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Basic design of a fermenter

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Basic design of a fermenter

  1. 1. Fermentation M.Sc. Biotechnology Part II (Sem III) Mumbai University Paper III - Unit I & II By: Mayur D. Chauhan 1
  2. 2. Fermentation • Fermentation is a metabolic process that converts sugar to acids, gases or alcohol. • It occurs in yeast and bacteria, but also in oxygen- starved muscle cells, as in the case of lactic acid fermentation. • Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium, often with the goal of producing a specific chemical product. • The science of fermentation is known as zymology 2
  3. 3. Range of Fermentation Processes • To Produce Microbial cells or Biomass • To Produce Microbial Enzymes • To Produce Microbial Metabolites • To Produce Recombinant Products • To modify a compound which is added to the fermentation (Transformation) 3
  4. 4. Steps to carry out a Fermentation • The formulation of media to be used in culturing the process organism during the development of the inoculum and in the production fermenter. • The sterilization of the medium, fermenters and ancillary equipment. • The production of an active, pure culture in sufficient quantity to inoculate the production vessel. 4
  5. 5. • The growth of the organism in the production fermenter under optimum conditions for product formation. • The extraction of the product and its purification. • The disposal of effluents produced by the process 5
  6. 6. Basic Design of a Fermenter 6
  7. 7. Various components of an ideal fermenter for batch process are: 7
  8. 8. Monitoring and controlling parts of fermenter are: 8
  9. 9. Basic Functions of a Fermenter • The vessel should be capable of being operated aseptically for a number of days and should be reliable in long-term operation and meet the requirements of containment regulations. • Adequate aeration and agitation should be provided to meet the metabolic requirements of the micro-organism. However, the mixing should not cause damage to the organism. • Power consumption should be as low as possible. • A system of temperature control should be provided. 9
  10. 10. • A system of pH control should be provided. • Sampling facilities should be provided. • Evaporation losses from the fermenter should not be excessive. • The vessel should be designed to require the minimal use of labour in operation, harvesting, cleaning and maintenance. • Ideally the vessel should be suitable for a range of processes, but this may be restricted because of containment regulations. 10
  11. 11. • The vessel should be constructed to ensure smooth internal surfaces, using welds instead of flange joints whenever possible. • The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant or plant to facilitate scale-up. • The cheapest materials which enable satisfactory results to be achieved should be used. • There should be adequate service provisions for individual plants 11
  12. 12. Hazard Assessment Systems • Once the organism has been allocated to a hazard group, the appropriate containment requirements can be applied. • Hazard group 1 organisms used on a large scale only require Good Industrial Large Scale Practice (GILSP). • Processes in this category need to be operated aseptically but no containment steps are necessary, including prevention of escape of organisms. • If the organism is placed in Hazard group 4 the stringent requirements of level 3 will have to be met before the process can be operated. 12
  13. 13. 13
  14. 14. Materials for Body Construction of a Fermenter • In fermentations with strict aseptic requirements it is important to select materials that can withstand repeated steam sterilization cycles. • On a small scale (1 to 30 dm3) it is possible to use glass and/or stainless steel. • Glass is useful because it gives smooth surfaces, is non-toxic, corrosion proof and it is usually easy to examine the interior of the vessel. 14
  15. 15. Two basic types of Fermenters • A glass vessel with a round or flat bottom and a top flanged carrying plate. • All vessels of this type have to be sterilized by autoclaving. 15
  16. 16. 16
  17. 17. • A glass cylinder with stainless-steel top and bottom plates. • Vessels with two stainless steel plates cost approximately 50% more than those with just a top plate. • At pilot and large scale, when all fermenters are sterilized in situ, any materials used will have to be assessed on their ability to withstand pressure sterilization and corrosion and on their potential toxicity and cost. 17
  18. 18. 18
