Biotechnology PM 50 Fermentation PDF

Biotechnology PM 50 Fermentation Biotechnological process is implemented in three stages: 1 2 3 Stage I : Upstream Stage II: Stage III: processing , which Fermentation , which Downstream involves pr...

Biotechnology PM 50 Fermentation Biotechnological process is implemented in three stages: 1 2 3 Stage I : Upstream Stage II: Stage III: processing , which Fermentation , which Downstream involves preparation involves the processing , which of liquid medium, conversion of involves separation separation of substrates to desired of cells from the particulate and product with the help fermentation broth, inhibitory chemicals of biological agents purification and from the medium, such as concentration of sterilization, air microorganisms desired product and purification waste disposal or recycle.  FERMENTER DESIGN The basic requirement of stage II is to provide a controlled environment for the growth of the microorganisms and production of the desired compound. Controlled environment includes providing the following facilities for the process: 1. Contamination free environment 2. Specific temperature and pH maintenance 3. Maintenance of agitation and aeration 4. Monitoring Dissolved Oxygen (DO) 5. Ports for nutrient and reagent feeding, ports for inoculation and sampling 6. Scope for scale up  FERMENTER DESIGN A fermenter is responsible for ensuring the maintenance of this controlled environment for product formation. The first truly large-scale aseptic anaerobic fermentation vessels were developed by Weizmann and co- workers for the production of acetone by a deep liquid fermentation using Clostridium acetobutylicum. For this, large cylindrical vessels of mild steel that permitted sterilization with steam under pressure were constructed, and piping, joints and valves were specially designed to maintain aseptic conditions Mixing was achieved by the large volumes of gas produced during fermentation. In later modifications, mechanical impellers were used to improve mixing of broth and dispersal of air bubbles. The basic function of a fermenter is to provide a controlled, aseptic environment for the growth of microorganism and subsequent production of desired product.  FERMENTER DESIGN While designing a fermenter, the following criteria should be kept into consideration: 1. It should provide a controlled environment for optimum biomass/product yields. 2. It should permit aseptic fermentation for a number of days reliably and dependably and meet the requirements of containment regulations. 3. It should provide adequate mixing and aeration for optimum growth and production, without damaging the microorganisms/cells. 4. The power consumption should be minimum. 5. It should provide easy and dependable temperature control. 6. Facility for sampling should be provided. 7. It should have a system for monitoring and regulating pH of the fermentation broth. 8. Evaporation losses should be as low as possible. 9. It should require a minimum of labor in maintenance, cleaning, operating and harvesting operations. 10. It should be suitable for a range of fermentation processes. But this range may often be restricted by the containment regulations. 11. It should have smooth internal surfaces, and joints should be welded wherever possible. 12. The pilot scale and production stage fermenters should have similar geometry to facilitate scale-up.  FERMENTER DESIGN The most commonly used fermenters for commercial purposes are stirred upright cylinder with sparger aeration. A typical fermenter/bioreactor comprises of different components for providing: (i) agitation. (ii) aeration. (iii) regulation of factors like temperature, pH, pressure, aeration, nutrient feeding, liquid level. (iv) sterilization and maintenance of sterility.  COMPONENTS OF A TYPICAL FERMENTER Fermenter Body Construction: Material used for the construction of fermenter body differs according to the scale (small, pilot and industrial scale) of the fermenter. At small scale, vessel made up of construction glass or stainless steel may be used. For pilot and large-scale process, stainless steel (>4% chromium), mild steel (coated with glass or epoxy material), wood, plastic or concrete may be used as vessel construction material depending upon the requirement of the fermentation process. One of the essential features of the used construction material is that it should be non- toxic and corrosion proof.  