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Chapter 5 Downstream Processing Downstream processing, what and why Downstream processing is any treatment of culture broth after fermentation to concentrate and purify products. 2 Separation Processes The challenge •Low product concentrations •Large number of impurities •Thermolabile bioproducts An ideal bioseparation process should combine high throughput with high selectivity, and should ensure stability of product. Nutrients Oxygen Genetic modification Microbes Fermentation Happy growing Drug producing Downstream Processing Remove cells/debris Concentrate and Purify the product Smash the microbes Formulate product Kill the microbes Market Biological products Product Nature of bioseparation required Alcoholic beverages: Beer, wine, spirits Clarification, distillation Organic acids: Acetic acid, citric acid Precipitation, filtration, adsorption Vitamins: Vitamin C, vitamin B12 Precipitation, filtration, adsorption Amino acids: Lysine, glycine, Precipitation, filtration, adsorption Antibiotics: Penicillins Precipitation, filtration, adsorption Enzyme,Polysaccharides Filtration, precipitation, centrifugation, adsorption, chromatography Distillation Separating components with different volatilities. Such as organic solvents , volatile flavors and aromas. Distillation Stages in Downstream Processing Removal of insoluble's Product Isolation Product Purification Product Polishing 1. Cell removal (filtration, centrifugation , coagulation and flocculation) 2. Primary isolation (adsorption, precipitation) Removing components with properties significantly different from those of the products Large volume, relatively non selective 3. Purification (chromatography, ultrafiltration) Removing components with properties similar to those of the products Highly selective 4. Product preparation (crystallization, drying) Removal of insoluble's Capture of the product as a solute in a particulate-free liquid Example Separation of cells, cell debris or other particulate matter from fermentation broth containing an antibiotic. Typical Operations Filtration A mechanical operation used for the separation of solids from fluids. Type of filtration unit Plate and frame filter 16 Rotary-drum vacuum filter Centrifugation Use of the centrifugal force for the separation of mixtures More-dense components migrate away from the axis of the centrifuge Less-dense components migrate towards the axis Centrifuges t t Decanter centrifuge Coagulation and Flocculation Coagulation: Forming semisolid lumps in a liquid. Flocculation: Forming woolly cloudlike aggregations. Product Isolation Reducing the volume of material to be handled and concentrating the product. 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 supernatant. 1. Isoelectric Precipitation pH GA+ GA± GA– GA= 2 3.22 7.0 >12 pI 2. Organic solvent precipitation By adding an organic solvent to an aqueous fermentation broth, the dielectric constant will decrease causing the solubility to decrease. Example Xanthan gum is recovered from an aqueous fermented broth containing the gum by adding to the broth organic solvent. 3. Insoluble salt precipitation Usually used in extraction of amino acid and organic acid. e.g. Citric acid extraction and purification process Heating (destroy the microorganisms ) Neutralizition (forming calcium citrate ) Acidifying (liberate the citric acid from its salt ) Disadvantages high chemical cost relatively complex technology Ion Exchange Resins Resins Organic or inorganic polymer used to exchange cations ( positive ions) or anions ( negative ions) from a solution phase General Structure Polymer backbone not involved in bonding Functional group for complexing anion or cation 30 Organic Resin Groups SO3 H Linkage group CH2 Cl Chloride Cation exchange CH2 N(CH3 )3 Cl Anion exchange e.g. Purification of Lactic Acid from Fermentation Broths by Ion-Exchange Resins First, a strong cation resin was used to reduce the broth pH and remove the cations present in the solution; Then the acidified broth was purified by an anion exchanger. Membrane Separation Function : clarification and sterilization Advantages No phase change Energy efficient A higher quality product Environmentally friendly Easy to scale-up Limitations Long-term reliability not proven Excessive pretreatment Concentration polarization and membrane fouling Replacement of membranes Membrane Fraction Technologies Nanofiltration (200 - 700 MW) Reverse Osmosis (<200 MW) Ultrafiltration (2000 - 50,000 MW) Microfiltration (100,000 MW) Semi-permeable Membrane Wine Filtration –Why? Filtration Objectives/Purposes Include Remove insoluble contaminants Improve clarity, brightness Extend shelf life Electrodialysis Product Polishing Final processing steps which end with packaging of the product in a form that is stable, easily transportable and convenient Crystallization, concentration and drying are typical unit operations Crystallization Formation of a crystalline phase from a parent phase, e.g. solution One of the oldest and most important unit operations, e.g. extracting salt crystals from sea water Definition of Crystal A substance in which the constituent atoms, molecules, or ions are packed in a regularly ordered, repeating three-dimensional pattern. The distinction between a crystal and an amorphous solid is that between order and disorder over large distances Crystallization Process Liquid Mixture Generation of Supersaturation :Driving force Final Product Nucleation: Birth of Solid Phase Crystal Growth Supersaturation refers to a state in which the liquid (solvent) contains more dissolved solids (solute) than can ordinarily be accomodated at that temperature. Solubility & Supersolubility Diagram Generation of supersaturation Solvent evaporation Solution cooling Altering pH Crystallization - by evaporation of saturated solution Crystallization - by cooling saturated solution Concentration Filtration Centrifugation Reduced pressure concentration Drying Drying involves the transfer of heat to the wet material and removal of the moisture as water vapor. Usually, this must be performed in such a way as to retain the biological activity of the product. Drying Methods (1) Atmospheric drying (2)Vacuum drying (Decompression ) (3) Freeze-drying (lyophilization) (4) Spray drying 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. Review Questions 1. Show the importance and main procedures of the downstream processing of fermentation. State the principles and the characteristics of every separation methods. 2. Tell the difference between freeze drying and vacuum drying.