Bioprocess Design - Instrumentation and Control Systems PDF

Unit-3 Bioprocess design - Instrumentation and control systems  Contents covered in this unit Fermentation facility, equipment and space requirements Fermenter design and its configuration, Body construction, Agitators, Stirrer glands and bearings, Spargers and valves, Aseptic op...

Unit-3 Bioprocess design - Instrumentation and control systems  Contents covered in this unit Fermentation facility, equipment and space requirements Fermenter design and its configuration, Body construction, Agitators, Stirrer glands and bearings, Spargers and valves, Aseptic operation and containment, Bioinstrumentation and its control Methods of measuring process variables, Online analysis of chemical factors, Control systems, Combination of methods of the controller, Troubleshooting in a fermentation plant.  Fermentation Department, Equipment and space requirements  Microbiological laboratories Microbiological laboratories frequently do mutation and isolation work - to produce strains with higher yields - to suppress a by-product - to reduce the formation of a surfactant - to change the physical properties of the broth to facilitate the product recovery etc other on-going studies include new culture preservation techniques, culture stability testing, new procedures- development, media improvements, search for inducers, repressors, inhibitors, etc. lab space and equipment requirement: 1. Glassware and Equipment Washing Area: 2. Media Preparation Area(s): 3. Inoculation rooms: 4. Incubator Areas: 5. Office: 6. Laboratories:  Analytical Support Laboratories The functions of these laboratories usually are - sterility testing of production samples, - chemical assays of raw materials for approval to use in the processes, raw materials before sterilization, optimized samples of production batches, fermenter feeds, waste streams different sources In many instances the analytical work for the culture laboratories will also be performed. Typical laboratories have Auto-analyzers for each of the common repetitive assays: the product of the fermentations, carbohydrates, phosphate, various ions, specific enzymes, etc. Other equipment generally includes balances, gas chromatography (GC), high pressure liquid chromatography (HPLC), Kjeldahl equipment, titrimeters, UV-visible spectrophotometers, Atomic absorption spectrophotometer (AAS), pH meters, viscometer, refractometer, densitometer, etc. a room with a laminar flow hood - to prepare plates, tubes, and shaking flasks Typical overall space requirements are 450 ft2 of floor space per working chemical technician  Production: Raw Material Storage Raw material warehousing most often is a separate building from manufacturing. Its location should be on a rail siding (for larger plants) twenty-ton trailers –to carry large amount of raw materials Large volume dry raw materials should be purchased in bulk (trucks or rail cars) and stored in silos (a cylindrical tower-used for storage). Transferring the raw material from the silos to the mixing tanks can be controlled by the panel in the instrument control room after selecting the weight and positioning diverter valves Wherever possible, liquid raw materials should be purchased in bulk and pumped. For safety and environmental reasons, drummed, liquid raw materials should be avoided, if possible.  Usually, two or three different sized tanks are used; - smaller batching tanks are for inoculum tanks - the larger tanks for feed and fermenter media preparation. The type of agitation varies widely. Media preparation tanks -capacity- 10,000 gallons and smaller - equipped with - 304 stainless steel, - dished or flat bottom and heads, - a slow speed (60 to 90 rpm) top-entering agitator with airfoil type impellers, The tanks need to be equipped with submerged (bottom) nozzles to supply both steam and air. Hot and cold water are usually piped to the top. A temperature recorder  The Seed Fermenter Layout Some companies prefer to locate all the seed fermenters in one area so that a group of workmen become specialists in batch sterilizing, inoculating, and indulging the first (plant) inoculum stage to maturity. Other companies locate the seed fermenters adjacent to the fermenters. Small plants cannot be able to spare to isolate equipment and have a specialized work force, large plants can have isolate groups of similar equipment, and specialize the work force which often results in higher productivity.  Seed fermenters usually do not have sterile anti-foam and nutrient feeds pipes to the tanks as the main fermenters have. results foaming led to infection which is one of the reasons they need more attention. Therefore the workmen should aware of careful inoculation procedures, sampling and sterilizing the transfer lines from the seed fermenter to main fermentor. During sterilization - one foreign organism or spore - not killed in time, contaminate the fermenter Importance of inoculum volume Larger cell masses of inoculum can shorten the growth phase of the culture using this concept some companies make the inoculum volume larger than a tenth of the fermenter volume (since the higher volume may reduce the lag time in the fermenter) so that they are minimizing the number of transfers from laboratory flask to the final fermenter  Nutrient Feed Tanks for feeding the nutrients - sterilizable tanks - essential equipment - productive fermentation department. Multiproduct plants - require several different sizes of feed tanks: (i) a small volume of nitrogen source to be transferred once every 12 or 24 h (ii) a large volume carbohydrate solution – fed continuously - may varying with the fermenter volume (iii) a precursor feed, fed in small amounts relative to assay data (iv) anti-foam (v) other tanks for acids, bases, salts, etc.  