COMPONENTS OF A TYPICAL FERMENTER 1. Glass vessel: is generally made up of borosilicate glass. Glass vessels generally are of two types: Type I – glass vessels with round or flat bottom with top plate which can be sterilized by autoclaving. The largest type I glass vessels have a diameter of 60cm. Type II – glass vessel with flat bottom and with top and bottom stainless-steel plate. This type is used in in situ sterilization process and the largest vessel has a diameter of 30cm. 2. Stainless steel: Pilot scale and industrial scale vessels are normally constructed of stainless steel or at least have stainless steel cladding to limit corrosion. Stainless steel is corrosion resistant due to the presence of a thin hydrous oxide film on the surface of the metal. This film is stabilized by chromium and is non-porous, continuous and self-healing. Increasing chromium concentration enhances the resistance to corrosion, but only steels with 10-13% chromium are able to develop effective films. Inclusion of nickel into the stainless-steel increases carrion resistance and improves engineering. Presence of molybdenum provides resistance to halogen salts, brine, and sea water. COMPONENTS OF A TYPICAL FERMENTER Sealing: An important factor to be considered while designing a fermenter is to ensure proper sealing between the top plate and the vessel to maintain airtight and aseptic conditions. Sealing needs to be carried out between three types of surfaces between glass-glass, glass metal and metal-metal. There are three main types of sealing used in a fermenter. For glass and metal, a seal can be made with a compressible gasket, lipseal and O‘ ring For metal- metal sealings, only O ring is used. This sealing made up of fabric-nitryl or butyl rubbers ensures tight joint in spite of expansion of vessel material during fermentation.  COMPONENTS OF A TYPICAL FERMENTER Aeration and Agitation: The purpose of aeration is to provide oxygen to microorganism submerged in the media for carrying out various metabolic operations and the medium must be suitably stirred to keep the cells in suspension and to make the culture homogeneous. The objective of stirring is to achieve good mixing without causing damage to the cells. Efficient aeration is achieved by bubbling air through the medium (sparging), but this may damage animal cells due to the high surface energy of the bubble and on the cell membrane. The damage can be reduced by using larger bubbles, lower gassing rates and by adding non- nutritional supplements like Pluronic F-68 (polyglycol) and sodium carboxymethyl cellulose. O2 supply in the culture vessel can be enhanced from the normal 21% to a higher value and the air pressure can be increased by 1 atmosphere.  COMPONENTS OF A TYPICAL FERMENTER Aeration and Agitation: This increases the O2 solubility and diffusion rates in the medium, but there is a risk of O2 toxicity. The type of aeration-agitation system used in the fermenter is dictated by the characteristics of the fermentation process. The following components of the fermenter are a part of aeration and agitation system: a) Agitator or Impeller b) Stirrer glands and bearings c) Baffle d) Sparger or the aeration system.  COMPONENTS OF A TYPICAL FERMENTER a) Agitator Agitators are required in a fermenter for achieving the following objectives: Bulk fluid and gas-phase mixing, air dispersion, oxygen transfer, heat transfer, suspension of solid particles and maintenance of a uniform environment throughout the vessel. There are different types of agitators used in fermenters depending on the requirements of the fermentation process (i) Disc turbines: Disc turbine prevents flooding by air bubbles. Flooding occurs when the air bubble is not properly dispersed, and air pockets are formed. (ii) Vaned discs: Air from the sparger hits the underside of the disc and is displaced towards the vanes where air bubbles are broken up into smaller bubbles. (iii) Open turbines with variable pitch: In this case the air bubbles do not initially hit any surface before dispersion by the vanes or blades. (iv) Propellers: The marine propeller is similar to variable pitch open turbine, except that it has blades in the place of vanes. In this case also, air bubbles contact the vanes/blades directly and are broken up and dispersed by them.  COMPONENTS OF A TYPICAL FERMENTER b) Stirrer Glands and Bearings Maintenance of aseptic conditions over long periods is one of the most critical aspects of fermentation process which requires a satisfactory sealing of the stirrer shaft assembly. The entry point of stirrer into fermenter may be from top to bottom or sides. Mostly used entry point is from the bottom as it leaves more space for entry ports on top. There are three types of stirrer glands and bearings: (i) Stuffing box: Comprises of several layers of packing rings of asbestos or cotton yarn pressed against the shaft by a gland follower. The drawback of this type of seal is that at high speeds, the packing wears. The packing is difficult to sterilize due to unsatisfactory heat penetration. (ii) Mechanical seal: comprises of two parts stationary part in the bearing housing and the other rotates on the shaft.  