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1.1 The importance of microbial pesticides

Studies of natural epizootics of entomopathogenic bacteria and fungi during the latter half of the nineteenth century and the first half of the twentieth century, stimulated man`s interest in employing them as microbial pesticides and myco-insecticides to control agricultural pests. Mass production of the selected bacteria and fungi is a necessary prerequisite for any large-scale field application, and the methodology involved was developed at an early stage to suit a number of different pest-pathogen situations. However, this technology stagnated as disillusionment as to the practical value of virus, bacteria and fungi as biological control agents of arthropods and their efficacy against a range of pests became dominant in various parts of the world, following the initial overwhelming success of chemical pesticides.

The revival of interest in microbial insecticides over the last 20 years, has led to large-scale production of Bacillus thuringiensis [Berliner], a promising fungi candidate, and to the marketing of the first bacterio-insecticides DIPEL, THURICIDE, BACTOSPEINE, VECTOBAC, TECNAR, BACTIMOS, BATURIN 82 and myco-insecticides MYCOTAL, VERTALEX based on the formulation of Verticillium lecani and METAQUINO which is based on the formulation of Metarhizium anisopliae. While, the production of bacterio-insecticides is common worldwide, there is little information available on the biotechnology of entomopathogenic fungi, and their industrial production is still relatively unsophisticated.

A microbial toxin can be defined as a biological poison derived from a microorganism, such as a bacterium or fungus. Pathogenesis by microbial entomopathogens occurs by invasion through the integument or gut of the insect, followed by multiplication of the pathogen resulting in the death of the host, e.g., insects. Studies have demonstrated that the pathogens produce insecticidal toxin important in pathogenesis (Burges 1981). Most of the toxins produced by microbial pathogens which have been identified are peptides, but they vary greatly in terms of structure, toxicity and specificity.

Toxins of Bacillus thuringiensis are the most widely investigated example. The organism was first isolated in 1902 by Ishiwata, but it was not until the 1950`s that the diamond-shaped crystals or "parasporal bodies" in sporulating cells of B.t. were recognized as being involved in killing insect larvae. Later it was demonstrated that the crystals were mainly protein "polypeptides" and Angus (1956 a, b) showed that the crystal caused paralysis of the midgut and cessation of feeding in the silkworm, Bombyx mori, while the spores were necessary for the establishment of septicemia.

Many compounds, such as Beauvericin, and the destruxin of Metarhizium anisopliae, aphidicolin of Verticillium lecanii are highly toxic to insects.

The more widespread acceptance and use of myco-insecticides will depend on improvement in a number of key areas. Widespread use of myco-insecticides in China, Brazil and USSR may be repeated in the low-input labour-intensive agricultures around the world. In the short term, with our present level of knowledge, it seems likely that commercial production of myco-insecticides is going to be restricted to small-scale production in specific areas. At present there appears to be a situation in which biological control agents including fungi would provide a viable commercial option, where conventional chemical control gives insufficient control or where there is insecticide resistance; where conventional chemicals are too expensive; or where government restricts application of chemicals.

Despite these problems many of the multinationals are now pursuing projects on biological control. However, predictably, most interest appears to be focused on microbial toxins, with the view of expressing pathogen toxin genes in plants or using the toxins themselves as starter molecules for developing new chemical insecticides.

1.2 Production and Commercialization of Pathogens

The practical use of microbial agents which kill insects is being carried out by many scientists and horticulturists throughout the world. Other alternatives to chemical pesticides are also under investigation, including novel pest control systems based on parasitic and predatory insects, predatory mites, fungi, bacteria, viruses, protozoans, nematodes and pheromones which modify the behaviour of insects for man`s benefit. However, most of these novel systems have not yet been exploited in agricultural practice on a commercial scale. This is due to the series of sometimes lengthy and sometimes expensive steps which must be carried out before new pest control systems can be offered to the grower.