  19. 19. • Pilot scale and Industrial scale vessels are normally constructed of stainless steel or at least have a stainless-steel cladding to limit corrosion. • The American Iron and Steel Institute (AISI) states that steels containing less than 4% chromium are classified as steel alloys and those containing more than 4% are classified as stainless steels. • Mild steel coated with glass or phenolic epoxy materials has occasionally been used 19
  20. 20. • The corrosion resistance of stainless steel is thought to depend on the existence of a thin hydrous oxide film on the surface of the metal. • The composition of this film varies with different steel alloys and different manufacturing process treatments such as rolling, pickling or heat treatment. • The film is stabilized by chromium and is considered to be continuous, non-porous, insoluble and self healing. • If damaged, the film will repair itself when exposed to air or an oxidizing agent. 20
  21. 21. • The minimum amount of chromium needed to resist corrosion will depend on the corroding agent in a particular environment, such as acids, alkalis, gases, soil, salt or fresh water. • Increasing the chromium content enhances resistance to corrosion, but only grades of steel containing at least 10 to 13% chromium develop an effective film. 21
  22. 22. • The inclusion of nickel in high percent chromium steels enhances their resistance and improves their engineering properties. • The presence of molybdenum improves the resistance of stainless steels to solutions of halogen salts and pitting by chloride ions in brine or sea water. • Corrosion resistance can also be improved by tungsten, silicone and other elements. 22
  23. 23. • At this stage it is important to consider the ways in which a reliable aseptic seal is made between glass and glass, glass and metal or metal and metal joints such as between a fermenter vessel and a detachable top or base plate. 23
  24. 24. Types of Seals Gasket Seal Lip Seal O ring Seal 24
  25. 25. 25
  26. 26. • With glass and metal, a seal can be made with a compressible gasket, a lip seal or an '0' ring. • With metal to metal joints only an '0' ring is suitable. • Nitryl or butyl rubbers are normally used for these seals as they will withstand fermentation process conditions. • A single '0' ring seal is adequate for GILSP and levels 1 and B2, a double '0' ring seal is required for levels 2 and B3 and a double '0' ring seal with steam between the seals (steam tracing) is necessary for levels 3 and B4 26
  27. 27. Aeration and Agitation • Primary purpose of aeration is to provide microorganisms in submerged culture with sufficient oxygen for metabolic requirements. • While agitation should ensure that a uniform suspension of microbial cells is achieved in a homogenous nutrient medium. 27
  28. 28. Agitator (Impellers) • The agitator is required to achieve a number of mixing objectives, e.g. bulk fluid and gas- phase mixing, air dispersion, oxygen transfer, heat transfer, suspension of solid particles and maintaining a uniform environment throughout the vessel contents. • Agitators may be classified as disc turbines, vaned discs, open turbines of variable pitch and propellers. 28
  29. 29. 29
  30. 30. • The disc turbine consists of a disc with a series of rectangular vanes set in a vertical plane around the circumference. • The vaned disc has a series of rectangular vanes attached vertically to the underside. • Air from the sparger hits the underside of the disc and is displaced towards the vanes where the air bubbles are broken up into smaller bubbles. 30
  31. 31. Modern Agitator Develoments • Four other modern agitator developments, the Scaba 6SRGT, the Prochem Maxflo T, the Lightning A315 and the Ekato Intermig are derived from open turbines 31
  32. 32. 32
  33. 33. • The Scaba 6SRGT agitator is one which, at a given power input, can handle a high air flow rate before flooding. • This radial-flow agitator is also better for bulk blending than a Rushton turbine, but does not give good top to bottom blending in a large fermenter which leads to lower concentrations of oxygen in broth away from the agitators and higher concentrations of nutrients, acid or alkali, or antifoams near the feed forts. 33
  34. 34. • Another is the Prochem Maxflo agitator. It of four, five or six hydrofoil blades set at a critical on a central hollow hub. • A high hydrodynamic thrust is created during rotation, increasing the downwards pumping capacity of the blades. • Good mixing and aeration in high viscosity broths may also be achieved by a dual impeller combination, where the lower impeller acts as the gas disperser and the upper impeller acts primarily as a device for aiding circulation of vessel contents. 34
  35. 35. Stirrer Glands and Bearings • The satisfactory sealing of the stirrer shaft assembly top plate has been one of the most difficult problems to overcome in the construction of fermentation equipment which can be operated aseptically for long periods. • The stirrer shaft can enter the vessel from the top, side or bottom of the vessel. 35