COMPONENTS OF A TYPICAL FERMENTER The two parts are pressed together by springs or expanding bellows. Steam condensate is used to lubricate and cool the seals during operation and servers as a contaminant barrier. Most modern fermenters use mechanical seals; these seals are more expensive, but they are more durable and less prone to leakage or contaminant entry. (iii) Magnetic drives: comprise of two magnets one driving and the other driven. The driving magnet is held in the bearing in the housing on outside of head plate and connected to drive shaft. The driven one is placed on one end of impeller shaft held in bearing in suitable housing. Magnetic drives, although quite expensive, are being used in some animal cell culture vessels. 3. Baffles: Baffles are metal strips roughly one-tenth of the vessel diameter and attached radially to the fermenter wall. They are normally used in fermenters having agitators to prevent vortex formation and to improve aeration efficiency.  COMPONENTS OF A TYPICAL FERMENTER Four baffles are used, but larger fermenters may have 6 or 8 baffles. Extra cooling coils may be attached to baffles to improve cooling. The baffles may be installed in such a way that a gap exists between the baffles and the fermenter wall. This would lead to a scouring action around and behind the baffles, which would minimize microbial growth on the baffles and the fermenter wall. 4. Aeration System (Sparger): Spargers are used for introducing air into the fermenter. Fine bubble aerators must be used for introducing air which will facilitate oxygen transfer to a greater extent as large bubbles will have less surface area than smaller bubbles. Agitation is not required when aeration provides enough agitation which is the case in Air lift fermenter. For aeration to provide agitation, the vessel height/diameter ratio (aspect ratio) should be 5:1.  COMPONENTS OF A TYPICAL FERMENTER There are three types of sparger viz. porous sparger, orifice sparger and nozzle sparger. (i) Porous sparger: made of sintered glass, ceramics or metal. It is used only in lab scale-non agitated vessel. The size of the bubble formed is 10-100 times larger than pore size. There is a pressure drop across the sparger and the holes tend to be blocked by growth which is the limitation of porous sparger. (ii) Orifice sparger: used in small stirred fermenters. It is a perforated pipe kept below the impeller in the form of crosses or rings. The size should be ~ ¾ of impeller diameter. Air holes are drilled on the under surfaces of the tubes and the holes are at least 6mm in diameter. This type of sparger is used mostly with agitation. It is also used without agitation in some cases like yeast manufacture, effluent treatment and production of SCP.  COMPONENTS OF A TYPICAL FERMENTER iii) Nozzle sparger: Mostly used in large scale fermenters. It is single open/partially Closed pipe positioned centrally below the impeller. When air is passed through this pipe there is lower pressure loss, and it does not get blocked. In small fermenters, a combined sparger-agitator may be used. In this case, the air is introduced via a hollow agitator shaft, and it comes out through holes drilled in the disc between the blades and connected to the base of the main shaft. Temperature Regulation The fermenter must have an adequate provision for temperature control. Metabolic activities of the microorganism and agitation lead to generation of heat. Temperature control may be considered at laboratory scale, and pilot and production scales.  COMPONENTS OF A TYPICAL FERMENTER iii) Nozzle sparger: Mostly used in large scale fermenters. It is single open/partially Closed pipe positioned centrally below the impeller. When air is passed through this pipe there is lower pressure loss, and it does not get blocked. In small fermenters, a combined sparger-agitator may be used. In this case, the air is introduced via a hollow agitator shaft, and it comes out through holes drilled in the disc between the blades and connected to the base of the main shaft. Temperature Regulation The fermenter must have an adequate provision for temperature control. Metabolic activities of the microorganism and agitation lead to generation of heat. Temperature control may be considered at laboratory scale, and pilot and production scales. Internal coils have to be used to circulate cold water through them for removing the excess heat.  