fermentation

RECOVERY & PURIFICATION OF

FERMENTATION PRODUCTS

Aqeela Ashraf

Introduction to Purification

Many biological processes

require a purification scheme to

reduce the fermentation broth to

its pure final product. Once

product is made in the

production fermenter, the broth

is still highly contaminated.

Factors for deciding the extraction method

• The value of the final product.• The degree of purity required.• 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.

Extraction

Extraction is used to liberate a product of microbial growth from the cells or cellular constituents that served as the enzyme source either by mechanical or non-mechanical means. Mechanical Extraction

• Mechanical disruption of the cell is easy to achieve on a small scale but can fail when used industrially

• High Pressure Homogenizer- A positive displacement pump with an adjustable valve, has been used to

break microorganisms like Aspergillus niger, Escherichia coli, and Bacillus megatherium.

- When cell concentration is high, the spores or mycelia from the microorganism can clog the valve

• High Speed Ball Mill - Used for release of proteins within yeast

Extraction

Non-mechanical Extraction• Desiccation

- Air drying that must be followed by buffer extraction

• Physical and Chemical Lysis

- Osmotic shock produced by an abrupt change in salt

concentration of the medium

• Solvent Extraction- Liquid extraction of a product from soluble particles within the

cell

- Must choose solvent accordingly, and purification efforts will follow to recover product from solvent

Cell disruption (for intracellular enzymes)

• Sonication– Use of high frequency sound waves to disrupt cell walls and

membranes• Can be used as continuous lysis method• Better suited to small (lab-scale) operations• Can damage sensitive proteins

• Pressure cells– Apply apply high pressure to cells; cells fracture as pressure is

abruptly released• Readily adapted to large-scale and continuous operations• Industry standard (Manton-Gaulin cell disruptor)

• Enzymic lysis– Certain enzymes lyse cell walls

• Lysozyme for bacteria; chitinase for fungi• Only useful on small laboratory scale

Cell Disruption

Mechanical

Algae, bacteria and fungiLarge scale, up to 2000kg/h liquid and solid

Principle of operation

• A grinding chamber filled with about 80% beads.• A shaft with designed discs or impellers is within the chamber.• The shift rotates at high speeds, high shearing and impact forces

from the beads break the cell wall.

http://www.cbmills.com/Products/horizontalmills.htm

Dyno-Mill(liquid)

Cell Disruption

Mechanical

Ball Mill Solid Frozen cell paste, cells attached to or within a solid

matrix. Large scale

http://www.unitednuclear.com/mills.htm

Cell Disruption

Challenge: Damage to the product

- Heat denaturation

- Oxidation of the product

- Unhindered release of all intracellular products

Precipitation

Precipitation is a procedure where the addition of a ionic solution

to an ionic fermentation broth forms insoluble particles, where

the desired product is usually contained within those particles.

Ionic fermentation broths usually consist of enzymes or proteins.

The ways to precipitate out a product can vary from simple pH

and temperature changes to chemical reactions involving metal

ions. Precipitation reactions are carried out in reactors,

continuous and batch.

Precipitation

Precipitation by Organic Solvents• By adding an organic solvent to an aqueous fermentation broth,

the dielectric constant will decrease causing the solubility to decrease

• Often used industrially because it’s inexpensive and simple

Precipitation by Metal Ions• Metal salts with lower solubilities can formed by enzymes and

proteins• Nucleic acids, which are present in microbial cells, must be

removed prior to this type of precipitation because they reduce the resolution of separation

• Salts can be used to selectively precipitate out those nucleic acids

Coagulation and Flocculation

Coagulation is defined for biological processes to be when small particles directly adhere to each other, while flocculation is when

an agent acts as a bridge that joins particles together. Coagulation and flocculation techniques are usually applied to

either whole cells, cell debris, or soluble proteins.