Usually feed tanks are not designed with high volume air flow but designed with only sufficient air pressure for the transfer air – needed only to transfer the feed, Since the air requirements – needed only to transfer the feed, the air piping design is different and the sterile air filter is smaller in size. For dissolvable nutrients - agitator - anti-foam system not required. Instrumentation is provided only to measure - temperature, - pressure - volume  Sterile Filters Today, the sterile air filtration is simple – providing with a commercial units readily available. However some companies still design their own to use a variety of filter media such as carbon, cotton, glass staple, etc. packed-bed or cartridge filters are used – - to obtain sterile air - to reduce the humidity of the air after compression so that the filter material always remains dry Careful selection of the cartridge design or the design of packed-bed filters improve the lifetime of filter for excess of 3 years  If a fiber material is used in a packed-bed type filter fine fiber diameter – get - efficient filtration. bed depth of filters is - 10 to 18 inches and10 microns. Other filter materials are less common and tend to have special problems and/or shorter life. Some plants have a separate filter for each sterile vessel. Others place filters in a central group which feeds all the vessels  Air Compressors It is ideal to have oil-free compressed air provided by "Oil free" screw air compressors -available in smaller sizes. The air should have the relative humidity of about 85%. The air from the compressors requires heat exchangers to lower the air temperature below the dew point, and additional heat exchangers to reheat and control the air to have the relative humidity at about 85%.  Valves (To Maintain Sterility) gate, diaphragm, ball plug valves In general, valves are less of a sterility problem diaphragm and ball valves - require considerable maintenance, but it is more useful for batch sterilizing operations plug type valves are used for continuous sterilizing operations Plug or diaphragm valves are commonly used for inoculum transfer and sterile feed piping. All the process valves and piping today are 316 S/S.  Pumps Use of peristaltic pump is an simple way to transfer inoculum from a large laboratory flask to a seed fermenter and to the fermenter. Connect the sterile adapter (which is attached to the flask) to the seed fermenter by sterile technique Install the rubber tubing (taigon and silicon tube types) in the pump open the hose clamp and start the pump Inoculum from seed fermenters and sterile feeds are transferred to the fermenter. Centrifugal pumps (316 S/S) are used to pump non-sterile raw materials, slurries, harvested broth, etc.  Cooling Equipment Cooling is required - to cool media from sterilizing temperatures, - to remove the exothermic heat of fermentation, - to cool broth before harvesting, - to cool the compressed air. Cooling water is provided from cooling towers chilled water (5º-15ºC) is produced by steam vacuum, or refrigeration units. the fermentation department should always be concerned about its cooling water supply, i.e., the temperature and chloride content.  When the Chloride ions above 150 ppm in the water which is being used for sterilization, at the same time if the stainless steel (seed fermenter or fermenter) is at above 80ºC (while sterilizing) will cause stress corrosion cracking of stainless steel A conductivity probe should be in the cooling water line it may indicate that the salt level is too high when the dissolved solids (due to salts) get too high some water must be discharged out and fresh water should be added.  After discharging the water from the sterilization process, If cooling water is discharged to a stream, river, etc., an pollution control agencies permit may be needed and special monitoring required. In the water (which is used for cooling), the chloride content should be determined analytically every two weeks to control the chloride to less than 100 ppm. The dischargeable level of chloride content in cooling water is less than 100 ppm. Environmental Control Odor Certain raw materials smell when sterilized. Each fermentation process tends to have its own unique odor ranging from mild to strong and from almost pleasant to absolutely foul. Because the high volume of air is being discharged from a large fermenter house. So the odor is unavoidable.  