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.

purification of products in the soluble portion

Filtration

1.The rotary vacuum filter:

• Consists of a hollow rotating cylinder divided 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 which the rotating cylinder is immersed.

• For thick slurries which are difficult to filter (e.g. aminoglycoside broths) a thin layer of filter aid (e.g. Kiesselghur) is first allowed to be absorbed on the cylinder.

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

• The rotating cylinder, the vacuum still on, is washed with a sprinkle of water; a knife whose edge is positioned just short of the layer of filter aid scrapes off the solids picked up from the broth.

• 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 a release of the vacuum in the segment of the cylinder helps release the material picked up from the broth.

2 .Plate and Frame Filters

3 .Pressure leaf filters

Horizontal

Vertical

Centrifugation

• Centrifuge when filtration is not a satisfactory separation method. Although expensive 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 standard

of hygiene is required.

• In the enzyme isolation industry, however, centrifugation is preferred to filtration, probably because unwanted cell debris are quite efficiently removed by this method.

• A large number of centrifuges are available in the market and a new fermentation industry or a change in the production method of old processes may require the use of centrifuges for primary separation.

1 .The Basket Centrifuge

2.The Tubular-bowl Centrifuge

3.The Multichamber Centrifuge

Harvesting of Microbial Cells

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

• Filter aids to improve filtration rates, while heat and flocculation treatments are employed as techniques for increasing sedimentation rates in centrifugation.

• Some potential developments in cell recovery include the use of:

• electrophoresis• Ultrasonic treatment to improve flocculation

characteristics

Coagulation and Flocculation

• Coagulation is the cohesion of dispersed colloids into small flocs

• flocs aggregate to form larger masses.• Induced by electrolytes (clay or activated

charcoal) • Bacteria and proteins being negatively

charged colloids are easily flocculated by electrolytes or polyelectrolyte

Flocculants should have the following 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.

4. Foam Fractionation

• The principle of foam fractionation is that in a liquid foam system the chemical composition of a given substance in the bulk liquid is usually different from the chemical composition of some substance in the foam.

• Foam is formed by sparging the bulk liquid containing 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 it the solutes to be fractionated.

• This method has been used to collect a wide range of microorganisms and although mainly experimental it may be used on a large scale in industry.

5.Whole-broth Treatment• acetonebutanol fermentation, the whole

unseparated broth is stripped of its content of the required product.

• Antibiotics streptomycin (using cationic-exchange resin)

• and novobiocin (on an anionic resin.) The antibiotics are eluted from the resins and then crystallized.

• This process saves the capital and recurrent expense of the initial separation of solids from the broth.

2 .PRIMARY PRODUCT ISOLATION

• After separation of the broth into soluble and insoluble fractions, the next process depends on the location of desired product as follows:

A. The cells themselves as in yeasts• B. Bound to the mycelia or to bacterial cells as in the

case of bound enzymes or antibiotics…. The cells then have to be disrupted with any of the several ways available – heat, mechanical disruption, etc.

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

Liquid extraction, dissociation extraction, sorption, or precipitation.

2.1 Cell disruption

• Cell disruption is a sensitive process because of the cell wall’s resistance to the high osmotic pressure inside them.

• Difficulties arise from a non-controlled cell disruption, that results from an unhindered release of all intracellular products (proteins nucleic acids, cell debris)

• Cell disruption without the desired product’s denaturation.

Mechanical Methods

i) Homogenizers.The method is applied mainly for the release of

intracellular molecules.ii) Ball Mills In 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.

Non-mechanical methods(i) Chemical Permeabilization. with organic solvents that act

by the creation of canals through the cell membrane: toluene, ether, phenylethyl alcohol or antibiotics…

(ii) Mechanical Permeabilization. One method of mechanical permeabilization is osmotic shock. The cells equilibrate internal and external osmotic pressure in a high sucrose medium, and then rapidly diluting away the sucrose. The resulting immediate overpressure of the cytosol is assumed to damage the cell membrane.

(iii) Enzymatic Permeabilization. This method is often limited to releasing periplasmic or surface enzymes. Firstly EDTA is used then enzymes as:

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

Liquid Extraction

• Also known as solvent extraction, or liquid-liquid extraction

• It is used to transfer a solute from one solvent into another in which it is more soluble.

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 so

selected that the material to be isolated is unionized since the ionized form is soluble in the aqueous phase and the unionized form is soluble in the solvent phase.

• Weak bases are therefore extracted under high pH conditions and weak acids under low pH conditions. The result is a rapid and complete extraction of the solute and materials similar to it.

Ion-exchange Adsorption• Ionic filtrates of fermentation broths can be

purified and concentrated using ion exchange resins packed in columns.

• An ion exchange resin is a polymer (normally polystyrene) with electrically charged sites at which one ion may replace another.

Functional groups that havecharges

• -COOH which is weakly ionized to -COO¯• -SO3H which is strongly ionized to -SO3¯• -NH2 that weakly attracts protons to form

NH3+• -secondary and tertiary amines that also

attract protons weakly• -NR3+ that has a strong, permanent

charge (R stands for some organic group)

The efficiency of the exchange depends on

• The capacity of the resin for the ion to be adsorbed, usually expressed in milliequivalents.

• 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 rapid the exchange.

Precipitation• It is particularly useful in the elimination of

proteinaceous impurities or in the isolation of enzymes.

• 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 of enzymes.

• Lactate and oxalate salts of erythromycin have been so isolated

• Citric acid has been isolated with its calcium salt.

3 .PURIFICATION

1. Chromatography(Adsorption, partition, ion-exchange, gel

filtration and affinity chromatography)2. Carbon Decolorization

3. Crystallization

PRODUCT ISOLATION

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

• Processing of crystalline products.• Drying of products direct from solution.

Crystalline Processing• Crystalline products are free-filtering and non-

compressible and therefore may be filtered on thick beds under high pressure.

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

• The crystals are washed to remove adhering mother liquor.

• After washing they are dried by spinning for further drying or solvent removal.

Drying• Drying consists of liquid removal (either

organic solvent or water) from wet crystals such as was described above from a solution, or from solids or cells isolated from the very earliest operation.

• Drying can be considered under two heads:

(i) liquid-phase moisture removal, and (ii) solid-phase moisture removal.

Liquid-phase moisture removal

• Liquid-phase moisture removal involves drying by heat.

• The simplest method is by direct heating in which heated atmospheric air both heats the material and removes the water vapor.

• In others, the heating is done at reduced pressure to facilitate evaluation of the water vapor.

)i (Tray Driers• The most commonly used in some

fermentation industries is the vacuum tray drier.

• It consists simply of heated shelves in a single cabinet which can be vacuum evacuated.

• As it can be evacuated, heating at fairly low temperature is possible and hence it is useful for heat-labile materials.

)ii (Drum dryers

• In this method the broth or slurry is applied to the periphery of a revolving heated drum.

• The drum may be single or in pairs. • High temperature is applied though for a

short time on the material to be dried and some destruction may occur.

)iii (Spray drying• This method is used extensively in the food and

fermentation industries for drying heat-sensitive materials such as drugs, plasma and milk.

• The conventional spray consists of an arrangement for introducing a fine spray of the liquid to be dried against a counter-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

http://www.made-in-china.com/image/2f0j00MTaQvnYRRtVqM/Spray-Drying-Equipment.jpg

Solid-phase moisture removal (freeze-drying)

• Same as in the vacuum drier described earlier.

• The main difference is that the material is first 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.