2.1 The Microbiological Laboratories
Isolation of organisms for new products normally does not occur in laboratories associated with production cultures, however, production (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. The experience, imagination and personal skill of the individual is fundamental for success. The results of mutation work have been of great economic value to the fermentation industry, therefore, the methods used remain closely guarded and are almost never published. Other on-going studies include new culture preservation techniques; improved culture storage methods; culture stability testing; new propagation procedures; media improvements; search for inducers, repressors, inhibitors, etc. Here again, the imagination of the researcher is essential to success because specific research methods are commonly nontraditional.
The highly developed production cultures must be preserved from degradation, contamination and loss of viability. Every conceivable method is being used and supported by experimental data—sand, soil, lyophils, spore and vegetative suspensions, slants and roux bottles, surface colonies under oil, etc. The temperature for culture storage varies from -196°C (liquid nitrogen) up to +2°C and above. The containers generally are glass, but vary from tubing, to test tubes, flasks (any shape and size), roux bottles, serum bottles, etc. A good argument can be made that the only important variable is to select the correct medium to grow the organism in or on before it is stored. Obviously, carbon, nitrogen, water and minerals are required for growth, but sometimes high concentrations of salts, polyols or other chemicals are needed to prevent a high loss of viability during storage. Frequently, a natural product (oat meal, tomato juice, etc.) is helpful for stability compared to a totally synthetic medium. Under the right conditions, procedures based on vegetative growth can be more stable than ones based on spores.
Submerged fermentation procedures are used almost exclusively today. A few surface fermentation processes (on liquids or solids) are still used. Cost comparisons of labor, air compression, infection, etc., can be made, but modern batch fed, highly instrumented and computerized submerged methods predominate. Submerged methods are also the predominant culture propagation technique. The general principle is to have the fewest possible transfers from the primary culture stock to the fermenter. This is based on the assumptions that transferring and media sterilization are the main infection risks. Generally, a lyophilized or frozen culture is used to inoculate a flask of liquid medium which is then shaken until sufficient cell mass has been produced. (Some prefer solid media, in which case a sterile solution must be added to suspend the culture in order to transfer the culture to the seed tank.) The medium in the seed flask frequently contains production raw materials rather than microbiological preparations used in research laboratories . (For a general description of various microbiological tasks performed in industry, see Peppier and Perlman.)
After the culture is grown, the flask (fitted with a hose and tank coupling device) is used to inoculate the seed fermenter. However, some transfer the culture from the seed flask to a sterile metal container (in the laboratory) which has a special attachment for the seed fermenter. This technique is usually abandoned in time. Ingenuity for the minimum transfers in the simplest manner will usually give the best results.
The space requirements and the equipment necessary for designing a culture maintenance lab vary so widely, from simple laminar flow hoods to air locked sterile rooms, that only each company can specify the details. The number of rooms and work areas depend upon the number of types of cultures maintained, as well as the variety of techniques for mutation, isolation and testing. Therefore, lab space and equipment might include:
1. Glassware and Equipment Washing Area. Washing and drying equipment, benches, carts.
2. Media Preparation Areafs). Space must be provided for large raw material lots, not only for growth in flasks, but testing of cultures in very small glass fermenters, large statistically designed shake flask experiments, serial growth experiments in Petri dishes for stability experiments and others. Equipment will be required to hydrolyze starch and proteins, to process molasses, in addition to kettles, homogenizers, centrifuges, sterilizers and large benches.
3. Inoculation Rooms. Frequently, separate rooms are used for work with bacteria, actinomycetes, molds, and sterility testing. High intensity UV lighting is commonly used when the rooms are unoccupied. These rooms generally have only work benches (or hoods) for easy cleaning.
4. Incubator Areas. Space is required for incubators (various temperatures), some of which could be the walk-in type, and/or floor cabinet models. Shaker cabinets at various temperatures are also needed.
5. Office. Record keeping and administration will require one or more offices, depending upon the size of the staff.
6. Laboratories. Depending upon the size of the facility, separate laboratories could be required for culture mutation, culture isolation, and testing in bench top fermenters. Space must be provided for microscopes, special analytical equipment for DNA, ATP, Coulter counters, water baths, pH and DO instruments, laminar flow hoods, balances, lyophilization equipment, etc.
