1. Downstream processing :-
Definition :-
The various stages of processing that occur after
completion of fermentation or bioconversion stage of
seperation, purification and packaging of products.
2. Stages in downstream processing :-
Removal of insoluble
Product isolation
Product purification
Product polishing.
The recovery and the purification of biosynthetic products, particularly
pharmaceuticals, from natural sources such as animal or plant tissue
or fermentation broth, including the recycling of salvageable components
and the proper treatment and disposal of waste.
3. Removal of insolubles:-
Seperation of cells , cell debris or other particulate matter.
Stages :-
Filtration
Centrifugation
Sedimentation
Floccuation
Gravity setting.
4. Filtration :-
This is a kind of mechanical operation that used for seperation of
solids from fluids (liquids or gases )by interposing a medium to
porous membrane through which the fluid can pass but solids in
the fluuds are retained .
The solids particles that deposited on the filter form of layer which
is known as filter cake.
All the solids particles are feeded and stopped by the cake and the
cake grows at qhich particles are brought to its surface .
All the fluids goes through the cake and fluid medium..
5.
6. Continous Rotary Vaccum Filter :-
It is one of the most commonly used type of filter
fermentation..
The drum is pre coated prior to filtration.
A small agent of coagulating is added to the broth before it is
pumped before it is pumped into the filter.
The drum rotates under vaccum and a thin layer of cells sticks
to the drum.
The thickness of the layer increases in the section designed
for forming the cakes.
7.
8. Points to be considered while
selecting the filter medium :-
Ability to build the soild.
Minimum resistance to flow the filtrate.
Resistance to chemical attack.
Minimum cost.
Long life.
9. Centrifugation :-
Centrifugation is used to separate particles of 100 – 0.1 micrometer
from liquid by gravitational forces.
It depends on particles size , density difference between the cells and
the broth and viscosity of broth.
Use of the centrifugal force for the seperation of mixtures.
More dense components migrate away from the axis of centrifuge .
Less dense components migrate towards the axis .
Types of centrifuges used are
Tubular bowl centrifuge
Multichamber centrifuge
Disc bowl centrifuge etc………….
10.
11. Sedimentation :-
It is applicable only for large particles greater than 100
micrometer flocs.
It is slow process and takes ~ 3hrs.
It is used in process like activated sludge effluent treatment.
It is free settling process depends only on gravity.
Particles settling is a high particle density suspension
(hindered settling ).
Mostly applicable in every fermentors industries which are
large scale or small scale……
12.
13. Flocculation :-
Process where a solute comes out of solution in the form of
flocs or flakes.
Particles finer than 0.1 micrometer in water remain
continoulsy in motion due to electrostatic charge which cause
them to repel each other.
Once their electrostatic charge in neutralized ( use of
coagulant ) the finer particles start to collide and combine
together.
These larger and heavier flocs of particels are called flocs.
14. Cell disruption :-
Some product are intracellular including many enzymes and
proteins required to disrupt the cell amd rrlease these products.
E.g :- yeast
Cell disruption can be achieved by both mechanical and non
mechanical methods.
Mechanical :-
Sonication and liquid shear homogenization etc…..
Non mechanical methods :-
Autolysis and osmosis shock etc…….
15. Solvent extraction :-
Reducing the volume of material to be handled and
concentrating the product.
Removal of those components whose properties vary
markedly from that of desired product …
Water Is the Che if impurity .
Liquid liquid extraction
Adsorption
Precipitation
Are some of the unit operations involved in them….
16. Liquid–liquid extraction (LLE) :-
Liquid–liquid extraction (LLE), also known as solvent
extraction and partitioning, is a method to separate
compounds or metal complexes, based on their
relative solubilities in two different immiscible liquids, usually
water (polar) and an organic solvent (non-polar).
There is a net transfer of one or more species from one liquid
into another liquid phase, generally from aqueous to organic.
The transfer is driven by chemical potential, i.e. once the
transfer is complete, the overall system of protons and
electrons that make up the solutes and the solvents are in a
more stable configuration (lower free energy). The solvent
that is enriched in solute(s) is called extract.
The feed solution that is depleted in solute(s) is called
17.
18. Adsorption
Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or
dissolved solid to a surface.
This process creates a film of the adsorbate on the surface of the adsorbent.
