INDUSTRIAL MICROBIOLOGY Industrial microbiology deals with all forms of microbiology that have an economic aspect. It deals with those areas of microbiology on which, in some manner, a monetary value can be placed, regardless of whether the. microbiology. involves a fermentation product or some form of deterioration, disease or waste disposal Industrial microbiology is an important branch of microbiology dealing with those areas of microbiology involving economic aspects, where valuable products are prepared from cheaper and often disposable substrates. OR Industrial microbiology may be defined as the study of the large scale and profit motivated production of microorganisms or their products for direct use or as inputs in the manufacture of other goods. Hence it has become possible for the industrial microbiologist to compare with the industrial chemist. eg. fermentative production costs of all antibiotics, except one or two are appreciably less than the synthetic production costs of the same. « oc" \), ‘Scope of Industrial Microbiology: Industrial microbiology is a very broad area for study. In fact, many nonindustrial areas of 0 microbiology are important to industrial microbiology and should be taken into consideration in wr understans Se ee pee agricultural microbiology, medical microbiology, microbial physiology, cytology and morphology, Seas penal ae a gy ood Ses aby eae acy Eee teed i enact oa industal oe ache SERRE oe Garon inl gg eee a engineering, medicine, economics, sales and law, particularly patent law and labor law, governmental Pactra agee care are pemeyem i Sanee A aa industrial microbiology, as a consideration of space and marine exploration. eee ein ae en microbiology, under the proper conditions easily can become a matter for consideration eg. An Dee ar eee eae ta eae aati Scone at tran EE tee Seine Sp aetnaary ace saucer alge iota! Pe ee ea aaiiee ak Oe Get Gia ler eae asa gy at onsg tines! aaa getEapOO gy ted soci etapa fields are directly or indirectly involved in the study of industrial microbiology. eee ee os aaetateal eins wa sieoasoe form the cultural responsibilities; the remaining bioparameters of the fermentation process are to be controlled by the biochemical engineer. Thus economization of a fermentation process requires both a Fourie | oar ere i (61) remavron: DESIGN AND ROLE OF DIFFERENT PARTS OF FERMENTOR 5Y'°%lin fermentation industries, microbes are to be grown in specially designed vessels loaded with Particular type of nutritive media, These vessels are referred to as Fermentor or Bioreactors Bioreactors or fermentors are complicated in design, because they must provide for the control ancl observation of many facts of microbial growth and biosynthesis. The design of fermentor depends upon the purpose for which itis to be utilized. Industrial fermentors are designed to provide the best possible growth and biosynthesis conditions for industrially important microorganisms and allows ease of manipulation for all operations associated with the use of the fermentors. The fermentor used for a particular process should possess following characters: Characteristics of and Ideal Fermentor or bioreactor: ‘There cannot be a fermentor ideal for all most all fermentation processes, but if there is then it should following characteristics: 1. Material used in the fabrication of fermentor should be strong enough to withstand the interior pressure due to the fermentation media, it should be resistant to corrosion and free form any toxic effect for the microbial culture and the product formed by the microbial culture. 2. A fermentor should permit easy control of contaminating microbes. PENSION POINT 9014171040310. u, 2 13. 14. It should be provided with the inoculation point for aseptic transfer of inoculum. Should be equipped with the aerating device (Spargers). Should be equipped with a stirring device for uniform distribution of air, nutrients and microbes (Impellers). . ‘There should be provision of baffles to avoid vortex formation. Fermentor should be provided with a sampling valve for aseptic withdrawing of sample for different laboratory tests, Fermentor should possess a device for controlling temperature (Temperature sensor and ‘water jacket internally fitted with heating coil) Fermentor should be provided with pH controlling device for monitoring and maintaining, pH of media during, fermentation process (pE probe and Acid base reservior) Should be provided with a facility for intermittent addition of antifoam agents for controlling foam formation (Reservior of sterile Antifoming agents or mechanical foam breakers) There should be provision fro feeding certain media components during the progress of fermentation (Precursors) ‘A drain at the bottom is essential for the removal of the completed fermentation broth for further processing A man hole should be provided at the top of fermentor for acess inside the fermentor for different purposes like repairing and thorough cleaning of fermentor between runs. A exit valve should be provided at the top for the exit of metabolic gases produced during fermentation processes, ‘TYPES OF FERMENTORS: Batch Fermentors are used to carry out microbiological processes ‘on batch basis. They are available with varying capacities. ‘The capacity of the fermentor may range form a few hundred to several thousand gallons. The capacity of the fermentor is usrally stated on the basis of the total volume capacity of the same. Thus, based on total volume capacity the fermentors are of following types: 4) Small Laboratory fermentors (i) Pilot plant fermentors (ii) Large industrial fermentors (iv) Horton spheres. + The small laboratory fermentor ranges from 1-2 rs with a