DOWNSTREAM PROCESSING

Presented By:Dakshita Narvekar

Introduction
Ideally, extraction and purification process is obtaining
a high quality product as quickly as possible at an efficient recovery rate using minimum plant investment operated at minimal costs. Intracellular or extracellular location of product Concentration of product in broth Physical & Chemical properties of product Use of that product Minimal acceptable standard of purity Magnitude of Bio-Hazard of the product Impurities in broth Marketable price for the product

Choice of recovery product is based of following criteria:-

Removal of Microbial Cells & Other Solid Matter

Microbial cells, etc are separated from harvested broth by Filteration or Centrifugation. Filteration is used to remove cells while heat and floculation treatments are used to increase sedimentation rates. Method of removal of cell & cell debris include Electrophoresis Dielectrophoresis Ultrasonic treatment Magnetic separation

Most frequently used procedure is 2 phase liquid extraction.

Foam Separation
It depends on using methods that exploit differences in surface activity of materials. The material may be selectively attached or adsorbed to surface of gas bubbles , to be concentrated or separated and finally removed by skimming. Some materials(Colligends) may be made surface active by surfactants(Collectors).

Precipitation
It can be conducted at various stages of recovery process. Typical agents used in precipitation Acids and Bases Salts Organic solvents Non Ionic Polymers Polyelectrolytes Protein Binding Dyes Affinity Precipitants

Filtration
Variety of factors influence choice of equipment to be used for filtration Properties of filtrate, particularly Viscosity & Density. Nature of solid particles The Solid: Liquid ratio Need for recovery of Solid or Liquid or both Scale of operation Need for Batch or Continuous operation Need for aseptic operation Need for pressure or vacuum suction to ensure adequate flow rate of liquid.

Centrifugation
Used when filtration is not satisfactory. According to Stokes Law, rate of sedimentation of spherical particles suspended in fluid of Newtonian viscosity characteristics is proportional to the square of diameter of particles, thus the rate of sedimentation of particle under gravitational force is:

Contd.
Cell aggregation and flocculation
Flocculation is used in effluent treatment for removal of cells and suspended colloidal matter. Aggregates of cells will sediment fast due to increased diameter of particles.(Stokes Law) During flocculation 1 or more mechanisms besides temperature induce cell flocculation Neutralization of anionic charges Reduction in surface hydrophilicity Use of high molecular weight polymerbridges.

Range of Centrifuges
Basket Centrifuge
Separating mould mycelia Separating crystalline compounds It is having perforated bowl lined with filter bag of nylon, cotton, etc. A continuous feed is used, & when basket is filled with filter cake, the cake is washed before removing it.

Contd.
Tubular-Bowl Centrifuge
The main component of the centrifuge is a cylindrical bowl (or rotor) suspended by a flexible shaft (B), driven by an overhead motor or air turbine (C). The inlet to the bowl is via a nozzle attached to the bottom bearing (D). The feed consist of solids and light and heavy liquid phases is introduced by the nozzle (E). During operation solids sediment on the bowl wall while the liquids separate into the heavy phase in zone (G) and the light phase in the central zone (H).

Thus the centrifuge may be altered to use for:

Light-phase/heavy-phase liquid separation. Solids/light-liquid phase/heavy-liquid phase separation. Solids/liquid separation

 Solid-Bowl Scroll Centrifuge

Contd.

Used for continuous broths, cell lysates & coarse materials. The slurry is fed through spindle of screw There is a differential speed between bowl & screw(0.5~100rpm). Solids settling on walls of bowl are scraped to the end of bowl. Slope of cone helps to remove excess liquid from solids. Liquid phase is discharged from opposite end of bowl.

Contd.
Multichamber Centrifuge
Used for slurry of upto 5% solids of particle size 0.1-200um diameter. In this, a series of concentric chambers are mounted within rotor chamber. Broth enters via central spindle and takes circuitous route through chambers. Solids are collected on outer faces of each chamber. The smaller particles collect in outer chambers where they are subjected to greater centrifugal forces.

