1. BTC 504-3 Unit USP and DSP Operations in Fermentations

2. What is fermentation? Fermentation is a process of chemical change caused by organisms or their products, usually producing effervescence and heat. It is done by means of mass culture of micro organisms. It is the oldest of all biotechnological processes. Fermentation is derived from the Latin verb FEVER To boil the appearance of fruit extracts or malted Grain acted upon by yeast, during the production of alcohol.

3. Unit operations in USP and DSP in fermentation Fermentation is a process which is combination of many more processing units leading to Unit Operations. Unit operation in fermentation means “separate operation processes needed for making products from fermentation procedure”. Fully separate operational unit or zone No linkage in between the activity or area of each unit

4. Two types: First part- Upstream unit operation  Media sterilization Media Formulation Inoculum development Fermentation Second part- Downstream unit operation  Solid-liquid separation ( cell-media separation) Decantation Filtration Centrifugation crystallization Inter-cellular product collection by cell breakage Contd……

5. Media Sterilization: Mostly Media is sterilized by continuous flow through a heat exchanger and holding tank in shorter time at a given temperature. Steam is applied and the less drastic heating minimize changes in the media, giving higher product titres. Sterilization Procedures - Sterilization involves either inactivation or removal of living organisms. This may be achieved by (i) Heating  BY using dry heat or steam (commonly used) (ii) Irradiation  High energy X-rays are used (iii) Chemicals  Formaldehyde, H2O2, ethylene oxide, propylene oxide etc. which cause inactivation by alkylation or oxidation. (iv) Filtration  Depth filter or screen filter removes fungal spores or bacteria from air used in aerobic process.

6. Media formulation: The role of media in fermentation: In inoculum (starter culture) propagation steps, pilot-scale fermentations and the main production fermentation. Provide a production medium that allows optimal growth of the micro organisms to produce biomass or primary metabolites. Provide an initial period of cell growth for production of secondary metabolites like antibiotics where the nutrient content is limited to cease the rapid growth after log phase. The table which applies to various differential media in which certain microbial activities are shown:

7. Inoculum development: The process adopted to produce an inoculum meeting some defined developed criteria is called “inoculum development”. A critical factor in obtaining a suitable inoculum is the choice of the culture medium. The developed media in inoculum not necessarily support the production of product rather give support the growth of organisms. In big fermenter, we first need a stock culture. Starting from the stock, the inoculum must be built up in a number of stages to produce sufficient biomass to inoculate the production-stage fermenter. Examples of commonly used inoclum development medium are: Whey powder, lactose, corn-steep, soybean flour, sugar-beet, cane- molasses, etc.

8. The basic inoculum development flow chart: Stock culture Maintenance culture Seed culture-second stage Production culture Seed culture-First stage

9. Fermentation:

10. 10 Bioreactor configurations Stirred tank bioreactor Similar to CSTR; this requires a relatively high input of energy per unit volume. Baffles are used to reduce vortexing. A wide variety of impeller sizes and shapes is available to produce different flow patterns inside the vessel; in tall fermenters, installation of multiple impellers improves mixing. Typically, only 70-80 % of the volume of stirred reactors is filled with liquid; this allows adequate headspace for disengagement of droplets from exhaust gas and to accommodate any foam which may develop. Foam breaker may be necessary if foaming is a problem. It is preferred than chemical antifoam because the chemicals reduce the rate of oxygen transfer. The aspect ratio (H/D) of stirred vessels vary over a wide range. When aeration is required, the aspect ratio is usually increased. This provides for longer contact times between the rising bubbles and liquid and produces a greater hydrostatic pressure at the bottom of the vessel. Care is required with particular catalysts or cells which may be damaged or destroyed by the impeller at high speeds.

11. 11 Bioreactor configurations

12. 12 Bioreactor configurations Bubble column In bubble-column reactors, aeration and mixing are achieved by gas sparging; this requires less energy than mechanical stirring. Bubble columns are applied industrially for production of bakers’ yeast, beer and vinegar, and for treatment of wastewater. A height-to-diameter ratio of 3:1 is common in bakers’ yeast production; for other applications, towers with H/D of 6:1 have been used. The advantages are low capital cost, lack of moving parts, and satisfactory heat and mass transfer performance. Foaming can be problem. Homogeneous flow: all bubbles rise with the same upward velocity and there is no back-mixing of the gas phase. Heterogeneous flow: At higher gas velocity. Bubbles and liquid tend to rise up in the center of the column while a corresponding down flow of liquid occurs near the walls.