7. Other. Space must be provided for refrigerators and freezers, which are the repositories of the production culture collection. Normally, toilets, showers and a coffee break room are provided since the total work areas are "restricted" to laboratory employees only.
The square feet of floor space per technician required for these laboratories will be four to eight times that required for the analytical laboratories of the fermentation department. The reason for this is cleanliness, and the rooms have specific purposes for which they may not be used every day. The work force moves from room to room depending upon the task scheduled. Also, the total work area depends upon the variety of microbiological tasks performed. A large plant may even have a pilot plant.
The functions of these laboratories usually are sterility testing of production samples, and chemical assays of: raw materials for approval to use in the processes, blends or batches of raw materials before sterilization, scheduled samples of production batches, fermenter feeds, waste streams and miscellaneous sources. In many instances the analytical work for the culture laboratories will also be performed.
Typical laboratories have Technicon 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 chromatographs, high pressure liquid chromato-
graphs, Kjeldahl equipment, titrimeters, UV/visible spectrophotometers, an atomic absorption spectrophotometer, pH meters, viscosimeter, refractome-ter, densitometer, etc. The cell mass is usually followed for its intrinsic value as well as to calculate specific uptake rates or production rates in the fermenter. Therefore, centrifuges and various types of ovens are required for drying in addition to ashing.
Fermenter sterility testing requires a room with a laminar flow hood to prepare plates, tubes and shake flasks. Space needs to be provided for incubators and microscopes. Since it is very important to identify when infection occurs in large scale production, microscopic examination of shake flasks is usually preferred because a large sample can be used, and it gives the fastest response. Similarly, stereo microscopes are used for reading spiral streaks on agar plates before the naked eye can see colonies.
Chemical and glassware storage, dish washing, sample refrigerators, glassware dryers, autoclaves for the preparation of sterile sample bottles for the plant, computer(s) for assay calculations, water baths, fume hoods, etc., are additional basic equipment items needed. Typical overall space requirements are 450 ft2 of floor space per working chemical technician.
2.3 Production: Raw Material Storage
Raw material warehousing most often is a separate building from manufacturing. Its location should be on a rail siding (for large plants) and have easy access by twenty-ton trailers. The dimensions of the building should make it easy to stack a palletized forty-ton rail car's contents—two pallets wide and three or four pallets high, from the main aisle to the wall. In this manner, raw material lots can be easily identified and used when approved.
Large volume dry raw materials should be purchased in bulk (trucks or rail cars) and stored in silos. Pneumatic conveying from the silos to the mixing tanks can be controlled from 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, The silos and bulk liquid tanks can usually be placed close to the batching area, whereas the warehouse can be some distance away. Since large volume materials are pneumatically conveyed or pumped, the floor space of the batching area for storing miscellaneous materials can be relatively small.
The equipment needed in warehousing are fork lift trucks, floor-washing machines, etc. Special materials must be on hand to clean up spills quickly, according to federal regulations. Good housekeeping and pest control are essential.
For good housekeeping, all equipment should be on or above the floor and no pits should be used. On the other hand, grated trenches make it easy to clean the floors, and minimize the number of floor drains.
The number, shape and volume of batching tanks that different companies use show personal preference and are not very important. Usually two or three different sized tanks are used; smaller batching tanks are for inoculum tanks and the larger tanks for feed and fermenter media preparation. The type of agitation varies widely. Batching tanks, 10,000 gallons and smaller, could be specified as 304 stainless steel, dished or flat bottom and heads, H/D ratio about 0.7 to keep a working platform low, a slow speed (60 to 90 rpm) top-entering agitator with airfoil type impellers, horsepower approximately 1.25 per 1000 gallons. The tanks need to be equipped with submerged (bottom) nozzles which are supplied with both steam and air. Hot and cold water are usually piped to the top. The hatch, with a removable grate of 'A" S/S rod on 6" x 6" centers, should be as large as a 100 lb. bag of raw materials. A temperature recorder is the minimum instrumentation. The cyclone, with a rotary air lock valve to permit material additions from the bulk storage silos, is normally located above the tank(s). For tanks larger than 10,000 gallons, the bottom head should be dished, the H/D ratio made 1 to 2, and airfoil type agitators used.
