DESCRIPTION OF THE PROCESSING
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The main task of a microbial small scale production is the verification of laboratory procedures and their modification to permit the scale-up of the pilot plant procedures to commercial scale.
The tasks of a microbial pilot plant include:
a) Scale-up of results from laboratory to pilot plant level. A pilot plant procedure
should be elaborated so that the process can be tested on industrial scale.
b) Modification of technology ensuring an increased production of the substance under study, minimum cultivation time of the production strain and an economical cultivation and isolation procedure.
c) Standardization of the product is the most important part of pilot scale production. Every rank of the product must be tested for biological activity and compared with international standard.
3.1 Small-Scale Processing of Submerged Fermentation
3.1.1 Growth Kinetics in Submerged Culture
The use of stirred fermenters with automatic control of the culture environment is the most suitable technique to evaluate bacterial or fungal kinetics. Cultures can be discontinuous (batch cultures).
Fig. 3.1 Batch growth curve with six phases; g = generation time
The batch culture growth curve of a microorganism can be divided into six phases (Fig. 3.1); log phase; accelerating growth; exponential growth; declearing growth; stationary; lytic decline phase. Growth has been often represented by mathematical models. In the case of media limitation, the equation of Monod is most often used (Fig.3.1)
u = um ----------------
Ks + S
um = u maximum specific growth rate
K = Saturation constant
If Ks <<S, ---------------- ~ 1 and the growth is exponential;
K + S
if the value of Ks is relatively high in comparison to S, when S decrease, a decelerating growth phase is reached. Values of u are dependent on the combination media-fungus but are most often between 0.1 and 0.4 h-1.
Values Ksa can be determined by plotting 1/m against 1/S. The equation becoming 1/m = Ks/mm. 1/S + 1/mm.
In the case of nonexponential growth the Ks value can be approximated from the curve m = f(s) (Fig.3.2). Within fungi in batch culture, it is difficult to observe exponential increases in biomass for more then five doubling times. After a certain growth time, the fungus will eventually modify the physicochemical condition of its environment and corresponding growth slow down will occur due to limitation in nutrient concentration or oxygen transfer or accumulation of staling products. Morphologically, linear growth can be correlated with a blockage of the branching whereas branching of the mycelium or the fermentation of blastospores "yeast-like cells" induce an exponential increase of the biomass.
3.1.2 Factors Governing Sporulation
It is well documented that conditions favouring spore formation are usually different and more restricted than those controlling mycelial growth (Hawker, 1966). Although spores are the main target propagule in the production of mycoinsecticides, the environmental factors governing the sporulation of entomopathogenic fungi have been poorly studied. In particular, the optimum conditions for sporulation and for enhancing the pathogenicity or viability of the spores produced need to be determined, since it has been established that the environmental factors during fermentation influence both the aggressiveness of the spores and their survival (Aoki, 1967; Fargues, 1981).
A period of vegetative growth invariably precedes sporulation and the production of a large number of spores normally requires a well-nourished mycelium. The nutrient concentration and quality that favour sporogenesis are often highly specific. For Beauveria bassiana, maximum sporulation is attained with glucose and vegetable oil plus either glutamine, lysine or serine. Starvation or reduction in food supply usually stimulates sporulation, nitrogen being the first nutrient to be exhausted, which appears to be a defence against autolysis or the formation of nonviable spores. However, sporulation can occur without any starvation of the mycelium, the production of conidia of Hirsutella thompsonii and blastospores of Verticillium lecanii in batch culture being parallel to mycelia growth (Lattgé et al. 1986.)
A sexual spores are formed predominantly in aerated conditions and this has led to classic two-step production procedure for most of the Deuteromycotina: submerge culture of the mycelium and solid media fermentation for sporulation. Nevertheless, there are no rules concerning the degree of aeration in order to reach a satisfactory sporulation level amongst the entomopathogenic fungi. Conidia of B. bassiana and Hirsutella thompsoni can all be obtained in submerged culture (Lattgé et al., 1988).
3.1.3 Mass Production
After the growth and sporulation processes have been thoroughly investigated and tested at laboratory level, mass production of the fungus can be undertaken on an industrial scale with various raw materials. Until now, entomopathogenic fungi have been produced in liquid or solid media.