maximum upto 12-15 liters, ‘+ Pilot plant fermentors have a total volume of 25 ~100 gallons upto 2000 gallons total volume, Larger fermentors range form 5,000 or 10,000 gallons total volume to approximately 1,00,000 gallons, Horton spheres are rarely employed with a size range of 2,50,000 to 5,00,000 gallons total capacity. Actually the working volume in a fermentor is always less than that of the total volume. In other words, a ‘head space’ is left at the top of the fermentor above the level of fermentation media. The PENSION-POINT 9011710403reason for keeping a head space is to allow aeration, splashing, and foaming of the aqueous medium, This head space usually occupies a fifth to a quarter or more of the volume of the fermentor. pl Control: pil control is achieved by acid or alkali addition, which is controlled by an auto-titratér. The autotitrator in turn is connected to a pH probe. ‘Temperature control: ‘Temperature contr ie achieved. by a woter joctet atu! the vessel, This is often supplemented by the use of internal coils, in order to provide sufficient heat-transfer surface. Agitation: ‘The agitating device consists of a strong, and straight shaft to which impellors are fitted. An impeller, in turn consists of a circular disc to which blades are fitted with bolts. Different types of blades are available and are used according-to the requirements. The shaft passes through a bearing in the lid of the fermentation tank. It is rotated with the help of an electric motor mounted externally at the top of the tank. The liquid medium is thrown up towards the walls of the fermentor while rotating the impeller blades at a high speed. This results in the formation of a vortex, which is climinated, usually by four equally spaced baffles attached to the walls of the fermentor. Aeration: Usually, the aerating device consists of a pipe with minute holes, through which pressurized air escapes into the aqueous medium in the form of tiny air bubbles. This aeration device is called a “SPARGER”. The size of the holes in a sparger ranges from 1/64 to 1/32 of an inch or larger. Holes smaller than this requires too high air pressure for economical bubble formation. One should always remember that the smaller the air bubbles, the greater is the bubble surface area. It is desirable to adjust the size of the air bubbles to give the greatest possible aeration without greatly increasing the ‘overall cost of the fermentation process, The reason for this is that sterile air is a costly item for large- scale fermentation. Foam Control: ‘Aeration and agitation of a liquid medium can cause the production of foam. This is particularly true for the media containing high levels of proteins or peptides. If the foam is not controlled, it will rise in the head space of the tank and be forced from the tank along with the exit valve. This condition often causes contamination of the fermentation from organisms picked up by breaking of some of the foam which then drains back into the tank, Excessive foaming also causes other problems for fermentation. ‘The usual procedure for controlling foam is to add an antifoaming agent, although a supplementary impeller blade mounted high in the tank may at times be effective. An antifoam agent Jowers surface tension and in the process decreases the stability of the foam bubbles so that they burst. The antifoam may be added at media makeup or may be added after sterilization or as called for during the fermentation process ‘There are two types of antifoam agents: () Antifoam agents (inert) (ii) defoamers, eg. Animal and vegetable oils , lard oil, corn and soubean oil, long chain alcohols such as ‘octadecanol. In addition mixtures of oils and alcohols are effective in controlling foam. Silicone ‘compounds are ideal inert antifoam agents but are too expensive. SCREENING ‘The most important factor for the success of any fermentation industry is of a production strain. Itis highly desirable to use a production strain possessing the following four characteristics: 1. Itshould be high-yielding strain. 2. Itshould have stable biochemical characteristics. 3. Itshould not produce undesirable substances. 4. Ttshould be easily cultivated on large-scale. Detection and isolation of high-yielding species form the natural sources material, such as soil, containing a heteerogenous microbial population is called screening. PENSIOQN-POINT 9041710403OR Screening may be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from among a large microbial population. ‘+ Thus to be effective, screening must in one or a few steps allow the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the smal percentage of useful microorganisms that are present in the population. + The concept of screening will be illustrated by citing specific examples of screening procedures that are or have been commonly employed in industrial research programs. + During screening programs except crowded plate technique a natural source such as soil is diluted to provide a cell concentration such that aliquots spread, sprayed or applied in some manner to the surface of the agar plates will yield well isolated colonies (30-300). Primary screening of Organic acid amine producer:~ For primary screening of organic acid or organic amine producers, soil sample is taken as a source of microorganism. It is diluted serially to an extent to get well-isolated colonies on the plate when spread or applied in any form. After preparation of dilution these dilutions are applied on a ‘media incorporated with a pH indicating dye such as neutral red or bromothymol blue, into a poorly buffered agar nutrient medium. The production of these compounds is indicated by a change in the color of