down stream processing- ankit.pptx baca aau

down stream processing, alcohol, beer, centrifugation, filteration, packaging

1. Down stream processing Submitted to: Dr. R. V. VYAS HOD, Microbiology Dept., AAU, ANAND. Submitted by: Ankit S Patel M.Sc. Agril. Microbiology, 04-1203-2010

2. Down stream 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 Stages in down stream processing Removal of insoluble's Product Isolation Product Purification Product Polishing

3. Steps in down stream processing

4. Cell harvesting Solid–liquid separation to remove the cells from the spent medium. Each fraction can then undergo further processing, depending on whether the product is intracellularly located, or has been secreted into the periplasmic space or the medium. Choice of solid–liquid separation method is influenced by The size and morphology of the microorganism (single cells, aggregates or mycelia) The specific gravity, viscosity and rheology of the spent fermentation medium.

5. Process where a solute comes out of solution in the form of flocs or flakes. Particles finer than 0.1 µm in water remain continuously in motion due to electrostatic charge which causes them to repel each other. Once their electrostatic charge is neutralized (use of coagulant) the finer particles start to collide and combine together. These larger and heavier particles are called flocs . Coagulation can be promoted using coagulating agents (simple electrolytes, acids, bases, salts, multivalent ions and polyelectrolytes ). In subsequent flocculation , smaller flocs are converted into larger settleable particles, is often aided by inorganic salts (e.g. calcium chloride) or polyelectrolytes such as polyacrylamide and polystyrene sulphate They are mostly used in association with sedimentation and centrifugation for the separation of cells from liquid media. Major advantages of these techniques are their low cost and ability to separate microbial cells from large volumes of medium. Flocculation

6. sedimentation Extensively used for primary yeast separation in the production of alcoholic beverages, and in waste-water treatment. Low-cost and slow technology is suitable only for large flocs (greater than 100µm diameter). The rate of particle sedimentation is a function of both size and density. The larger the particle and the greater its density the faster the rate of sedimentation. The basis of this method of separation is sedimentation under gravity, which for a spherical particle can be represented by Stokes’ Law: For rapid sedimentation the difference in density between the particle and the medium needs to be large, and the medium viscosity must be low. Vg = rate of particle sedimentation (m/s) dp = diameter of the particle (m) ps – pl = difference in density between the particle and surrounding medium (kg/m 3 ) g = gravitational acceleration (m/s 2 ); and h= viscosity (Pascal seconds (Pa s))

7. Centrifugation Used to separate particles as small as 0.1µm diameter and for some liquid–liquid separations. Its effectiveness depends on particle size, density difference between the cells and the medium, and medium viscosity. In a centrifuge, the terminal velocity of a particle is The faster the operating speed (w) and the greater the distance from the centre of rotation, the faster the sedimentation rate ( Vc ). Centrifuges can be compared using the relative centrifugal force (RCF). Higher-speed centrifuges achieving RCF of 20000g may be required to recover suspended bacterial cells, cell debris and protein precipitates from liquid media. Vc = centrifugal sedimentation rate or particle velocity (m/s) w= angular velocity of the centrifuge ( rad /s); and r = distance of the particle from the centre of rotation (m) dp = diameter of the particle (m) ps – pl = difference in density between the particle and surrounding medium (kg/m 3 ) h= viscosity (Pascal seconds (Pa s))

8. Advantages The availability of fully continuous systems that can rapidly process large volumes in small volume centrifuges. Centrifuges are steam sterilizable, allowing aseptic processing. Disadvantages High initial capital costs Noise generated during operation Cost of electricity. Physical rupture of cells may occur due to high shear and the temperature may not be closely controllable, which can affect temperature-sensitive products.

9. INDUSTRIAL CENTRIFUGES Centrifuges can be divided into small-scale laboratory units and larger pilot- and industrial-scale centrifuges. For most industrial purposes semicontinuous and continuous centrifuges are required to process the large volumes involved. However, the RCFs achieved are relatively low. Four main types of industrial centrifuge are commonly used Tubular centrifuges Multichamber bowl centrifuges Disc stack centrifuges Screw-decanter centrifuges

10. Tubular centrifuges Produce the highest centrifugal force of 13000–17000g. Particulate material is thrown to the side of the bowl Clarified liquid passes out at the top for continuous collection. As the particulate material accumulates on the inside of the bowl, the operating diameter becomes reduced. Cleaning is required

11. Multi chamber bowl centrifuges It consist of a bowl that is divided by vertically mounted interconnecting cylinders Capable of operating at 5000–10000 g. The liquid feed passes from the centre through each chamber in turn, and the smaller particles collect in the outer chambers.