COMPONENTS OF A TYPICAL FERMENTER 1. In laboratory scale fermentations: normally little heat is generated. Therefore, heat has to be added to the system; this can be achieved in the following ways: (a) The fermenter may be placed in thermostatically controlled bath (b) internal heating coils may be used (c) water may be circulated through a heating jacket (d) a silicone healing jacket may be used. The silicone jacket consists of two silicone rubber mats, and heating wires between these mats. 2. In case of larger fermenters beyond a certain size: excess heat is generated, and the fermenter surface becomes inadequate for heat removal. The size at which fermenter surface becomes inadequate for heat removal will depend on the fermentation process and the ambient temperature at which fermentation is being carried out.  COMPONENTS OF A TYPICAL FERMENTER Internal coils have to be used to circulate cold water through them for removing the excess heat. The cooling surface area necessary for temperature control will depend mainly on the following factors: (i) temperature of cooling water. (ii) the culture temperature. (iii) the type of microorganism. (iv) the energy provided by stirring. The cooling water consumed during bacterial fermentation in a vessel of a size around 55,000 L would range between 500 to 2,000 L/h Fungal fermentation, however, may need 2,000 to 10,000 L cooling water per hour as they have a lower optimum temperature for growth.  COMPONENTS OF A TYPICAL FERMENTER Maintenance of Aseptic Conditions 1. Sterilization of the Fermenter: The fermenter should be designed in such a manner that it can be sterilised under pressure. The media may be sterilised in the fermenter or separately. In case of in situ sterilization of the medium, its temperature should be raised before injection of the live steam to prevent formation of large amount of condensate. This can be achieved by introducing steam in fermenter coils or steam jackets. Every point of entry and exit in a fermenter is a potential source of contamination; therefore, steam should be introduced through every entry and exit point. All the pipes should be constructed so as to ensure that steam reaches each and every part of the fermenter.  COMPONENTS OF A TYPICAL FERMENTER Maintenance of Aseptic Conditions 2. Sterilization of the air supply: Aerobic fermentations require a very large volume of sterile air. There are two main ways of sterilizing air: by heat which is not economically viable at industrial scale, and filtration. Glass wool, mineral slag wool or glass fiber were used for filtration of air but now most of the fermenters use cartridge-type filters with a membrane pore size of 0.2 to 0.3 μm for sterilization. 3. Sterilization of exhaust gases: Normally carried out by using a 0.2μm filter on the outlet pipe. One of the problems encountered in this method is plugging of the filter due to the moisture and solid matter released during fermentation in the form of an aerosol. To avoid this, cyclone separators for solids and coalesce for liquids are included in the upstream of the filters in series.  COMPONENTS OF A TYPICAL FERMENTER Addition of Inoculum, Nutrients and Other Supplements : The fermenter and the addition vessel are maintained at positive pressure and the addition ports are sterilised with steam supply prior to release of inoculum or nutrients into the fermenter. Addition of nutrients, acid or alkali in small fermenters is normally done with the help of silicone tubes which are sterilised separately and pumped by a peristaltic pump. In large fermenters, the feed reservoirs and associated piping is an integrated part of the fermenter and is sterilised along with the vessel by using steam. Foam Control: Foam is produced during most microbial fermentations. Foaming may occur either due to a medium component, e.g., protein present in the medium, or due to some compound produced by the microorganism. Foaming can cause removal of cells from the medium; such cells undergo autolysis and release more proteins into the medium which in turn, further stabilizes the foam.  COMPONENTS OF A TYPICAL FERMENTER Five different patterns of foaming that are recognized are listed below: 1. Foaming remains at a constant level throughout the fermentation. Initial foaming is due to the medium, but later microbial activity contributes to it. 2. Foaming declines steadily in the initial stages but remains constant thereafter this type of foaming is due to the medium. 