  36. 36. A simple Stirrer Seal 36
  37. 37. • A porous bronze bearing for a 13-mm shaft was fitted in the centre of the fermenter top and another in a yoke directly above it. • The bearings were pressed into steel housings, which screwed into position in the yoke and the fermenter top. • The lower bearing and housing were covered with a skirt-like shield having a 6.5 mm overhang which rotated with the shaft and prevented air- borne contaminants from settling on the bearing and working their way through it into the fermenter. 37
  38. 38. Four Main Types The Stuffing Box (Packed Gland seal) The Mechanical Seal Simple Bush Seal The Magnetic Drive Seal 38
  39. 39. The Stuffing Box (Packed Gland Seal) • The shaft is sealed by several layers of packing rings of asbestos or cotton yarn, pressed against the shaft by a gland follower. • Chain et al. (1954) used two stuffing boxes on the agitator shaft with a space in between kept filled with steam. • These seals are sufficient for the requirements of GILSP containment. 39
  40. 40. 40
  41. 41. The Mechanical Seal • The seal is composed of two parts, one part is stationary in the bearing housing, the other rotates on the shaft, and the two components are pressed together by springs or expanding bellows. • The two meeting surfaces have to be precision machined, the moving surface normally consists of a carbon-faced unit while the stationary unit is of stellite-faced stainless steel. 41
  42. 42. 42
  43. 43. Magnetic Drive • The problems of providing a satisfactory seal when the impeller shaft passes through the top or bottom plate of the fermenter may be solved by the use of a magnetic drive in which the impeller shaft does not pierce the vessel. • A magnetic drive consists of two magnets: one driving and one driven. 43
  44. 44. • The driving magnet is held in bearings in a housing on the outside of the head plate and connected to a drive shaft. • The internal driven magnet is placed on one end of the impeller shaft and held in bearings in a suitable housing on the inner surface of the headplate. 44
  45. 45. Baffles • Four baffles are normally incorporated into agitated vessels of all sizes to prevent a vortex and to improve aeration efficiency. • Baffles are metal strips roughly one-tenth of the vessel diameter and attached radially to the wall. • The agitation effect is only slightly increased with wider baffles, but drops sharply with narrower baffles. 45
  46. 46. • It is recommended that baffles should be installed so that a gap existed between them and the vessel wall, so that there was a scouring action around and behind the baffles thus minimizing microbial growth on the baffles and the fermenter walls. • Extra cooling coils may be attached to baffles to improve the cooling capacity of a fermenter without unduly affecting the geometry. 46
  47. 47. Aeration System (Spargers) • A sparger may be defined as a device for introducing air into the liquid in a fermenter. • Three basic types of sparger have been used and may be described as the Porous sparger, the Orifice sparger (a perforated pipe) and the Nozzle sparger (an open or partially closed pipe). 47
  48. 48. Porous Sparger • The porous sparger of sintered glass, ceramics or metal, has been used primarily on a laboratory scale in non-agitated vessels. • The bubble size produced from such spargers is always 10 to 100 times larger than the pore size of the aerator block. • There is also the problem of the fine holes becoming blocked by growth of the microbial culture. 48
  49. 49. Orifice Sparger • In small stirred fermenters the perforated pipes were arranged below the impeller in the form of crosses or rings (ring sparger), approximately three-quarters of the impeller diameter. • In most designs the air holes were drilled on the under surfaces of the tubes making up the ring or cross. 49
  50. 50. • Sparger holes should be at least 6 mm (1/4 inch) diameter because of the tendency of smaller holes to block and to minimize the pressure drop. 50
  51. 51. Nozzle Sparger • Single open or partially closed pipe as a sparger to provide the stream of air bubbles • Ideally the pipe should be positioned centrally below the impeller and as far away as possible from it to ensure that the impeller is not flooded by the air stream. 51
  52. 52. Sterilization of Air Supply for Fermentation • Sterile air will be required in very large volumes in many aerobic fermentation processes. • Heating and Filtration are the main methods for sterilization. Heat is generally too costly for full scale operation. • Glass wool, glass fibre or mineral slag wool have been used as filter material, but currently most fermenters are fitted with cartridge-type filters. 52