12. Disc stack centrifuges Operate at 5000–13000g . The centrifuge bowl contains a stack of conical discs whose close packing aids separation. As liquid enters the centrifuge particulate material is thrown outwards. These centrifuges usually have the facility to discharge the collected material periodically during operation.

13. Screw-decanter centrifuges They operate continuously at 1500–5000g Suitable for dewatering coarse solid materials at high solids concentrations. Used in sewage systems for the separation of sludge, and for harvesting yeasts and fungal mycelium.

14. Filtration Conventional filtration of liquids containing suspended solids involves depth filters composed of porous media (cloth, glass wool or cellulose) that retain the solids and allow the clarified liquid filtrate to pass through. As filtration proceeds collected solids accumulate above the filter medium, resistance to filtration increases and flow through the filter decreases. These techniques are generally useful for harvesting filamentous fungi, but are less effective for collecting bacteria. The two main types of conventional filtration commonly used in industry are Plate and frame filters or filter presses Rotary vacuum filters

15. Plate and frame filters or filter presses They are industrial batch filtration systems. Here a series of cloth-lined chambers are formed into which the cell suspension is forced under pressure. These systems are used for harvesting microorganisms from fermentations, including the preparation of blocks of baker’s yeast, the recovery of protein precipitates and the dewatering of sewage sludge.

16. Rotary vacuum filters They are simple continuous filtration systems that are used in several industrial processes, particularly for harvesting fungal mycelium during antibiotic manufacture, for baker’s yeast production and in dewatering sludge during waste-water treatment. The device comprises a hollow perforated drum that supports the filter medium. This drum slowly rotates in a continuously agitated tank containing the suspension to be filtered . Solids accumulate on the filter medium as liquid filtrate is drawn, under vacuum, through the filter medium into the hollow drum to a receiving vessel. As the drum rotates, collected solids held on the filter medium are removed by a knife that cuts/sloughs them off into a collection vessel. SLURRY CLEAR FILTRATE FILTER MEDIA

17. FILTER PRESS ROTARY VACUUM FILTER

18. MEMBRANE FILTRATION Modern methods of filtration involve absolute filters rather than depth filters . These consist of supported membranes with specified pore sizes that can be divided into three main categories. 1.Microfiltration 2. Ultrafiltration 3. Reverse osmosis membranes. As filtration progresses, the flux across the membrane can slow due to membrane fouling. The suspension to be filtered is pumped across the membrane (cross-/tangential-flow) rather than at a right angle to it, as occurs with conventional filtration methods. This retards fouling of the membrane by particulate materials.

19. Microfiltration Microfiltration is used to separate particles of 2µm to 10µm , including removal of microbial cells from the fermentation medium. This method is relatively expensive due to the high cost of membranes, but it has several advantages compared with centrifugation. They include quiet operation, lower energy requirements, the product can be easily washed, good temperature control is possible. Consequently, it is suitable for handling pathogens and recombinant microorganisms.

20. Ultra filtration It is similar to microfiltration except that the membranes have smaller pore sizes, and are used to fractionate solutions according to molecular weight, normally within the range 2000–500000Da. The membranes are composed of a thin membrane with pores of specified diameter providing selectivity, lying on top of a thick, highly porous, support structure. Several of these ultrafiltration units can be linked together to produce a sophisticated purification system. These methods are extensively employed for the purification of proteins, and for separating and concentrating materials. Ultrafiltration is also effective in removing pyrogens (bacterial cell wall lipopolysaccharides ), cell debris and viruses from media, and for whey processing. Another variation on the ultrafiltration system is diafiltration , where water or other liquid is filtered to remove unwanted low molecular weight contaminants.