13. 13 Bioreactor configurations Airlift reactor Airlift reactors are often chosen for culture of plant and animal cells and immobilized catalyst because shear level are low. Gas is sparged into only part of the vessel cross section called the riser. Gas hold-up and decreased liquid fluid density cause liquid in the riser to move upwards. Gas disengages at the top of the vessel leaving heavier bubble-free liquid to recirculate through the downcomer. Airlift reactors configurations are internal-loop vessels and external-loop vessels. In the internal-loop vessels, the riser and downcomer are separated by an internal baffle or draft tube. Air may be sparged into either the draft tube or the annulus. In the external-loop vessels, separated vertical tubes are connected by short horizontal section at the top and bottom. Because the riser and downcomer are further apart in external-loop vessels, gas disengagement is more effective than in internal-loop devices. Fewer bubbles are carried into the downcomer, the density difference between fluids in the riser and downcomer is greater, and circulation of liquid in the vessel is faster. Accordingly, mixing is usually better in external-loop than internal- loop reactors.

14. 14 Bioreactor configurations

15. 15 Stirred and air-driven reactors: comparison of operating characteristic For low-viscosity fluids, adequate mixing and mass transfer can be achieved in stirred tanks, bubble columns and airlift vessels. When a large fermenter (50- 500 m 3 ) is required for low-viscosity culture, a bubble column is an attractive choice because it is simple and cheap to install and operate. Mechanical- agitated reactors are impractical at volumes greater than about 500 m 3 as the power required to achieve adequate mixing becomes extremely high. Stirred reactor is chosen for high-viscosity culture. Nevertheless, mass transfer rates decline sharply in stirred vessels at viscosities > 50-100 cP. Mechanical-agitation generates much more heat than sparging of compressed gas. When the heat of reaction is high, such as in production of single cells protein from methanol, removal of frictional stirrer heat can be problem so that air-driven reactors may be preferred. Stirred-tank and air-driven vessels account for the vast majority of bioreactor configurations used for aerobic culture. However, other reactor configurations may be used in particular processes

16. 16 Other bioreactors Packed bed Used with immobilized or particulate biocatalysts, for example during the production of aspartate and fumarate, conversion of penicillin to 6- aminopenicillanic acid, and resolution of amino acid isomers. Damaged due to particle attrition is minimal in packed beds compared with stirred reactors. Mass transfer between the liquid medium and solid catalyst is facilitated at high liquid flow rate through the bed. To achieve this, packed are often operated with liquid recycle. The catalyst is prevented from leaving the columns by screens at the liquid exit. Aeration is generally accomplished in a separated vessel because if air is sparged directly into the bed, bubble coalescence produces gas pockets and flow channeling or misdistribution. Packed beds are unsuitable for processes which produce large quantities of carbon dioxide or other gases which can become trapped in the packing.

17. 17 Other bioreactors Fluidized bed To overcome the disadvantages of packed bed, fluidized bed may be preferred. Because particles are in constant motion, channeling and clogging of the bed are avoided and air can be introduced directly into the column. Fluidized bed reactors are used in waste water treatment with sand or similar material supporting mixed microbial populations, and with flocculating organisms in brewing and production of vinegar. Trickle bed Is another variation of the packed bed. Liquid is sprayed onto top of the packing and trickles down through the bed in small rivulets. Air may be introduced at the base; because the liquid phase is not continuous throughout the column, air and other gases move with relative ease around the packing. Trickle-bed reactors are used widely for aerobic wastewater treatment.

18. 18 Other bioreactors

19. OPTIMIZATION OF FERMENTATION PROCESS Fermenter design Process optimisation- Monitor and Control

20. FERMENTOR DESIGN Choice of reactor configuration depends on; (a) BIOCATALYST; Animal/ plant cells Microbial cells; Growing Non-growing Enzymes; Soluble Immobilised

21. Choice of reactor configuration depends on (b) Reactor configuration; Batch, Semi-, Continuous, Plug-flow Free, Immobilised (c) Economics; Value of product Degree of process control Product parameters

22. DESCRIPTION OF MAJOR FERMENTOR CONFIGURATIONS Laboratory vs Industrial scale Batch Continuous Tower / loop, air-lift Plug-flow Immobilised Geometry / shape Types of aerators and agitators Generalised difference between animal, plant and microbial cells

23. Downstream Processing-What and Why Downstream processing is any treatment of culture broth after fermentation to concentrate and purify products. It follows a general sequence of steps: Cell removal (filtration, centrifugation) Primary isolation to remove components with properties significantly different from those of the products (adsorption, liquid extraction, precipitation). Large volume, relatively non selective

24. Downstream processing, what and why Purification-Highly selective (chromatography, ultra filtration, fractional precipitation) Final isolation (crystallization, followed by centrifugation or filtration and drying). Typical for high-quality products such as pharmaceuticals. Downstream processing mostly contributes 40-90 % of total cost.

