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Factors for deciding the

extraction method

The value of the final product. The degree of purity required to make the final product

acceptable, bearing in mind its revenue-yielding potential. The chemical and physical properties of the product. The location of the product in the mixture i.e. whether it is

free within the medium or is cell-bound. The location and properties of the impurities. The cost-effectiveness of the available alternate isolation

procedures.


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purification of products in the

soluble portion


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Filtration

1.The rotary vacuum filter:


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Consists of a hollow rotating cylinderdivided into four partitions and covered

with a metal or cloth gauze. A vacuum is applied in the cylinder and as

it rotates the vacuum sucks liquid

materials from the shallow trough in whichthe rotating cylinder is immersed.

For thick slurries which are difficult to filter

(e.g. aminoglycoside broths) a thin layer offilter aid (e.g. Kiesselghur) is first allowedto be absorbed on the cylinder.


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Later the filter cylinder with its thin coating of thefilter aid is allowed to rotate in the trough inwhich the broth is now placed.

The rotating cylinder, the vacuum still on, iswashed with a sprinkle of water; a knife whoseedge is positioned just short of the layer of filter

aid scrapes off the solids picked up from thebroth. When it is used for easily filtered broth such as

in penicillin broth no filter aid is used.

Instead an arrangement of strings coupled with arelease of the vacuum in the segment of thecylinder helps release the material picked upfrom the broth.


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2.Plate and Frame Filters


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3.Pressure leaf filters


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Horizontal


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Vertical


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Centrifugation

Centrifuge when filtration is not asatisfactory separation method. Althoughexpensive when compared with a filter it

may be essential when:1. Filtration is slow and difficult.

2. The cells or other suspended matter must

be obtained free of filter aids.3. Continuous separation to a high standardof hygiene is required.


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In the enzyme isolation industry, however,centrifugation is preferred to filtration,

probably because unwanted cell debrisare quite efficiently removed by thismethod.

A large number of centrifuges areavailable in the market and a newfermentation industry or a change in the

production method of old processes mayrequire the use of centrifuges for primaryseparation.


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1.The Basket Centrifuge


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2.The Tubular-bowl Centrifuge


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3.The Multichamber Centrifuge


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Harvesting of Microbial Cells

Filtration or centrifugation. Because of its smallsize it will be necessary to consider the use:

Filter aids to improve filtration rates, while heat

and flocculation treatments are employed astechniques for increasing sedimentation rates incentrifugation.

Some potential developments in cell recovery

include the use of: electrophoresis Ultrasonic treatment to improve flocculation

characteristics


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Coagulation and Flocculation

Coagulation is the cohesion of dispersedcolloids into small flocs

flocs aggregate to form larger masses.

Induced by electrolytes (clay or activatedcharcoal)

Bacteria and proteins being negativelycharged colloids are easily flocculated byelectrolytes or polyelectrolyte


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Flocculants should have thefollowing properties

They must react rapidly with the cells. They must be non-toxic. They should not alter the chemical constituents of the cell. They should have a minimum cohesive power in order to

allow for effective subsequent water removal by filtration. Neither high acidity nor high alkalinity should result from

their addition. They should be effective in small amounts and be low in

cost. They should preferably be washable for reuse.


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4. Foam Fractionation


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The principle of foam fractionation is that in aliquid foam system the chemical composition of

a given substance in the bulk liquid is usuallydifferent from the chemical composition of somesubstance in the foam.

Foam is formed by sparging the bulk liquidcontaining the substance to be fractionated with

an inert gas. The gas is fed at the bottom of a tower and the

foam created overflows at the top carrying with itthe solutes to be fractionated.

This method has been used to collect a widerange of microorganisms and although mainlyexperimental it may be used on a large scale inindustry.


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5.Whole-broth Treatment

acetonebutanol fermentation, the wholeunseparated broth is stripped of its content ofthe required product.

Antibiotics streptomycin (using cationic-

exchange resin) and novobiocin (on an anionic resin.) The

antibiotics are eluted from the resins and thencrystallized.

This process saves the capital and recurrentexpense of the initial separation of solids fromthe broth.


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2.PRIMARY PRODUCT

ISOLATION

After separation of the broth into soluble andinsoluble fractions, the next process depends on thelocation of desired product as follows:

A. The cells themselves as in yeasts B. Bound to the mycelia or to bacterial cells as in thecase of bound enzymes or antibiotics. The cellsthen have to be disrupted with any of the severalways available heat, mechanical disruption, etc.

C. Extracellularly available or if it has been obtained byleaching with or without cell disruption then it istreated by one of the following methods:

Liquid extraction, dissociation extraction, sorption, or

precipitation.


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2.1Cell disruption

Cell disruption is a sensitive processbecause of the cell walls resistance to thehigh osmotic pressure inside them.

Difficulties arise from a non-controlled celldisruption, that results from an unhinderedrelease of all intracellular products(proteins nucleic acids, cell debris)

Cell disruption without the desiredproducts denaturation.


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Mechanical Methods

i) Homogenizers.

The method is applied mainly for the release ofintracellular molecules.

ii) Ball MillsIn a ball mill, cells are agitated in suspension with

small abrasive particles. The beads disrupt the

cells to release biomolecules.iii) Ultrasonic disruption.