the indicating dye in the close vicinity of the colony to a color representing an acidic or alkaline reaction ‘The usefulness of this procedure is increased if media of greater butfer capacity are utilized 50 that only those microorganisms that produce considerable quantities of the acid or amine can induce changes in the color of the dye. An alternative procedure for detecting organic acid production involves the incorporation of calcium carbonate in the medium so that organic acid production is indicated by a cleared zone of dissolved caleium carbonate around the colony. These procedures are not foolproof, however, since inorganic acids or bases also are potential products of microbial growth. For instance, if the nitrogen source of the medium is the nitrogen of ammonium sulfate the organism may utilize the ammonium ion, leaving behind the sulfate ion as sulfuric acid, a condition indistinguishable form organic acid production, Thns culttres yielding positive reactions require further testing to be sure that an organic acid or base actually has been produced. Primary screening of antibiotic producer (Crowded plate technique): ‘The crowded plate technique is the simplest screening technique employed in detecting and. isolating antibiotic producers. It consists of preparing a series of dilution of the source material for the antibiotic producing microorganisms, followed by spreading the dilution on the agar plates. The agar plates having 300- 400 or more colonies per plate are considered since they are helpful in locating the colonies proclucing antibiotic activity. The of a colony to exhibit antibiotic activity is indicated by the presence of a zone of growth inhibition surrounding the colony. Such a colony is subcultured to a similar medium and purified. It is necessary to carry on further testing to confirm the antibiotic activity associated with a microorganism since zone of inhibition surrounding the colony may sometimes be due to other causes, Notable among these are a marked change in the pH value of the medium resulting from the metabolism of the colony, or rapid utilization of critical nutrients in the immediate vicinity of the colony. ‘Thus, further testing again is required to prove that the inhibitory activity associated with a microorganism can really be attributed to the presence of an antibiotic. The crowded plate technique has limited application, since usually we are interested in finding a microorganism producing antibiotic activity against specific microorgnism and not against the unknown microorganism that were by chance on the plate in the inity of an antibiotic producing organism. Antibiotic -PENSION.POINT 9011710403screening is improved, therefore by the imcorporation into the procedure of a “Test organism’ that is an organism used as an indicator for the presence of specific antibiotic activity. Dilutions of soil or of other microbial sources are applied to the surface of agar plates so that well isolated colonies will develop. The plates are incubated until the colonies are a few millimeters in diameter and so that antibiotic production will have occurred for those organisms having this potential. A suspension of test organism is then sprayed or applied in some manner to the surface of the agar and the plates are further incubated to allow growth of the test organism. Antibiotic activity is indicated by zones of inhibited growth of the organism around antibiotic producing colonies. In addition a rough approximation of the relative amount of antibiotic produced by barious colonies can be gained by ‘measuring in mm the diameters of the zones of inhibited test organism growth. Antibiotic producing, colonies again must be isolated and purified before further testing. Primary screening of growth factor (Amino acid Vit) producer (Auxanography): This technique is largely employed for detecting microorganisms able to produce growth factors (eg. Amino acid and Vitamins) extracellularly. The two major steps are as follows: Step 1 ‘+ A filter paper strip is kept across the bottom of a petri dish in such a way that the two ends pass over the edge of the dish. ‘+ A filter paper disc of petri dish size is placed over paper strip on the bottom of the plate. ‘+ The nutrient agar is poured on the paper dlisc in the dish and allowed to solidify. + Microbial source material such as soil, is subjected to-dilution such that aliquots on plating, ‘will produce well isolated colonies, ‘+ Plating of aliquots of properly diluted soil sample is done. * A minimal medium lacking the growth factor under consideration is seeded with the test organism, + The seeded medium is poured on the surface of a fresh petri dish and allowed to solidify. The agar in the first plate as prepared in step- I is carefully and aseptically lifted out with the help of tweezers and a spatula and placed without inverting on the surface of the second plate as prepared in the second step. ‘The growth factor(s) produced by colonies present on the surface of the first layer of agar can diffuse into the lower layer of agar containing the test organism, The zones of stimulated growth of the test organism around the colonies is an indication that they produce growth factor(s) extracellularly. Productive colonies are sub cultured and are further tested. OR A similar screening approach can be used to find microorganisms capable of synthesizing ‘extracellular vitamins, amino acids or other metabolites. However, the medium at makeup must be totally lacking in the metabolite under consideration. Again the microbial source is diluted and plated to provide well-isolated colonies and the test organism is applied to the plates before further incubation. The choice of