21. Reverse osmosis Reverse osmosis is used for dewatering or concentration steps and has been employed to desalinate sea water for drinking. In osmosis water will cross a semipermeable membrane if the concentration of osmotically active solutes, such as salt, is higher on the opposite side of the membrane. However, if pressure is applied to the ‘salt side’ then reverse osmosis will occur, and water will be driven across the membrane from the salt side. This reversal of osmosis requires a high pressure, e.g. a pressure of 30–40 bar is needed to dewater a 0.6mmol/L salt solution (note: 1 bar=100kPa=0.987atm). A strong metal casing is required to house this equipment. As the membranes have pore sizes of only 10-2 to 10-4 µm diameter, solute molecules can deposit on the surface, causing a large resistance to solvent flow.

22. MECHANICAL CELL DISRUPTION Currently, intracellular products are released from microorganisms mainly by mechanical disruption of the cells. In this process, the cell envelope is physically broken, releasing all intracellular components into the surrounding medium. Physico mechanical method Liquid shear Solid shear Agitation with abbressive Freeze thawing Ultrasonication

23. HOMOGINISER In these devices the cell suspension is drawn through a check valve into a pump cylinder. At this point, it is forced under pressure (up to 1500 bar) through a very narrow annulus or discharge valve, over which the pressure drops to atmospheric. Cell disruption is accomplished by two different mechanisms: 1. High liquid shear in the orifice 2. Sudden pressure drop upon discharge causing finally an explosion of the cell. The method is applied mainly for the release of intracellular enzymes

24. Solid shear Pressure extrusion of frozen microorganisms around - 25°C through small orifice is well established technique at laboratory scale using X press or hughes press. Disruption is due to combination of liquid shear through a narrow orifice and the presence of ice crystals It was possible to obtain 90 % disruption with a single passage of S. cerevisiae using throughput of 10 kg yeast cells paste per hour This technique might be ideal for extraction of products which might be temperature labile

25. Grinding On a small scale, manual grinding of cells with abrasives,usually alumina, glass beads or silica can be an effective means of disruption, but results may not be reproducible. In industry, high-speed bead mills, equipped with cooling jackets, are often used to agitate a cell suspension with small beads (0.5–0.9 μm diameter) of glass, zirconium oxide or titanium carbide. Cell breakage results from shear forces, grinding between beads and collisions with beads. The efficiency of cell breakage is a function of agitation speed, concentration of beads, bead density and diameter, broth density, flow rate and temperature. Maximum throughput in these systems is about 2000 L/h.

26. Cascading beads Cells being disrupted Rolling beads

27. Ultrasonic disruption Ultrasonic disruption is performed by ultrasonic vibrators that produce a high-frequency sound with a wave density of approximately 20 kilohertz/s. A transducer converts the waves into mechanical oscillations via a titanium probe immersed in the concentrated cell suspension. Cell suspension Ultrasound tip Ultrasound generator

28. Draw backs Highly effective at lab scale Power requirement is high Large heating effects that effects thermolabile proteins Noice generation Short working life of probe Continuous operation is not possible

29. Non-mechanical cell disruption methods An alternative to mechanical methods of cell disruption is to cause their permeabilization . This can be accomplished by autolysis, osmotic shock, rupture with ice crystals (freezing/thawing) or heat shock. AUTOLYSIS Used for the production of yeast extract and other yeast products. It has the advantages of lower cost and uses the microbes’ own enzymes, so that no foreign substances are introduced into the product.

30. Freezing and thawing Freezing and thawing of microbial paste will cause ice crystal formation and their expansion followed by thawing will cause disruption of cell DRAWBACKS 1. Slow technique 2. Limited release of cellular material Use : To obtain β glucosidase from S. cerevisiae

31. Detergents Components use for cell disruption includes quaternary ammonium compound, sodium lauryl sulphate , SDS, Triton X 100 etc USES To extract pollulanase from cell wall of K. pneumoniae cell were suspended in 1 % sodium cholate and stirred for 1 hour to solubilize enzyme Triton X 100 in combination with guanidine-HCL can release 75 % proteins in less than one hour from E. coli LIMITATIONS Detergent may cause denaturation of protein Detergent must be removed before further purification stages

