DOWN STREAM PROCESSING

Sanjeet Kumar
Ravenshaw University

sanjeet.biotech@gmail.com

·        Downstream processing refers to the recovery and purification of biosynthetic products. The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing (DSP) or product recovery.

·        DSP is as complex and important as fermentation process.

·        The methodology adopted for downstream processing depends on the nature of the end product, its concentration, stability and the degree of purification required, besides on the presence of other products.

·         The desired products for isolation by DSP are most frequently metabolites which may be present as follows:-

1.      Intracellular metabolites- Vitamins

2.      Extracellular metabolites- amino acids

3.      Both Intra- and Extra- cellular- Flavomycin

Stages in Downstream processing

Downstream processing of metabolites is a multistage operation and broadly divided into the following stages:

1.      Release of intracellular products

2.      Solid-liquid separation

3.      Concentration

4.      Purification

5.      Formulation

Solid-liquid Separation

The first step in product recovery is the separation ofwhole cells and other insoluble ingradients from the culture broth.

Several methods are useful for solid- liquid separation such as:

a)      Flotation

b)      Filtration

c)      Flocculation

Filtration

Filtration is the most commonly used technique for separating the biomass and culture filtrate. Several types of filters are in use.

1)      Depth Filters:- Filamentous fungi are removed

2)      Absolute Filters:- Removal of bacteria from culture medium

3)      Rotary Drum Vacuum Filters:- Filtration of yeast cells

4)      Membrane Filters:- Reduces Clogging

Ø  Static Flow

Ø  Cross Flow

In cross-flow filtration, the culture broth is pumped in a crosswise fashion across the membrane.

Ther are 3 types of filtration used:

a)      Microfiltration

b)      Ultrafiltration

c)      Reverse Osmosis

Type	Applications
a)      Microfiltration	Separation of cells or cell fractions, viruses
b)      Ultrafiltration	Separation of compounds with molecular weight greater than 1000 μg
c)      Reverse Osmosis	Separation of compounds with molecular weight less than 1000 μg

Release of Intracellular Product

·         There are several biotechnological products such as vitamins, enzymes etc. are located within the cells. Such compounds have to be first released for their further processing and final isolation.

·         The selection of a particular methods for released of intracellular products, depends on the nature of the cells.

·         Gram-negative bacteria and filamentous fungi can be more easily broken compared to Gram-positive bacteria and yeasts.

·         In order to increase the efficiency of cell disintegration in a cost-effective manner, a combination of physical, chemical and enzymatic methods are employed.

Concentration

The filtrate that is free from suspended particles usually contains 80-98 % of H₂O. The desired product is a very minor constituent. The H₂O has to be removed to achieve the product concentration. The commonly used techniques are:

a)      Evaporation

b)      Liquid-liquid extraction

c)      Membrane filtration

d)      Precipitation

e)      Adsorption

    Evaporation can be done by evaporated such as plate evaporators, Falling film evaporator, Forced film evaporators and Centrifugal forced film evaporators.

Liquid-liquid extraction is useful for partial purification of a product, which is categorized as:

a)      Extraction of low molecular weight products using Dissociation, supercritical fluid etc.

b)      Extraction of high molecular weight compounds using Aqueous 2-phase systems (ATPs), and reverse miceller systems.

Precipitation is the most commonly used technique for the concentration of macromolecules using neutral salts, organic solvents etc.

Purification

The biological products of fermentation are very effectively purified by chromatography. It is basically an analytical technique dealing with the separation of closely related compounds from a mixture.

The different types of chromatographic techniques are used for such as

a)      Gel-filtration chromatography

b)      Ion-exchange chromatography

c)      Affinity chromatography and

d)      Hydrophobic interaction chromatography

CHROMATOGRAPHY	PRINCIPLE
Gel-filtration	Size and Shape
Ion-exchange	Net charge
Affinity	Molecular recognition
Hydrophobic Interaction	Polarity
Immobilized metal-ion affinity	Metal ion-binding

Formulation

Formulation broadly refers to the maintenance of activity and stability of biotechnological products during storage and distribution.

·         The formulation of low molecular weight products can be achieved by concentration them with removal of most of the water.

·         For certain small molecules such as citric acid, antibiotic etc., formulation can be done by crystallization by adding salts.

LIGAND	TYPES OF PROTEIN
Antibody	Antigen
Cofactor	Enzyme
Hapten	Antibody
Lectins	Glycoproteins

                       (Ligands use for separation of proteins by Affinity Chromatography)

Drying is an essential component of product formulation. It basically involves the transfer of heat to a product for removal of moisture. Freeze-drying or Lyophilization is the most preferred method for drying and formulation of wide-range of products such as food stuffs, pharmaceuticals, bacteria, viruses. Lyophiization is based on the principle of sublimation of liquid from a frozen state. In this technique, the liquid containing the product is frozen and then dried in a freeze-dryer under vacuum. The vacuum released and the product containing vials are sealed.  Penicillin can be freeze dried directly in ampoules. For good output can integrate the fermentation and downstream processing to finally get the desired product.