25. Unit operations after completion of fermentations: There are specific operational units after fermentation has been completed which involve product harvesting & recovery Separation of the cell from the ambient fluid : 1. Screening through a sieve 2. Dead-end filtration 3. Cross flow filtration 4. Decantation 5. Centrifugation 6. Combination of two or more operations - Sometimes coagulants or filter aids may be used to assist processing.

26. Isolation of a rather impure product from the fluid Purification of the crude impure product  These two steps involve potential unit operations like: 1. Solvent extraction 2. Ion exchange chromatography 3. Absorption 4. Gel & affinity chromatography 5. Precipitation 6. Crystallization 7. Spray-drying 8. Freeze drying 9. Electrophoresis - Specific processes are chosen taking into account the physical and chemical properties of the required product and the impurities present. Contd……

27. -With all the different possibilities of ion exchange, reverse-phase, affinity, size-exclusion, ion-pairing and any other of the numerous techniques, this liquid chromatography offers. - NPC, RPC, IEC, SEC all can be done by this in a large scale.

28. Microbial cell disintegration methods: If the product is intracellular, before purification, the cells need disruption or lysis. 2 types: 1.Non-mechanical: Chemical treatment (acids,bases,detergents,solvents) Physical treatment (freeze thawing, osmotic shock) Enzymatic degradation: Lytic enzymes, phages 2. Mechanical: Wet milling High pressure homogenization Pressure exclusion Sonication

29. Cell separation: After the fermentation is completed, one of the following operations facilitate cell separation: 1. Sedimentation 2. Floatation 3. Filtration and ultra-filtration (vaccines, viruses, proteins of m.w 500-100,000) 4. Centrifugation 5. Cross flow membrane filtration Filtration

30. Type of filtration unit: Plate and frame filter. For small fermentation batches Rotary-drum vacuum filter. Continuous filtration that is widely used in the fermentation industry. A horizontal drum 0.5-3 m in diameter is covered with filter cloth and rotated slowly at 0.1-2 rpm.

32. Centrifugation is used to separate materials of different density when a force greater than gravity is desired The type of industrial centrifugation unit: Tubular bowl centrifuge (Narrow tubular bowl centrifuge or ultracentrifuge, decanter centrifuge, etc). Simple and widely applied in food and pharmaceutical industry. Operates at 13000-16000 G, 105-106 G for ultracentrifuge Centrifugation

33. Disc-stack bowl centrifuge. This type is common in bioprocess. The developed forces is 5000-15000 G with minimal density difference between solid and liquid is 0.01-0.03 kg/m3. The minimum particle diameter is 5 µm

34. Mechanical cell disruption methods press (pressure cell) and high-pressure homogenizers. 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 primary achieved by high liquid shear in the orifice and the sudden pressure drop upon discharge causes explosion of the cells.

35. Ultrasonic disruption It is performed by ultrasonic vibrators that produce a high- frequency sound with a wave density of approximately 20 kHz/s. A transducer convert the waves into mechanical oscillations via a titanium probe immersed in the concentrated cell suspension. For small scale

36. Cell disruption

37. Non mechanical cell disruption methods Autolysis, use microbe own enzyme for cell disruption Osmotic shock. Equilibrating the cells in 20% w/v buffered sucrose, then rapidly harvesting and resuspending in water at 4oC. Addition of chemicals (EDTA, Triton X- 100), enzymes (hydrolyses, b-glucanases), antibiotics (penicillin, cycloserine) Cell disruption

38. Strategy Move from organism to pure protein in as few steps as possible with as little loss of activity (assayable quality) as possible Time and temperature are factors

39. Protocols for Protein Purification Highly individualized Use a common approach Fractionate crude extract in a way that protein of interest always goes into the pellet or the supernatant. Follow progress with functional assay

40. Lactate Dehydrogenase NADH + H + + Pyruvate  =  NAD + + Lactate Enzyme clears lactic acid from working muscles The obvious source of enzyme is muscle tissue (heart & skeletal muscle, H&M, isomers) We will assay for the enzymes ability to convert

