Summer Training Report

Objective:

To study the working of a pilot plant (fermentation) and utilities.

Pilot plant: Intermediate plant between Laboratory-scale and Large-scale plants. It comprises fermenter vessels of volume intermediate between laboratory-scale and large-scale plants, a network of pipelines (carrying steam, hot water, cold water, chilled water, inert gases like Nitrogen and Carbon dioxide etc.) and electric lines, PLC and a small-scale microbiology-based laboratory.

Fermenter vessel: vessel in which fermentation (defined by industrial microbiologists as any process for the production of product by the mass culture of a micro-organism) is carried out. Batch process is the most popular mode for carrying out fermentation. Most commonly used fermenter is called Aerated Stirred Batch Fermenter , also known as Classical Fermenter . Design features: y Stainless steel body to sustain hydrostatic pressure of the broth and avoid corrosion. SS316 grade is a preferred grade of stainless steel for use in fermenters because of its added advantages such as resistance to leaching and corrosion caused by chloride ions and increased durability over other grades. Top stirrer with dry mechanical seal essential for homogenization of the fermentation broth and most importantly for meeting microbes biological Oxygen demand. Baffles (usually 4 in number and equally spaced) to prevent vertex formation by liquid broth (due to agitation). Ports for probes (Temperature,pH, DO2, CO2 etc.) for inserting probes so that system variables such as Temperature, pH, DO2, CO2 etc. can be measured. Impellers embedded on the stirrer and create agitation in the broth as the stirrer rotates. Different combinations of impellers are used to suit various needs. Rushton turbine is used for radial flow while pitched blades are used for axial flow. Marine propellers can be used for mixed flow i.e. radial and axial flow both. Sparger for aerating the broth with compressed air to continuously meet microbes biological Oxygen demand.

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Water Jacket To achieve temperature control in the vessel by circulation of hot water, cold water or chilled water. Inoculation port, sampling point and harvesting line. Transfer line for transfer of broth from one vessel to another (usually a seed culture into a larger vessel for production). Vent valve To vent out or hold back air present in the vessel so as to increase or decrease the pressure inside the vessel.

The line carrying compressed air has two filters embedded: a pre-filter (Stainless Steel or PTFE, 1.0 micron) and a membrane filter (PTFE, 0.2 microns). The membrane filter prevents most of the micro-organisms present in the compressed air from entering the vessel. The membrane filter has finite number of cycles (typically 90 cycles) after which it should be replaced. The lines carrying steam are properly insulated to avoid any physical contact (temperature being as high as 185oC).

Fig. 1. Schematic diagram of a classical fermeter vessel

Valves: Valves may provide various kinds of control to fluid flow be it ON/OFF, coarse adjustment or fine adjustment. Diaphragm and Ball valves are widely used in fermenters and associated lines. They provide coarse adjustment of fluid flow but are good for maintain sterile conditions and suitable for GILSP containment level process. Needle valves provide fine tuning of fluid flow and used in filters, rotameters etc. Gate valves are used for ON/OFF control generally in the pipelines. Steam traps are also of particular interest owing to its ability to allow only condensate to flow, checking the flow of the steam through it in the steam line. It is present at the end of the steam line and used to remove condensate formed in the steam line without any loss of steam.

Autoclaving: It is a routine operation carried out at 15-20 PSIG, 121 oC for around 30-45 minutes depending upon nature (heat-sensitivity) and volume of the material to be autoclaved. Packaging of autoclavable materials is important. All the open ends are closed with cotton plugs and wrapped around by Aluminum foil and paper. Autoclaves are broadly of two types: horizontal and vertical. To insure proper sterilization, temperature of different sections of the autoclave can be electronically monitored. There is also provision for steam and condensate outlets and safety valve.

Notes: 1. All the glasswares and tubes used in fermentation batch are autoclaved prior to being employed in the operations. 2. Thermo-labile medium components such as Vitamins, sucrose, minerals, growth factors etc. are autoclaved separately and inoculated later.