  53. 53. Two Procedures based on the construction of filter unit. • During sterilization the main nonsterile air- inlet valve A is shut, and initially the sterile air valve B is closed. • Steam is applied at valve C and air is purged downwards through the filter to a bleed valve at the base. 53
  54. 54. • When the steam is issuing freely through the bleed valve, the valve B is opened to allow steam to pass into the fermenter as well as the filter. • It is essential to adjust the bleed valve to ensure that the correct sterilization pressure is maintained in the fermenter and filter for the remainder of the sterilization cycle. 54
  55. 55. 55
  56. 56. Use of Steam jacketed Air Filter • At the beginning of a sterilization cycle the valve A will be closed and steam passed through valves B and C, and bled out of D. • Simultaneously steam will be passed into the steam jacket through valve F and out of G. • When steam is issuing freely from valve D, valve F, may be opened and steam circulated into the fermenter. 56
  57. 57. • The bleed valve D will have to be adjusted to ensure that the correct pressure is maintained. • Once the sterilization cycle is complete, valves Band E are closed and A is opened to allow air to pass through the heated filter and out of valve D to dry the filter. • Finally the steam supply to the steam jacket is stopped. • Valve D is closed and valve E opened, thus introducing sterile air into the fermenter to achieve a slight positive pressure in the vessel. 57
  58. 58. 58
  59. 59. Valves and Steam Traps • Valves attached to fermenters and ancillary equipment are used for controlling the flow of liquids and gases in a variety of ways. • There are four main types of valves, 59
  60. 60. • Simple ON/OFF valves which are either fully open or fully closed. • Valves which provide coarse control of flow rates. • Valves which may be adjusted very precisely so that flow rates may be accurately controlled. • Safety valves which are constructed in such a way that liquids or gases will flow in only one direction. 60
  61. 61. Gate Valves • In this valve, a sliding disc is moved in or out of the flow path by turning the stem of the valve. • It is suitable for general purposes on a steam or a waterline for use when fully open or fully closed and therefore should not be used for regulating flow. • Not suitable for aseptic conditions 61
  62. 62. • there may be leakage round the stem of the valve which is sealed by a simple stuffing box. • This means that the nut around the stem and the packing must be checked regularly. 62
  63. 63. 63
  64. 64. Globe Valves • In this valve, a horizontal disc or plug is raised or lowered in its seating to control the rate of flow. • It is not suitable for aseptic operation because of potential leakage round the valve stem which is similar in design to that of the gate valve. • There is a high pressure drop across the valve because of the flow path. 64
  65. 65. 65
  66. 66. Piston Valves • The piston valve is similar to a globe valve except that flow is controlled by a piston passing between two packing rings. • This design has proved in practice to be very efficient under aseptic operation. • There may be blockage problems with mycelial culture and the pressure drop is similar to a globe valve. 66
  67. 67. 67
  68. 68. Needle Valve • The needle valve is similar to the globe valve, except that the disc is replaced by a tapered plug or needle fitting into a tapered valve seat. • The valve body can be used to give fine control of steam or liquid flow. • Accurate control of flow is possible because of the variable orifice formed between the tapered plug and the tapered seat. • The aseptic applications are very limited. 68
  69. 69. 69
  70. 70. Plug Valves • In this valve there is a parallel or tapered plug sitting in a housing through which an orifice, A, has been been machined. • When the plug is turned through 90° the valve is fully open and the flow path is determined by the cross-sectional area of A, which may not be as large as that of the pipeline. 70
  71. 71. 71
  72. 72. Ball Valve • This valve has been developed from the plug valve. • The valve element is a stainless-steel ball through which an orifice is machined. • The ball is sealed between two wiping surfaces which wipe the surface and prevent deposition of matter at this point. • The valve is suitable for aseptic operation, can handle mycelial broths and can be operated under high temperatures and pressures. 72
  73. 73. 73