the particular test organism to be used is critical. It must possess a definite ‘growth requirement for the particular metabolite and for that metabolite only, so that production of this compound will be indicated by zones of growth or at least increased growth of the test organism. adjacent to colonies that have produced the metabolite. Enrichment culture technique: This technique was designed by a soul microbiologist, Beijerinck, to isolate the desired microorganisms form a heterogeneous microbial population present in soil. Either medium or incubation conditions are adjusted so as to favors the growth of the desired microorganism. On the other hand, unwanted microbes are eliminated or develop poorly since they do not find suitable growth conditions in the newly created environment. Today this technique has become a valuable tool in many screening programs mean for isolating industrially important strains.Secondary screening: Secondary screening, is strictly essential in any systematic screening, programme intended to isolate industrially useful microorganisms, since primary screening merely allows the detection and isolation of microbes that possess potentially interesting industrial applications. Moreover, primary screening does not provide much information needed in setting up a new fermentation process. Secondary screening helps in detecting really useful microorganisms in fermentation processes. This ‘can be realized by a careful understanding of the following points associated with secondary 1. “ts very useful in sorting our microorganisms that have real Son Sauna on Esany isolates obtained during primary screening. At the same time, microbes that have poor applicability in a fermentation process are discarded. It is advisable to discard poor cultures {as soon as possible since such studies involve much labour and high expense. 2. It proviles information whether the product produced by a microorganism is a new one or not. This may be accomplished by paper, thin layer or other chromatographic techniques. 3. Itgives an idea about the economic position of the fermentation process involving the use of a newly discovered culture, Thus one may have a comparative study of this process with processes that are already known, so far as the economic status picture is concerned, 4. It helps in providing information regarding the product yield potentials of different isolates. ‘Thus this is useful in selecting efficient cultures for the fermentation processes. 5, It determines the optimum conditions for growth or accumulation of a product associated with a particular culture, 6. It provites information pertaining to the effect of different components of a medium. This is valuable in designing the medium that may be attractive so far as economic consideration is concerned 7. It detects gross genetic instability in microbial cultures. This type of information is very important, since microorganisms tending to undergo mutation or alteration is some way may lose their capability for maximum accumulation of the fermentation products. 8. It gives information about the number of products produced in a single fermentation. Additional major or minor products are of distinct value, since their recovery and sale as by- products can markedly improve the economic status of the prime fermentation. 8; Information about the solubility of the product in various organic solvents is made available. (useful in product recovery operation and purification) 10, Chemical, physical and biological properties of a product are also determined during secondary screening. Moreover, it reveals whether a product produced in the culture broth ‘occurs in more than one chemical form. 11, Itreveals whether the culture is homofermentative or heterofermentative. 12. Determination of the structure of product is done. The product may have a simple, complex or even a macromolecular structure. 13. With certain types of products (e.g. antibiotics) determination of the toxicity for animals, plants or man are made if they are to be used for therapeutic purpose. 14, It reveals whether microorganisms are capable of chemical change or of even destroying their own fermentation products. E.g. microorganism that produce the adaptive enzyme, decarboxylase can remove carbon dioxide from amino acid, leaving behind an organic 15, It tells us something about the chemical stability of the fermentation product. PIS ovens ev my sn, et arise during final sorting out of industrially useful microorganisms. a z ‘This is accomplished by performing experiments on agar plates, in flasks or small bioreactors containing, liquid media, or a combination of these approaches, A specific example of antibiotic producing Streptomyces species may be taken for an understanding of the sequence of events during a screening programme. ‘Those streptomycetes able to produce antibiotics are detected | and isolated in a primary screening programme. These streptomycetes exhibiting antimicrobial activity are subjected to aninitial secondary screening where their inhibition spectra are determined. A simple “Giant - Colony technique” is used to do this. Each of the streptomycal isolates is streaked in a narrow band across the centers of the nutritious agar plates. ‘Then, these plates are incubated until growth of a streptomycete occurs. Now, the test organisms are streaked from the edges of the plates upto bur not touching the streptomycete growth, Again, the plates are incubated, At the end of incubation, growth inhibitory zones for each test organism are measured in millimeters. Thus, the microbial inhibition spectrum study extensively helps in discarding poor cultures. Ultimately, streptomycete isolates that have