Sterilization:Carried out in the vessel itself at 1.2-1.3 Bars, 121 oC for 30-45 minutes depending on nature and volume of the medium. Sterilization is carried out twice: firstly, an empty vessel sterilization to reduce existing microbial burden and secondly, sterilization of medium in the vessel. Notes: 1. Atmospheric pressure is considered to be zero since all the pressure gauges zero is calibrated at atmospheric pressure. 2. Positive pressure means the pressure is greater than the atmospheric pressure.

3. Back pressure is the pressure created inside the vessel in the head space by holding back some air in it. At steady-state condition, air flow rate inside the vessel equals air flow rate outside the vessel. 4. Air flow rate (In Liters per minute) is determined by a Rotameter present in the air line prior to the filters. 5. Air flow rate per unit volume of the broth is a more preferred parameter popularly known as VVM i.e. volume of air per unit volume of broth per minute.

Empty vessel sterilization: y y y y y y Firstly, membrane filter is sterilized (mostly when the filters have not been kept at positive pressure) with steam. Compressed air is then flown in the filter to remove the condensate. At the same time, the harvest and other lines are opened to remove condensate. The vessel is depressurized and afterwards steam is passed through the sparger from bypass line. All the vents are partially opened so that sterilization is achieved in all the ports and lines. Steam is passed so that the gauge pressure inside the vessel reaches 1.2 bars. Sterilization is carried out for 45 minutes after the temperature reaches 121 oC. During sterilization, Tempilstik (commercial name) is rubbed at various parts of the vessel to check for melting. Melting of Tempilstik ensures a temperature of 121 oC or above. If Tempilstik doesn t melt somewhere, more steam is availed to that portion. After 45 minutes of sterilization, steam supply is cut off. The vessel is allowed to cool off by running cold water in the jacket. The vessel is maintained at positive gauge pressure (nearly 1 bar).

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Notes: 1.Prior to carrying out empty vessel sterilization, the vessel is first washed with water under agitation. Then, the vessel is washed with 1% (w/w) NaOH solution under agitation. 2. NaOH solution acts on the cell wall components of the culture from previous batch attached on the walls of the vessel and the ports and detaches them. 3. NaOH solution is heated up to 100 oC by passing steam through sparger and then allowed to cool. The solution is discharged from the vessel prior to sterilization.

Calibration of probes: After empty vessel sterilization, pH and DO2 transmitters are calibrated and their probes are inserted in the vessel. pH transmitter is calibrated by two standard pH buffers: one having pH 7.0 and the other having pH 4.0. DO2 transmitter is calibrated by two liquid samples: one having a solution of sodium meta-bisulfite (% saturation is ideally 0) and other solution completely saturated with oxygen. Medium is poured in the vessel after the pH and DO2 probes have been inserted (after pH and DO2 transmitters have been calibrated) in the vessel through the ports designed specifically for the probes. Medium is poured through the watch glass located in the head of the fermenter.

Air Pressure Test: The head is separated from rest of the vessel by a gasket ring. Tear and wear in the gasket may lead to leakage and subsequent contamination. To avoid that an Air Pressure Test (APT) is performed at a pressure significantly greater than the pressure at which the batch is intended to be run. Usually APT is carried out at 1.5 bars for the reason that any leakage that manifests at a pressure less than 1.5 bars will surely manifest at 1.5 bars. This provides a larger window for operating pressure and adds to surety of detection of any leakage present. The test is simply sprinkling of soap water over all the seals and gaskets and checking for formation of air bubbles. If no bubbles are formed, the vessel has passed the APT.

In situ medium sterilization: APT is usually performed before starting sterilization. Pouring medium is followed by in situmedium sterilization (sterilization of the medium in the vessel itself) which encompasses the following procedures. y y y y Removing all the condensate present in all the lines, in the water jacket and in the vessel. Passing steam in the water jacket and letting out steam through the jacket outlet for condensate and steam. Checking for building up of pressure inside the jacket (the gauge pressure should not exceed 1 bar) and rise in the temperature up to 100 oC. After the temperature has reached 100 oC, steam flow through the jacket is stopped and steam is directly introduced in the vessel through the sparger, the sampling pipe and the transfer pipe (the last two being dipped deep inside the vessel in the medium) and all of the vents are opened to let the steam out. The sterilization is carried out henceforth in the same manner as in the empty vessel sterilization.

