Title of Invention

"A COMPOSITION USEFUL AS MEDIUM FOR MASS PRODUCTION OF FUNGI"

Abstract

The present invention provides a novel medium based on inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor, molasses and some inorganic salts in a definite proportion useful for fungus growth. Existing processes take 5-9 days for the production of 10 fold increase in the number of spore of Trichoderma species from the initial inoculum as against in the present novel medium wherein 100 fold increase in the number of spores is achieved in 48 hours.

Full Text

The present invention relates to a composition useful as medium for mass production of fungi The present invention particularly relates to the development of a novel medium composition based on inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor and molasses useful for the production of spores of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and any other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions. Trichoderma species are used as efficient biocontrol agents against the soil-borne Wilt and Root-rot diseases of various crop plants. With the growing awareness of the harmful effects of many synthetic chemical pesticides on humans, non-target animals and plant species, and the environment as a whole, there has been a shift in attention to the research and development of more environment-friendly methods of pest and disease control. Use of environmentally safe Disease Management Technologies based on Biopesticide and Biocontrol agents through natural enemies - parasitoids, predators and pathogens in integrated pest management programmes are ever increasing. Biocontrol agents like Trichoderma species and Gliocladium species are examples of some of the biocontrol agents used in the management of root diseases of chickpea, peas, groundnut, soybean spices and vegetables. Root-rot diseases of crops in general and pulses in particular are a serious problem in rain-fed areas caused mostly by species of Fusarium and Rhizoctonia etc. Chemical control of these diseases requires a continuous use of fungicides, which is not economical and causes deleterious effects on the rhizosphere microflora and also have the residual problems. WHO now has banned even some of the commonly used fungicides due to their severe toxicity. Under these circumstances the biocontrol agents can be used for the management of Root-rot diseases at comparatively cheaper cost with long term antagonistic effects against the pathogens because these biocontrol agents multiply and remain near the root zone of the plant giving protection to these plants throughout the growth period. Investigations carried out world over have confirmed, that synthetic plant protection chemicals such as fungicides, insecticides and herbicides deteriorate soil fertility. Such deterioration occurs through disappearance of bio-diversity in soils. On account of this, several strains of soil microorganisms have been used which are able to suppress a wide range of disease causing microorganisms. Jackson et al., (Enzyme-Microb. Technol.; (1991) 13, 6, 456-61) reported the culture media optimisation for production of the biological control agents Gliocladium virens G20, Trichoderma pseudokoningii IMI 322662, and T. viride IMI 322659 and IMI 322663. In glucose-alanine medium, the optimum dry wt./g carbon occurred with a C:N ratio of 15:1. Addition of 3.28-mg atoms iron/1 to the medium increased biomass production of all isolates, but a concentration of 164-mg atoms/1 was toxic to the Trichoderma species. Growth decreased in media lacking Mg, P, K or S, but the amount of the decrease differed between the four isolates. Sporulation in agar was reduced in the absence of Mg, P, K and N. Addition of biotin, p-amino - benzoic acid and thiamine-HCl increased biomass production slightly. The glucose-alanine basal medium supported better growth of all 4 isolates than a commercial molasses-yeast medium; conidia production was greater in the molasses-yeast medium. The pH of the glucose-alanine medium remained constant at 4.5, whereas the pH of the yeast-molasses medium (initially 5.5) increased to 8.0-8.6. Chlamydospores were produced by all isolates, but the numbers varied according to the culture conditions used. Gervais and Sarrette ( J.Fement.Bioeng. (1990, 69, 1, 46-50) reported the Emerson agar medium (1 8 g/1 agar) containing sodium octanoate (1 g/1) as a 2-heptanone precursor and glycerol as a water activity depressor for the solid-state fermentation of T. viride for cheese aroma production. Cultures were grown in Petri dishes at 20 °C. Sporulation was visible on the 9th day. Toyama et al (J. Ferment. Technol.; (1983, 61, 4, 409-11) reported a sporulation medium for T. reesei QM 9414, to be used for the production of protoplasts. Kennedy-M-J; Davies-R-J; Surrey-M-R; Reader-S-L; Hoefakker-P-C, Australia's. Biotechnol.; (1995) 5, 6, 349-54 reported preparation of biological control agents based on Serratia entomophila, Bacillus thuringiensis, Pestalotia species, Truncatella augustata, Trichoderma viride, Trichoderma species., cricket-paralysis virus, flock-house virus. The report focuses on the 4 areas of process expertise required to develop and produce a commercial biological control agent: (a) culture medium design, (b) scale-up of the biological production system, (c) downstream processing of the product and (d) extending the shelf life of the biological control agent. Nigam P. (Process-Biochem.; (1994, 29, 5, 337-42) reported a medium containing diluted molasses solutions of 3-4% sugar concentration for T. viride QM 9414, T. reesei Rut-C-30 NRRL 11460 under submerged fermentation conditions (SF) performed in flasks in 100 ml medium with agitation at 180 rpm for 5 days. Culture media for two-stage culture of T. longibrachiatum, T. viride, T. aureoviride, AN: 91-13819, CA: Moscow-Tech.lnst.Food-Ind. were reported. The culture medium (pH 4.0-4.2) used in stage I had the following composition (wt.