The size and number of batching tanks depend upon whether the plant uses continuous sterilizers or batch sterilization. The difference is that in the latter case, the tanks can be large (50 to 80% of the size of the fermenter), and usually all the materials are mixed together. For continuous sterilizers, there is usually a minimum of four smaller tanks so that proteins, carbohydrates and salts can be batched and sterilized separately. In this case, the tanks are considerably smaller than the fermenter.
The media preparation area is also where hydrolysates of proteins, and starches, as well as special processing of steep liquor, molasses and other crude materials takes place. Very strict accuracy of weights, volumes, pH adjustments and processing instructions are the first step to reproducible fermentation results. A well-run batching area depends upon purchasing a uniform quality of raw materials, adequate equipment, detailed batching instructions and well trained, reliable personnel. Record keeping of batch quantities, lot numbers, pH, temperatures, etc. are necessary for quality and good manufacturing practices.
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 coddling the first (plant) inoculum stage to maturity. Other companies locate the seed fermenters adjacent to the fermenters. Small plants cannot afford to isolate equipment and have a specialized work force, however, large plants do isolate groups of similar equipment, and specialize the work force, which often results in higher productivity.
The operation of fermenters is basically the same regardless of size, but seed fermenters usually do not have sterile anti-foam and nutrient feeds piped to the tanks as the main fermenters have. Therefore, foaming in the seed fermenters can lead to infection, which is one of the reasons they need more attention. Careful inoculation procedures, sampling and sterilizing the transfer lines from the seed fermenter require alert personnel. Careful attention to these details is more important than the proximity of the seed and main fermenters.
The number of inoculum stages or scale-up is traditional. The rule of a tenfold volume increase per stage is followed by some companies, but is not critical. The multiplication rate of an organism is constant after the lag phase so the amount of cell mass developed to inoculate the next stage, minus the starting amount, is a matter of time, providing, of course, there is sufficient substrate and environmental conditions are reasonable. After all, the theory is that one foreign organism or spore, if not killed during sterilization, will, in time, contaminate the fermenter. Larger cell masses of inoculum can shorten the growth phase of the next larger stage. Using this concept, some companies make the inoculum volume larger than a tenth of the fermenter volume so that the number of transfers from laboratory flask to the final fermenter is minimum. This also assumes there is a higher risk of infection during transfers as well as a certain viability loss. A higher inoculum cell mass may reduce the lag time in the fermenter. This, combined with using continuous sterilization for a short "turn around" time of the fermenter, can increase productivity for little or no cost.
For simplicity of piping, especially the utility piping, the fermenters are usually placed in a straight line, sometimes two or more parallel lines. In this manner the plant is easily expanded, and other tank layouts do not seem as convincing. It is desirable to have the working platform extend completely around the circumference of the top dish, and to have enough room between tanks for maintenance carts (1 to 1.5 meters). Good lighting and ventilation on the working platform should not be overlooked. Using water from hoses for cleaning is common so care must be taken to have nonskid floors with adequate drains, especially at the top of stairs. Open floor grating is not desirable. All structural steel should be well primed to prevent corrosion from the very humid atmosphere. Electronic instrumentation and computers must be placed in control rooms which run at constant (HVAC) temperature. Most fermenter buildings are between 40 and 100 feet high, making it possible to have one or more floors between the ground floor and the main fermenter working platform. The intermediate floors can be used for the utility and process piping, sterile air filters, the sterile anti-foam system, instrumentation sensors (temperature, pH, DO, etc.), heat exchangers, motor control center, laboratories and offices. Buildings 40 feet or more high frequently have elevators installed.
Fermenters can be located outdoors in most countries of the world. The working platforms usually are enclosed and heated in temperate zones, and only shaded in more tropical zones. In more populated areas, open fermenter buildings make too much noise for local residents. The environmental awareness, or the tolerance of the public, could preclude open fermenter buildings in the future. Odor is also offensive to the public. The environmental authorities are demanding that equipment be installed to eliminate the offensive odor of the off-gases. (Noise levels inside a fermenter building will be greater than 90 dBA if no preventive measures are taken.)