Production in Liquid Media
Three types of reactors or fermenters are commonly used: the stirred tank; the tower and the loop fermenters (Kristiansen & Chamberlein, 1983). The stirred tank fermenter has the form of a vertical cylinder with the agitator mechanism centrally placed (Fig.3-3A). These reactors produce a violent agitation of the culture medium with good homogenization of the broth and a high gas transfer coefficient whilst at the same time avoiding mycelial aggregation and subsequent pellet formation. One drawback is damage to the mycelium on contact with the stirring mechanism. Stirred tank fermenters have been employed for the production of the production of both mycelium and yeast-like cells of all the common entomopathogenic fungi. Blastospores of Deuteromycotina, conidia of B.bassiana and H. thompsoni and resting spores of Conidiobolus obscurus, C. thromboides and Lagenidium giganteum have also been produced in such tanks (Lattgé et al.,1977). The tower fermenter is a vertical cylinder with a height/diameter ratio greater than six and lack any mechanical agitation (Fig. 3-3B). Nutrient mixing is promoted by the injection of gas at the base of the reactor. Most fungi produce mycelial aggregates in this type of fermenter, which can also be used for those species producing spores by conjugation. The loop fermenter is a modification of the latter in which the culture medium is forced back down to the bottom of the reactor. The recycling of the medium is achieved by the incorporation of a draught tube (internal recirculating, (Fig. 3-3C) or by a pipe (external recirculating, (Fig. 3-3D) in the design. The mass transfer at the gas-liquid interface can be as efficient as the stirred tank fermenter but with an important saving of energy. Resting spores of C. obscurus can be produced in this type of fermenter.
The presence of oxygen could not be detected more than 0,2 mm from the pellet surface.
Fig. 3.3.: Diagram of fermenters suitable for the production of entomopathogenic fungi. A - Stirred tank fermenter, B - Tower tank fermenter, C and D loop fermenter with internal (C) or axternal (D) recirculating of medium, E - Tray reactor, F - homogeneous solid reactor, G - rotating disk fermenter: a - motor, b - air sparger, c - heating ang cooling system, d - impeller, e - baffle, f - foam breaker, g - rotating discs, h - granular media.
3.1.4 Production of Bacillus thuringiensis
B.t. is an ideal microorganism for large scale cultivation. Present commercial production turns out B.t. based formulations in submerged cultivation conditions, in fermenter or chemostats.
126.96.36.199 Storage of Production Strain
Conserved cultures of B.t. can be kept in lyofilized condition in sealed ampoules for decades. The culture is suspended into sterile skimmed milk, is filled into ampoules drop by drop (cc 0.2 ml), and is lyofilized. The sealed ampoule is vacuum tested. When revitalizing the culture the outside of the ampoule is wipedwith disinfectant solution (e.g., 70% ethyl alcohol) to preventpossible contamination on opening the ampoule. Then the ampouleis cut to facilitate rupture, and 1-2 ml of sterile nutrientmedium is added into it. The suspension obtained is transferredonto the surface of slanted agar or of Petri dish agar, and isallowed to cultivate for 24 - 48 hours at 28oC.
188.8.131.52 Cultivation Media
The selection of nutrients for cultivation depends onavailability, price, and suitability for B.t. The media used,however, represent only a small share of the costs of equipment,servicing, and utilities required for operation.
Source of carbon
When formulating the nutrient medium, carbon is provided by mono,di, and polysaccharides such as glucose, starch, molasses, etc.If their concentration is too high the pH will drop below 5.6 -5.8, and acidity may prevent growth. It depends on balancing thelevel of saccharides and the sources of nitrogen, inasmuch as B.t. is producing alkaline components from the nitrogen-bearingmaterial and these can neutralize the acidic products. As long asthe medium is properly chosen the initial pH will drop fromneutral to 5.8 - 6.0 and then will rise slowly toward 8.0 - 8.3.