%): wheat bran, 0.3-0.4; additional hydrolysed cotton cake, 0.3-0.4; gibbersib (sic) biomass, 0.45-0.50; NH4H2P04, 0.25-0.30; K2S04, 0.20-0.22; and MgSO4, 0.025-0.030. The culture medium used in stage 11 (pH 5.0-5.2) had the following composition (wt.%): beet-root pulp, 2.5-2.8; wheat bran, 0.3-0.4; additional hydrolysed cotton cake, 0.3-0.4; K2S04, 0.25-0.30; NH4NO3, 0.25-0.30 and stomach contents hydrolyzate containing reducing compounds, 0.20-0.25. Abou-Zeid (Bioresource-Technol.; (1991) 37, 3, 239-42) reported the following medium (pH 6) (g/1): cellulosic-source, 10.0; (NH4)2S04, 2.0; KH2P04, 1.0; KC1, 0.5; MgS04.7H20, 0.5; MnSO4.4H20, 0.05; and FeSO4.7H20, 0.005, for the Fermentation of T. viride using leaflets and midribs of date palm (Phoenix dactylifera) leaves dried at 100-105 ° C, 144 hr at 30° C with 200 rpm agitation. Pre-treatment of palm leaflets with 1% NaOH and 5% H2S04 improved their suitability as C-sources for T. viride growth. The conventional media reported earlier are based on expensive gradients such as yeast extract, malt extract, protein hydrolysates and the like. Such a media are not suited for the economic mass production of these biocontrol agents by fermentation at technical scales. We designed the medium for the mass production of spores of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and other scantily sporulating species of fungi under solid state,- surface and submerged fermentation conditions based on inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor and molasses. These agro-waste industrial by products are rich in micro-nutrients and other essential elements like sugars, amino acids providing synergistic effects on the growth and sporulation of these fungi. The present invention relates to the development of a novel medium based on inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor, molasses and some inorganic salts in a definite proportion useful for the production of viable spores of Trichoderma species (used as biocontrol agents) and a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and other scantily sporulating species of fungi under solid state and submerged fermentation . Existing processes take 5 - 9 days for the production of 10 fold increase in the number of spore of Trichoderma species from the initial inoculum as against in the present novel medium wherein 100 fold increase in the number of spores is achieved in 48 hours. This, therefore, makes the present invented process an economical , fast and highly advantageous from the commercial viewpoint. The above novel medium works equally efficiently for other ascomycetes also, though not exemplified herein. The main objective of the present invention is to provide a novel composition of fermentation medium useful for mass production of spores of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and any other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions. Another objective of the present invention is to prepare a medium based on inexpensive agro-based industrial by-products like beetroot pulp, wheat bran, corn-steep liquor and molasses, preferably com-steep liquor and molasses. Accordingly the present invention provides a composition useful as growth medium for fungi essentially comprising of carbon and nitrogen rich agro-based industrial by-products selected from beet-root pulp, wheat bran, corn-steep liquor and molasses preferably corn-steep liquor and molasses , said composition comprising Molasses 5-20 (g/l), Corn Steep liquor (CSL) 10- 25 (g/l), Sodium chloride 5-15 (g/l), CaSO4 0.1 - 0.5 (g/l), KH2PO4 0.001-0.01 (g/l), MgS04.7H2O 0.001-0.01 (g/l), CuSO4 0.001-0.0050 (g/l), FeSO4 0.0009 - 0.005 (g/l) and silicone oil 0.1 - 0.5 (g/l). Fermentation procedure: The composition of the present invention has been described, which is useful for the production of viable spores of spores of Trichoderma species and a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions based on the inexpensive agro-waste industrial by products like beet-root pulp, wheat bran, corn-steep liquor and molasses, preferably corn-steep liquor and molasses. Ingredients based on agro-waste industrial by-products are high energy and protein rich natural sources containing large number of trace elements and are very often used in culture media. However, these ingredients have not been used for inducing early sporulation in ascomycetous fungi. These ingredients in the medium result in very fast and better sporulation under submerged fermentation conditions when compared to conventional medium (Molasses-yeast extract medium) containing yeast extract, peptone, malt extract and refined sugars like glucose, & sucrose. The composition of the specially designed fermentation medium (FM1) has the following constituents: Constituents Amount (g/I) Molasses 