Once any new system has been identified and characterized in the laboratory, the following steps must be completed before it can be successfully commercialized.

Process Development. A process must be developed which can be carried out on a large enough scale to ensure that an adequate amount of the material can be made. This process must be sufficiently reliable to provide a product which is both safe and effective. In addition, the production cost must allow manufacturers to make a profit.

Product Development. Many systems for controlling pests are successful in the laboratory. However they fail when tried in the greenhouse or in the field. This is one of the many problems which must be solved by product development. Products must be manufactured and formulated in a way which makes them stable for the longest possible time, as convenient as possible to use, and as immune as possible to "use and abuse" i.e. the failure of many people to store or use the product as directed and then to blame the product for poor performance.

Recommendations must be developed for the use of the product in actual agricultural practice. Normally these recommendations cannot be so novel or so complex or require such unconventional equipment that people refuse to use the product. Often the recommendations are developed in collaboration with the appropriate advisory service to ensure that they will be accepted by the trade.

For novel products, testing is necessary to establish that they are safe to use; in addition, quality control tests must be devised which will ensure that every batch will be safe and effective. Once any testing is completed and quality control protocols developed, the appropriate government authority must be approached for permission to sell the product; at present, this must be done individually for every country in the world.

Once a product has been sold, it is often helpful to visit growers who have used it, either to confirm its success or if it has failed, to investigate and determine the cause. Further development work can than be carried out to find ways of avoiding product failure in the future.

Local Marketing and Sales. Once made, the product must be sold and used if all the effort spent in development is to be worthwhile. There are many obstacles to selling alternative pesticides, especially fungi. Users are unfamiliar with how to use these types of products and how they work. Marketing requires extra efforts both to familiarize growers with the new product and to provide a substantial back-up service for the product.

The proceeding sections outline the problems which must be solved for any novel pest control system. We shall now discuss the particular problems which must be solved during the development and commercialization of fungal pathogens for the control of insects. (Quinlan, R.J. and S.G. Lisansky, 1983).

Before one can sell a live micro-organism as a pest control agent, a reliable method of production must be developed which yields large quantities for which a product specification can be drawn up.

1.2.2.1 Organism Storage.

The first problem in producing a micro-organism is storing it in a way that ensures the retention of desirable features. The most obvious feature to be retained is pathogenicity; the second is productivity in terms of yield in the commercial production process. This is typically measured either by total biomass, or by number of infective propagules, i.e. spores or fragments of mycelium , produced per litre-hour of fermentation time. Many microorganisms are known to lose desirable features either on storage or after repeated sub-culturing. Among insect pathogens, Beauveria bassiana has been reported to have reduced virulence after subculturing whereas both Verticillium lecanii and Metarhizium anisopliae are reported as being undiminished in virulence after many passages. Organisms which lose virulence may sometimes be restored to their former potency by passing them through their normal host, i.e. the target insect; however, such a technique would be cumbersome as a routine part of a production process and always presents the risk of contamination. Therefore the problem is often avoided by storing a large number of elements of a single spore isolate in a deep frozen or freeze-dried condition. Samples are checked periodically to make certain that virulence and productivity have not diminished with time.

The standard method of production of microorganisms is the process of fermentation. There are many types of fermentation; the two most common are "submerged" and "semi-solid".

Submerged or deep-tank fermentation is, as the name implies, a growth of micro-organisms in a fully liquid system. There are a number of advantages to fully liquid systems which include the ability to hold temperature and pH constant, the ability to pump large quantities of air into the system and disperse it by means of stirring impellers, and the ability to generate reasonably homogeneous conditions to maximize the growth of micro-organism.