  74. 74. Butterfly Valve • The butterfly valve consists of a disc which rotates about a shaft in a housing. • The disc closes against a seal to stop the flow of liquid. • This type of valve is normally used in large diameter pipes operating under low pressure where absolute closure is not essential. It is not suitable for aseptic operation. 74
  75. 75. 75
  76. 76. Pinch Valve • In the pinch valve a flexible sleeve is closed by a pair of pinch bars or some other mechanism which can be operated by compressed air remotely or automatically. • The valve is suitable for aseptic operation with fermentation broths, even when mycelial, as there are no dead spaces in the valve structure, and the closing mechanism is isolated from the contents of the piping. 76
  77. 77. 77
  78. 78. Diaphragm Valve • Like the pinch valve, the diaphragm valve makes use of a flexible closure, with or without a weir. • Suitable for aseptic operation. 78
  79. 79. Most Suitable Valve • Among these group of valves which have just been described, globe and butterfly valves are most commonly used for ON/OFF applications, gate valves for crude flow control, needle valves for accurate flow control and ball, pinch or diaphragm valves for all sterile uses. 79
  80. 80. Check Valves • The purpose of the check valve is to prevent accidental reversal of flow of liquid or gas in a pipe due to breakdown in some part of the equipment. • There are three basic types of valve: swing check, lift check and combined stop and check with a number of variants. • The swing check valve is most commonly used in fermenter designs. • The functional part is a hinged disc which closes against a seat ring when the intended direction of flow is accidentally reversed. 80
  81. 81. 81
  82. 82. Pressure Control Valves • When planning the design of a plant for a specific process, the water, steam and air should be at different, but specified pressures and flow rates in different parts of the equipment. • For this reason it is essential to control pressures precisely and this can be done using reduction or retaining valve. 82
  83. 83. Two Main Types Pressure Reduction Valve Pressure Retaining Valve 83
  84. 84. Pressure Reduction Valve • Pressure-reduction valves are incorporated into pipelines when it is necessary to reduce from a higher to a lower pressure, and be able to maintain the lower pressure in the downstream side within defined irrespective of changes in the inlet pressure or changes in demand for gas, steam or water. 84
  85. 85. Pressure Retaining Valve • A pressure retaining valve will maintain pressure in the pipeline upstream of itself and the valve is designed to open with a rising upstream pressure. 85
  86. 86. Steam Traps • In all steam lines it is essential to remove any steam condensate which accumulates in the piping to ensure optimum process conditions • This may be achieved by incorporating steam traps, which will collect and remove automatically any condensate at appropriate points in steam lines 86
  87. 87. • A steam trap has two elements. One is a valve and seat assembly which provides an opening, which may be of variable size, to ensure effective removal of any condensate. • The second element is a device which will open or close the valve by measuring some parameter of the condensate reaching it to determine whether it should be discharged. 87
  88. 88. Types of Fermenter The Waldhof type The Tower Fermenter Cylindro Conical Vessel Air-lift Fermenter 88
  89. 89. Deep Jet Fermenter Cyclone column The packed tower Rotating Disc Fermenter 89
  90. 90. The Waldhof Fermenter • The fermenter was of carbon steel, clad in stainless steel, 7.9 m in diameter and 4.3-m high with a centre draught tube 1.2m in diameter. • Trade Name of such fermenters are Acetators and Cavitators. 90
  91. 91. • Fundamental studies by Hromatka and Ebner on vinegar production showed that if Acetobacter cells were to remain active in a stirred aerated fermenter, the distribution of air had to be almost perfect within the entire contents of the vessel. • They solved the full-scale problem by the use of a self-aspirating rotor. 91
  92. 92. 92
  93. 93. • In this design, the turning rotor sucked in air and broth and dispersed the mixture through the rotating stator (d). • The aerator also worked without a compressor and was self-priming. • Vinegar fermentations often foam and chemical antifoams were not thought feasible because they would decrease aeration efficiency and additives were not desirable in vinegar. 93
  94. 94. • A mechanical defoamer therefore had to be incorporated into the vessel and as foam builds up it is forced into a chamber in which a rotor turns at 1,000 to 1,450 rpm. • The centrifugal force breaks the foam and separates it into gas and liquid. • The liquid is pumped back into the fermenter and the gas escapes by a venting mechanism. 94