At the end of the sterilization, air is slowly introduced in the vessel through the sparger such that positive gauge pressure (roughly 1 bar) is maintained in the vessel. As the steam cools down, the pressure decreases and vacuum is created, sucking air from outside if not maintained at positive gauge pressure.

Notes: 1.The medium is heated to 100 oC through jacket and afterwards directly through steam so that formation of condensate (increases overall volume of the medium) is relatively less. 2. pH of the medium is adjusted before sterilization by adding alkali or acid. 3. pH of the medium decreases after sterilization due to release of proton from all the acidic groups present in the medium under high temperature. It springs back to the original value when the medium cools down. 4. Jacket is very thin as compared to the vessel s body. It can t sustain very high pressure. So, it is imperative that the gauge pressure inside the vessel due to steam doesn t exceed very much beyond 1 bar. Safety valve is present to avert damage to the jacket in case pressure exceeds safety limit.

Inoculation: Many additives (sucrose, mineral solution, growth factors, antifoams etc.) and inoculum/seed need to be added to the sterilized medium. Inoculum port is used for addition of the additives and the inoculum/seed. Near the opening in the port, flame is used to maintain sterile condition. Autoclaved silicone tubes are used for addition. Autoclaved glass vessels which carry the additives or the inoculum/seed is tightly fitted with a neck assembly which has a filter PTFE membrane on one end and silicone tube on the other. The silicone tube is dipped deep inside the additives or the inoculum/seedon the one end and the other end is capped over the opening of the inoculation port. Mechanism: The silicone tube is wrapped around a rotor in the peristaltic pump which presses against the tube as it rotates and creates a suction which sucks the additives or the inoculum/seed, resulting in drop in pressure in the vessel containing additives or the inoculum/seed. As a result, air from outside is sucked through the PTFE filter membrane inside the vessel. Note: The vessel is depressurized i.e. back pressure is zero, before the addition of the additives or the inoculum/seed.

Withdrawal of sample: The sampling port is first sterilized with steam. Then the broth is initially done away with for a while. The sample is withdrawn under sterile condition by using flame in close vicinity of the mouth of the port. Note: Broth is initially done away with in order that the sampling port cools down (after having been sterilized with steam).

Transfer of seed:Transfer of seed batch to the production vessel is a routine operation. The back pressure in the vessel to which the seed is to be transferred is kept lower than in the other vessel during the transfer. Transfer port or discharge line (sterilized with steam before the seed is withdrawn) can be used. Autoclaved silicone tube is used for transfer of the seed. Notes: 1. Seed provides fresh, healthy micro-organisms for sake of optimum yield. Medium for the seed culture is highly nutritive. 2. In addition, duration of the lag phase is considerably reduced, reducing overall batch time.

Centrifugation: This is a routine procedure of downstream processing of a batch. After a batch is complete i.e. desired batch characteristics (cell mass concentration, extent and stages of sporulation etc.) have been achieved, the broth is collected into a sterilized vessel through the discharge line. The broth is then fed into a tubular centrifuge which contains a bowl rotating at 15000 rpm. The cells stick on the wall of the bowl and scraped later for further downstream processing. Basket centrifuge is employed for refinement of corn steep liquor (CSL) or any such liquor which is further used as a media component. A bag is employed to act as a filter under high centrifugal force. The filtrate passes through the bag and is collected in a can. Before using the bag for filtration, the bag is washed (by filling it with water and centrifuging it) to remove remnants from the previous use.

Anaerobic fermentation:For anaerobic micro-organisms (facultative or obligate), Nitrogen and Carbon Dioxide is used in various proportions (N2 and CO2 can be used absolutely) instead of air. The medium can be flushed with N2to expel out the dissolved oxygen. Ball valves are employed along these gas lines to control the flow of these gases in the vessel. N2 and CO2 are directly availed through the respective gas cylinders. These gas cylinders are mouthed with an assembly having pressure gauges, an output line and a regulator which

regulates the flow of the gas in a manner reverse (clockwise for opening and anticlockwise for closing) to that for commonly used valves (clockwise for closing and anticlockwise for opening).