odors have to be avoided ▪ may use - wet scrubbing towers with sodium hypochlorite expensive, and will discharge Na+ and Cl2, to the waste system not suitable ▪ In some cases, a very tall exhaust stack can be fixed and used for the dilution of the released gas with the atmosphere before the odor reaches the ground but is not considered as an acceptable solution by pollution control board. ▪ Ozone treatment can be effective. Organic Pollution The fermentation department should monitor and control the COD/BOD of its liquid waste before discharging to the sewer. Procedures for cleaning up spills and reporting should be Standard Operating Procedure. Using a primary aeration basin will reduce the COD to 80-90 ppm. Using secondary aeration lagoons will reduce the BOD to acceptable levels. Noise Noise levels are very difficult to reduce to the standard levels. Hearing protection for employees is essential. If providing automatic processing of fermentation processes in the fermentation department operators can have less exposure to noisy work areas Fermenter design and its configuration A typical fermentor used for microbial fermentations is shown in the following figure: Laboratory scale fermentors with liquid volumes of less than 10 litres are constructed out of Pyrex glass. For larger reactors,stainless steel is used. Various components of an ideal fermenter for batch process are: Monitoring and controlling parts of fermenter are:  Fermenter A fermenter is basically a device in which the organisms are cultivated to form the desired products. It is a containment system designed to give right environment for optimal growth and metabolic activity of the organism.  Basic Fermenter Design Criteria Microbiological and biochemical characteristics of the cell system (microbial, mammalian, plant) Hydrodynamic characteristics of the bioreactor Mass and heat transfer characteristics of the bioreactor Kinetics of the cell growth and product formation Genetic stability characteristics of the cell system Aseptic equipment design Control of bioreactor environment (both macro and micro-environment) Implications of bioreactor design on downstream products separation Capital and operating costs of the bioreactor Potential for bioreactor scale-up  Fermenter Design The basic points of consideration while designing a fermenter: Productivity and yield Fermenter operability and reliability Product purification Water management Energy requirements Waste treatment Other few significant things to be taken in account: Design in features so that process control will be possible over reasonable ranges of process variables. Operation should be reliable Operation should be contamination free  Fermenter Design To achieve these the fermenter should have: Heat and oxygen transfer configuration Sterilization procedures Foam control Fast and thorough cleaning system Proper monitoring and control system Traditional design is open cylindrical or rectangular vessels made from wood or stone. Most fermentations are now performed in close system to avoid contamination. It should be constructed from non-toxic, corrosion-resistant materials. Small fermentation vessels of a few liters capacity are constructed from glass and/or stainless steel. Basic design and construction of fermenter and its ancillaries  Aeration and Agitator The 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 homogeneous nutrient medium. The structural components of the fermenter involved in aeration and agitation are: a. The agitator (impeller) b. Stirrer glands and bearings c. Baffles d. The aeration system (sparger).  AGITATOR (IMPELLER) The agitator is required to achieve a number of mixing objectives, for example, 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 are classified as having radial flow (perpendicular to impeller shaft) or axial flow (parallel to impeller shaft) characteristics. With radial flow mixing, the liquid flow from the impeller is initially directed towards the wall of the reactor; ie. along the radius of the tank. With axial flow mixing, the liquid flow from the impeller is directed downwards towards the base of the reactor, ie. in the direction of the axis of the tank. Radial flow impellers are primarily used for gas-liquid contacting (such as in the mixing of sparged bioreactors) and blending processes. Axial flow impellers provide more gentle but efficient mixing and are used for reactions involving shear sensitive cells and particles.  Agitator design and operation - Radial flow impellers Radial flow impellers contain two or more impeller blades which are set at a vertical pitch:  Agitator design The high shear is effective at breaking up bubbles. For this reason, radial flow impellers are used for the culture of aerobic bacteria. High shear can also damage shear sensitive materials such as crystals and precipitates and shear sensitive cells such as filamentous fungi and animal cells. With radial flow impellers, vertical (or axial) mixing is achieved with the use of baffles Radial flow impellers  Axial flow impellers Low shear conditions are achieved by pitching the impeller blades at an angle and by making the edges of the impeller blades thing and smooth.  Geometric Ratios for a Standard Bioreactor Vessel Impeller Di/Dt HL/Dt Li/Di Wi/Di Hb/Di Wb/Dt No. type Baffles Flat-Blade 0.33 1.0 0.25 0.2 1.0 0.1 4 Turbine Paddle 0. 