Sources of nitrogen
In the fermentation of B.t. there are variegated sources ofnitrogen; these can include albuminous materials containing vitamins and various factors such as yeast autolyzate, yeast extract, dried yeasts, peptone, soya meal, maize meal, maize extract, residues from the production of alcoholic beverages,fish meal, etc.
Some ions which supplement nutrition are indispensable for the growth and sporulation of B.t. Mostly, they are supplied in the form of MgSO4.7H2O, MnSO4.4H2O, FeSO4.7H2O, ZnSO4.7H2O, CaCO3(0.001-0.005 %).
Agar medium for B. thuringiensis (pH 5.7 ...8.1)
Tryptosephosphate agar (TPA) g/l
Accord. Dulmage, 1982 NYSMA g/l Bactopepton 3.0
Beef extract 5.0
Yeast extract 0.5
Beef pepton agar BPA SEVAC g/l
Beef extract 10.0
According de Barjac (1979) Yeast extract 0.5%
K2HPO4. 7H2O 0.01%
MgSO4 . 7H2O 0.05%
184.108.40.206 Laboratory Scale Cultivation
For cultivation on a shaker, 500 ml flasks are used, filled with 50 - 100 ml of nutrient medium (e.g., tryptosophosphate media) containing tryptose 20.0 g, glucose 2.0 g, NaCl 5.0 g, Na2HPO4 2.5 g per 1 liter of distilled water. The flasks are inoculated with culture from slant agar and are put on a shaker for 24 hoursat 28-32oC.
The first passaging follows, and inoculation is done using 2% of volume of the flask contained culture. After 12-24 hours theinoculum thus obtained is used to inoculate the seed fermenter. Even the flasks from the first passaging can be used for that. The final development of the culture on the shaker, i.e., for sporulation and liberation of the spores and of crystalline inclusions, takes 24-48 hours.
220.127.116.11 Cultivation in Fermentation Tanks
In the beginning of small scale processing the fermenters used are the seed fermenters of 20-40 litres capacity which contain 10-25 litres of nutrient medium. Culture from the shaker or from laboratory fermenter (1-3 litres), to the amount of 1-3% of volume of the medium, serves for inoculation.
The seed tanks can be made of glass or stainless steel; the fermentation tanks are made of stainless. An aeration ring isused to aerate the culture, together with an agitator or an air outlet under a propeller. The air volume used for aeration should correspond to between 1/2 and full volume of the cultivation medium.
The changes of pH during fermentation depend on the composition of the medium used. After sterilization of the fermentation medium the pH should be 6.8-7.2. After inoculation, with acids being formed from the saccharides, pH will drop to 5.8-6.0 (after 10-12 hours) and then will rise again to about 7.5 (25 hours) and further on to 8.0 (ca 30 hours) and, ultimately, 8.8 (50-60 hours).
Unless foam suppressing agents are used, the fermentation liquid will be subject to intensive foaming at the beginning of fermentation and also at the sporulation time (after about 24 hours). Silicone defoaming agents can be used to mitigate foam formation.
After B.t. inoculation and the lag phase there follows the exponential phase where an intensive growth and separation of the culture takes place. This phase persists up to the 16th - 18th hour. At the end of the exponential phase, spores start appearing within the cells, together with inclusions of crystalline toxin. Sporulation is complete after 20-24 hours. Subsequently, the sporangia become subject to lysis, liberating spores an crystalline inclusions of the fermentation liquid. Some 90-98 %of all spores and inclusions are liberated after 32-42 hours. Prior to stopping the fermentation, it is recommended to add another 10-12 hours in order to maximize toxin production.
18.104.22.168 Harvesting of Active Biomass
Separation and drying
The sporulated culture of B.t. consisting of spores and crystal inclusions must be separated from the fermentation media with its residues of nutrients, dissolved metabolites, enzymes and possibly exotoxins. From small volumes the B.t. can easily be separated by precipitation with acetone, using a procedure described by Dulmage et al. (1970). The biomass is centrifuged from the fermentation liquid and is suspended in 1/10 - 1/20 of volume of 4-6 % lactose. Under constant stirring, 4 - 5 parts of acetone are added and the mix is blended in dish for 30 minutes. The product is kept on hold for 10-30 minutes and is filtered by suction (using a water vacuum pump) in a Buchner filter using filtering paper. The biomass on the paper filter is triply flushed with acetone. The second and third filtrations with acetone serve to remove water from the biomass. Then the biomass is allowed to dry on the filter overnight. On the next day the dry powder is transferred into a dish and aggregates are removed by slight wiping action. The culture grown and sporulated on solid agar medium can be processed in the same way after being transferred into acetone by a wire scraper.