It is expensive and is used mainly in laboratories.


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Non-mechanical methods

(i) Chemical Permeabilization. with organic solvents that actby the creation of canals through the cell membrane:toluene, ether, phenylethyl alcohol or antibiotics

(ii) Mechanical Permeabilization. One method of mechanicalpermeabilization is osmotic shock. The cells equilibrate

internal and external osmotic pressure in a high sucrosemedium, and then rapidly diluting away the sucrose. Theresulting immediate overpressure of the cytosol isassumed to damage the cell membrane.

(iii) Enzymatic Permeabilization. This method is often limited

to releasing periplasmic or surface enzymes. Firstly EDTAis used then enzymes as:

beta(1-6) and beta(1-3) glycanases, proteases, andmannase.


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Liquid Extraction

Also known as solvent extraction, or liquid-liquid extraction

It is used to transfer a solute from onesolvent into another in which it is moresoluble.


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Dissociation Extraction

A special case of liquid-liquid extraction. Many fermentation products are either weak bases

or acids.

When solvent extraction is employed the pH is soselected that the material to be isolated isunionized since the ionized form is soluble in theaqueous phase and the unionized form is soluble

in the solvent phase. Weak bases are therefore extracted under high pHconditions and weak acids under low pHconditions. The result is a rapid and complete

extraction of the solute and materials similar to it.


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Ion-exchange Adsorption

Ionic filtrates of fermentation broths can bepurified and concentrated using ionexchange resins packed in columns.

An ion exchange resin is a polymer(normally polystyrene) with electricallycharged sites at which one ion may

replace another.


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Functional groups that havecharges

-COOH which is weakly ionized to -COO

-SO3H which is strongly ionized to -SO3

-NH2 that weakly attracts protons to formNH3+

-secondary and tertiary amines that alsoattract protons weakly

-NR3+ that has a strong, permanentcharge (R stands for some organic group)


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The efficiency of the exchangedepends on

The capacity of the resin for the ion to beadsorbed, usually expressed inmilliequivalents.

The size of the resin spheres: the smaller,the more the exchange.

The flow rate; the slower, the greater the

adsorption. Temperature: the higher, the more rapidthe exchange.


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Precipitation

It is particularly useful in the elimination ofproteinaceous impurities or in the isolation ofenzymes.

Salts are precipitated by one of several methods:

Adding inorganic salts and (or) Reducing the solubility with the addition of

organic solvents such as alcohol in the case ofenzymes.

Lactate and oxalate salts of erythromycin havebeen so isolated Citric acid has been isolated with its calcium salt.


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3.PURIFICATION

1. Chromatography

(Adsorption, partition, ion-exchange, gel

filtration and affinity chromatography)

2. Carbon Decolorization

3. Crystallization


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PRODUCT ISOLATION

The final isolation of the product is done inone of the two following ways:

Processing of crystalline products.

Drying of products direct from solution.


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Crystalline Processing

Crystalline products are free-filtering and non-compressible and therefore may be filtered onthick beds under high pressure.

This is usually done on a centrifugal machinecapable of developing very high (about 1,000fold) gravitational force.

The crystals are washed to remove adhering

mother liquor. After washing they are dried by spinning forfurther drying or solvent removal.


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Drying

Drying consists of liquid removal (eitherorganic solvent or water) from wet crystalssuch as was described above from a

solution, or from solids or cells isolatedfrom the very earliest operation. Drying can be considered under two

heads:

(i) liquid-phase moisture removal, and

(ii) solid-phase moisture removal.


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Liquid-phase moisture removal

Liquid-phase moisture removal involvesdrying by heat.

The simplest method is by direct heating inwhich heated atmospheric air both heatsthe material and removes the water vapor.

In others, the heating is done at reduced

pressure to facilitate evaluation of thewater vapor.


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(i) Tray Driers

The most commonly used in somefermentation industries is the vacuum traydrier.

It consists simply of heated shelves in asingle cabinet which can be vacuumevacuated.

As it can be evacuated, heating at fairlylow temperature is possible and hence it isuseful for heat-labile materials.


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(ii) Drum dryers

In this method the broth or slurry is appliedto the periphery of a revolving heateddrum.

The drum may be single or in pairs.

High temperature is applied though for ashort time on the material to be dried and

some destruction may occur.


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(iii) Spray drying

This method is used extensively in the food andfermentation industries for drying heat-sensitive materialssuch as drugs, plasma and milk.

The conventional spray consists of an arrangement for

introducing a fine spray of the liquid to be dried against acounter-current of hot air. As the material is exposed to high temperature for only a

short while very little damage usually occurs. It is convenient because of its continuous nature. Sometimes the material is introduced simultaneously with

air


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S lid h i t l
http://www.made-in-china.com/image/2f0j00MTaQvnYRRtVqM/Spray-Drying-Equipment.jpg


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Solid-phase moisture removal

(freeze-drying)

Same as in the vacuum drier describedearlier.

The main difference is that the material isfirst frozen.

In this frozen state, the water evaporates

straight from the material. It is useful for heat-labile materials such as

enzymes, bacteria, and antibiotics.

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