3. The foaming increases after a slight initial fall‘, in this case, microbial activity is the major cause of foaming. 4. The foaming level increases with fermentation duration; such foaming pattern is solely due to microbial activity. 5. A complex foaming pattern that combines features of two or more of the above patterns. Foaming can lead to several physical and biological problems such as: 1. Decrease in the working volume of the fermenter caused by circulation of oxygen-depleted gas bubbles in the system. 2. Decline in the heat and mass transfer rates.  COMPONENTS OF A TYPICAL FERMENTER Foaming may interfere with the functioning of sensing electrodes resulting in invalid process data, and incorrect monitoring and control of pH, temperature. The biological problems of foaming include: 1. Deposition of cells in the upper parts of the fermenter. 2. Interference in sterile operation as the air filter exits of the fermenter becomes wet, and increased risk of contamination. 3. Product loss due to siphoning of the culture broth. The problem of excessive foam formation can be resolved by adopting the following strategies: 1. A defined medium may be used to avoid foam formation. This may be combined with modifications in physical parameters like pH, temperature, aeration and agitation. This approach will be successful in such cases where medium is the main culprit but will fail whenever microbial activity is the main contributor.  COMPONENTS OF A TYPICAL FERMENTER 2. Antifoam should be used this is the most standard approach to combat foaming. Antifoams are surface active agents; they reduce surface tension in the foams and destabilize protein film by the following effects: (a) hydrophobic bridges between two surfaces. (b) displacement of the absorbed protein. (c) rapid spreading of the surface film. Several compounds have been found to be suitable antifoam agents for different fermentation processes; these include: alcohols (stearyl and octyldecanol), esters, fatty acids and their derivatives (especially, triglycerides like cottonseed oil, linseed oil, soybean oil, sunflower oil, etc.), silicones, sulfonates, and miscellaneous compounds like oxaline, Alkaterge C polypropylene glycol.  COMPONENTS OF A TYPICAL FERMENTER Many of the antifoams are of low solubility; therefore, they are added with a carrier like lard oil, liquid paraffin and castor oil. Many antifoams would reduce oxygen transfer by up to 50% when used at effective concentrations. Antifoams are generally added when foaming occurs during fermentation. But foam control in fermentation industry is still an empirical art. 3. A mechanical foam breaker may also be used. Valves and Steam traps: Valves: attached to fermenters and additional vessel are required to control the flow of liquids and gases. There are four types of addition valves: (a) Simple ON and OFF: which are either fully open or closed (b) coarse control of flow rates (c) Accurate adjustment valves for precise adjustment of flow rates (d) Safety valve: which is constructed in such a way that they allow flow of liquids and gases in one direction only.  COMPONENTS OF A TYPICAL FERMENTER Check valves: Valves used to prevent accidental reversal flow of liquid or gas due to break down. Pressure control valves: These types of valves are used for two purposes. a) Pressure reduction. b) Pressure retaining. Safety valve: These are types of safety valves by which the increase in pressure is released. Steam traps: are important for the removal of steam condensates. Control and Monitoring of Fermentation System: There are three types of sensors used in fermenter: In-line sensors form integral part of fermenter. The directly measured value controls the process. eg. Antifoam probe. On-line sensors form integral part of fermenter. The measured value must be entered into control system to control process eg. Ion specific sensors, mass spectrophotometer. Off-line sensors do not form integral part of fermenter. The measured value must be entered into control system for data collection.  COMPONENTS OF A TYPICAL FERMENTER 1. (a) Temperature Measuring Devices Temperature is an important parameter of fermentation, since, in the cultivation of many microorganisms, the temperature deviation by a couple of degrees can diminish dramatically the growth and biosynthetic productivity Mercury-in-glass thermometers: Mercury enclosed in bulb expands with increase in temperature. Expansion is read as measure of temperature and is used only as indication. Electrical resistance: The property of some metals whose resistance changes with change in temperature are used to measure temperature. Bulb with mica is used for accurate measurement and ceramic for less accurate measurement. This is covered by temperature sensing platinum. Thermistors: These are semi-conductors of pure oxides of iron, nickel. They exhibit large change in resistance with a small temperature change and hence are highly sensitive even with little temperature change.  