32. Osmotic shock Dramatic change in the solute concentration of the liquid surrounding the microorganism – can cause the cell to burst Osmotic shock is often useful for releasing products from the periplasmic space. This may be achieved by equilibrating the cells in 20% (w/v) buffered sucrose, then rapidly harvesting and resuspending in water at 4°C. USE Extraction of luciferase from Photobacterium fischeri Only low levels of soluble proteins were released using this technique

33. Enzyme treatment Several cell wall degrading enzymes have been successfully employed in cell disruption. For example, lysozyme , which hydrolyses β -1,4 glycosidic linkages within the peptidoglycan of bacterial cell walls, is useful for lysing Gram-positive organisms. Addition of ethylene diamine tetraacetic acid (EDTA) to chelate metal ions also improves the effectiveness of lysozyme and other treatments on Gram-negative bacteria. This is because EDTA has the ability to sequester the divalent cations that stabilize the structure of their outer membranes. Enzymic destruction of yeast cell walls can be achieved with snail gut enzymes that contain a mixture of β glucanases . These enzyme preparations are also useful for producing living yeast protoplasts.

34. Antibiotic treatment The antibiotics penicillin and cycloserine may be used to lyse actively growing bacterial cells, often in combination with an osmotic shock. Other permeabilization techniques include the use of basic proteins such as protamine ; the cationic polysaccharide chitosan is effective for yeast cells.

35. Product recovery Recovery of extracellular proteins is from the clarified medium Disrupted cell preparations are used for both intracellular proteins and those held within the periplasmic space Following cell disruption, soluble proteins are usually separated from cell debris by centrifugation. The resultant supernatant, containing the proteins, is then processed. Products can be recovered by Chromatography Dialysis and electro dialysis Distillation

36. Chromatography Chromatographic techniques are usually employed for higher-value products normally involving columns of chromatographic media (stationary phase). In choosing a chromatographic technique for protein products molecular weight, isoelectric point, hydrophobicity and biological affinity should be considered. Each of these properties can be exploited by specific chromatographic methods that may be scaled up to form an industrial unit process.

37. Adsorption chromatography Involves binding of solute molecules to solid phase primarily by weak van der Waals forces. Separates according to the affinity of the protein, or other material, for the surfaces of the solid matrix. The material used to pack the column for chromatography includes active carbon, Aluminium oxide, magnesium oxide, silica etc. USES Purification of antibiotics and removal of pigments

38. Affinity chromatography The technique involves specific chemical interactions between solute molecules, such as proteins, and an immobilized ligand (functional molecule). Ligands are covalently linked to the matrix material, e.g. agarose . Some ligands interact with a group of proteins, notably nicotinamide adenine dinucleotide , adenosine monophosphate ; other ligands are highly specific, especially substrates, substrate analogues and antibodies. Elution is achieved using specific cofactors or substrates; alternatively, non-specific elution may be performed with salt or pH change.

39. ADVANTAGES Give up to several thousand-fold purification in a single step Large sample volumes can be purified High speed technique DISADVANTAGES Expensive on an industrial scale Some ligands are heat sensitive so it may cause problem during sterilization USES Enzyme purification Antigen purification Since monoclonal antibodies have become more readily available, immunoaffinity chromatography methods have been developed for the purification of various antigens.

40. Gel filtration chromatography It essentially involves separation on the basis of molecular size (molecular sieving). The stationary phase consists of porous beads composed of acrylic polymers, agarose , cellulose, cross-linked dextran , Solute molecules below the exclusion size of the support material pass in and out of the beads. Molecules above the exclusion size pass only around the outside of the beads through the interstitial spaces and the apparent volume of the column is smaller for these larger excluded molecules. As a result, they flow faster down the column, separating from smaller molecules and eluting first. Smaller molecules able to enter the pores are then eluted in decreasing order of size. USES It is particularly useful for desalting protein preparations.