  95. 95. • Vinegator is a self-aspirating stirrer and a central suction tube which aerates a good recirculation of liquid. • Additional air is provided by a compressor. • Foam is broken down by a mechanical defoamer 95
  96. 96. Tower Fermenter • The main feature appears to be their height:diameter ratio or aspect ratio. • Tower fermenter as an elongated non- mechanically stirred fermenter with an aspect ratio of at least 6:1 for the tubular section or 10:1 overall, through which there is a unidirectional flow of gases. 96
  97. 97. Examples • Pfizer Ltd has always used non-agitated tower vessels for a range of mycelial fermentation processes including citric acid and tetracyclines. • A vertical-tower beer fermenter design was patented by Shore et al. .(1964). • Perforated plates were positioned at intervals in the tower to maintain maximum yeast production 97
  98. 98. Cylindro Conical Vessels • The vessel consists of a stainless-steel vertical tube with a hemispherical top and a conical base with an included angle of approximately 70°. • Aspect ratios are usually 3:1 and fermenter heights are 10 to 20 m. • Operating volumes are chosen to suit the individual brewery requirements, but are often 150,000 to 200,000 dm3. • Vessels are not normally agitated unless a particularly flocculant yeast is used, but small impellers may be used to ensure homogeneity when filling with wort 98
  99. 99. 99
  100. 100. Advantages of Cylindro conical vessels • Reduced process times may be achieved due to increased movement within the vessel. • Primary fermentation and conditioning may be carried out in the same vessel. • The sedimented yeast may be easily removed since yeast separation is good. • The maturing time may be reduced by gas washing with carbon dioxide. 100
  101. 101. Air Lift Fermenter • An air-lift fermenter is essentially a gastight baffled riser tube (liquid ascending) connected to a downcomer tube (liquid descending). • The driving force for circulation of medium in the vessel is produced by the difference in density between the liquid column in the riser (excess air bubbles in the medium) and the liquid column in the downcomer (depleted in air bubbles after release at the top of the loop). 101
  102. 102. 102
  103. 103. • Circulation times in loops of 45-m height may be 120 seconds. • This type of vessel can be used for continuous culture. • It would be uneconomical to use a mechanically stirred fermenter to produce SCP (single-cell protein) from methanol as a carbon substrate, as heat removal would be needed in external cooling loops because of the high rate of aeration and agitation required to operate the process. • To overcome these problems, particularly that of cooling the medium when mechanical agitation is used, air-lift fermenters with outer or inner loops were chosen. 103
  104. 104. 104
  105. 105. Deep Jet Fermenter • Some designs of continuous culture fermenter achieve the necessary mechanical power input with a pump to circulate the liquid medium from the fermenter through a gas entrainer and back to the fermenter. • Two basic construction principles have been used for the gas entrainer nozzles- Injector and the Ejector. 105
  106. 106. 106
  107. 107. • In an injector a jet of medium is surrounded by a jet of compressed air. • In an ejector the liquid jet enters into a larger converging-diverging nozzle and entrains the gas around the jet. • The gas which is sucked into the converging- diverging jet is dispersed in that zone. 107
  108. 108. Cyclone Column • Dawson (1974) developed the cyclone column, particularly for the growth of filamentous cultures. • The culture liquid was pumped from the bottom to the top of the cyclone column through a closed loop. • The descending liquid ran down the walls of the column in a relatively thin film. • Nutrients and air were fed in near the base of the column whilst the exhaust gases left at the top of the column. • Good gas exchange, lack of foaming and limited wall growth have been claimed with this fermenter. 108
  109. 109. 109
  110. 110. Oxygen requirements of Fermentation • A microbial culture must be supplied with oxygen during growth at a rate sufficient to satisfy the organisms' demand. • The oxygen demand of an industrial fermentation process is normally satisfied by aerating and agitating the fermentation broth. 110
  111. 111. Important Terms • Specific Oxygen Rate (Qo2)- It is the milli moles (mm) of oxygen consumed per gram dry weight of cells per hour. • Dissolved Oxygen Concentration – Amount of Oxygen in the fermentation medium. • Ccrit – It is the maximum concentration of oxygen that can be utilized by an organism. Below the Ccrit Value, there is no change in the oxygen uptake rate. 111
  112. 112. 112