COMPONENTS OF A TYPICAL FERMENTER (b) Temperature Controlling Device: The temperature in laboratory bioreactors is controlled by one of the following ways: 1. A heater is located inside the bioreactor vessel, and cooling is ensured by thin-wall pipes located in the upper cover, which are connected with an electromagnetic valve with the cooling water it ensures a more economic constructive solution. 2. Heating and cooling proceed in a thermostat, and this water, with the help of a pump, circulates through the bioreactor jacket ensures a more even distribution of heat throughout the bioreactor volume, which is essential in microorganisms cultivation. 2. (a) Gas Flow Rate Measuring Device: Flow rate can be measured by simple variable area meter Rotameter: is a vertically mounted glass tube with an increasing bore size and enclosing a free moving float the position of float indicates the flow rate. COMPONENTS OF A TYPICAL FERMENTER (b)Gas Flow Rate Controlling Device Needle valve is used to control the gas flow rate. Piston movement of the valve is controlled by fluctuations in pressure in flow measuring device. This should be placed upstream of supply when regulated air flow rate is required and downstream when fluctuations and back pressure is constant. 3. (a) Liquid Flow Rate Measuring Device: (a) Liquid Flow Rate Measuring Device 1. Electric flow transducer 2. Rotameter Load cells b) Liquid Flow Rate Controlling Device Syringe pump: is useful in case of fed batch for controlling the liquid flow rate. Peristaltic pump: is also used to control flow rate by squeezing and releasing pulse flow. Diaphragm pump: The liquid is allowed to flow through a flexible tube which utilizes a piston and pump controls the flow  COMPONENTS OF A TYPICAL FERMENTER 4. Pressure Measuring and Controlling Devices : Bourbon tube pressure gauge is used to measure the pressure changes Diaphragm gauge: used to measure the pressure under aseptic condition. Pressure bellows: connected to core of transformer. Movement of core generates voltage output which can be measured. Strain gauge: a wire subjected to strain results in change of electrical resistance which can be measured. Piezo electric transducer: a solid crystal (quartz), has asymmetrical charge distribution. Any change in shape due to pressure produce equal external electric charge on the opposite face of crystal (piezo electric effect). 5. Agitation Measuring and Controlling Device: Agitation speed can be measured by power consumed by agitator shaft. Wattmeter is usually used in large scale process. It is a measure of power consumed for rotation of agitator shaft. This measure is less accurate because power required to rotate against friction in the bearing is taken into consideration.  COMPONENTS OF A TYPICAL FERMENTER Torsion dynamometer is used in small scale. This has to be placed outside the vessel and is less accurate due to friction. Tachometer can be used to control the agitation speed 6. Foam Sensing: For elimination of foam, 2 methods or their combinations are commonly used: Additional metering of antifoam: based on the information provided by the foam sensor. The given impulses are relatively low, with long pauses and a limited metering time. Mechanical metering of foam: Foam formation can be sensed by a probe which is a stainless-steel rod insulated except at tip and set at a defined level. When foam touches the probe tip, current passes through, with foam as electrolyte and vessel. 7. Dissolved Oxygen Monitoring: One of the most specific aspects of the fermentation monitoring is pO2 measurement and control. pO2 control is characteristic only for fermentation processes.  COMPONENTS OF A TYPICAL FERMENTER Dissolved oxygen can be measured by: Galvanic electrodes: which consist of KCl or KOH or bicarbonate or acetate as an electrolyte. Lead is used as anode and silver acts as cathode. The electrodes measure the partial pressure of the dissolved oxygen concentration. Polarographic electrode : consists of silver as anode, Pt or gold as cathode and KCl as the electrolyte. Fluorometric sterilizable oxygen sensors : are now being developed which are based on the fact that change in oxygen partial pressure quenches the fluorescence lifetime of a chromophore. Tubing method: It comprises of a probe made up of a coil of permeable membrane tubing which is placed inside the fermenter. Through this membrane helium or nitrogen is passed. Oxygen that diffuses into tubing from the medium is then measured by Paramagnetic gas analyzer.  