33 1.0 - 0.25 1.0 0.1 4 Impeller Marine 0.33 1.0 pitch 1.0 0.1 4 Propeller = Di Where: Dt = tank diameter, HL = liquid height Di = impeller diameter Hb = impeller distance from bottom of vessel Wb = baffle width Li = impeller blade length Wi = impeller blade height Different Impeller Types. (a) Marine-type propellers; (b) Flat-blade turbine, Wi = Di/5. © Disk flat-blade turbine, Wi = Di/5, Di = 2Dt/3, Li = Di/4; (d) Curved-blade turbine, Wi = Di/3; (e) Pitched-blade turbine, Wi = Di/8; and (f) Shrouded turbine, Wi = Di/8. Mixing Patterns for Flat-Blade Turbine Impeller. Effect of Baffles. Liquid agitation in presence of a gas-liquid interface, with and without wail baffles: (a) Marine impeller and (b) Disk flat-blade turbines; (c) in full vessels without a gas-liquid interface (continuous flow) and without baffles.  STIRRER GLANDS AND BEARINGS The stirrer shaft can enter the vessel from the top side or bottom of the vessel. Top side entry is commonly used, but bottom entry may be advantageous Mechanical seals can be used for bottom entry and should be maintained periodically and replaced at recommended intervals Four basic types of seal assembly have been used: the stuffing box (packed-gland seal), the simple bush seal, the mechanical seal, and the magnetic drive. Most modern fermenter stirrer mechanisms now incorporate mechanical seals instead of stuffing boxes and packed glands. Mechanical seals are more expensive but are more durable and less likely to be an entry point for contaminants or a leakage point for organisms or products which should be contained. Magnetic drives are also quite expensive and are widely used in animal cell culture vessels.  BAFFLES Four baffles are normally incorporated into agitated vessels of all sizes to prevent a vortex and to improve aeration and agitation efficiency and to prevent vortexing. 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 The 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  AERATION SYSTEM (SPARGER) 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). A combined sparger-agitator may be used in laboratory fermenters 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 10–100 times larger than the pore size of the aerator block. The throughput of air is low because of the pressure drop across the sparger and there is also the problem of the fine holes becoming blocked by growth of the microbial culture. Orifice sparger: It is used in small stirred fermenter. 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 drilled on the under surfaces of the tubes and the holes should be atleast 6mm diameter. Orifice spargers without agitation have been used to a limited extent in yeast manufacture, effluent treatment, and later in the production of single-cell protein in the air-lift fermenter, Nozzle sparger: Point or nozzle spargers are used in many agitated fermenters from laboratory to production scale. These spargers consist of a single open pipe or partially closed pipe providing a point-source stream of air bubbles. The single-nozzle sparger causes a lower pressure loss than any other sparger and normally does not get blocked. Combined sparger–agitator On a small scale (1 dm3), Herbert, Phipps, and Tempest (1965) developed the combined sparger-agitator design, introducing the air via a hollow agitator shaft and emitting it through holes drilled in the disc between the blades and connected to the base of the main shaft. The design gives good aeration in a baffled vessel when the agitator is operated at a range of rpm Aseptic operation and containment  Aseptic Operation and Containment Aseptic Operation Involves protection against contamination Containment Involves in prevention of escape of viable cells from a fermenter or downstream equipment Containment guidelines were initiated during 1970s To establish the appropriate degree of containment which will be necessary to grow microorganism, it, and in fact the entire process must be carefully assessed for potential hazards that could occur should there be an accidental release. Once the hazards are assessed, an organism can be classified into hazard group for which there is an appropriate level of containment. Outline of European community adopted Procedure Nongenetically engineered organisms may be placed into a hazard group (1-4) using criteria to assess risk those given by Collins: 1. The known pathogenicity of the microorganism 2. The virulence or level of pathogenicity of the microorganism – are the diseases it causes mild or serious? 