  113. 113. Effects of Dissolved Oxygen concentration • Hirose and Shibai’s (1980) investigations of amino acid biosynthesis by Brevibacterium flavum provides an excellent example of the effects of the dissolved oxygen concentration on the production of range of closely related metabolites. 113
  114. 114. • These workers demonstrated the critical dissolved oxygen concentration for B. flavum to be 0.01 mg dm-3 and considered the extent of oxygen supply to the culture in terms of the degree of 'oxygen satisfaction’, that is the respiratory rate of the culture expressed as a fraction of the maximum respiratory. • A value of oxygen satisfaction below unity implied that the dissolved oxygen concentration was below the critical level. 114
  115. 115. 115
  116. 116. • It may be seen that the production of members of the glutamate and aspartate families of amino acids was affected detrimentally by levels of oxygen satisfaction below 1.0, whereas optimum production of phenylalanine, valine and leucine occurred at oxygen satisfaction levels of 0.55, 0.60 and 0.85, respectively. 116
  117. 117. • It may be seen that the glutamate and aspartate families are all produced from tricarboxylic acid (TCA) cycle intermediates, whereas phenylalanine, valine and leucine are produced from the glycolysis intermediates, pyruvate and phosphoenol pyruvate. • Oxygen excess should give rise to abundant TCA cycle intermediates, whereas oxygen limitation should result in less glucose being oxidized via the TCA cycle, allowing more intermediates to be available for phenylalanine, valine and leucine biosynthesis. • Thus, some degree of metabolic disruption results in greater production of pyruvate derived amino acids. 117
  118. 118. • An example of the effect of dissolved oxygen on secondary metabolism is provided by Zhou et al. 's (1992) work on cephalosporin C synthesis by Cephalosporium acremonium. • These workers demonstrated that the critical oxygen concentration for cephalosporin C synthesis during the production phase was 20% saturation. • At dissolved oxygen concentrations below 20% cephalosporin C concentration declined and penicillin N increased. 118
  119. 119. Oxygen Supply • Bartholomew et at. (1950) represented the transfer of oxygen from air to the cell, during a fermentation, as occurring in a number of steps: • The transfer of oxygen from an air bubble into solution. • The transfer of the dissolved oxygen through the fermentation medium to the microbial cell. • The uptake of the dissolved oxygen by the cell 119
  120. 120. • The rate of oxygen transfer from air bubble to the liquid phase may be described by the equation: 120
  121. 121. • CL – It is the concentration of dissolved oxygen in the fermentation broth (mmoles dm-3) • t – It is time (hour) • dCL /dt – It is the change in oxygen concentration over a time period, i.e. the oxygen transfer rate (mmoles O2 dm-3 h-1), • KL - is the mass transfer coefficient (cm-1), • a - It is the gas/liquid interface area per liquid volume (cm2 cm-3), • C* - It is the saturated dissolved oxygen concentration (mmoles dm-3 ). 121
  122. 122. • KL may be considered as the sum of the reciprocals of the resistances to the transfer of oxygen from gas to liquid and (C* - CL ) may be considered as the 'driving force' across the resistances. • The volumetric mass-transfer coefficient (KLa) is used as a measure of the aeration capacity of a fermenter. • The larger the KLa, the higher the aeration capacity of the system. 122
  123. 123. Methods of Determining KLa • The sulphite oxidation technique • Gassing out techniques: The static method and The dynamic method • Oxygen Balance technique 123
  124. 124. The Sulphite Oxidation technique • Cooper et at. (1944) were the first to describe the determination of oxygen-transfer rates in aerated vessels by the oxidation of sodium sulphite solution. • This technique does not require the measurement of dissolved oxygen concentrations but relies on the rate of conversion of a 0.5 M solution of sodium sulphite to sodium sulphate in the presence of a copper or cobalt catalyst. 124
  125. 125. • Na2SO3 + 0.5 O2 = Na2SO4 • The rate of reaction is such that as oxygen enters solution it is immediately consumed in the oxidation of sulphite, so that the sulphite oxidation rate is equivalent to the oxygen- transfer rate. • The dissolved oxygen concentration, for all practical purposes, will be zero and the KLa may then be calculated from the equation: • OTR = KLa x C* 125
  126. 126. • The procedure is carried out as follows: the fermenter is batched with a 0.5 M solution of sodium sulphite containing 10-3 M Cuz+ ions and aerated and agitated at fixed rates; samples are removed at set time intervals (depending on the aeration and agitation rates) and added to excess iodine solution which reacts with the unconsumed sulphite, the level of which may be determined by a back titration with standard sodium thiosulphate solution. 126
