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UPSTREAM in bioprocess: STERILIZATION: It is used so only the biocatalyst grows. In the sterilization vapors are introduced through the equipment increasing the temperature. The sterilization cycle implies a rise in temperature, which is why the temperature drop must be taken into account. There are different levels of sterility: 1) Disinfection: destruction or removal of vegetative pathogens but not bacterial endospores. Usually used obly on inanimate objects. 2)Sterilization: complete removal or distruction of all viable microorganism. Also used on inanimate objects. 3) Aseptic: chemicals applied to body surfaces to destroy or inhibit vegetative pathogens. MEDIA FORMULATION: culture media should contained the required nutrients in the metabolism pathways involving the bioprocess. The media should be previous optimized and sterilized. FEEDSTOCK PREPARATION: such us the state of matter, particle size, T, pH, purity and concentration. INOCULUM PREPARATION: required steps for the cell growth conducted to obtain the best biocatalyst for the bioprocess: yield, productivity, strain manteinance and proper procedure.
DOWNSTREAM in bioprocess: BROTH: complex mixtures of components containing products in dilute solution. DOWNSTREAM: any treatment of the culture broth after fermentation. OBJETIVE: concentrate and purify the product for sale. MULTI‐STEP PROCESS: Minimize the number of steps maintaining the yield. The final product obtained with the downstream processes are:
– Complete cells, Intracellular productà disruption and purification, Extracellular productà concentration and purification
OVERALL PROCESS: 1) Nature of the starting material 2) Initial localization of the target product (whole cells, extra‐, intra‐cellular) 3) Physical/chemical properties of the target product. Impurities / by products àsimilar properties 4) Concentration, volume or flow‐rate of the starting material 5) Concentration of the target product in the starting material 6) Susceptibility to denature / (thermal) degrade àgentle conditions, relative usage of organic acids (promote degradation), sub-ambient temperatures 7) The desire physical form of the final producto (lyophilized powder, sterile solution, suspension, …) 8) Stringent quality requirements for products used for prophylactic, diagnosis, therapeutic purposes (purity, absence of endotoxins or aggregates, …) 9) Product market price, process costing and economics.
WHOLE CELLS:The product are the cells. A solid-liquid separation is produced from the broth by microfiltration, centrifugation and sedimentation. The liquid is discarded or recycled and Biomass is the product
EXTRACELLULAR PRODUCTS:the product is outside the cells A solid-liquid separation is carried out from the broth using microfiltration, centrifugation and sedimentation. The biomass is disposable or recycled. The liquid (cell-free liquor) is first concentrated (primary insulation) then purified (enrichment of the product) and the final product is obtained (final isolation)
INTRACELLULAR PRODUCTS:The product is inside the cells. From the broth a solid-liquid separation is carried out by microfiltration, centrifugation and sedimentation. The liquid obtained is discarded or recycled. The biomass undergoes cell disruption to obtain a homogenate and gives rise to the cell debris, that is discarded, and a liquid (cell-free liquor) that is first concentrated (primary isolation), then purified (enrichment of the product) and finally obtains the final product (final isolation).
SOLID-LIQUID SEPARATION:
The difference between solid and liquid fractions is based on the physico-chemical properties such as size and morphology. This process includes the fractionation of sub‐cellular organelle, the separation of plasmid DNA from chromosomal DNA, and the separation of mature cells from young cells. Filtration or micro-filtration: Separation is achieved by forcing the suspension through a porous medium or a membrane which hold back the solid particles. Precoat filtration is a type of mechanical filtration that can be used to clarify liquids. It is used when the broth has high viscosity. Sedimentation: separation is achieved by subjecting the broth to natural gravitational fields. It is used when: Depending on the size and density, when the diameter of the solid is> 100 micromol, Low viscosity of the broth, Because is cheap Centrifugation: the separation is achieved by subjecting the broth to gravitational fields induced by rapid rotation. It is used when: Depending on the size and density, when the diameter of the solid is <0.01 micromol, Low viscosity of the broth, It is also used for non-cellular solids such as enzymes, substrates.
CELL DISRUPTION: The aim is to release the maximum amount of product in active state. It consists of cell lysis and the method can be mechanical, physical, chemical or enzymatic.Mechanical: widely used in laboratory and industrial scale. Are: 1) Liquid shear 2)Solid shear 3)Agitation with abrasives 4)Ultrasonication. It releases DNA, proteases which with heat are denaturalized. Non-mechanical: 1)Physical: heatshock or osmotic shock 2) Chemical: cationic detergents and anions, organic solvents 3) Enzymatic: it is mild and selective with lysozyme enzymes for example
PRIMARY ISOLATION: After the initial separation solid-liquid the filtrate contains between 85-90% of water. The water must be removed and it can be done in different ways: 1) Evaporation: simple but has a great expenditure of energy. Normally using steam as the heat source in large scale. 2) Extraction of the solvent: the concentration is based on the change of solubility. This liquid must be immiscible with the fermentation broth. In this technique you can also extract small hydrophobic molecules. 3) Aqueous system of two phases: when we have two immiscible liquids of the same nature. they can be separated by density and solubility if both are different. 4) Filtration according to what is wanted: cell, viruses, small molecules… 5)Precipitation (salt-out): the solubility of organic compounds depends on the concentration of salts.
PURIFICATION:It is usually 50-70% of the production cost. A chromatography is performed. The chemical species found in the bulk liquid phase adhere to the porous surface of a solid adsorbent. This adsorbent has to be selective for the product. The resin can then be recovered by elution. It can be exclusion, ion exchange …
FINAL ISOLATION: Stabilization of the final product. The agent that is added must guarantee the quality / activity of the product during the storage period. 1) Distillation: Different boiling point of each compound in the concentrate solution.Eliminate the solvent and concentrate the product. 2)Crystallization: evaporation of the solvent at low temperature and slowly. In order to reduce product solubility: pH changes, adding of solvents, salts, electrolytes, … 3)Drying: reduce the moisture content. Heat could be applied by means of conduction, convection and radiation. Vacuum is to reduce operational temperature. 4) Freeze drying (lyophilization): It is based on a previous freezing of the broth and then sublimation for the conservation of live cells / labile molecules for a long period of time. The sublimation is to move from a solid state to gas without going through the liquid by vacuum. It is used to preserve cell viability.