Despite the many advantages of submerged fermentation some fungi will not yield a satisfactory product by this technique. Semi-solid fermentation offers an alternative in which the fungi grow primarily on the wet surface of a solid material, often some form of processed cereal grain to which nutritional adjuvants have been added, though attempts are made frequently to use "waste" materials or media of low value, such as straw. This allows fungi to grow in conditions more similar to those found in nature; spores, the infective propagules by which the fungus survives and infects insects, are produced in the air and are consequently more durable. Semi solid fermentations are relatively easy to develop on a small scale. Scaling them up to the sizes necessary for commercial product presents numerous problems; aeration becomes a major difficulty as the volume of a semi-solid mass increases more rapidly than the available surface area. This requires either a very large area of relatively shallow media, e.g. on trays, or in a vessel which can agitate or tumble the media. On any scale, trays are very difficult to sterilize and keep sterile. The development of large vessels for semi-solid media fermentation requires the invention of a number of techniques or pieces of equipment for:

a. keeping the media friable after sterilization. Its tendency is to set solid when it cools, rather like oatmeal.
b. inoculation with the desired fungus without contamination
c. aeration and agitation during fermentation
d. drying the material prior to opening the fermenter in order to avoid contamination, etc.

Medium Development. Which ever type of fermentation is chosen, nutrients must be provided so that the micro-organism can grow. Which nutrients are chosen will markedly affect how fast the organism grows, how much is produced and often, how infective the final product is. Nutrients to be provided include a carbon source, e.g. glucose or molasses, nitrogen source, e.g. soybean meal or yeast extract, and a "defined", in which case the precise nature and quantity of every nutrient is known, or, alternatively, they can contain ingredients of an indeterminate and occasionally variable nature e.g. molasses; most commercial media are latter type.

1.3 Theoretical Background of Industrial Processing in Biotechnology

The two main disciplines which determine the trends of small and large-scale industrial microbiology are theoretical micro-biology (including microbial genetics, physiology and bio-chemistry) which forms the theoretical ground, and microbial engineering which creates the basis for the application of engineering aspects in microbial processes. A successful microbial process on a small or large scale requires the research and development [R&D] of methods satisfying providing for the following demands:

Maintenance of stability of the production culture, both in its initial form (preservation) and as subcultures infrequently propagated via sporulation or vegetative generations.

Maintenance of suitable conditions ensuring reproducible yields. This entails the use of row materials of appropriate quality, especially complex organic compounds, and the development of analytical methods for controlling both culture processes and the quality of the raw materials used.

Application of the technology for maintaining strictly aseptic conditions; this factor is important in the biotechnology of most products, in the isolation steps, and in the conversion of the product to the final form.

Increase in production yields of a particular product by modifying the medium composition and the genetic properties of the production strain (Sikyta, 1983).

1.3.1 Laboratory Processing

1.3.1.1 Cleanliness and Safety

The normal technical procedures designed to keep cultures sterile or pure and prevent cross-infection of laboratory materials contribute considerably to the safety of the individuals an most laboratory workers observe reasonably high standards of personal hygiene and wash their hands after handling cultures of any kind and before touching the rest of their person, handling their food or smoking.

The maximum standards of safety can be achieved only if every laboratory worker has a real understanding of the basic principles and is able to apply them to reduce the risk in each particular circumstance.

There are cabinets (Microflow Ltd.) which are fitted with filters which remove particles down to 0.01 mm and discharge their effluence into the laboratory air.

Continuous irradiation of the air above eye level is possible and reduces the bacterial content. Ultra-violet light can be used to disinfect the insides of protective cabinets but to much reliance should not be placed on this as a disinfecting agent. Chemical methods should also be used (75 % ethylalcohol). Ultra-violet light does not penetrate surfaces.

One important microbiological technique will be introduced: the use of the oilimmersion objective is essential to most microscopic studies of bacterial cells. The best type of microscope is with Phase-contrast.