41. Begin with intact tissue Disrupt Blender, homoginizer Remove debris Centrifugation Precipitate/concentrate Ammonium sulfate Remove salt dialysis Purify Chromatography Analyze Activity, molecular weight

42. Ammonium Sulfate precipitation Has a wide range of application Relies on fact that proteins loose solubility as concentration of salt is increased Is characteristic of particular protein Results in a partial purification of all proteins with similar solubility characteristics Must determine [amm sulf] to precipitate your protein empirically. Produces “salt cuts”

43. Salting in / Salting out Salting IN At low concentrations, added salt usually increases the solubility of charged macromolecules because the salt screens out charge- charge interactions. So low [salt] prevents aggregation and therefore precipitation or “crashing.” Salting OUT At high concentrations added salt lowers the solubility of macromolecules because it competes for the solvent (H 2 O) needed to solvate the macromolecules. So high [salt] removes the solvation sphere from the protein molecules and they come out of solution.

44. Kosmotrope vs. Chaotrope Ammonium Sulfate Increasing conc causes proteins to precipitate stably. Kosmotropic ion = stabilizing ion. Urea Increasing conc denatures proteins; when they finally do precipitate, it is random and aggregated. Chaotropic ion = denaturing ion.

45. The terms 'kosmotrope' (order- maker) and 'chaotrope' (disorder-maker) originally denoted solutes that stabilized, or destabilized respectively, proteins and membranes; kosmotropes stabilize proteins and hydrophobic aggregates in solution and reduce the solubility of hydrophobes.

46. thus chaotropes unfold proteins, destabilize hydrophobic aggregates and increase the solubility of hydrophobes

47. Dialysis Passage of solutes through a semi-permeable membrane. Pores in the dialysis membrane are of a certain size. Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.

48. Column Chromatography

49. Gel Filtration

50. Principles of gel filtration (molecular sieving) 10 6 Da 3x10 5 Da 10 5 Da 10 4 Da 1. Apply a mixture of proteins on a gel filtration column (Sepharose, Sephacryl, etc) 2. Collect fractions, typically 120 from a 1.5x100 cm column. Do not change buffer composition 3. High molecular weight macromolecules (higher Stoke’s radius) elute first 4. Determine proteins in eluate using suitable assay 5. Estimate approximate molecular weight of unknown proteins and/or protein complexes using calibration curve with pre-run standard proteins of known M.Wt. and the following formula: Kav = Ve -Vo Vt - Vo Ve – elution volume Vo – void volume Vt – total volume Kav Log M.Wt.

51. Ion Exchange

52. Affinity Chromatography We will use bound Adenosine -5’-monophosphate. This is part Of NAD +. LDH will Bind. Release LDH by adding NADH

53. Affinity chromatography Remember: NADH is a co-substrate for lactate dehydrogenase. We use AMP-Sepharose: AMP is covalently bound to the affinity gel, which will not pass through the filter. LDH binds to the AMP b/c it looks like half an NADH. Thus LDH remains immobilized in the column until we ad NADH which binds tighter to the LDH.

54. Protein Purification A 280 Activity NADH

55. Protein Concentration Lowry ( most cited reference in biology) Color assay A 280 Intrinsic absorbance Relies on aromatic amino acids BCA Modification of Lowry: increased sensitivity and consistency Bradford Shifts A max of dye from 465nm to 595nm

56. A 280 Uses intrinsic absorbance Detects aromatic residues Resonating bonds Depends on protein structure, native state and AA composition Retains protein function

57. PAGE Apparatus (purity and MW)

58. Protein separation using SDS-PAGE (Laemmli system) Stacking gel Resolving gel 1. Apply protein/dye samples into polyacrylamide gel wells 2. Run the electrophoresis until dye reaches the end of the gel 3. Remove the gel from the apparatus and stain for proteins

59. SDS PAGE of Purification 1.Complete mix of proteins 2.High Salt 3.Ion exchange 4.Gel-filtratio 5.Affinity 10micrograms loaded in each lane

60. Chromatographic techniques usually employed for high value products. These methods, normally involving columns of chromatographic media (stationary phase), are used for desalting, concentration and purification of protein preparations. Several important aspects are molecular weight, isoelectric point, hydrophobicity and biological affinity. Chromatography

62. Crystallization 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, and polyelectrolytes, or by altering pH. Finishing steps (final isolation)

63. 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. The equipment could be rotary drum drier, vacuum tray drier, or freeze-drier.