From large volumes the B.t. is separated by spray drying. This drying can be preceded by thickening of the fermentation liquid with the biomass, in order to reduce volume. The solution can be thickened by centrifugation or by evaporation. In either process the temperature must not be allowed to rise above 80 oC. In the spray drier the liquid is sprayed in droplets onto heated walls of the drying chamber, and the dry particles are collected at the bottom. The drying time on the walls is no more than several seconds, so that no overheating occurs even with entry temperatures of 150-200oC. The outlet temperature should be less than 80oC.
22.214.171.124 Final Adjustment
The final product, a microbial insecticide of B.t., is of the form of wettable powder, aqueous or oil concentrate (flowable),spraying powder, or granulate. The formulation is so prepared as to afford optimum efficiency toward a specific pest on a specific host (crop).
The formulations contain inert filler for adjustment to the desired activity level. Further, they contain wetting agents and adhesives. The wettable powders contain wetting agents such ascasein, gelatine, lactose, NaCl, Triton-100, soaps, and modern detergents. Inasmuch as these substances also accelerate flushing of the preparations by rainfall or dew, adhesives must also be added such as molasses, syrups, methyl cellulose dried milk, dried blood, latexes, dextrins etc. The objective is to form a water-resistant film on the plant. In no case must these substances repel the pests as this would impair the pathogenicity of the bioinsecticide. Substances added for taste include glucose, lactose, Gustol, molasses, etc.
The emulsified preparations are either water concentrates or oil concentrates. On completion of the fermentation, the spores and crystal are thickened and stabilized by addition of various protective and conserving such as sorbitol, sodium benzoate, xylol, etc. The oil emulsions are suitable for low-volume spray apparatuses. These oil formulations make use of edible oils which are harmless when sprayed onto vegetables and fruits. Granulates represent yet another form of the B.t. formulations. Granulates are made by impregnation of organic or inorganic granules with a thick suspension of bacteria, with or without emulsifier. In water or dew the bacteria will liberate from the granules and exert a long term effect. The media for organic granules can be grains or coarse particles of maize; as mineral granules are used crushed polystyrene or polypropylene or sifted zeolites (bentonite, diatomaceous earth).
In case of field application, the granules will fall into leaf troughs and there they will serve as a permanent source of infection to caterpillars (e.g., corn borer) which tend to penetrate the stalk near the bottom of a walled-in leaf. When applied against mosquitoes, the preferred granules are those anorganic media which will stay on the surface of water fora period of time and later, at the bottom, is used for nutrition by larvae which will thus become infected. Quality inspection, in the form of an assay of biological effectiveness, e.g., against Trichoplusia caterpillars, is done prior to separation by centrifugation, at the time when the semi-product is scaled-down for standard efficiency, i.e., prior to formulating the final product.
3.2 Small-Scale Processing of Semi-Solid Fermentation
Small-Scale Processing in Plastic Fermenters
Small-scale processing of microbial pesticides based on filamentous fungi can be done in plastic fermenters with surface cultivation. The aerial spores "conidia" realized on the surface of the mycelial mat are a homogeneous population of vegetative cells.
The fungi Metarhizium anisopliae = M.a., Beauveria bassiana =B.b. and Verticillium lecanii = V.l. can successfully be culti-vated using the surface technique on a liquid nutrient medium, in polyethylene bags additionally aerated by sterile air.
The production strain is obtained by reverse passaging via a target pest (e.g., with V.l. = Aphids, White fly; with B.b. and M.a. = larvae of beetles - Leptinotarsa decemlineata, Otiorr-hynchus sulcatus) and its cultivation is done on slantagar or on agar in Petri dish.