Contd.
Disc Bowl Centrifuge
Its efficiency relies on the presence of discs in rotor or bowl. Central inlet pipe is surrounded by stack of stainless steel conical discs. Each disc has spacers so that a stack can build up. The broth flows outwards from central feed pipe, then upwards & inwards between discs at 450 angle to axis of rotation. Close packing of discs assists rapid sedimentation and solids then slide to edge of bowl and eventually accumulate on the inner wall of the bowl.

Cell Disruption
Is required to release cell components. Number of methods have been developed for this purpose. This process must ensure that liable molecules are not denatured, or hydrolyzed by enzymes present in cell.
Physio-mechanical methods Liquid shear Solid shear Agitation with abrasives Freeze- thawing Ultra sonication
Chemical methods Detergents Osmotic shock Alkali treatment Enzyme treatment

Liquid-Liquid Extraction
Separation of component from liquid mixture by treatment with a solvent in which the desired component is soluble. The specific requirement is that a high percentage extraction of product must be concentrated in smaller volume of solvent. Solubility characteristic- like dissolves like. Polar liquids mix with each other and dissolve solids and salts & vice versa.

Contd.
The dielectric constant D is a measure of the degree of molar polarization of a compound. This value predicts whether compound is polar or non polar. D can be measured by determining the electrostatic capacity C of condenser containing the substance between plates. If Co is the value of same condenser when it is completely evacuated then,

Contd.
If D1 & D2 are dielectric constants of experimental liquids and standard liquids & C1 & C2 are electrostatic capacities of condenser when filled with each liquids, then The final choice of solvent will be influenced by the distribution or partition coefficient K where

The value of K defines the ease of extraction.

Contd.
Liquid-liquid extraction is done by two systems :Co-current System, & Counter-current System. Co-current System- there are n no. of mixer/separator vessels in line and the raffinate goes from vessel 1 to vessel n. Fresh solvent is added to each stage, the feed & extracting solvent pass through the cascade in the same direction. Extract is recovered at each stage. Although a relatively large amount of solvent is used, a high degree of extraction is obtained.

Contd.
Counter-Current System- There are a number of mixer/separators connected in series. The extracted raffinate passes from vessel 1 to vessel n while product-enriched solvent is flowing from vessel n to vessel 1. The feed & extracting solvent pass through the cascade in opposite directions.

Solvent Recovery
It is usually a distillation unit. It is not essential to remove all the raffinate from solvent as this will be recycled through this system. Distillation may be achieved by
Evaporation (removal of solvent as vapour) Vapour-liquid separation in column Condensation of vapour

Two Phase Aqueous Extraction

Large varieties of natural & synthetic hydrophilic polymers are used to create two (or more) phases. Phase separation occurs when hydrophilic polymers are added to an aqueous solution and when the concentrations exceed a certain value two immiscible aqueous phases are formed. Settling time for the two phases can be prolonged, depending on the components used and vessel geometry. Phase separation can be improved by using centrifugal separators or magnetic separators. many systems are: Non-ionic polymer/ Non-ionic polymer/ water(PEG/dextran) Polyelectrolyte / Non-ionic polymer/ water(SCMC/PEG) Polyelectrolyte/ Polyelectrolyte/ water(SDS/SCMC)

Chromatography
Used to isolate and purify relatively low concentrations of metabolic products. Depending upon the mechanism used it is divided into following types Adsorption Chromatography Ion Exchange Chromatography Gel permeation Chromatography Affinity Chromatography Reverse Phase Chromatography High Performance Liquid Chromatography

Drying & Crystallization

Drying is done by
Spray drier Freeze Drier Fluidized Bed Driers Crystallization- used in initial recovery of organic acids and amino acids, more widely used for final purification of range of compounds.