This process differs from absorption, in which a fluid (the absorbate)
is dissolved by or permeates a liquid or solid (the absorbent), respectively.
Adsorption is a surface phenomenon, while absorption involves the whole
volume of the material. The term sorption encompasses both processes,
while desorption is the reverse of it.
The separation is based on the interaction of the adsorbate with the
adsorbent. The adsorbent is the surface and adsorbate is the molecules of
interest which are getting adsorbed on the adsorbent.
19. Ultrafiltration
Ultrafiltration (UF) is a pressure-driven barrier to suspended solids,
bacteria, viruses, endotoxins and other pathogens to produce water with
very high purity and low silt density.
Ultrafiltration (UF) is a variety of membrane filtration in which hydrostatic
pressure forces a liquid against a semi permeable membrane.
The variety of membrane filtration in which forces
like pressure or concentration gradients lead to a separation through
a semipermeable membrane.
Suspended solids and solutes of high molecular weight are retained in
them called retentate, while water and low molecular weight solutes pass
through the membrane in the permeate (filtrate).
This separation process is used in industry and research for purifying and
concentrating macromolecular (103 - 106 Da) solutions,
especially protein solutions.
20. Precipitation :-
Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides.
Further, precipitation technique can also be employed for the removal of certain
unwanted byproducts e.g. nucleic acids, pigments.
Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic),
besides alteration in temperature and pH are used in precipitation.
In addition to these non-specific protein precipitation reactions (i.e. the nature of the
protein is unimportant), there are some protein specific precipitations e.g., affinity
precipitation, ligand precipitation.
The most commonly used salt is ammonium sulfate, since it is highly soluble, nontoxic to
proteins and low-priced.
Ammonium sulfate increases hydrophobic interactions between protein molecules that
result in their precipitation.
The precipitation of proteins is dependent on several factors such as protein
concentration, pH and temperature.
22. Gel-filtration chromatography:
This is also referred to as size-exclusion chromatography. In this technique,
the separation of molecules is based on the size, shape and molecular
weight.
The sponge-like gel beads with pores serve as molecular sieves for
separation of smaller and bigger molecules.
A solution mixture containing molecules of different sizes (e.g. different
proteins) is applied to the column and eluted.
The smaller molecules enter the gel beads through their pores and get
trapped.
On the other hand, the larger molecules cannot pass through the pores
and therefore come out first with the mobile liquid (Fig. 20.7).
At the industrial scale, gel-filtration is particularly useful to remove salts
and low molecular weight compounds from high molecular weight
products.
23.
24. Ion-exchange chromatography:
It involves the separation of molecules based on their surface charges.
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).
The most commonly used cation-exchangers are Dowex HCR and
Amberlite IR, the anion-exchangers are Dowex SAR and Amberlite IRA.
In ion-exchange chromatography, the pH of the medium is very crucial,
since the net charge varies with pH.
In other words, the pH determines the effective charge on both the
target molecule and the ion-exchanger.
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.
Ion-exchange chromatography is useful for the purification of antibiotics,
besides the purification of proteins.
25. Affinity chromatography:
This is an elegant method for the purification of proteins from a complex
mixture.
Affinity chromatography is based on an interaction of a protein with an
immobilized ligand.
The ligand can be a specific antibody, substrate, substrate analogue or an
inhibitor. The immobilized ligand on a solid matrix can be effectively used
to fish out complementary structures.
The protein bound to the ligand can be eluted by reducing their
interaction.
This can be achieved by changing the pH of the buffer, altering the ionic
strength or by using another free ligand molecule.
The fresh ligand used has to be removed in the subsequent steps
26. Product formulations :-
Formulation broadly refers to the maintenance of activity and stability of a
biotechnological products during storage and distribution.
The formulation of low molecular weight products (solvents, organic acids) can be
achieved by concentrating them with removal of most of the water.
For certain small molecules, (antibiotics, citric acid), formulation can be done by
crystallization by adding salts.
Proteins are highly susceptible for loss of biological activity; hence their formulation
requires special care.
Certain stabilizing additives are added to prolong the shelf life of protein. The stabilizers
of protein formulation include sugars (sucrose, lactose), salts (sodium chloride,
ammonium sulfate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol).
Proteins may be formulated in the form of solutions, suspensions or dry powders.