1 x 10^-2or 1 : 100; 1.0 ml + 99.0 ml or 1.0 ml: (99 ml) = 1/100
2 x 10^-2 or 1 : 50; 2.0 ml + 98.0 ml
5 x 10^-3 or 1 : 200; 0.5 ml + 99.5 ml
2 x 10^-4 means 2/ 10.000 = 1/5.000

Dilution Plate Counting and Distribution of Bacteria

Preparation of Dilutions: Shake each stock dilution well. Using aseptic techniques, transfer 0.1 ml of the 100 stock dilution to a 9.9 ml dilution blank (1:10,000 dilution). Mix well to obtain even distribution of organisms. With a pure pipette, place 1.0 ml of the 1:10,000 dilution into a sterile Petri plate and with the same pipette transfer 1.0 ml to a 9.0 ml dilution blank (1:100,000). Discard the pipette and mix the dilution to a fresh 9.0 ml dilution blank (1:1,000,000). Mix. with a fresh pipette transfer 1.0 ml of the final dilution to a sterile Petri dish. Pour approximately 15-20 ml of nutrient agar which has been steam-pressure sterilized (autoclaved) and cooled to 45^oC in a water bath; then rotate each plate to mix the inoculum and medium.

After the agar has completely solidified (15-30 minutes) invert the plates and place in incubator space to dry off excess surface moisture. Time of incubation is 2 days at 25 or 28^oC. After incubation count the number of colonies.

Transfers from agar plates to tubes, and from tube culture to tube is a common and simple procedure, but requires careful attention to certain details. Transfer from a colony on an agar plate to an agar slant or tube of broth may be made either a straight wire or a loop.

Pure cultures of different microbial species and subspecies may be isolated from the highly complex mixed populations in nature by a variety of methods, the most common of which will be used is a dilution plating. Each discrete colony which arises in or on a well prepared dilution plate is assumed to be composed of cells, all of which are descendant of a single cell. Sub-cultures from such a colony should produce colonies all of the same kind, any one of which is composed all of the same kind of cells as the original colony.

After culture purity has been established in subcultures, a stock culture of the organism is established. The stock culture may be maintained on an agar slant kept in the refrigerator, but unless it is transferred at suitable intervals to establish a fresh stock, it may loss its viability (its capacity to reproduce). Viability of agar slant cultures may be prolonged by covering the slant with a layer of sterile mineral oil to exclude oxygen and prevent evaporation. Since frequent transfers of stock cultures encourage accumulation and selection of mutations, microbiologists often preserve pure cultures virtually unchanged for identifinite periods by lyophilization (a suspension of cells is quick-frozen at about -60^oC, rapidly dried under high vacuum, then hermetically sealed in a glass tube) or by immersion in liquid nitrogen (-195^oC).

Copper or zinc containers must not be used. Small quantities of these metals will dissolve in the culture media and are bactericidal and fungicidal. Large quantities of media can be made in stainless steel buckets such as are used in dairy and food trades. Smaller amounts can be made in resistance glass laboratory flasks.

Distilled water of commercial bulk supplier quality may contain substances which inhibit bacterial growth, e.g. oil materials from stripper stills attached to steam lines. Manesty-type distilled water, glass-distil water and deionized water should be used, although it must be realized that some "trace elements" necessary to bacterial growth are in fact supplied by the distilled water, by glassware, or by recognized impurities analytical quality reagents.

Dextrose, when autoclaved with salts such as phosphate may yield inhibitory substances. It is best to add this and other carbohydrates as sterile solutions after the medium has been sterilized. Excessive heating, and remelting may destroy growth factors and gelling capacity and cause darkening or pH drift of culture media.

Pipette 10 ml of the medium into a 152 mm x 16 mm test-tube and measure pH with indicator test-paper; according to scale can be seen the pH value, and can be corrected by N/20 HCl or N/20 NaOH.

Example :10 ml of medium at pH 6.4 requires 0.6 ml of N/20 NaOH to give the required pH 7.2, than the bulk medium will require

_{19. x 1000 ml}
------------    of  N.NaOH per liter = 3.0 ml
20 x 10

All final readings of pH must be made with the medium at room temperature because hot medium will give a false reaction with some indicators.