126.96.36.199 Laboratory Cultivation of Inocula (seed culture)
The strain being passaged is cultivated on agar media and then inoculated into a liquid nutrient medium in Erlenmeyer flasks; then it is cultivated for 3-5 days on a shaker at the temperature of 22 - 25oC or in a laboratory fermenter (1 - 3 litres) by submerged fermentation for the same time and at the same temperature. The contents of the laboratory fermenter is transferred under sterile conditions into a larger inoculating fermenter (15 - 20 litres) where submerged fermentation takes place on another nutrient soil, for the same period of time.
The contents of these inoculation fermenters (submerged blastospores and mycelium) serve for the inoculation of small-scale fermenters of 250 - 1000 litres capacity. In these latter fermenters, prior to inoculation, the nutrient medium is sterilized at 130oC for 30 minutes, whereupon it must be cooled down to 35oC C over about 2 hours; only then can the inoculation take place where the nutrient media is inoculated in small-scale fermenters. The submerged cultivation proper takes place there for 3 to 4 days. The inoculated nutrient medium from the fermenters is transferred to the plastic fermenters via sterile tubing.
Fermentation in plastic fermenters takes place without aeration for the first two days and starting the third day, air is introduced by plastic piping; then the cultivation takes 14- 16 days, at temperatures of 22 - 25oC. The plastic fermenters must be positioned absolutely horizontally, because the layer depth to which they are filled is 1 cm.
188.8.131.52 Small-Scale Processing
(a) The nutrient medium in fermenters is sterilized directly by steam at 130oC
for 30 minutes.
(b) Then the sterile nutrient medium is cooled down to 35oC over 2 hours.
(c) Fermenter inoculation using submerged inoculum (1-3%) from seed fermenters.
(d) Submerged fermentation in the fermenters, for 3-5 days.
(e) Sterilization of the tubing whereby the inoculated medium is to be transferred from above fermenters to the plastic fermenters
(f) Pumping the inoculated nutrient medium from above fermenters into plastic fermenters.
(g) Surface air fermentation in polyethylene bags (cushion) for the first two days, without aeration. Subsequently, surface fermentation for 14-16 days at daylight.
(h) "Recovery" harvesting where a platform 3-5 meters long is tilted and the nutrient medium discharged at the other end. The plastic bag is splitted longitudinally and a wetting agent added (Tween 80), whereupon the contents is centri-fuged using a dense filter cloth. The slurry is mixed with "Siloxyde" filler/dispergator of which the individual particles, of 10 nm in size, are adsorbed on the surface of the conidia having the size of 15-25 æm.
(i) Drying either in air at 25-30oC or in an oven where the temperature of about 35oC is alternated with undercooling; this will condense out the atmospheric humidity thus accelerating the drying on such days when the relative atmospheric humidity is high.
(j) Grinding/milling, taking place in a meat mincer while the harvested conidia with filler still are wet; after-drying in air. This produces coarse granules dispersible in water. Fine milling of the granules to obtain wettable powder is done using a milling machine by CONDUX Co. (Universal Mühle Type 150/S-D) which prevents any heating up of the particles, incl. the conidia. The contents of declared bioactive units is adjusted before packaging the finished product.
(k) Quality inspection (i.e., check on the biological activity) is done after fermentation in the plastic fermenter, in the case of standardization before final formulation, and with the formulated product. The germination and count of the conidia are examined.
3.3 Small-Scale Processing of Microbial Insecticides
The mass production of entomopathogenic fungi is usually undertaken using primitive reactors, in which the solid media is stirred only weekly, or not at all. The media used industrially are mainly cereal grains, broken or not, supplemented with specific nutrients or inert clays, such as vermiculite, impregnated with the culture medium.
Tray reactors consist of several trays containing the media, with or without stage wise transport from one tray to another (Fig. 3-3E). Constant temperature and humidity are maintained by the addition of water or nutrient and the system can be illuminated if this is essential for sporulation. Conidia of B. bassiana, H. thompsonii are produced in such tray fermenters, the inoculum liquid coming from stirred tank reactors. This technology has been adopted in Brazil for the large scale production of M. anisopliae, where rice is fermented in autoclavable plastic bags (Margues et al. 1981).
