2 Fermentation Design Allan C Soderberg 1.0 INTRODUCTION Industrial scale fermentation technology tends to be a " proprietary science. The industries with submerged liquid fermentation processes as a synthetic"step for producing a commercial product generally have devel oped their own technology and have not shared developments with their competitors, academe, or the public. If major fermentation industries decided to openly discuss the criteria of their procedures and processes for their fermentation departments, they would not agree on most systems and equipment, from culture storage methods to valves, from lab culture propa gation to fermenter design, from scale-up to sterile filters, or from tank inoculation methods to continuous sterilizers. The experience ofevery author or speaker, though he may have years of practical knowledge, is probably regarded as inferior to the experience of the reader or listener. That is, the subjective analysis of the data by each company has resulted in different solutions to common problems, or each company has a customized plant suited to its procedures and products
2 Fermentation Design Allan C, Soderberg 1.0 INTRODUCTION Industrial scale fermentation technology tends to be a “proprietary science. ” The industries with submerged liquid fermentation processes as a “synthetic” step for producing a commercial product generally have developed their own technology and have not shared developments with their competitors, academe, or the public. Ifmajor fermentation industries decided to openly discuss the criteria of their procedures and processes for their fermentation departments, they would not agree on most systems and equipment, from culture storage methods to valves, from lab culture propagation to fermenter design, from scale-up to sterile filters, or from tank inoculation methods to continuous sterilizers. The experience ofevery author or speaker, though he may have years of practical knowledge, is probably regarded as inferior to the experience of the reader or listener. That is, the subjective analysis of the data by each company has resulted in different solutions to common problems, or each company has a customized plant suited to its procedures and products. 67
68 Fermentation and Biochemical Engineering Handbook 2.0 FERMENTATION DEPARTMENT, EQUIPMENT AND SPACE REQUIREMENTS 2.1 The Microbiological Laboratories Isolation of organisms for new products normally does not occur in laboratories associated with production cultures, however, production(mi crobiological) 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. Theexperience, 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 tech niques;improved culture storage methods; culture stability testing;new propagation procedures; media improvements; search for inducers, repres sors,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 datasand, soil, lyophils, spore and vegetative suspensions, slants and roux bottles, surface colonies under oil, etc. The temperature for culture storage varies from.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 itis 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 to day. Afew 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 propa- gation technique. The general principle is to have the fewest possible
68 Fermentation and Biochemical Engineering Handbook 2.0 FERMENTATION DEPARTMENT, EQUIPMENT AND SPACE REQUIREMENTS 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 ofthe 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
ermentation Design 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 labora- tories.( For a general description of various microbiological tasks performed in industry, see Peppler and Perlman. y) After the culture is grown, the flask(fitted with a hose and coupling device) is used to inoculate the seed fermenter. However, 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 Area(s). 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 statisticallydesigned shake flask experiments, serial growth experiments in Petri dishes for stability experiments and others. Equipment will be required to hydrolyze starch proteins, to pre in addition to ke homogenizers, centrifuges, sterilizers and large benches 3. Inoculation Rooms. Frequently, separate rooms are used for work with bacteria, actinomycetes, molds, and steril ity 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
Fermentation Design 69 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 Peppler 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 Area@). 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
70 Fermentation and Biochemical Engineering Handbook 4. Incubator Areas. pace is required for incubators(vari ous 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 muta- tion,culture isolation, and testing in bench top fermenters Space must be provided for microscopes, special analyti cal 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 tech laboratories will be four to eight times that required for the analytical laboratories of the fermentation department. The reason for this is cleanli ness, 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 microbio logical tasks performed. A large plant may even have a pilot plant 2.2 Analytical Support Laboratories 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-
70 Fermentation and Biochemical Engineering Handbook 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. Ofice. 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. 2.2 Analytical Support Laboratories 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 ofproduction 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-
Fermentation Design graphs, Kjeldahl equipment, titrimeter, UV/visible spectrophotometers, an atomic absorption spectrophotometer, pHmeter, 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 require nents are 450 ft 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 cars 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 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
Fermentation Design 71 graphs, Kjeldahl equipment, titrimeters, Wkisible spectrophotometers, an atomic absorption spectrophotometer, pH meters, viscosimeter, refiactometer, 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