exhibited interesting microbial inhibition spectra need further testing. With streptomycetes suspected to produce antibiotics with poor solubility in water, the initial secondary screening is done in some different way. Further screening is carried our employing liquid! media in flask, since such studies give more information than that which can be obtained on agar media. At the same time, itis advisable to use accurate assay technique (e-g. paper disc agar diffusion assay) to exactly determine the amounts of antibiotic present in samples of cutture fluids. Thus , each of the streptomycete isolates is stuctied by using several different liquid media in Erlenmeyer flasks provided with baffles, These streptomycete cultures are inoculated into sterilized liquid media. Then , such seeded flasks are incubated at a constant temperature. Usually such cultures are incubated at near room temperature. Moreover, stich flasks are aerated by keeping them on mechanical shaker, since the growth of streptomycetes and production of antibiotics occur better in aerated flasks than in stationary ones. Samples are Withdrawn at regular intervals under aseptic conditions and are tested in a quality control laboratory, Important tests to be carried out include: i, Checking for contamination, ii, Checking of pH iii, Estimation of critical nutrients iv. Assaying of the antibiotic, and v. Other determinations, if necessary ‘The result of the above tests points out which medium is the best for antibiotic formation and at Which stage the antibiotic yields are greatest during the growth of the culture on the various media. After performing all necessary routine tests in the screening of an actually useful Streptomycete for the fermentation process, other additional determinations are macle. They are: i. Screening of fermentation media through the exploitation of which the highest antibiotic yields may be obtained. ii, Determination of whether the antibiotic is new. iii, Determination of the number of antibiotics accumulated in the culture broth is made. iv. _ Effect of different bioparameters on the growth of streptomycete culture, fermentation process and accumulation of antibiotic. v. Solubility picture of antibiotic in various organic solvents. Also, it is to be determined. whether antibiotic is adsorbed by adsorbent materials. vi, Toxicity tests are conducted on mice or other laboratory animals. An antibiotic is also tested for the adverse effects if any, on man, animal or plant. vii, The streptomycete culture is characterized and is classified upto species. viii, Further studies are made on a selected individual streptomycete culture. For example mutation and other genetic studies For strain improvement are carried out. In conclusion, tests are designed and conducted in such a way that production streptomycete strains may be obtained with least expenses, Similar screening and analytical techniques could be ‘employed for the isolation of microbial isolates important in the production of other industrial chemical substances. STOCK CULTURE AND ITS MAINTENANCE (5 ) INTRODUCTION: infty 202% All practicing microbiologists have felt the need to preserve the viability of microorganisms with which they work. In addition, all the cultural characteristics of a culture, as they were at the time of preservation, must be conserved. ‘The nature of work being done will determine whether the ipreservation requirement is only very short-term or for an unlimited time period. Long-term preservation of a culture is required if a culture is to be deposited in one of the service culture collections with a view to preserving something of scientific value “for perpetuity”. Many methods of preservation for microorganisms have been developed. Here, itis to be noted that there exist different types of microorganisms (bacteria, viruses, algae, protozoa, yeasts and mould). Therefore, there are two criteria for selecting a method of preservation for a given culture, They are: he period of preservation desired, and ie nature of a culture to be preserved. Definition: - Stock cultures are those cultures of microorganisms that are stored or maintained for future use in such a fashion that their growth and productive capacities remains unaltered, There are two types of stock cultures: (i) working stocks and (ii) Primary stocks. + ‘The working stock cultures are those which are used frequently and they must be maintained in a vigorous and uncontaminated condition. These cultures are maintained as agar slants, agar stabs, spore preparations or broth cultures and they are held under refrigeration. They must be checked constantly for possible changes in growth characteristics, nutrition, productive capacity and contamination + Primary stocks are cultures that are held in reserve for practical or new fermentations, for ‘comparative purposes, for biological assays or for possible later screening programs. These cultures are not maintained in a state of high physiological activity and they are delved into only rarely. Transfers from these cultures are made only when a new working-stock is required, or when the primary stock culture must be subcultured to avoid death of the cells. ‘Thus, primary stock cultures are stored in such a manner as to require the least possible numbers of transfers over a period of time. Death of a high percent of cells in a primary stock culture is not particularly serious, if viable cells can still be recovered for subculture to fresh medium. Primary stock cultures stored at room temperature are maintained in sterile soil or in agar or broth that is provided with an overlay of sterile mineral oil. Agar and broth cultures without mineral il also are refrigerated