The usual objective in heat sterilization is the destruction of all microorganisms with the least possible damage to the material being sterilized. "Sterilization", as commonly understood, is not necessarily absolute; for example, instrument sterilizers may kill all infection - causing bacteria, but not all sporeforming bacteria. The aim in sterilization of culture media is the destruction of all life without rendering the medium ineffective for cultivation because of physical and/or chemical damage to its ingredients. Methods employing moist or dry heat, or filtration are used in the sterilization of culture media and of materials and equipment used in the bacteriological laboratory.

Steam under pressure in a closed system (autoclave) is frequently used to sterilize heat stable culture media, heat stable chemicals, dilution blanks, rubber articles, and to sterilized heat stable culture media (antibiotic) must be sterilized before being added aseptically to be sterilized, cooled media.

A temperature of 121^oC at 1.5 MPa pressure for 15 minutes generally is required for moist heat sterilization. The most important point in the operation of the autoclave is the removal of all air from the chamber. The temperature rather than the pressure is the lethal agent. Therefore, it is important that the desired temperature is reached before timing the sterilization cycle. Adequate time must be allowed for heat convection current to penetrate the centre of the material. Preheating large quantities of media is often desirable to reduce the over-exposure to sterilization temperature. Viscosity reduces convection, and therefore, a long period of time is required for sterilization of agar than of broth. Two to five minutes must be added for agar sterilization. Never autoclave solidified agar in a flask; most of the autoclaving time is required to melt the agar. Premelt the agar in a boiling water bath prior to sterilization.

Although not achieving red heat, flaming is a method commonly used for decontaminating the mouths of bottles, flasks, culture tubes, glass slides, scalpels etc. by passing them though a Bunsen flame without allowing them to become red hot.

The process of sterilization by hot air ovens is simple in principle and the materials to be processed can be prepacked in craft paper or sealed in metal containers. The method is used for sterilizing such articles as glass syringe etc. Material such as dry powders, fats, oils and petroleum jelly in small shallow containers can also be sterilized by this method.

Air is not a good conductor of heat so that hot air sterilization poses problems of penetration. Special apparatus "fan" is needed to ensure that all parts of the load have been maintained at the required temperature. The hot air must circulate between the packages.

The Heating-Up Period is the time taken to reach sterilization temperature; this may take about 1 hour.

The Holding Periods at different sterilization temperatures recommended are:

160^oC for 45 min.
170^oC for 18 min
180^oC for 7 min

The Cooling-Down Period is carried out gradually to prevent glassware from cracking as a result of a too rapid fall in temperature. This period may take up to 2 hours.

The full sterilizing process from loading the oven to unloading it may therefore take about 3-4 hours, depending on the size of load.

Long exposure to a lower dry heat temperature has been a method of sterilization recommended like a dry heat cycle of 135^oC for 22 hours.

Variability, one of the characteristic features of microorganisms, tends to increase with increasing productivity of producer strains. The variability can result in transient, non-hereditary changes, or in permanent changes of hereditary character. In some cases the properties of the whole culture change, in others the culture becomes heterogeneous. Heterogeneous cultures comprise a number of cell variants with diverse morphological, physiological or biochemical properties. The heterogeneity or homogeneity of a culture can usually be examined by plating individual spores or cells onto a solid medium and comparing the properties of the resulting cultures. Transient changes on the cultures are usually easily eliminated since they depend mostly on the composition of the media used for culture and on other ambient conditions.

Close attention must be paid to the permanent, hereditary changes in the cultures. In most cases the change does not affect the whole culture and the homogeneous parent culture thus becomes heterogeneous. The strain heterogeneity is currently thought to be brought about by uncontrolled environmental factors. Its appearance depends on the ambient conditions in the broadest sense of the word; for instance, certain sporulation media give rise to a larger number of variants than others. Strain heterogeneity is more conspicuous with a spore inoculum than with a vegetative one.