Maintenance of Filters: Membrane filters are sensitive to prolonged exposure of compressed air (decreases longevity of a filter). Therefore, many a times the filters are depressurized. Once a filter has beendepressurized, it is highly likely that it gets contaminated by air (which can make its way through the air line much more easily). Therefore, it is required that the filter is sterilized before use. Sterilization of filter is a necessary precaution which also wards off chances of contamination arising due to cross-contamination of the filter from the precious batch. Integrity test: Also known as Bubble Test . The integrity of the membrane filter is tested after every 5 cycles. The integrity test determines whether the filter is suitable for further use or not (pores may get much bigger than 0.2 microns in size due to wear and tear). The filter is dipped into 70% iso-propanol for half an hour and then replaced in its housing. Air is released into the filter and inlet air pressure is gradually increased. A silicone tube is connected at one end to the bleeder present at the bottom of the filter housing. The other end is dipped into water. The pressure at which air bubbles start to form should not be less than 1.2 bars for filter to pass the integrity test.

Microbiology-based laboratory:equipped with relevant and essential modern instruments such as Laminar Flow Hood, pHmeter, colorimeter, light microscope, centrifuges, weighing balance etc. Laminar Hood: an absolute necessity for preparation of neck assemblies and other operations under sterile condition. Laminar hood s floor is first wiped with 70% ethanol or iso-propanol and then sterilized by UV exposure for 15 minutes (later can be skipped). Laminar air flow is switched on and UV light is switched off. One should be careful to not appear in front of the Laminar Hood while UV light is on. pH meter: another important instrument routinely used for pHdetermination. Apart from the reading displayed by pHtransmitter, the pHof the broth is separately determined using the pHmeter. The pHprobe is dipped in KCl solution when not in use.

Colorimeter: used for determining optical density (absorbance) of a sample indicative of cell density. Colorimeter is calibrated by setting absorbance value of zero for a control. The optical density for cells is usually measured at 620 nm. Calorimeter can measure the optical density at many wavelengths within visible light range (that is why the name colorimeter ).

Light Microscope: Maximum 1000 (typical one) times magnification (10x at objective and 10x or 40x or 100x at eyepiece) can be achieved. The morphology of the cells is continuously checked for by observing samples drawn intermittently from a batch.

Centrifuge: pellets of cells can be obtained by centrifuging (1 ml of a sample) in an Eppendorf at 6000 rpm for 10 minutes. The supernatant is thrown and the pellet can be resuspended in 0.85% NaCl solution(for osmotic balance). The above procedure can be performed for many practical purposes (to determine cell dry weight, to simply wash the cells to inoculate in other medium etc.).

Utilities
Utilities are essential for a fermentation process. Demineralized water is essentially required because impurities present in the tap water may interfere in the growth of the micro-organisms and other processes. Steam is an absolute requirement since majority of the processes are carried out in aseptic conditions. Chilled and cooling water is needed to cool down the vessel after sterilization which is carried out at a temperature as high as 121 oC. Hot water is required to raise the temperature of the vessel if the temperature lowers down the incubating temperature. Compressed air is required to meet continuously the BOD of the micro-organism used in the process.

Boiler: Generates steam at 185 oC, 10 bar pressure from soft water. Soft water is used to avoid damage to the coil in which water flows and gets heated up by the flames produced by burning small droplets of diesel.

Fig. 2. Revomax Steam generator Major components: 1. Blower: blows air in the inner jacket which flows along the sides of the coil and direct the flames towards the coil. Hot gases rise up and enter the outer jacket.

2. Economizer: hot gases from outer jacket enter tubes present in the outer jacket. The heated gases exchange their heat with the water present outside the tubes in the economizer. 3. Optimizer: consists of two tubes one carrying water from the supply tank to the economizer and the other carrying the heated water from the economizer into the coils in the heater. 4. Heater: chamber in which flames are produced and water is further heated by the flames in the coils. 5. Burner: has two electrodes with a potential difference of 14000 volts creating spark which generates flame on coming into contact with small droplets of diesel being sparkled through nozzle. 6. Moisture collector: moisture present in the steam produced settles down in this chamber due to gravity. 7. Control Panel: To control the processes in the boiler such as filling water in the coil, setting flame (fire), setting temperature etc. 8. Chimney: The flue gas makes its way from the outer jacket to the atmosphere via chimney.

