtration. In cross-flow filtration, the culture broth is pumped in a crosswise fashion
across the membrane. This reduces the clogging process and hence better than the static
filtration.

Centrifugation:
The technique of centrifugation is based on the principle of density differences between the
particles to be separated and the medium. Thus, centrifugation is mostly used for separating solid
particles from liquid phase (fluid/particle separation). Unlike the centrifugation that is
conveniently carried out in the laboratory scale, there are certain limitations for large scale
industrial centrifugation. There is a continuous feeding of the slurry and collection of clarified
fluid, while the solids deposited can be removed intermittently.

Tu
bular bowl centrifuge:
This is a simple and a small centrifuge, commonly used in pilot plants. Tubular bowl centrifuge
can be operated at a high centrifugal speed, and can be run in both batch or continuous mode.
The solids are removed manually.
Disc centrifuge:
It consists of several discs that separate the bowl into settling zones. The feed/slurry is fed
through a central tube. The clarified fluid moves upwards while the solids settle at the lower
surface.
Multi-chamber centrifuge:
This is basically a modification of tubular bowl type of centrifuge. It consists of several
chambers connected in such a way that the feed flows in a zigzag fashion. There is a variation in
the centrifugal force in different chambers. The force is much higher in the periphery chambers,
as a result smallest particles settle down in the outermost chamber.
Scroll centrifuge or decanter:
It is composed of a rotating horizontal bowl tapered at one end. The decanter is generally used to
concentrate fluids with high solid concentration (biomass content 5-80%). The solids are
deposited on the wall of the bowl which can be scrapped and removed from the narrow end.
Cell Disruption:
Physical methods of cell disruption:
The microorganisms or cells can be disrupted by certain physical methods to release the
intracellular products.
Ultra sonication:
Ultrasonic disintegration is widely employed in the laboratory. However, due to high cost, it is
not suitable for large-scale use in industries.
Osmotic shock:
This method involves the suspension of cells (free from growth medium) in 20% buffered
sucrose. The cells are then transferred to water at about 4°C. Osmotic shock is used for the
release of hydrolytic enzymes and binding proteins from Gram-negative bacteria.
Heat shock (thermolysis):
Breakage of cells by subjecting them to heat is relatively easy and cheap. But this technique can
be used only for a very few heat-stable intracellular products.
High pressure homogenization:
This technique involves forcing of cell suspension at high pressure through a very narrow orifice
to come out to atmospheric pressure. This sudden release of high pressure creates a liquid shear
that can break the cells.
Impingement:
In this procedure, a stream of suspended cells at high velocity and pressure are forced to hit
either a stationary surface or a second stream of suspended cells (impinge literally means to
strike or hit).
The cells are disrupted by the forces created at the point of contact.
The advantage with impingement technique is that it can be effectively used for disrupting cells
even at a low concentration.
Grinding with glass beads:
The cells mixed with glass beads are subjected to a very high speed in a reaction vessel. The
cells break as they are forced against the wall of the vessel by the beads. Several factors
influence the cell breakage-size and quantity of the glass beads, concentration and age of cells,
temperature and agitator speed. Under optimal conditions, one can expect a maximal breakage of
about 80% of the cells.
Chemical methods of cell disruption:
Treatment with alkalies, organic solvents and detergents can lyse the cells to release the contents.
Alkalies:
Alkali treatment has been used for the extraction of some bacterial proteins.
Organic solvents:
Several water miscible organic solvents can be used to disrupt the cells e.g., methanol, ethanol,
isopropanol, butanol. These compounds are inflammable; hence require specialised equipment
for fire safety. The organic solvent toluene is frequently used. It is believed that toluene dissolves
membrane phospholipids and creates membrane pores for release of intracellular contents.
Detergents:
Detergents that are ionic in nature, cationic-cetyl trimethyl ammonium bromide or anionic-
sodium lauryl sulphate can denature membrane proteins and lyse the cells.
Non-ionic detergents (although less reactive than ionic ones) are also used to some extent e.g.,
Triton X-100 or Tween.
The problem with the use of detergents is that they affect purification steps, particularly the salt
precipitation.
This limitation can be overcome by using ultrafiltration or ion-exchange chromatography for
purification.
Enzymatic methods of cell disruption:
Cell disruption by enzymatic methods has certain advantages i.e., lysis of cells occurs under mild
conditions in a selective manner. This is quite advantageous for product recovery.
Lysozyme is the most frequently used enzyme and is commercially available (produced from hen
egg white). It hydrolyses β-1, 4-glycosidic bonds of the mucopeptide in bacterial cell walls.