The culture requires a strict regulation and control of ambient conditions (temperature, aeration and mixing intensity, composition of medium etc.); equal care should be exercised to ensure the highest yields and the highest stability of the strain. A variety of methods have therefore been developed for culture preservation.

To preserve a strain properly means to maintain it under conditions ensuring the longest possible stability of the culture; in other words, under conditions minimizing the occurrence of permanent hereditary changes leading to strain heterogeneity.

Production strains can be preserved in several ways depending on the type of microorganism:

a) Preservation of cultures on sporulation agar slopes at low temperatures.

b) Preservation of cultures on agar slopes which are, after inoculation, overlaid with sterile liquid paraffin. This technique is suitable for preserving nonsporulating fungi and yeasts.

c) Lyophilisation or freeze-drying is based on freezing vegetative cells or spores to temperatures far below the freezing point and drying them under a high vacuum. The suspension obtained by washing the culture from the surface of the medium which contains agar is transferred to test tubes containing either a protective colloid such as broth, blood serum, whey, or a mixture of soil and sand. The samples are frozen in solid carbon dioxide in ethanol and then dried in a vacuum of about 7 - 70 Pa. The test tubes are then sealed under vacuum. The resulting stock cultures so preserved (about 1 g) can serve to inoculate only a small number of subcultures.

Preservation of cultures in liquid nitrogen. Cultures sealed in glass ampoules are rapidly frozen by placing them in a container with liquid nitrogen.

The development of a process includes three steps: elaboration on laboratory scale, small scale and pilot large scale production and finalization of product e.g. formulation of microbial pesticides.

The goal of the laboratory research is to develop

a) preservation of the culture,
b) culture of the strain on solid media and its propagation,
c) nutrient medium of a suitable composition,
d) techniques of laboratory submerged or semi-solid scale culture.

The medium for propagation of the strain has to meet several demands. It has to ensure perfect growth and sporulation and, at the same time, guarantee a satisfactory stability of the strain. Both the strain and its culture conditions, i.e. the composition of the nutrient medium, incubation temperature, time of incu-bation, etc., should be conceived as forming an integral system which can be severely disturbed by changing only some of the conditions. The appropriate preservation techniques are to be determined by studying the stability of the production strain preserved by different techniques, in long-term experiments. Each type of stock culture should be prepared in a sufficient amount even when the preserved strain is not yet optimal for production. This procedure ensures the supply of a uniform inoculation material for sufficient periods of time; the stability of the strain in the spectrum activity to target organisms and the efficiency of preservation must be examined in long-term studies over several years.

The composition of the nutrient medium is determined at the initial stages of research; the medium should, above all, ensure reproducible yields. The quality and quantity of raw materials used, and the technique of media preparation (sterilization, etc.) should be adjusted accordingly.

The next step is a detailed study of the composition of the medium which should give the optimum quantitative ratios of individual components and ensure the maintenance of their optimum proportions throughout the process. Apart from productivity, a number of additional factors determine the medium composition suitable for scale-up purposes. These include the accessibility and price of individual raw materials, their sterilizability, requirements for an economical isolation procedure and final formulation of the product.

The next step is the elaboration of the process on a laboratory scale when the culture is usually grown on reciprocal or rotary shakers, the latter affording a more intensive aeration of the culture. The optimum aeration regime for laboratory cultures can be attained by using shakers with different types of motions, changing the shape of the culture flasks and the volume of the nutrient medium. The aeration intensity is to some extent inversely proportional to the amount of the medium in the flask. With optimum aeration intensity defined, other factors are determined which could affect the product yields and the length of time necessary to attain maximum yield. Another important facet is the determination of a strain stability during vegetative transfers.

Experiments conducted in flasks on laboratory shakers provide fundamental information on the production strain, the composition of the medium, suitable type of inoculum, etc., but they provide meagre data on technological factors such as the choice of construction materials, aeration, measuring and control devices, etc. All this information is obtained from experiments with laboratory and seed fermenters.