3. The number of organisms required to initiate an infection 4. The routes of infection 5. The known incidence of infection in the community and the existence locally of vectors ad potential reserves 6. The amounts or volumes of organisms used in the fermentation process 7. The techniques or processes used 8. Ease of prophylaxis and treatment  Hazard group 1 organisms used on a large scale require only Good Industrial Large-Scale Practice (GILSP). Processes in this category need to be operated aseptically but no containment steps are required. If the organism is placed in Hazard group 2,3,4 the stringent requirement levels of 1,2,3 respectively have to be met before the process operation. Genetically modified orgarnisms (GMOs) were classified as either harmless (Group I) or potentially harmful (Group II). The process is classified as either small scale or large scale. Again, large scale processes fell into two categories IB or IIB. IB processes require containment level B1 and are subject GILSP, and IIB processes were further assessed to determine the most suitable containment level ranging from B2-B4. Levels B2 to B4 correspond to levels 1 to 3 or nongenetically engineered organisms. The UK’s Health and Safety Executive issued the “The GMO (Contained Use) regulations 2014,” in October 2014. The 2014 regulations (Schedule 1) describe the classes of contained use: Class 1. Contained use of no or negligible risk. Containment level 1 is appropriate to protect human health and the environment Class 2 contained use of low risk. Containment level 2 is appropriate to protect human health and the environment Class 3 Contained use of moderate risk. Containment level 3 is appropriate to protect human health and the environment Class 4 Contained use of high risk. Containment level 4 is appropriate to protect human health and the environment Bioinstrumentation and Control  Introduction Success of fermentation depends upon the existence of defined environmental conditions of biomass and product formation For this, it is important to understand what is happening to a fermentation process and how to control it to obtain the optimal operating conditions. The conditions include: temperature, pH, degree of agitation, oxygen concentration in the medium, and other factors such as nutrient/substrate concentration etc. The provision of such conditions requires careful monitoring of the fermentation so that any deviation from specified optimum can be corrected by control systems. Major Areas of Bioprocess Monitoring Methods of Monitoring OPERATING FEATURES Main criteria for a bioprocess monitoring equipment  Sensors A sensor is a device that detects and responds to some type of input from the physical environment. In bioreactors, the input from the above-mentioned table include temperature, pressure, Agitator shaft power, rpm, foam, flow rate, pH etc There are three main classes of sensors: 1. Sensors which penetrate into the interior of the fermenter, Eg: pH electrodes, DO electrodes 2. Sensors which operate on samples which are continuously withdrawn from the fermenter. Eg: exhaust- gas analyzers 3. Sensors which do not come into contact with the fermentation broth or gases. Eg: tachometers, load cells  It is also possible to characterize a sensor in relation to its application for process control: 1. In line sensor: The sensor is an integrated part of the fermentation equipment and the measured value obtained from it is used directly for process control 2. On line sensor: Although the sensor is an integral part of the fermentation equipment, the measure value cannot be used directly for control. An operator must enter measured values into the control system if the data is to be used in the process control 3. Offline sensor: The sensor is not a part of the fermentation equipment. The measured value cannot be used directly for process control. An operator is needed for actual measurement (e.g. medium analysis of dry weight sample) and for entering the measured values into the control system for process control Methods of measuring Process variables Temperature Temperature in a vessel or pipeline is one of the most important parameters to monitor and control in any process. Measured by mercury-in-glass thermometers, bimetallic thermometers, pressure bulb thermometers, thermocouples, metal-resistance thermometers or thermistors. Metal-resistance thermometers and thermistors are used in most fermentation applications Accurate mercury-in-glass thermometers are used to check and calibrate the other forms of temperature sensors and cheapest thermometers being used in laboratory fermenters Mercury-in-glass thermometers: It may be used directly in small laboratory scale fermenters It fragility is only the disadvantage In large fermenters, it is inserted into the thermometer pocket in vessel which introduces a time lag in registering the vessel temperature It can be used solely for indication not for automatic control or recording Electrical resistance thermometers: It is known that the electrical resistance of metals changes with change in temperature. This property is used in the design of resistance thermometers The bulb of the instrument contains a resistance element, a mica framework (for very accurate measurement) or a ceramic framework around which the sensing element is wound. A platinum wire of 100Ω resistance is normally used. The reading normally obtained by the use of Wheatstone bridge circuit and is a measure of the average temperature of the sensing element This type of thermometer have a greater accuracy (± 0.25%) than some of the other measuring devices and is more sensitive to small temperature changes. These thermometers are normally enclosed in stainless-steel sheaths if they are to be used in large vessels and ancillary equipment. Thermistors semiconductors