and cultures in milk or agar are maintained frozen at low temperatures..Finally, primary stock cultures are lyophilized or freeze-clried and stored at low temperatures, Often mote than one of this procedure is employed to insure against loss of the cultures 6r changes in the cells ‘There are three hasic aims in maintaining and preserving the microorganisms, They are: - i, Tokeep culture alive Uncontaminated and As healthy as possible, both physically and physiologically, preserving their original properties until they are deposited in any major collections. iv. To have adequate stocks and appropriate sustems for replenishing stocks when necessary. Serial subeulture:- ‘This is the simplest and most common method of maintaining microbial cultures. Microbes are grown on agar slants and are transferred to fresh media before they exhaust all the nutrients or dry out. An exception to this is aerobic Streptomyces spp. Where drying up of the medium has been found successful, provided the initial growth showed the production of serial hyphae. The drying of ‘medium appeared to encourage good sporulation and the preserved specimen became simply a dried out strand of agar coated with spores, which remained viable for a few years at room temperature. For some microbial cultures no other methods have been found satisfactory, but for the majority of species other methods are available. ‘The maintenance of refrigerated stock cultures on agar or in broth is the least desirable of these procedures. Although the cultures may survive six months or more under refrigeration, usually they are transferred more frequently. These frequent culture transfers and the many cell generations accompanying these transfers allow the possible occurrence of and selection for undesired genetic changes in the organisms. Also the potential for contamination is markedly increased with frequent transfer of the cultures. Certain microorganisms, such as Blakeslea trispora used in P-carotene production, cannot be stored at refrigeration temperatures, because they die out relatively quickly atthese temperatures. However, such cultures can be held as agar slants at room temperature with transfers being made to fresh medium when the culture have become nearly dried out. ‘Overlaying cultures with mineral oj . ‘Agar slant and stab cultures of many microorganisms will survive several years at room temperature if the growth is submerged under sterile mineral oil. The oil overlay provides dissolved ‘oxygen, prevents drying of the agar and apparently decreases the metabolic activity of the cells to an almost negligible rate. However, genetic changes do occur in cultures stored in this manner. ‘The steps involved in this method are: + Inoculation of agar saint/stab with the culture to be maintained. + Inoculated agar slant/ stab is subjected to incubation until good growth appears. + Using sterile technique, a healthy agar slant/-tab culture is covered with sterile mineral oil to a depth of about 1 cm above the top of the agar slant. If a short slant is used, less oil is required. + Finally, oiled cultures can be stored at room temperature. But better viability is obtained when stored at lower temperatures (15 °C). Note: The depth of oil of 1 cm is fairly critical, as the oxygen transmission by layers of mineral oil in ‘excess of 11cm becomes less favorable. If less oil is used, strands of mycelium may be exposed which allows the cultures to dry out: Ifthe bottles or screw capped tubes are used, the rubber liners should. bbe removed form the caps as the oil tends to dissolve the rubber and this can be toxic to the culture. ‘This method has following advantages: i. Practically all bacterial species or strains tested live longer under oul than in the control tubes without oil. Some bacterial species have been preserved satisfactorily for 15-20 years. ‘Transplants may be prepared when desired without affecting the preservatio of the stock cultures. iii, ‘The method is especially advantageous when working with unstable variants where ‘occasional transfers to fresh media or growth in mass cultures results in changes in the developmental stages of the strains iv, This method also appears to be an ideal method of storage for a busy laboratory with limited funds and a relatively small collection Soil culture (Soil stock): Sterile soil has found wide use for the stock culture maintenance of microorganisms that form spores. This method is particularly applied for the preservation of sporing microbes specially fungi In fact, microorganisms that do not form spores also will survive in sterile soil, bur they may die out unexpectedly after a period of time. Soil stocks are prepared as follows iA mixture of soil (20 %), sand (78%) and calcium carbonate (2%) is prepared and distributed into tubes (a few grams per tube). They are sterilezed for 8-15 hours at 130°C and then cooled. fi, _A.small volume of thick suspension of spores or of an actively growing culture is then added to the sterile soil and incubated till good growth. fii, ‘The inoculated tubes are kept in desiccators under vacuum. The reason behind this is to evaporate the excess water. Then the tubes are sealed. iv. Soil stocks, thus prepared are stored at room temperatures with cotton plugs or screw caps protected from dust. These cultures can be stored in refrigerators at about 5-8 °C ‘temperature. Lyophilization or freeze-drying: Lyophilization is the most satisfactory method of long-term preservation of microorganisms. It is universally used for the preservation of bacteria, viruses, fungi, sera, toxins, enzymes and other biological materials. Lyophilization is the most popular form of suspended metabolism. It consists of drying of cultures or a spore suspension from the frozen state under reduced pressure. This can be accomplished in several ‘ways. The major steps involve in this techniques are:i. A thick cell or spore suspension is prepared in a suitable protective medium (10% skimmed ilk or bovine serum, 5% inositol in distilled! water). ii, Using sterile techniques, this thick suspension is clistributed in small quantities into glass ampoules. 