the various processes used for the actual recovery of useful products from fermentation or any other...

• The various processes used for the actual recovery of useful products from fermentation or any other process together constitute ‘downstream processing’

• Mainly employed for isolation and purification of biomolecules

• It Can be divided into following stages:1)Separation of particles2)Disintegration of cells3)Extraction 4)Concentration 5)Purification6)Drying

Primary recovery operation Separate whole cells from culture broth,

removal of cell debris , collection of protein ppt. etc.

Includes

a)Filtration

b)Centrifugation

c)Flocculation

d)Floatation

A mechanical operation used for the separation of solids from fluids (liquids or gases) by interposing a medium to fluid flow through which the fluid can pass, but the solids in the fluid are retained.

Used for separation of filamentous fungi and filamentous bacteria eg.streptomycetes and often yeast flocs.

Uses pressure created by overpressure or vacuum. Rate depends on: filter area, fluid viscosity and

resistance generated by filter cake

Commonly used: Rotary drum filters Rotating drum with partial vacuum Drum rotates through vacuum Cells form coating on drum Cells scraped off to prevent blocking

ROTARY DRUM VACUUM FILTERS

Use of the centrifugal force for the separation of mixtures

More-dense components migrate away from the axis of the centrifuge

Less-dense components migrate towards the axis.

Used to separate bacteria and protein

Used when bacterial cells are not separated by centrifugation

Flocculation –sticking together of cells Induced by:- inorganic salts and organic Poly

electrolytes Floatation – used where flocculation is not

effective

It involves introduction of fine bubbles Fine gas bubbles are created by: Sparger Release of over pressure Electrolysis Gas bubbles adsorb to and surround the cells,

raising them to surface of medium in form of foam (floatation)

Fatty acids and amines promote stable foam formation

Disruption of microbial cells is difficult due to:

Small size Strong cell wall High osmotic pressure

It is achieved by : Mechanical means Lysis Drying

Uses shear ,pressure and pressure release

Shear –grinding in ball mill

--colloid mill Pressure –homogenizer

-ultrasound Cell suspension is forced through fine

nozzle Cells disintegrate due to shear

Widely used method Dried by adding cells into large excess of

cold acetone Followed by extraction Extracted using buffer or salt solution Induces changes in cell wall structure

which facilitate extraction

Lysis can be achieved by two means:-

a)chemical means

b)lytic enzymes Chemical means:-salts, surfactants,

osmotic shock, freezing Lytic enzymes:-lysozymes

Process of recovering a compound or group of compound from a mixture or from cells into a solvent phase is called extraction

Involves both separation as well as concentration of product

Useful for recovery of lipophilic substances and in antibiotic recovery

Early step after cell separation

Employs two immiscible liquids into which product is differentially soluble

Smaller volumes of solvent are used for repeated extraction of a given sample

Back extraction tends to increase sensitivity of extraction

Some concentration occur during extraction process

Final concentration may be achieved by:- Evaporation Membrane filteration Ion exchange methods Adsorption methods

Used in case of solvent extraction Efficient arrangements are made for recovery of

evaporated solvent to reduce cost

For low grade products evaporation of whole broth is done using spray drier

Spray drying

Requires use of heat to evaporate water – unsuitable for most proteins

Generally achieves both concentration and separation of the products on the basis of size

It includes:- microfiltration ,ultra filtration , reverse osmosis and electro dialysis

Micro and ultra filtration separates molecules of diff sizes

Reverse osmosis $ electro dialysis separate molecules of same size

On basis of charge these are of two types:-

a)Anion exchangers –have positively charged groups e.g. Diethyl aminoethyl, diethyl ammonium salt etc.

b) Cation exchangers:-having negatively charged groups e.g. Caboxymethyl sulfonate

Porous polymers Without ionization Compounds adsorb to resin in non ionised state Porosity determines surface availability for

adsorption Resins may be :-a) Apolar – e.g. Styrene divinyl benzene b)Polar : e.g. Sulphoxide , amide etc.c)Semipolar :- eg. Acrylic esters The product from such resins are recovered by

solvent extraction , changed pH etc.

Aims at obtaining product in highly purified state

It is achieved by:- Crystallisation Chromatographic procedures

Process of formation of solid crystals precipitating from a solution, melt or more rarely deposited directly from a gas.

Chemical solid-liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs.

Mainly used for purification of low molecular weight compounds eg. In case of antibiotics like penicillin G

Final stage in purification of products

Used for purification of low molecular wt. compounds from mixture of similar molecules

It includes:-a)Adsorption chromatographyb)Ion exchange chromatographyc)Gel permeation chromatographyd)Affinity chromatography

Separates molecules due to their differential affinities for the surface of solid matrix

Eg. Of solid matrix are- silica gel, hydroxyapetite or an organic polymer

Ion exchange chromatography

• Involves binding and separation of proteins based on charge-charge interactions

• Proteins bind at low ionic strength, and are eluted at high ionic strength

Use molecules called effectors Product has high and specific affinity for

effectors Effector is immobilised on water insoluble

carrier Carrier is packed in column through which

mixture is passed Effector binds to only those molecules for which it is specific and retains in the column Later recovered by elution using buffer

solution of specific pH

Also known as ‘size exclusion chromatography’ and ‘gel filtration chromatography’

Separates molecules on the basis of molecular size

Separation is based on the use of a porous matrix. Small molecules penetrate into the matrix more, and their path length of elution is longer.

Large molecules appear first, smaller molecules later

Last step in DSP Makes product suitable for handling and

storage Should be accomplished with minimum rise in

temperature It involves

a)Vaccum dryingb)Spray drying

c)Freeze drying

Solution or slurry to be dried is atomized by nozzle or rotating disc

Hot air current is passed (150-250.c) Drying is so rapid that temp. of particles

remain low Used for enzymes ,food products and

antibiotics

Uses both heat and vacuum Can be applied both in batch mode and

continuous mode More effective

Liquor to be dried is first frozen Water is sublimed from frozen mass Low pressure is maintained to promote sublimation Energy for sublimation is provided by heated plates and

radiation onto the surface Temperature is regulated by regulating pressure in drying

chamber Most gentle method of drying Used mainly out of all Used for many pharmaceutical products like vaccines ,

enzymes etc.

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