made from specific mixtures of pure oxides of Iron, Nickel and other metals. main characteristic is a large change in resistance with a small change in temperature The change in resistance is a function of absolute temperature The temperature reading is obtained with a Wheatstone bridge or a simpler or more complex circuit depending on the appicaton Thermistors are relatively cheap, proved to be very stable, give reproducible readings and can be sited remotely from read-out point The main disadvantage is the marked nonlinear temperature vs resistance curve Temperature control Water jackets or pipe coils in fermenter are used as temperature controls In small systems a heating element and a cooling water supply are present and are on or off depending on the need for heating or cooling. The heating element should be as small as possible to reduce the size of the “heat sink” and result in overshoot when heating is no longer required In some cases, it is advisable to run cooling water continuously at a low and steady rate and to have only the heating element connected to the control unit. In larger fermenters, where heating is not required for fermentation process, a regulatory valve at the cooling-water inlet may be sufficient to control the temperature Steam inlets to the coil and jacket must be present if a fermenter is being used for batch sterilization of media  Flow measurement and control In the process management, flow measurement and control of both gases and liquid is important. Gases Simplest method for measuring gas flow to a fermenter is by means of variable area meter Rotameter is the mostly used. It consists of a vertically mounted glass tube with an increasing bore and enclosing a free-moving float which may be a ball or a hollow thimble. Rotameters should not be sterilized and are normally placed between a gas inlet and a sterile filter. No provision for online data logging with the simple rotameters. Rotameters can be used to measure both gas flow rates and liquid flow rates. While it is used to measure liquid flow rate, make sure that no abrasive or fibrous particles are present The use of oxygen and carbon di oxide gas analyzers for effluent gas analysis requires the provision of very accurate gas-flow measurement if the analyzers are being used effectively These instruments have a ±1% full-scale accuracy and work on the principle of measuring a temperature difference across a heating device placed in the path of the gas flow  Temperature probes such as thermistors are placed upstream and downstream of the heat source, which may be inside or outside of the pipeline. The mass flow rate of the gas, Q, can be calculated from the specific heat equation: 𝐻 = 𝑄𝐶𝑝(𝑇2 − 𝑇1) H = Heat transferred Q = mass flow rate of the gas Cp = Specific heat of the gas T1 = Temperature of the gas before heat is transferred to it T2 = Temperature of the gas after the heat is transferred to it This equation can be rearranged for Q: 𝑄 = 𝐻/𝐶𝑝(𝑇2 − 𝑇1) Control of gas flow is carried out by needle valves. Fluctuations in pressure in a flow-measuring orifice cause a valve or piston pressing against a spring to gradually open or close so that the original flow rate is restored Liquids Measurement of the flow rates of sterile liquids presents a number of problems. On a laboratory scale flow rates may be measured manually by using sterile burette connected to the feed pipe and timing the exit of measure volume. Rotameters and electrical flow transducer can also be used to measure the flow rate of the liquids. In batch and fed-batch fermenters, an alternative is to measure the flow rates indirectly by load cells. The fermenter and all ancillary reservoirs are attached to the load cells, which monitor the increase and decrease in weight of the various vessels at regular time intervals. If the specific gravity of the liquids are known, it is also possible to estimate the flow rates accurately in different feed pipes  Pressure measurement and control Pressure is also another crucial measurement to be considered for operating different processes and also for media sterilization In a fermenter, pressure influences the solubility of gases and contribute to the maintenance of sterility in the presence of positive pressure Bourdon tube pressure gauge is used as direct indicating gauge for measuring pressure Pressure is also another crucial measurement to be considered for operating different processes and also for media sterilization In a fermenter, pressure influences the solubility of gases and contribute to the maintenance of sterility in the presence of positive pressure Bourdon tube pressure gauge is used as direct indicating gauge for measuring pressure. In this type, with the increase in pressure , the partial coil with elliptical cross-section tends to become circular, and the difference between internal and external radii gradually straightens out.  