Whole Cells• Many flocculation agents are used to separate products, such as anionic

and cationic electrolytes, polyamines, alumina, and synthetic polymers• Less information is known about coagulants, but some studied

inorganic coagulants have been alum, ferric salts, and calcium salts

Coagulation and Flocculation

Cell Debris and Proteins

• Coagulation and flocculation are useful techniques in removing

the cell debris that can be produced during mechanical agitation

• Coagulation and flocculation can be used alternatively to

precipitation methods to remove enzymes

• The same agents for whole cell removal can be applied to cell

debris and protein removal

Centrifugation

Centrifugation involves separation of liquids and particles

based on density. Centrifugation can be used to separate cells

from a culture liquid, cell debris from a broth, and a group of

precipitates

• Tubular Bowl Centrifuge

• Disc Bowl Centrifuge

• Perforate Bowl Basket Centrifuge

Filtration

Filters use a filter cloth or some porous material along with

applied pressure to push smaller particles through the filter, thus

separating elements of the solution based on size. Filtration for

biological materials is generally completed using batch filtration,

rotary drum filtration, or ultra filtration methods e.g..,

Batch Filtration

Rotary Drum Filtration

Ultra filtration

purification of products in the soluble portion

Separation of Soluble Products

:

Reduce the product solubility in the fermentation broth by adding chemicals.

Applicable: separate proteins or antibiotics from fermentation broth.


Membrane separation- Microfiltration: 0.1 - 10 µm, bacterial and yeast cells.

- Ultrafiltration: macromolecules (2000 <MW< 500,000)

- Dialysis: removal of low-MW solutes: organic acids (100<MW<500) and inorganic ions (10<MW<100).

- Reverse osmosis: a pressure is applied onto a salt-containing phase, which drives water from a low to a high concentration region. MW < 300.

ChromatographyTo separate the solutes based on the different rate of movement

of the solutes in the column with adsorbent materials.

PrinciplesChromatographic processes involve a stationary phase and a

mobile phase.Stationary phase can be adsorbent, ion-exchange resin, porous

solid, or gel usually packed in a cylindrical column.Mobile phase is the solution containing solutes to be separated

and the eluant that carriers the solution through the stationary phase.

Applicable for protein, organics separation.


• Adsorption chromatography– Ion exchange chromatography – binding

and separation of proteins based on charge-charge interactions

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

Protein purification

Affinity chromatography

Binding of a protein to a matrix via a protein-specific ligand

• Substrate or product analogue• Antibody• Inhibitor analogue• Cofactor/coenzyme

Specific protein is eluted by adding reagent which competes with binding

Gel permeation chromatography (GPC)

• 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


Electrophoresis

To separate charged solutes based on their specific migration rates in an electrical field.

Positive charged solutes are attracted to anode and negative charged solutes to cathode.Factors: electric field strength, electric charge of the solutes, viscosity of liquid and the particles size.

Applicable for protein separation.

Recovery and Purification of Bio-Products

Crystallization Last step in producing highly purified products such as antibiotics.

Supersaturated solution, low temperature, Crystals are separated by filters.

Drying

To remove solvent from purified wet product such as crystal or dissolved solute.Vaccum-tray dryers: pharmaceutical productsFreezing drying: by sublimation (from solid ice to vapor), antibiotics, enzyme, bacteriaSpray dryer: heat-sensitive materials

Downstream processing depends on product use

1. Enzyme preparations for animal feed

supplementation (e.g., phytase) are not purified

2. Enzymes for industrial use may be partially

purified (e.g., amylase for starch industry)

3. Enzymes for analytical use (e.g., glucose

oxidase) and pharmaceutical proteins (e.g.,

TPA) are very highly purified

Summary of separation and purification

• Liquid-Solid Separation

- Filtration: micro- and ultrafiltration

- Centrifugation

• Cell disruption

- Mechanical: ultrasonication, milling, homogenization

- Nonmechanical: chemicals, enzyme and osmotic shock

Summary of separation and purification

• Separation of soluble products

- Precipitation

- Adsorption

- Membrane separation: ultra filtration, dialysis, reverse osmosis

- Chromatography

- Electrophoresis

• Crystallization and drying

fermentation

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