Notes: 1.Economizer is important in saving fuel consumption as it employs heated effluent gases in heating water to some extent. 2. The effluent gases are discharged in the atmosphere through chimneys as specified by the state laws.

Air compressor: It consists of motor-driven uniquely designed pump(s) and a pressure switch. The pump has two openings (with one way valves) of different diameters. Air is sucked in from the larger opening as piston moves backward creating suction. When the piston moves forward the extra air sucked in is forced to be expelled through the smaller opening (compressing the air due to squeezing). There is a provision to collect moisture from air and to remove them. Pressure switch is essential in that it shuts the motor when the pressure exceeds a stipulated value and switched the motor on when it falls below another stipulated value, thereby averting continuous consumption of power. The compressed air is temporarily stored in a receiver tank from where it is stored in another common tank. The inlet pipe to the latter has Pressure Reduction Valve (reduces the air pressure downstream to a fixed value).

Air dryers are present at places in the compressed air line to remove moisture.

Demineralized water plant: It has following components. 1. 2. 3. 4. 5. Sand Filter: Carbon Filter: Softener: Reverse Osmosis Filter: Demineralizer (Cation and Anion bed):

Cation and anion beds remove all the charged particles. Softener removes salts of Calcium and Magnesium. Carbon filter removes all the carbon impurities while sand filter removes coarse particles.

Chiller: It has four major components: compressor, condenser, expansion valve and evaporator. Compressor: It takes the lower-pressure vaporized refrigerant coming out of the evaporator,
compresses it to a higher pressure, and discharges it into the condenser.

Fig. 3. Schematic diagram of working of a chiller plant.

The condenser is where the refrigerant rejects heat (energy) to the condenser water or air, causing refrigerant phase change from gas to liquid.

Expansion Valve: High pressure liquid refrigerant passes through expansion valves, reducing pressure
and flashing to a gas within the evaporator, absorbing energy from the chilled water. The evaporator is where the refrigerant removes heat (energy) from the chilled water.

Water cooling tower: Cools the inlet water to room temperature. The inlet water is pumped through sprinklers. The water droplet thus formed has higher velocity than the water before being sprinkled thus losing in pressure according to Bernoulli s theorem. The air from surrounding rushes in and evaporates the surface of the water droplet thus cooling it.

Water heater:It is required to heat water to moderately high temperature electrically while water flows through coils. The desired temperature can be set and the heater is switched on only when it is required.

Effluent Treatment Plant:The effluent can t be directly discharged into sewers as it is polluted with potentially harmful aliquot of micro-organisms and chemicals. So, the effluent is treated by ETP before finally discharging it. ETP is a series of tanks which filters water at various levels. Oil tank: The effluent water is fed into oil tank first where oil and water form separate layer due to density differences. Grease Tank: The water present on the top passes to the Grease tank where grease settles down and water further flows down to equalization tank. Equalization tank: Water from grease tank and sludge bed is fed to this tank from where sludge is separated intermittently and water is pumped to tube settler tank. This tube is constantly aerated to separate sludge. Tube-settler tank: Here water is treated with Alum (10%) which settles down certain impurities along with it. The processed water is further fed to aeration tank. Aeration tank: Organic impurities are degraded microbially here. Various layers of aerobic and anaerobic micro-organisms are formed. The tube is constantly aerated by an air pump. Microorganisms are introduced by feeding cow dung into the aeration tank.

Clarifier tank: Water from the aeration tank is fed into clarifier tank.Sludge is intermittently removes from the bottom of the tank. Sludge Bed: The sludge from clarifier tank is fed to sludge bed (usually bed of sand). The water from the sludge percolates down and is finally poured into equalization tank. The water from clarifier tank is collected in another tank and further purified by sand and carbon filters (not always essential). The treated water is finally discharged into drains.