Nested Diaphragm-Type pressure sensor is used when a vessel or pipeline is to be operated under aseptic condition. The changes in the pressure cause movements of diaphragm capsule being monitored by mechanically levered pointer The pressures can be measured remotely using pressure bellows connected to the core of a variable transformer Piezoelectric transducers: Pressure can be measured by means of electrodes attached to the opposite surfaces of the crystal with the application of Piezoelectric transducers. Any change in shape if the crystal produces equal, external, unlike electric charges on the opposite faces of the crystal → Piezoelctric effect Pressure control The correct pressure in different components should be maintained by regulatory valves controlled by associated pressure gauges. Safety valves incorporated in all vessels and pipelines are likely operated under pressure. The valve should be set to release the pressure as soon as it increases markedly above the specific working pressure  Foam sensing and control Foam formation during microbial fermentation can cause serious problems if not controlled. It mainly influences the concentrations of the dissolved oxygen Antifoam can be added to the fermenter when the culture starts foaming above the predetermined level. Methods used for foam sensing and antifoam additions will depend on the process and economic considerations. Other Mechanical antifoam devices include discs, propellers, brushes or hollow cone attached to the agitator shaft above the surface of the broth. Some of the manufactured devices include horizontal rotating shafts, centrifugal separators and Jets spraying on to deflector plates. Troubleshooting in a fermentation plant  Introduction Problems from electrical, instrumentation, mechanical and physical during or after the fermentation process have to resolved Other source to troubleshoot the process is the presence of microorganisms (foreign) in the media This can lead to rise few more problems: Inoculum cannot be used Inhibition of production formation in fermenters If product is produced, contaminated products cannot be separated Due to physical broth characteristics, the fermentation broth cannot be filtered or processed  Basic Sources of Contamination in Fermentation department Contamination of stock culture due to poor techniques Contamination by the raw materials used in the fermentation Contamination attributed to inadequate sterilization of equipment, air or media involved in a fermentation plant Contamination attributed to inadequate procedures or insufficient operator training Contamination by bacteriophages  Six categories to be checked regularly to prevent contamination 1. Contamination in the culture laboratory Check stock cultures for foreign organism Check sterilization procedures Checking the sterility of the sterile area Check the sterilizer including temperature, pressure gauages and operation Carry out all practical tests for contamination using best techniques such as agar plates etc and microscopic examination 2. Contamination in Raw materials Purchase all dry materials in finely ground form For undissolved materials, suspend them in the cold water first which usually prevents lump formation and then use adequate agitation in the mix tank Discard the oversized lumps  Six categories to be checked regularly to prevent contamination 3. Contamination from Equipment Contaminated inoculum tanks Checking inoculum, inspecting tanks regularly for cleanliness, checking tanks and its accessories for leaks, calibrating the temperature and pressure gauges Contaminated fermenters Inspecting the inoculum line or hoses Checking cleanliness of the fermenter Check for accessories for any leaks Check the anti-foam system and determine if anti-foam is sterile  Six categories to be checked regularly to prevent contamination 4. Operating Procedures Proper training has to be provided to all new operators and explain them how and why each operation is important. No change in the procedures by individuals should be permitted A basic operating manual should be available Regular safety meetings and technical meetings should be scheduled with the operators and update them any changes in technical and procedural changes Operators must be encouraged to give their feedbacks regarding their observations, ideas and errors without any fear. 5. Lack of Maintenance as a source of contamination 6. Bacteriophage contamination  Six categories to be checked regularly to prevent contamination 5. Lack of Maintenance as a source of contamination Braided packing on agitator shafts of sterile vessels is common problem. Germicidal solutions can be helpful Mechanical seals on agitator shafts of sterile vessels usually have a sterilizing liquid circulating which can act as a lubricant also Calibration of temperature and pressure gauges for accurate sterilizing temperatures are critical Check the flanged sterile piping for leaks 6. Bacteriophage contamination Bacteriophages affecting bacterial and fungal fermentations can be found in soils, water, air and even in raw materials. Their presence results in lysis of the cells which results in reduction of cell mass, no further product formation, no Oxygen uptake, no heat production etc Confirmation of presence of bacteriophage can be made with phage plaque plates or by using ultra-filters and isolating the specific virus itself and finally testing the filtrate in the laboratory Online analysis of chemical factors