Fermentation technology Down Stream Processing

Processing Basics of Fermentation

By

Chanakya Pachi

Basics of Fermentation

and Its Technology

Fermentation • Fermentation is the term used by microbiologists to describe any process for the production of a product by means of the mass culture of a microorganism.• The extraction and purification of a biotechnological product from fermentation.

Fermentation BasicsThe product can either be:

The cell

itself

•Refe

rred to

as Biom

ass

productio

n.

A microorga

nisms’ own metabolite• Referred to

as a product

from a natural strain.

A microorganisms foreign product• Referred to as a product from recombinant DNA technology or genetically engineered strain, i.e. recombinant strain.

Batch Fermentation• A batch fermentation can be considered to

be a closed system.

• At time t=0 the sterilized nutrient solution in the fermenter is inoculated with microorganisms and incubation is allowed to proceed.

• In the course of the entire fermentation, nothing is added, except oxygen (in case of aerobic microorganisms), and acid or base to control the pH

Batch Fermentation• The composition of the culture medium,

the biomass concentration, and the metabolite concentration generally change constantly as a result of the metabolism of the cells.

• After the inoculation of a sterile nutrient solution with microorganisms and cultivation under physiological conditions, four typical phases of growth are observed

Growth PhasesLag phase• Physicochemical equilibration between

microorganism and the environment.

Log phase• Growth of the cell mass can now be

described quantitatively as a doubling of cell number per unit time for bacteria.

Stationary phase• As soon as the substrate is metabolized or

toxic substances have been formed, growth slows down or is completely stopped.

Death phase• In this phase the energy reserves of the

cells are exhausted.

Bio Reactors…

Down Stream

Processing

General Steps in Downstream Purification

Downstream processing• The various stages of processing that

occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product.

Stages of Downstream Processing

Removal of Insolubles

Product Isolation

Product Purification

Product Polishing

To markets

Stages in DownstreamProcessing• A few product recovery methods may be

considered to combine two or more stages.• For example, expanded bed adsorption

accomplishes removal of insolubles and product isolation in a single step. Affinity chromatography often isolates and purifies in a single step.

Crystallization

Finishing/packagin

g

Chromatography

High resolution technique

s

Precipitation

Concentration

Centrifugation

Clarification

Homogenizers

Hydrolytic enzymes

Cell disruption

Flocculation

Centrifugation

Filtration

Cell Separatio

nUnit operations in downstream processing

Separation of cells and medium• Recovery of cells and/or medium (clarification)• For intracellular enzyme, the cell fraction is

required• For extracellular enzymes, the culture

medium is required•On an industrial scale, cell/medium separation is almost always performed by centrifugation• Industrial scale centrifuges may be batch,

continuous, or continuous with desludging

Solid liquid separation• Following steps are involved:• Floatation- gas is introduced into the

liquid it forms bubble. Cells and other solid particles are absorbed and removed.• Flocculation -cells and debris from large

aggregates and settles down. Addition of flocculating agents are often done.• Filtration- most commonly used to

separate biomass in culture medium.

Types of filtration usedDepth filters: they composed of filamentous

matrix. Particles are trapped in the matrix and fluid passes out.

Absolute filters: the are of specific pore size than the particles to be removed. Mostly used to remove bacteria.

Rotatory drum vacuum filters: consists of rotating drum immersed in tank of broth. As the drum rotates it picks up the mass which forms a cake.

Membrane filters:

Centrifugation• Tubular bowl centrifuge: small, commonly used in pilot plants, can be run at high speed and can be run in both batch or continuous fermentation.•Disc centrifuge: consists of several discs that separate bowl into settling zones. Slurry is fed through the centre.•Multichamber centrifuge: modification of tubular bowl type. consists of several chambers which allows zigzag movement of feed.

Release of intracellular productsPhysical methods of cell disruption:• Ultrasonication• Osmotic shock: used to separate

hydrolytic enzymes and binding proteins.• Heat shock: • High pressure homogenization:• Grinding with glass:

Chemical methods• Alkali treatment:• Organic solvents: toulene is very oftenly

used. It dissolves the membrane phospholipids.• Detergents: triton x-100 or tweed is used

Enzymatic method• Lysozymes: most frequently used for

gram +ve bacteria, for –ve in combination with EDTA it is used.

Combination metods• Concentration: the filtrate free from

suspended particles contains 80% water. The water is removed to achieve concentration. Commonly used techniques are:• Evaporation• Liquid-liquid extraction• Membrane filtration• Precipitation• adsorption

Evaporation• The evaporators in general , have a

heating device for supply of steam and unit for the seperation of concentrated product and vapour, a condenser, accessories and control equipment.

Dewatering • Precipitation

• Salting out – addition of a high concentration of a soluble salt (typically ammonium sulphate) causes proteins to aggregate and precipitate.

• Addition of organic solvents• Ultrafiltration

• The solution is forced under pressure through a membrane with micropores, which allows water, salts and small molecules to pass but retains large molecules (e.g., proteins)

• Spray drying• Requires use of heat to evaporate water –

unsuitable for most proteins

Protein purification• 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

++

+

+

++

+ ++

+

-

- -

-

++

+

+

+

++

+

+

+-

- -+

Positively charged(anionic) ion

exchange matrix

Net negatively charged (cationic)

protein at selected pHProtein binds to matrix

Typical ion exchange protein separation

Loading starts

Loading ends,Low salt wash begins

Protein absorbance

Peak of unbound protein

Salt gradient

0

1M

Salt gradient begins

Salt gradient ends

Eluted peaks of weakly bound (I), moderately bound (II)

and tightly bound (III) proteins

IIIII

I

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

Affinity chromatography

Matrix Spacer arm

Affinity ligand

+

Active-site-bound enzyme

1. Substrate analogue affinity chromatography

Matrix Spacer arm

Antibody ligand+

Antibody-bound enzyme

2. Immunoaffinity chromatography

Protein epitope

Enzyme

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

Precipitation• Formation of a solid in a solution during a

chemical reaction.• Solid formed is called the precipitate and

the liquid remaining above the solid is called the supernate.

Product Purification

• To separate contaminants that resemble the product very closely in physical and chemical properties.

• Expensive and require sensitive and sophisticated equipment.

Crystallization• 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.

Product Polishing

• Final processing steps which end with packaging of the product in a form that is stable, easily transportable and convenient.

• Crystallization, desiccation, lyophilization and spray drying are typical unit operations

lyophilization

• Freezing the material

• Reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas.

Downstream processing should be modified based on target product

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

Fermentation

Culture supernatant

Centrifugation to remove cells

Liquid preparation to animal feed

market

Animal feed enzyme

Fermentation

Culture supernatant

Centrifugation to remove cells

Proteinprecipitation

Protein fraction1 or 2 purification

stepsSemi-purified

proteinLyophilisationBottling

To chemicals market

Analytical enzyme

Fermentation

Cell pellet

Intracellular fractionTherapeuticProtein

Centrifugation to remove

medium

Cell lysis Centrifugation

Proteinprecipitation

Protein fraction3-4 purification

stepsHomogeneo

usproteinSterile

bottling

To pharmaceuticals market

References

Wikipedia

Microbial Biotechnology• Glazer

Lecture Notes• Dr.B.S.Vijayaku

mar• Head• Dept. of

Biosciences