The first task in small scale production is the development of a basic culture procedure affording reproducible results. Absolute production yields are of secondary importance, the main emphasis being put on the expected standard course of the microbial process with the attendant changes in growth, pH, consumption of nutrients, production of metabolites, and in the physiological state of the microorganisms. On attaining reproducible results the search for individual optimum conditions can begin.

The first factors to be explored are usually aeration and stirring. Next to be examined is the effect of changes in the ratio of individual components of the nutrient medium, with special reference to the source of carbon and nitrogen and their mutual proportions. The effect of different types and amounts of antifoam agents is also studied since the physical action of these agents can affect the dispersion of air in the culture and respiration of the microorganisms; some antifoam agents (vegetable oils, lard oil) can be utilized by the microorganisms, thus changing the metabolism of the culture. Fluid from laboratory and small scale fermenters can be used for tentative isolation of metabolites and preliminary tests of their quality.

Microbial contamination of fermentation processes and principles and techniques for its elimination should be appraised from several viewpoints:

a) Type of the metabolite produced;
b) Specific properties of the culture;
c) Machinery and equipment used in the process;
d) Nutrient medium and the raw materials used for its preparation;
e) Technology adopted in the process.

Contaminating microorganisms affect negative the microbial process by:

a) destroying the cells of the production strain,
b) inactivating the synthesized metabolites,
c) producing substances affecting the producer`s metabolism and thus decreasing the production of the required metabolite,
d) exhausting compounds crucial for growth and product synthesis from the medium.

However, the presence of a contaminating microorganism need not always results in a drop in metabolite or spores production; in the absence of this drop, it is often difficult to detect contamination. In semi-solid fermentation process, contamination need not always lead to failure.

The term "protected fermentation" is used for such processes in which foreign contamination is suppressed by the presence of an antimicrobial agent. This agent is added to the medium in suitable form.

Checks of the absence of alien microorganisms are required during all crucial stages of the process, culture, product isolation, and its final formulation.

Identification of microbial contamination is done by microscopic and culture methods. Microscopic examination represents a useful tool for immediate detection of massive contamination of cultures with an alien microflora. In most cases viable cells are difficult to distinguish from dead ones. The differentiation is somewhat aided by the so-called vital test which, however, is not always fully reliable. The most reliable method for identification of alien microorganisms is by culturing.

The following procedure is currently used in laboratories: Samples are transferred under aseptic conditions into sterile flasks. A volume of 1 ml serves to inoculate two tubes with 5 ml broth each and, using a loop, blood, blood agar in a Petri dish. Incubation is then carried out for 24-48 hours at 37^oC. Both microscopic and macroscopic counts of alien microorganisms are then taken. If the results are not unequivocal then 1 ml of culture from the test tubes and colonies on the Petri dishes is used to inoculate another four test tubes (2 + 2) and counts are taken again after 24 hours of incubation at 37^oC. A macroscopic indication of the presence of contaminating microorganisms is often a different growth form.

From this viewpoint, microbial processes can be divided into:

1. processes that do not require stringent control and protection against the proliferation of a foreign microflora in the medium,
2. processes requiring strictly aseptic conditions - the so-called aseptic processes - microbial insecticides.

Although crucial technological factors may be assessed in laboratory fermenters, it is often necessary to verify the laboratory results in devices more closely resembling industrial fermenters. These devices are pilot-plant fermenters; they serve to complete, modify and verify the data obtained in laboratory fermenters and to test the suitability of construction, regulation an monitoring devices. Since, with the same type of agitator with dimensions proportional to fermenter dimensions, laboratory fermenters require higher impeller speed to achieve the same aeration effect as in large fermenters, and since the aeration effect depends largely on the transfer of mechanical force into liquid via the impeller, the verification of agitation and aeration conditions is of utmost importance. Different types of agitators, their relative size, different types and dimensions of baffles, aeration systems, and the effect of their position inside the fermenter are also studied on pilot-plant scale.