There are two major disadvantages of the tray reactors: because of the long fermentation cycle (3-4 weeks), the risk of contamination is high; media utilization is inefficient. The homogeneous solid reactors are temperature-controlled vessels in which the medium is mixed by means of a simple rotating arm (Fig. 3-3F). The homogenization of the media is superior to that of the tray but the illumination is lower. Rotary fermenters have been used for the mass production of V. lecanii. The rotating disc fermenter (Fig. 3-3G) allows the development of a fungal biofilm on a series or partially submerged discs which rotate slowly in a trough of medium, exposing the film to the nutrient solution and air. Film thickness is self-regulating since excess growth sloughs from the carrier surface (Anderson, 1983). These fermenters would be suitable for the large scale production of sexual spores but costs would be high.
3.3.1 Processing of Microbial Insecticide on Solid Media
The fungi Beauveria bassiana and Metarhizium anisopliae have been known for a long time as insect pathogens with rather high pathogenous effect on diverse species of different Genus of insects. Amount the few examples of practical application could be named Leptinotarsa decemlineata, Mahanarva posticata, Othiorhynchus sulcatus and a products called "Boverin" and "Metaquino".
It is possible to produce B. bassiana and M. anisopliae quite simply on solid and sterilized media containing conidia. However, this technique is hardly applicable for industrial processing of "large-scale" type. Both fungi species grow successfully on liquid media, but they produce blastospores showing shorter viability than conidia.
The aim of production is technology processing for production of a sufficient amount of conidia B. bassiana and M. anisopliae to enable application in the field by smallholders.
184.108.40.206 Preparing Seed Inoculum
M. anisopliae a B. bassiana must be obtained from Research Stations or must be isolated from the nature where this fungi caused an endemic epidemic on the pest.
Both fungi are growing on Sabouraud`s agar. In the case of self isolation from green muscardine streptomycin (500 mg/l) must be added to agar, as soon as the temperature of the sterilizer medium dropped to approx. +40 ... 50oC. This treatment is sufficient to obtain a pure culture fully clear of any bacteria.
Seed inoculum is better prepared as a submersion culture in seed fermenter or in Erlenmeyer flasks on shaker.
220.127.116.11 Processing by Cultivation
B. bassiana is cultivated on different media: wheat bran, wheat hybrid, oat semolina.
The mass of 30g of media with 45 ml water was poured to glass bowls. The treatment was in a sterilizer for 30 minutes at 120oC and pressure 1 mPa/cm2. The media, after cooling to approx. 35oC, is inoculated by the spores from the agar or by blasto-spores and mycelium from a seed fermenter. The culture is stored in a thermostat at 25oC +/- 1oC and ca. 75 % R.H.
M. anisopliae is cultivated on rice or wheat bran: Rice should give good results and could be used in all following cultures. The mass of 300 g boiled rice (for 10 min.) is put into a bag with a synthetic sponge in its opening to enable air circulation. The bags are treated twice in a sterilizer in the interval of two days under the above mentioned conditions. The rice in bags is then inoculated by a single use syringe. The spores were taken from agar in Petri dish or from seed fermenter. To prevent tearing of a bug it is necessary to attach a piece of adhesive plastic tape at the place of injection, another piece being required to cover the hole after inoculation to prevent contamination. The bags are opened after sporulation (approx. after 15 ....20 days) and dried it at 30oC at 30 % R.H. for three days. After this treatment the cultures are stored in a refrigerator at approx. + 8oC before being counted and used. The test of germination of conidia is possible to be done on microscopy slides in a wet chamber. Orange juice (O.02 %) in a drop suspension in dist. water and conidia support the germination of conidia (Táborsky, unpubl. data).
18.104.22.168 Formulation of Product
(a) The "Tween 80" of 0.02 or 0.05 % concentration in dist. water is added to
(b) The dist. water with Tween 80 is poured into bags and is shaked in a shaker for 30 min.
(c) The suspension is filtered using a cloth filter.
(d) The liquid containing spores is centrifuged for 15 minutes at 1500 R.P.M..