3 iii, ‘These ampoules are subjected to deep-freezing by keeping, the cultures at lower temperature (200 iv. Then the chilled ampoules are connected with a high vacuum system usually incorporating a desiccant (e.g. phosphorous pentoxide, silica gel or a freezing trap), and immersed into a freezing mixture of dry ice and alcohol (-78°C). vy. The vacuum pump is tumed on and the ampoules are evacuated till drying is complete, vi. Freeze dried ampoules are then immediately sealed off and stored under refrigeration. If properly prepared and stored, most lyophilized cultures will remain viable for long periods (> 20 yrs.), without the occurrence of genetic changes. When needed, the cultures are recovered from the ampoules by suspending, the lyophilized cells in a minimal amount of growth medium and then incubating seat Vacuum pump = Aspestes is packing vias containing tube ee outer bacterial suspension tne vit — (cotton-plugaed Gtass task for pene tam containing Iyorvhe 30 10 100 specimen ‘small vials Advantages of lyophilization: i, As the ampoules are sealed there is no risk of contamination of infection with mites. ii, The prepared ampoules are easily stored, they are not readily broken and most species remain viable for many years. iii, ‘There is less opportunity for the cultures to undergo changes in characteristics (ie. they remain unchanged during storage period). iv. Owing to the small size of glass ampoules, hundreds of lyophilized cultures can be stored in a small storage space. In addition to this, the ampoules size makes them ideal for postage. v. Lyophilization cuts down the number of transfers. Liquid nitrogen storage: This method is also called as Cryogenic storage. It is like lyophilization, a satisfactory method for the long-term preservation of microorganisms. It has also been successful with many specimens which cannot be preserved by lyophilization. The maintenance of microbes is done by suspended metabolism. Life is regarded as “Stand still” at ~130°C and below, so at the temperature of liquid nitrogen (-196°C), provided the cultures survive the treatment, the period of preservation should be indefinite. Thus, long-term preservation without any change in the cultural characteristics is now attainable, Major steps involved in the methods are: PENSION-POINT 9044710403 _i. The culture to be maintained is suspended (thick suspension) as a cell or spore suspension in 10% glycerol. ii, This thick suspension in glycerol is distributed into ampoules (resistant to cold-shock). E iii, Ampoules filled with a culture suspension are frozen (at the rate of about 1°C per minute to ~ 20 to -35°C) and are sealed. iv. The frozen ampoules are then clipped on metal (aluminium) canes, one above the other and six to each cane. The canes in turn are packed in metal boxes or canisters (aluminium), which holds about 20 canes, These are perforated to allow the free running of the liquid nitrogen. v. The cultures are revived by removing form the container rapidly thawing, and culturing them in the usual manner. Advantages: i, tis effective method of preservation fi, Nosubculturing is required ‘The cultural characteristics remain unchanged. ‘The ampoules are not open to contamination or infection by mites, since they are sealed. v. The living material of a type, which would not normally grow in a culture and would not be preserved in a culture collection, can be retained in a viable state. Regardless of the method or methods chosen for preservation of primary stock cultures, it is of utmost importance that good, descriptive records be kept on these cultures and that the cultures be well labeled. If little is known or recorded about a newly isolated microbial strain, we cannot hope to be able to recognize changes that may have occurred in that culture after prolonged storage period. Role of Microbiologist in Industrial Microbiology: ‘The microbiologist has a central and key role in industrial organization. Some of his functions ncludes: ‘The selection of the organism to be used in the process. ‘The choice of the medium of growth of the organism. * The determination of the environmental conditions for the organisms optimum productivity (pH, temp, aeraton, DO, etc). + During actual production, the microbiologist must monitor the process for the absence of contaminants and participate in quality control to ensure uniformity of quality in the products. + The proper custody of the organisms usually in a culture collection, so that their desirable properties are retained (.e. stock culture maintenance). + The improvement of the performance of the microorganisms by genetic manipulation or by ‘medium reconstitution, ‘Types of Bioreactor: ‘+A batch reactor is an upright closed cylindrical tank fitted with four or more baffles attached to the side of wall, a water jacket or coil for heating and or cooling . a device for forcible aeration known as sparger, a mechanical agitator usually carrying a pair or more impellers, means of introducing organisms and nutrients and of taking samples and outlets for exhaust gases. In batch fermentor nothing is added nor any thing is removed till the fermentation is completed. ‘+ Fed batch reactor: This fermentor is cyclic in fed and batch phases with a short discharge phase in between