COMPONENTS OF A TYPICAL FERMENTER 8. pH Monitoring Devices: pH measurement is the determination of the activity of hydrogen ions in an aqueous solution. pH electrode used for estimating the pH of the media consists of two parts: a sensing electrode and a reference electrode. The sensing electrode consists of a thin hydrogen permeable membrane containing a solution and an electrode. The membrane of the sensing electrode allows hydrogen ions to slowly pass, creating a positive voltage across the membrane. The voltage created in this electrode is then compared to the voltage in the reference electrode. The voltage difference between the two electrodes is then used to determine the pH of the unknown solution.  TYPES OF FERMENTERS 1. Stirred Tank Fermenter The Stirred tank reactor comprises of baffles and a rotating stirrer attached either at the top or at the bottom of the bioreactor. The conventional fermentation is carried out in a batch mode. Since stirred tank reactors are commonly used for batch processes with slight modifications, these reactors are simple in design and easier to operate. reactor design, the industry, still prefers stirred tanks because in case of contamination or any other substandard product formation, the loss is minimal. The batch stirred tanks generally suffer due to their low volumetric productivity. The downtimes are quite large and unsteady state fermentation imposes stress to the microbial cultures due to nutritional limitations. The Stirred tank reactors offer excellent mixing and reasonably good mass transfer rates. The cost of operation is lower, and the reactors can be used with a variety of microbial species. TYPES OF FERMENTERS 2. Airlift Fermenter Airlift fermenter (ALF) is generally classified as pneumatic reactor without any mechanical stirring arrangements for mixing. The turbulence caused by the fluid flow ensures adequate mixing of the liquid. The advantages of Airlift reactors are the elimination of attrition effects generally encountered in mechanical agitated reactors. It is ideally suited for aerobic cultures since oxygen mass transfer coefficient is quite high in comparison to stirred tank reactors. This is ideal for SCP production from methanol as carbon substrate. This is used mainly to avoid excess heat produced during mechanical agitation.  TYPES OF FERMENTERS 3. Tower Fermenter: Tower fermenter has been defined as an elongated non-mechanically stirred fermenter that has an aspect ratio (height to diameter ratio) of at least 6:1 for the tubular section and 10:1 overall. There are several different types of tower fermenters, which are grouped based on their design: (i) Bubble Column Tower Fermenters: These are the simplest type of tower fermenters; they consist of glass or metal tubes into which air is introduced at the base. Fermenter volumes from 3 l to up to 950 l have been used and the aspect ratio may be up to 16:1. These tower fermenters have been used for citric acid and tetracycline production, and for a range of other fermentations based on mycelial fungi. (ii) Vertical-Tower Beer Fermenters: These fermenters were designed for beer production and to maximise yeast biomass yields. Tower of up to 20,000 l capacity and capable of producing up to 90,000 L beer per day have been installed. TYPES OF FERMENTERS (iii) Multistage Tower Fermenters: These fermenters have been used for continuous culture of E. coli, S. cerevisiae (baker‘s yeast), and activated sludge. 4. Fluidized Bed Bioreactor: Fluidized bed bioreactors (FBB) have received increased attention in the recent years due to their advantages over other types of reactors. Most of the FBBs developed for biological systems involving cells as biocatalysts are three phase systems (solid, liquid and gas). The FBBs are generally operated in co-current up flow with liquid as continuous phase. Usually, fluidization is obtained either by external liquid re-circulation or by gas fed to the reactor. In the case of immobilized enzymes, the usual situation is of two-phase systems involving solid and liquid, but the use of aerobic biocatalyst necessitates introduction of gas (air) as the third phase. A differentiation between the three-phase fluidized bed and the airlift bioreactor would be made on the basis that the latter have a physical internal arrangement (draft tube), which provides aerating and non-aerating zones.  