(e) Superrnatan is discarded and the sediment containing conidia is resuspended in dist. water, mixed with Siloxyl, and dried for 24 or 48 hours at 30oC. The spores powder in 1 gram contains 109 of conidia. The so called Burker chamber is used for counting of spores.
M. anisopliae is cultivated much more easily than B. bassiana on rice broth, without serious difficulties. Contamination can be avoided in most cases due to the fast growth of these fungi. The spores production of M. anisopliae is more than twice that of B. bassiana.
The cultivation in plastic bags is very practical and easily manageable when the bags are carefully handled to avoid tearing. The bags also enable an even division of the mycelium, easily transferred from one place to another. However, the flasks cultivation brings the advantage of an absolutely aseptic work, since the inoculation is carried out directly from agar tablets into flasks. On the other hand, the use of plastic bags requires first to get a water suspension of spores and then make use of disposable syringe as described above. Yet, this technique increases risk of contamination. The mixture of raw rice and water from water supply system at the rate of 2:1 was found sufficient, after sterilizing, to provide good growth and sporulation. Further, the suspension of spores is almost completely cleared of rice remainders thus making the drying of spores easier.
The powder production of spores is successful only in the case when we get a clear suspension of spores from the rice particles. The filling by dry and inert substances as kaolin, talc, Siloxyl makes the handling with the powder easier. The storage of spores` mixture with rice is also possible.
ANDERSON, J.G., 1983: Immobilized cells and film reactor systems for filamentous fungi. In: The filamentous fungi Vol. 4 Fungal Technology (Ed. J.E. Smith, D.R. Berry and Kristiansen), Edward-Arnold Publ., London, pp. 145-170.
AOKI, J., 1967: Some considerations on the infection mechanisms of insect pathogenic fungi: nitrogen utilization of Beauveria bassiana, Isaria farinosa and Isaria fumosa-rosea.Proc. US-Jap. Sem. Microbial Contr. Pests, pp. 107-113.
DULMAGE, H.T., 1970: (See chapter 2. )
FARGUES, J., l981: Spécificité des Hyphomycétes entomopathogénes et réesistance interspécifique des larves d`insectes. Doct. D`Etat., Univ. Paris 6, 2 vol.
HAVKER, L.E., 1966: Environmental influences on reproduction. In: The fungi; an advanced treatise (Eds. G.C. Ainsworth and A.S. Sussman), Acaddemic. Press. New York and London., pp. 435-469.
KRISTIANSEN, B. and CHAMBERLEIN, H.E. 1983: Fermenter design. In: The filamentous fungi. Vol. 4 Fungal technology (Eds. J.E.Smith, D.R. Berry and B. Kristiansen), Edward Arnold Publ. London, pp. 1-19.
LATTEG, J.P., SOPER, R.S., MADOR, C.D. 1977, Media suitable for industrial production of Entomophthora virulenta zygospores. Bio. Bioengin.. 19: 1269-1269-1284.
LATTGE, J.P., COLE, G.E., HORISBERGER, M. and PREVOST, M.C., 1986: Ultrastructure and chemical composition of the ballistospore wall of Conidiobolus obscurus. Exp. Mycol. 10:99-113.
LATTGE, J.P., CABRERA-CABRERA, R.I. and PREVOST, M.C. 1988: Microcycle conidiation in Hirsutella thompsoni. Can.J. Microbiol
MARGUES, E.J., VILLASBOAS, A.M., and PEREIRA, C.E.F. 1981: Orientacoes tecnicas para a producao do fungo entomogeno Metarhizium anisopliae em laboratorios setoiaris. Boletim tecnico Planalsucar, Piracicaba 3:5-23.
PIRT, S.J., 1975: Principles of microbe and cell cultivation. Blackwell Sc. Publication, Oxford, 274 pp.
SAMSON, R.A., EVANS, H.C. and LATTGE, J-PAUL. 1989: Atlas of Entomopathogenic Fungi. Springer Verlag. pp. 187. Sinclair,
MAVITUNA, C.G. AND F. 1983: Mass and energy transfer. In: The filamentous fungi. Vor 4. Fungal technology (Eds. J.E. Smith, D.R. Berry and B. Kristiansen). Edward Arnold Publ. London. pp. 20-76.
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