  127. 127. Advantages and Disadvantages • The sulphite oxidation method has the advantage of simplicity and, also, the technique involves sampling the bulk liquid in the fermenter and, therefore, removes some of the problems of conditions varying through the volume of the vessel. • However, the method is time consuming (one determination taking up to 3 hours, depending on the aeration and agitation rates) and is notoriously inaccurate 127
  128. 128. • The rheology of a sodium sulphite solution is completely different from that of a fermentation broth, especially a mycelial one so that it is impossible to relate the results of sodium sulphite determinations to real fermentations. 128
  129. 129. Gassing out techniqques • The estimation of the KLa of a fermentation system by gassing-out techniques depends upon monitoring the increase in dissolved oxygen concentration of a solution during aeration and agitation. • The oxygen transfer rate will decrease during the period of aeration as CL approaches C* due to the decline in the driving force (C* - CL) 129
  130. 130. • The oxygen transfer rate, at anyone time, will be equal to the slope of the tangent to the curve of values of dissolved oxygen concentration against time of aeration. • To monitor the increase in dissolved oxygen over an adequate range it is necessary first to decrease the oxygen level to a low value. • Two methods have been employed to achieve this lowering of the dissolved oxygen concentration - the static method and the dynamic method. 130
  131. 131. 131
  132. 132. The Static Method of Gassing out • Oxygen concentration of the solution is lowered by gassing the liquid out with nitrogen gas, so that the solution is 'scrubbed' free of oxygen. • The deoxygenated liquid is then aerated and agitated and the increase in dissolved oxygen monitored using some form of dissolved oxygen probe. 132
  133. 133. • The increase in dissolved oxygen concentration has already been described by the equation, • Integration of the equation yields, • In(C* - CL) = -KLat • A plot of In (C* - CL) against time will yield a straight line of slope Kla. 133
  134. 134. 134
  135. 135. Advantages and Disadvantages • This technique has the advantage over the sulphite oxidation method in that it is very rapid (15 mins) and may utilize the fermentation medium, to which may be added dead cells or mycelium at a concentration equal to that produced during the fermentation. 135
  136. 136. • However, employing th fermentation medium with, or without killed biomass necissitates the use of membrane type electrode, the response type of which may be inadequate to reflect the true change in the rate of oxygenation over a short period of time. 136
  137. 137. • Whilst the method is acceptable for small scale vessels, there are severe limitations to its use on large scale fermenters which have high gas residence times. • When the air supply to such a vessel is resumed after deoxygenation with nitrogen, the oxygen concentration in the gas phase may change with time as the nitrogen is replaced with air. • Thus, C* will no longer be constant. 137
  138. 138. The Dynamic Method of Gassing Out • The procedure involves stopping the supply of air to the fermentation which results in a linear decline in the dissolved oxygen concentration due to the respiration of the culture. 138
  139. 139. 139
  140. 140. • The slope of the line AB is a measure of the respiration rate of the culture. • At point B the aeration is resumed and the dissolved oxygen concentration increases until it reaches concentration X. • Over the period BC, the observed increase in dissolved oxygen concentration is the difference between he transfer of oxygen into solution and the uptake of oxygen by the respiring culture as expressed by the equation, 140
  141. 141. • dCL/ dt = Kla (C* - CL) - xQo2 • Where x is the concentration of the biomass and Qo2 is the specific respiration rate. • The above equation maybe rearranged as, • Plot of CL versus dCL/dt + xQo2 will yield a straight line, the slope of which will equal - l/KLa 141
  142. 142. 142
  143. 143. Advantages and Disadvantages • The dynamic gassing-out method has the advantage over the previous methods of determining the Kla during an actual fermentation and may be used to determine KLa values at different stages in the process. • It may be difficult to apply the technique a fermentation which has an oxygen demand close the supply capacity of the fermenter 143
  144. 144. References • Principles of Fermentation Technology by P.F. Stanbury 144
  145. 145. 145

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