TYPES OF FERMENTATION Any fermentation process can be carried in either of the three configurations: 1. Batch Fermentation: Is a closed culture system which contains initial, limited amount of medium that is not altered by further addition or removal. This form is simple and widely used in both laboratories and industries. As the growth of microorganism proceeds, the medium availability changes and the culture passes through a number of phases. After inoculation, there is a phase during which it appears that no growth occurs; this phase is called lag phase and is considered as the time of adaptation. This is followed by a phase when rate of cell growth gradually increases, and this period is called log phase. This growth results in the consumption of nutrients and excretion of microbial products, leading to a decrease in the growth rate. This cessation of growth is called the stationary phase.  TYPES OF FERMENTATION 2. Continuous Fermentation: In this, the exponential growth phase of organism may be prolonged by the addition of fresh medium to the vessel. The vessel should be designed in such a way that the added volume displaces an equal volume of culture from the vessel. If medium is fed continuously to such vessel at a suitable rate, a steady state is achieved eventually. Steady state occurs when formation of new biomass in the vessel is equivalent to the loss of cells from the vessel. The medium flow into the vessel is related to the total volume of the medium in the vessel expressed as dilution rate. Thus, under steady state conditions the specific growth rate is controlled by the dilution rate which is a controllable variable. An important objective of continuous culture operation is to control cell growth at a level at which productivity is optimum.  TYPES OF FERMENTATION A turbidostat is a continuous culturing method where the turbidity of the culture is held constant by manipulating the rate at which medium is fed. If the turbidity tends to increase, the feed rate is increased to dilute the turbidity back to its set point. When the turbidity tends to fall, the feed rate is lowered so that growth can restore the turbidity to its set point. The most widespread large-scale application of continuous culture reactors is in wastewater treatment. 3. Fed-Batch Fermentation: Two basic approaches to the fed-batch fermentation can be used: Fixed volume fed-batch: In this type of fed-batch, the limiting substrate is fed without diluting the culture. The culture volume can also be maintained practically constant by feeding the growth limiting substrate in undiluted form, for example, as a very concentrated liquid or gas.  TYPES OF FERMENTATION certain type of extended fed-batch - the cyclic fed-batch culture for fixed volume systems - refers to a periodic withdrawal of a portion of the culture and use of the residual culture as the starting point for a further fed- batch process. once the fermentation reaches a certain stage the culture is removed, and the biomass is diluted to the original volume with sterile water or medium containing the feed substrate. The dilution decreases the biomass concentration and result in an increase in the specific growth rate. As feeding continues, the growth rate will decline gradually as biomass increases and approaches the maximum sustainable in the vessel once more, at which point the culture may be diluted again. Variable volume fed-batch: A variable volume fed-batch is one in which the volume changes with the fermentation time due to the substrate feed. The way this volume changes it is dependent on the requirements, limitations and objectives of the operator. This type of fed-batch can still be further classified as repeated fed-batch process or cyclic fed-batch culture, and single fed-batch process.  TYPES OF FERMENTATION The former means that once the fermentation reached a certain stage after which is not effective anymore, a quantity of culture is removed from the vessel and replaced by fresh nutrient medium. The decrease in volume results in an increase in the specific growth rate. The latter type refers to a type of fed-batch in which supplementary growth medium is added during the fermentation, but no culture is removed until the end of the batch. This system presents a disadvantage over the fixed volume fed-batch and the repeated fed-batch process: much of the fermenter volume is not utilized until the end of the batch and consequently, the duration of the batch is limited by the fermenter volume.