Sterility criteria. A basic feature of most microbial technology is the stringent requirement for the absence of foreign microorganism during the process (without contamination). This has to be maintained throughout the process from the moment of opening of stock culture to the termination of growth. Large fermenters offer a greater possibility of culture contamination, being much more complicated than laboratory devices. The presence or absence of contaminating microorganisms is determined by the so-called sterility test. The fermenter is supplied with a relatively rich medium but not inoculated. A conventional process is then carried out including agitation, air feed and sampling and for duration at least equal to that of the real process. Samples of medium are then subjected to sterility tests in a laboratory. These tests are necessary since each unsuccessful pilot-plant and, especially, industrial scale run represents a considerable economical loss owing to the large volume of medium and high operating costs involved. To meet the demand for the absence of foreign microorganisms, the device has to be sterilized thoroughly and the possibility of contamination by undesirable microorganisms must be excluded.

Pilot-plant fermenters, their pipelines and fittings are sterilized prior to medium sterilization, sometimes together with air filters. After medium sterilization, the outlet valve at the bottom of the fermenter is briefly opened to remove air bubbles and the medium is cooled down. When the pressure inside the fermenter drops to 0.05 MPa, air is introduced by means of the aeration device. During sterilization all valves and connecting lines are flushed with steam. Flange packing and seals are used as rarely as possible and, wherever possible, are replaced by welding. When using flanges, the packing has to be selected carefully. The packing must not be made of porous materials but even the most common nonporous material, rubber, has to be of high quality, without textile ply, and has to be resistant to high sterilization temperatures without losing elasticity or cracking. Valves are mounted in such a way that their packing is located underneath the steam seal, usually in a horizontal position. The mounting of valves on a fermenter deviates from conventional rules; here the streaming liquid does not flow against the valve cone. The valves are mounted so that the cone shaft packing communicates with the valve steam seal, not with the fermenter interior. This is because of the risk of microbial contamination by growth through the packing, which is especially acute in valves with large construction lengths (Fig.1.2). The valves have to be placed so that no condensate is formed on the steam-blocked side since this would prevent a through heating by steam (Fig.1.3).

Valves communicate directly with the medium in the fermenter wall, with minimum length of connecting tubes. This holds especially for medium sample collectors (Fig.1.4). It is convenient to arrange valves into a system of valve sets which take up less room and are sealed by a common steam inlet. Manometers, valves and back pressure regulators are mounted on the descending part of tubes wherever possible.

Contamination may often be caused by minute leakages in cooling coils or jackets. The localization of these leakages is often difficult, testing for them should include a thorough mechanical removal of medium residue adhering to surfaces and the use of relatively high water pressures. The test for leaks may be carried out using air or ammonia, with detection of the ammonia leakage by an appropriate reagent. The correct position of packing in a flange is tested by smearing its circumference with a soap solution and observing the formation of soap bubbles at leakages points.

The contamination of medium by Gram-positive microorganisms from the air is usually due to faulty packing or air filter failure whereas Gram-negative microorganisms come from water through leakages in the cooling system.

The design and construction of pilot-plant fermenters has to meet the following requirements:

a) No connection should exist between sterile and nonsterile parts of tubes and fittings;
b) Connection and flange packing should be made of higher-quality rubber without textile ply;
c) Wherever possible, welded construction should be used;
d) Valves and other fittings should be easy to sterilize and should remain sterile throughout the culture process;
e) After sterilization all parts and tubes that have to remain sterile should be kept at a constant positive pressure of sterile air;
f) It is necessary that every part of the device can be separately sterilized without disturbing the operation of other parts;
g) Inlet and outlet valves and tubing which are not otherwise protected should be connected to a live steam inlet;
h) Prior to each new fermentation the tightness of fermenter seal, tube connections, valves and other fittings should be tested.

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