Harvest tanks can be justified as the responsibility of the fermentation or recovery department. They are economical (carbon or stainless steel) with a shape described by (H/D si) and should be insulated and equipped with cooling coils and agitator(s).
Essential equipment to a productive fermentation department are sterilizable tanks for nutrient feeds. Multiproduct plants usually require several different sizes of feed tanks: (i) a small volume to be transferred once every 12 or 24 hours such as a nitrogen source; (ii) a large volume carbohydrate solution fed continuously, perhaps varying with the fermenter volume; (in') a precursor feed, fed in small amounts relative to assay data; (iv) anti-foam (Some companies prefer a separate anti-foam feed system for each fermenter. A continuously sterilizing system for anti-foam is discussed below which is capable of servicing all the fermenters.); (v) other tanks for acids, bases, salts, etc. Many companies prefer to batch sterilize a known quantity and transfer the entire contents quickly. Sometimes, the feeds require programming the addition rate to achieve high productivity. In this latter case, large volume tanks are used and the contents are presterilized (batch or continuous) or the feed is continuously sterilized between the feed tank and the fermenter. Usually feed tanks are not designed as fermenters, even though they are sterilizable, and there is no need for high volume air flow, but only sufficient air pressure for the transfer. For solvable nutrients the agitator and anti-foam system are not required. Since the air requirements are needed only to transfer the feed, the air piping design is different and the sterile air filter is proportionately smaller. Instrumentation is usually limited to temperature, pressure and volume. The H/D ratio of the vessel can be near one for economy and need not be designed for the aeration/agitation requirements of a fermenter.
Sterile air filtration is simple today with the commercial units readily available. However, some companies still design their own (see Aiba, Humphrey and Millis) to use a variety of filter media such as carbon, cotton, glass staple, etc. (For recent papers about industrial applications of cartridge filters, see Bruno'3' and PerkowskiJ4')
The essential method to obtain sterile air, whether packed-bed or cartridge filters are used, is to reduce the humidity of the air after compression so that the filter material always remains dry. The unsterilized compressed air must never reach 100% relative humidity. Larger plants install instrumentation with alarms set at about 85% relative humidity. Careful selection of the cartridge design or the design of packed-bed filters will result in units that can operate in excess of three years without replacement of filter media. If a fiber material is used in a packed-bed type filter, the finer the fiber diameter the shallower the bed depth needs to be for efficient filtration. Other filter media are less common and tend to have special problems and/or shorter life. The bed depth of filters is only 10 to 18 inches for fibers of less than 10 microns. These filters run "clean" for 2 weeks or longer before being resterilized.
Some plants have a separate filter for each sterile vessel. Others place filters in a central group which feeds all the vessels. In this case, one filter, for example, might be taken out of service each day, sterilized and put back into service. If there were ten filters in the group, each one would be sterilized every tenth day. This system has the advantage that the filter can be blown dry after sterilization with sterile air before it is put into service again.
Figure 1. Domnick-Hunter sterile air filter.
It is ideal to have oil-free compressed air. Centrifugal machines generally are available up to 40,000 cfm. "Oil free" screw air compressors are available in smaller sizes. Regarding oil-free screw type compressors, it is necessary to read the fine print of the manufacturer. For example, one manufacturer uses no lubricant on the screws and another claims to be oil free, but does use a non-hydrocarbon liquid lubricant. Carbon ring reciprocating compressors are available and used, but maintenance is annoying.
For small plants, non-lubricated screw compressors with two-speed motors and constant pressure control will provide versatility. For large plants, centrifugal air compressors, driven by non-condensing steam turbines with 50 psig steam extraction for process requirements, are suitable. In all cases, extra considerations include locating the intake 20 feet or more above the ground level and installing filters on the intake to the compressors to prevent dirt accumulation on the sterile filters. Occasionally, the noise levels measured at the suction inlet exceed OSHA regulations and bother the neighbors of the plant. The air from the compressors requires heat exchangers to lower the air temperature below the dew point, plus additional heat exchangers to reheat and control the air to have the relative humidity at about 85%.
