Stages of downstreaming processing by using flowcharts and easy description.

1. Downstreaming processing
Presented by: Ayesha Mumtaz
Msc Microbiology

3. 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

5. Stages in Downstream Processing
 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.

7. 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

8. 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.

9. Types of filtration used
 Depth 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:

10. 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.

11. Release of intracellular products
 Physical methods of cell disruption:
 Ultrasonication
 Osmotic shock: used to separate hydrolytic enzymes and binding proteins.
 Heat shock:
 High pressure homogenization:
 Grinding with glass

12. Chemical methods
 Alkali treatment
 Organic solvents: toulene is very oftenly used. It dissolves the membrane phospholipids.
 Detergents: triton x-100 or tweed is used

13. Enzymatic method
 Lysozymes: most frequently used for gram +ve bacteria, for –ve in combination with EDTA it is
used.

14. 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

15. 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.

16. 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 protein

17. 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) Net negatively charged (cationic) protein at selected Protein binds to matrix
ion exchange matrix

19. 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

21. 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

22. 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.

23. Product Purification
 To separate contaminants that resemble the product very closely in physical and chemical
properties.
 Expensive and require sensitive and sophisticated equipment.

24. 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.

25. 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

26. 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

27. 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