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Down Stream Processing
This document provides an overview of fermentation technology and downstream processing. It defines fermentation as the production of a product by microorganism mass culture. It describes the basic stages of batch fermentation including lag, log, stationary and death phases. It then outlines the main steps in downstream processing including removal of insolubles, product isolation, purification, polishing and packaging. Specific unit operations used at each stage like centrifugation, filtration, chromatography are also explained. The document emphasizes that the level of downstream processing depends on the target product and its end use.
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Down Stream Processing
- 1. Fermentation technology Down StreamProcessing Processing Basics of Fermentation By Chanakya Pachi
- 2. Basics of Fermentation andIts Technology
- 3. Fermentation • Fermentation isthe 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.
- 4. Fermentation Basics The productcan either be: A microorganisms ’ own metabolite • Referred to as a product from a natural strain.
- 5. Batch Fermentation • Abatch 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
- 6. Batch Fermentation • Thecomposition 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
- 8. Growth Phases Lag 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.
- 9. Stationary phase • Assoon 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.
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- 13. General Steps inDownstream Purification
- 14. Downstream processing • Thevarious stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product.
- 15. Stages of Downstream Processing Removalof Insolubles Product Isolation Product Purification Product Polishing To markets
- 16. 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.
- 17. Crystallization Filtration Gel chromatography Drying Finishing/pack aging Chromatograph y Electrophoresis Dialysis High resolution techniques Precipitation Chromatograph y Ultrafiltration Partitioning Distillation Concentration Centrifugation Filtration Clarification Homogenizers Hydrolytic enzymes Cell disruption Flocculation Centrifugation Filtration CellSeparation Unit operations in downstream processing
- 18. Separation of cellsand 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
- 19. 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.
- 20. Types of filtrationused 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:
- 21. Centrifugation • Tubular bowlcentrifuge: 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.
- 22. Release of intracellularproducts Physical methods of cell disruption: • Ultrasonication • Osmotic shock: used to separate hydrolytic enzymes and binding proteins. • Heat shock: • High pressure homogenization: • Grinding with glass:
- 23. Chemical methods • Alkalitreatment: • Organic solvents: toulene is very oftenly used. It dissolves the membrane phospholipids. • Detergents: triton x-100 or tweed is used
- 24. Enzymatic method • Lysozymes:most frequently used for gram +ve bacteria, for –ve in combination with EDTA it is used.
- 25. 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
- 26. Evaporation • The evaporatorsin 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.
- 27. Dewatering • Precipitation • Saltingout – 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
- 28. Protein purification • Adsorptionchromatography • 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 pH Protein binds to matrix
- 29. Typical ion exchangeprotein 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 II III I
- 30. Affinity chromatography • Bindingof 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
- 31. Affinity chromatography Matrix Spacerarm 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
- 32. 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
- 33. Precipitation • Formation ofa 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.
- 34. Product Purification • Toseparate contaminants that resemble the product very closely in physical and chemical properties. • Expensive and require sensitive and sophisticated equipment.
- 35. Crystallization • Process offormation 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.
- 36. Product Polishing • Finalprocessing 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
- 37. lyophilization • Freezing thematerial • 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.
- 38. Downstream processing shouldbe 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
- 39. Fermentation Culture supernatant Centrifugation to removecells Liquid preparation to animal feed market Animal feed enzyme
- 40. Fermentation Culture supernatant Centrifugation to removecells Protein precipitation Protein fraction 1 or 2 purification steps Semi-purified protein Lyophilisation Bottling To chemicals market Analytical enzyme
- 41. Fermentation Cell pellet Intracellular fractionTherapeutic Protein Centrifugation toremove medium Cell lysis Centrifugation Protein precipitation Protein fraction 3-4 purification steps Homogeneous protein Sterile bottling To pharmaceuticals market
- 43. Reference s Wikipedia Microbial Biotechnology • Glazer Lecture Notes •Dr.B.S.Vijayakumar • Head • Dept. of Biosciences