2.10 Valves (To Maintain Sterility)
Most companies have tried gate, diaphragm, ball, and plug valves, to name a few. Some have designed and patented special valves for the bottom or sample positions. Some companies will disassemble all fermenter valves after an infected run. No companies use threaded nipples or valves on a fermenter because the threads are a site of potential infection. In general, valves are less of a sterility problem when a continuous sterilizer is used for the substrate than fermenters which batch sterilize the substrate. This is because, in the former case, the vessel is sterilized empty, and all valves are opened and sterilized in an outward direction so that a steam plume can be seen. The temperature of the valves during sterilization can be checked with a Tempilstik™. Batch sterilizing requires all valves below the liquid level to be sterilized with steam passing through the valve into the substrate. This depends upon steam pressure and how much the valve is opened (which might affect the PIT conditions of sterilization). This is much more subject to human error and infection. Most plants drill and tap the body of the valve near the valve seat in order to drain the condensate away from all sections of pipe where a steam seal is required for sterility. In general, diaphragm and ball valves require considerable maintenance, but tend to be popular in batch sterilizing operations, while plug type valves are more typical on fermenters where continuous sterilizers are used. 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. Utility piping remains carbon steel up to the first S/S valve on the fermenter. Valves used in non-process piping are selected for the best type of service and/or control. Butterfly valves have been used in applications where perfect closure is not essential, such as a vent valve.
In summary, the valves which maintain a sterile environment on one side and a non-sterile environment on the other side are the essential valves. They must be devoid of pockets, easily sterilized, maintained, and occasionally replaced.
Apart from continuous sterilizers, pumps are a minor concern in the fermentation department. A simple way to transfer inoculum from a large laboratory flask to a seed fermenter, without removing the back pressure on the vessel, is to use a peristaltic pump. Connect the sterile adapter (which is attached to the flask) to the seed fermenter by sterile technique. Install the gum rubber tubing in the pump, open the hose clamp and start the pump.
Inoculum from seed fermenters and sterile feeds are transferred to the fermenter by air pressure. Centrifugal pumps (316 S/S) are used to pump non-sterile raw materials, slurries, harvested broth, etc. The centrifugal pumps and piping should be cleaned immediately after a transfer has been completed. Occasionally a specialty pump may be required.
Cooling is required to cool media from sterilizing temperatures, to remove the exothermic heat of fermentation, to cool broth before harvesting, and to cool the compressed air. Some portion of the heat can be reclaimed to produce hot water for the preparation of new substrate, and for general cleaning of equipment, platforms and floors, however, the excess heat must be disposed to the environment. Cooling water is provided from cooling towers, but chilled water (5°-15°C) is produced by steam vacuum, or refrigeration units.
In any case, the fermentation department should always be concerned about its cooling water supply, i.e., the temperature and chloride content. Chloride ions above 150 ppm when stainless steel is above 80°C (while sterilizing) will cause stress corrosion cracking of stainless steel. A conductivity probe should be in the cooling water line. When the dissolved solids (salts) get too high, it may indicate a process leak, or that the salt level is too high and some water must be discharged and fresh water added. If cooling water is discharged to a stream, river, etc., an NPDES permit may be needed and special monitoring required. The chloride content should be determined analytically every two weeks to control the chloride to less than 100 ppm. This is done by draining water from the cooling tower and adding fresh water.
Stack odors have to be avoided. 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. Due to the high volume of air discharged from a large fermenter house, odor is neither easy nor cheap to eliminate. Carbon adsorption is impractical. Normally, more air is exhausted than required for steam production from the boilers which eliminates that route of disposal. Wet scrubbing towers with sodium hypochlorite are expensive ($1.50/yr. cfm), and discharge Na+ and Cl2 to the waste system which may preclude this method. Ozone treatment can be effective. A very tall exhaust stack for dilution of the off gas with the atmosphere before the odor reaches the ground is possible in some cases, but is not considered an acceptable solution by U. S. Authorities.
The fermentation department should monitor and control the COD/ BOD of its liquid waste to the sewer. Procedures for cleaning up spills and reporting should be Standard Operating Procedure. A primary aeration basin will reduce the COD to 80-90 ppm. Secondary aeration lagoons will reduce the BOD to acceptible levels which have no odor.
Noise levels are very difficult to reduce to Federal standards. Hearing protection for employees is essential. The move towards greater automation has resulted in operators having less exposure to noisy work areas.
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