41. Ion-exchange chromatography It involves the selective adsorption of ions or electrically charged compounds onto ion-exchange resin particles by electrostatic forces. The matrix material is often based on cellulose substituted with various charged groups, either cations or anions. A commonly used example is the anion-exchange resin diethylaminoethyl (DEAE) cellulose. Proteins possess positive, negative or no charge depending on their isoelectric point ( pI ) and the pH of the surrounding buffer

42. High-performance liquid chromatography (HPLC) It was originally developed for the separation of organic molecules in non-aqueous solvents, but is now used for proteins in aqueous solution. This method uses densely packed columns containing very small rigid particles, 5–50μm diameter, of silica or a cross-linked polymer. Consequently, high pressures are required. The method is fast and gives high resolution of solute molecules. Equipment for use in large-scale operations is now available.

43. Hydrophobic chromatography Relies on hydrophobic interaction between hydrophobic regions of a solute protein and hydrophobic functional groups of the support particles. These supports are often agarose substituted with octyl or phenyl groups. Elution from the column is usually achieved by altering the ionic strength, changing the pH or increasing the concentration of ions, e.g. thiocyanate . This technique provides good resolution and, like ion-exchange chromatography, has a very high capacity as it is not limited by sample volume.

44. Metal chelate chromatography It utilizes a matrix with attached metal ions, e.g. agarose containing calcium, copper or magnesium ions. The protein to be purified must have an affinity for this ion and binds to it by forming coordination complexes with groups such as the imidazole of histidine residues. Bound proteins are then eluted using solutions of free metal-binding ligands , e.g. amino acids.

45. Dialysis and electrodialysis Low molecular weight solutes move across the membrane by osmosis from a region of high concentration to one of low concentration. Membranes used contain ion-exchange groups and have a fixed charge; e.g. positively charged membranes allow the passage of anions and repel cations. ELECTRODIALYSIS methods separate charged molecules from a solution by the application of a direct electrical current carried by mobile counter-ions. USES Primarily used for the removal of low molecular weight solutes and inorganic ions from a solution.

46. Distillation Distillation is used to recover fuel alcohol, acetone and other solvents from fermentation media, and for the preparation of potable spirits. Batch distillation in pot stills continues to be used for the production of some whiskies but for most other purposes continuous distillation is the method of choice. With ethanol, for example, the continuous system produces a product with a maximum ethanol concentration of 96.5% (v/v). This mixture is the highest concentration that can be achieved from aqueous ethanol.

47. Finishing steps CRYSTALLIZATION Used in initial recovery of organic acids and amino acids Product crystallization may be achieved by evaporation, low-temperature treatment or the addition of a chemical reactive with the solute. The product’s solubility can be reduced by adding solvents, salts, polymers (e.g. Nonionic PEG) and polyelectrolytes , or by altering the pH.

48. Drying Drying involves the transfer of heat to the wet material and removal of the moisture as water vapour . This must be performed in such a way as to retain the biological activity of the product. Parameters affecting drying physical properties of the solid–liquid system intrinsic properties of the solute conditions of the drying environment and heat transfer parameters Heat transfer may be by direct contact, convection or radiation.

49. Driers ROTARY DRUM DRIERS remove water by heat conduction. A thin film of solution is applied to the steam heated surface of the drum, which is scraped with a knife to recover the dried product. In VACUUM TRAY DRIERS the material to be dried is placed on heated shelves within a chamber to which a vacuum is applied. This allows lower temperatures to be used due to the lower boiling point of water at reduced pressure. The method is suitable for small batches of expensive materials, such as some pharmaceuticals. SPRAY DRYING involves spraying of product solution into a heated chamber, and resultant dried particles are separated from gases using cyclones.

50. Freeze-drying (lyophilization) In this method, frozen solutions of antibiotics, enzymes or microbial cell suspensions are prepared and the water is removed by sublimation under vacuum, directly from solid to vapour state. This method eliminates thermal and osmotic damage. USES Often used where the final products are live cells, as in starter culture preparations, or for thermo labile products. This is especially useful for some enzymes, vaccines and other pharmaceuticals, where retention of biological activity is of major importance.

51. References Industrial Microbiology- an introduction by- Michael J. Waites , Neil C. Morgan, John S. Rockey , Gary Higton Industrial Microbiology by Lester Earl Casida Principal of fermentation technology 3 rd edition by P. F. Stanbury , A. Whitakar and S.J. Hall. Krishna Prashad 2010- downstream processing a new horizon in biotechnology

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