cycles. Fed- batch reactor is a batch reactor which is initially charged partially full with broth, After a controlled length of time liquid feed is added continuously at a controlled rate (till it is full). After which the fermentor is allowed to run as a batch and then harvested. And the cycle is repeated, + Continuous reactor: The tank used in this system is essentially similar to that of the batch fermentor. It differs only in so far as there is provision for the inlet of medium and the outlet of the broth. In continuous reactor throughout the fermentation process the flow rate is maintained which is very essential to maintain the continuity of the process. During, continuous fermentation the Input rate is kept equal to output rate (In put = out put). PENSION.POINT 9011710403 , ’ 3+ Tubular fermentors: The tubular fermentor was originally so named because it resembled a tube. In general tubular fermentors are continuous unstrirred fermentors in which the reactants move ina general direction. Reactants enter at one end and! leave form the other and no attempt is made to mix them. Due to the absence of mixing, there is a gradual fall in the substrate concentration between the entry point and the outlet while there is an increase in the product in the same direction. A tubular reactor may be visualized a a column of soldier marching with a uniform pace down an avenue through a city block. Each soldier remains a separate entity and spends an equal length of time passing through the particular city block. + The Fluidized bed fermentor: This is essentially similar to the tubular fermentor, in bothe the : continuous stirred fermentor and the tubular fermentor there is a real danger of the organisms being washed out. The fluidized bed reactor is an answer to this problem because it sis intermediate in nature between the stirred tank and the tubular fermentor. The microorganisms which are in a fluidized bed fermentor are kept in suspension by a medium flow rate whose force just balances the gravitational force. Ifthe flow were lower, the bed would remain fixed and it the flow rate was at a force higher than the weight of the cells then elutriation would occur with the particles being washed away from the tube. (eg; the tower fermentor for the brewing, of peer and production of vinegar is an example of a gluidized bed fermentor). ‘+ Tower fermentors: Tower fermentors (developed for continuous production of beer). In this process yeast flocs are maintained in suspension by the upward movement of the nutrient medium. Moreover, any entrained particles are returned by means of a sedimentation device at the top of the tower. The fermentor consists of a vertical cylinder with an specific ratio (length Diameter) (101). At the top of tower a separator is provided to induce the gas bubble produced by the reaction and escape from the liqitid phase. Within the seperater, there is a quiescent zone, free of the rising gas, so that the yeast may seitie and return fo the main body of the tower and clear beer can be removed. A flocculent yest (capable of forming, lange floc) is essential for alcoholic fermentation in the FBF at acceptable flow rates otherwise a large population of the yeast would be washed out. Computer Application in Fermentation Technology: + Computers are’ being used since 1960's for modeling fermentation processes and in process control (for production of glutamic acie and penicillin), ‘+ The application of computers to fermentation technology is very dependent upon the end use of the computer system. For e.g, the requirements for a pilot plant system are quite different from those for a production plant. + Ina pilot plant, the computer is used primarily as a research tool where as for full scale production plant, the requirements for a computer coupled system are quite different. The primary objective is to produce a product in an optimal manner. +. Asa result, computer is used for process control and optimization aimed at maintaining quality control and product uniformity. + There are two levels in which computer system can be utilized in a fermentation plant : (i) the first level involves operator-oriented programs which can be utilized by a technician. To run those programs is is necessary for a technician to know only the computer commond necessary to start them, these programs include three recognized distinct areas of computer application and function in fermentation process: (i) Logging of process data (ii) Data analysis (Reduction of logged data) (ii) Process control. + The second level of computer utilization requires and engineer, with programming abil develop and insert into the operator-oriented programme new reutines to be run by the technician. Also engineer interacts with the computer to develop control strategies and. process ‘optimization routines. Data Logging: Data logging is performed by the data acquisition system which has both hardware and. software components, There is an interface between the sensors and the computer. The software should include the computer programme for sequential scanning of the sensor signals and the Procedure of data storage PENSION-POINT 9011710403Data analysis: (reduction of logged data) Data analysis is performed by data analysis system, which is a computer programme based ona series of selected mathematical equations. The analysed information may then be put on a print ‘out, fed into a data bank or utilized for process control. Process control: It is performed using a computer programme. Signals from the computer are fed to pumps, valves or switches via the interface. In addition the computer program may contain instructions to display devices or teletypes to indicate alarms etc