5-20 Corn Steep Liquor (CSL) 10-25 Sodium chloride 5-15 CaSO4 0.1-0.5 KH2PO4 0.001-0.01 MgSO4.7H20 0.001-0.01 CuSO4 0.001-0.0050 FeSO4 0.0009-0.005 silicon oil 0.1-0.5 The above ingredients were dissolved in de-ionised water and steam sterilised at 121 °C / 15 Ib for 15 min. The sterilisation of the medium is done ex-situ for the operation of small bench fermentors and in-situ when large vessels are operated. For initiation of the fermentation process, spores from the a week old culture of Trichoderma species raised on solid medium (FM1 medium with agar) are eluted with pre-sterile Tween 80 solution (0.02% v/v in water) and inoculated in the fermentor to the final concentration of Ix 106 conidia /ml. Fermentation is carried out in FM1 medium in cylindrical glass jars or stainless steel vessels at 29 +1°C. The vessels are agitated with standard stirrers at a moderate rate of 200-400 rpm and aerated at a constant air flow of 0.5 to 1.0 volumes per volume of liquid per minute at Ibar (kg) over pressure. All the inoculated spores germinate and vegetative growth of the ftingus with a numerous chlamydospores emerges within 24-h fermentation period. Thereafter, 3porulation sets in and chlamydospores disappear completely within next 24-h fermentation period. The fermentation is terminated after an incubation period of 48-72 h or until the maximum conidiospore concentration of 1-5 x108/ml is obtained and no chlamydospores are spotted under microscopic examination. The whole culture broth is thereafter thoroughly mixed with pre-sterilised lignite (dry heated at 120° C for 6-8 h. and later cooled to room temperature) of mesh size 200-250 in the ratio of 1: 2 and packed in 200 g packets in milky white pollywogs (size 6"xlO") of 50-75 jag thickness. The biocontrol formulation thus prepared can be stored at 4°C for a period of 12 months and at 30°C upto 6 months, without significant loss of viability. The Following examples are given by way of illustration and should not construe the scope of the invention. Example 1: Production of spores of Trichoderma species in shake flasks using FM1 medium. Strains of Trichoderma species were obtained from culture repository of Indian Agriculture Research Institute New Delhi. The cultures are maintained on standard Potato-dextrose medium (Potatoes 300 (g/1), glucose 20 (g/1) agar 17 (g/1)). For the purpose of mass propagation of its spores, the culture is pre-grown for a week in test tubes or large suitable containers like Roux bottles on FM1 medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2PO4 0.0060 (g/1), MgS04.7 H20 0.0050 (g/1), CuS04 0.0010 (g/1), FeS04 0.0016 (g/1), and additional Agar 15.00. For the production of the spores of Trichoderma species at the shake flask scale, the culture is raised in an Erlenmeyer flask of 500 ml capacity containing 100 ml of above described FM1 medium without agar, autoclaved at 121 °C /15 Ib for 15 min. The medium was inoculated with the spores of Trichoderma species with final concentration of Ix 106 conidia /ml from one week old slant eluted in pre-sterile Tween 80 (0.02 % v/v in water) solution and incubated on rotary shaker agitated at 180 revolutions/ minute and 2.5 cm throw at 30°C for 4 days. At the end of the fermentation time no vegetative cells or chlamydospores were seen under microscope. An average conidia count of 5x 108/ml broth was obtained. The viability of spores as determined by colony forming units on standard Potato Dextrose Agar (PDA) was of the order of 95 to 100 %. Spore count (5x 108/ml) is achieved in this novel medium is 100 times from the initial inoculum during the above incubation period. On the other hand in conventional medium (Molasses-yeast extract medium containing Molasses 7.50 (g/1), Glycerin 7.50 (g/1), Sodium Chloride 10.00 (g/1), Yeast Extract 5.00 (g/1), CaSO4 0.25 (g/1), KH2PO4 0.006 (g/1), MgS04.7 H2O 0.005 (g/1), CuS04 0.001 (g/1), and Trace Elements 0.0016 (g/1)), it is only five fold during the same period. Besides, the number of chlamydospores in the conventional medium is much higher at any given time as compared to the new medium. Chlamydospores are not considered useful for packaging the fungi and only the conidiospores are used for downstream processing due to the better viability of the latter. The higher number of Chlamydospores in the conventional medium (Molasses-yeast extract) make it much inferior compared to the designed novel medium (FMl). The viable conidia thus generated under submerged conditions are mixed with pre-sterilised lignite of 200 - 250 mesh size by doing so the spores remain viable for 6-8 months at 30°C. This lignite carrier based spores of Trichoderma species preparation was field tested on several crops with satisfactory results. Example 2: Production of spores of Trichoderma species in shake flasks using FMl medium with higher concentration of ingredients. The seed of spores of Trichoderma species was raised in Erlenmeyer flasks in FMl medium (medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSC-4 0.2500 (g/1), KH2PO4 0.0060 (g/1), MgS04.7 H2O 0.0050 (g/1), CuS04 0.0010 (g/1), FeSO4 0.0016 (g/1)) as described in example 1. For the production of spores of Trichoderma species at the shake flask scale, the culture is raised in an Erlenmeyer flask of 500 ml capacity containing 100 ml of FMl medium with following ingredients Molasses 20.00 (g/1), Corn Steep Liquor (CSL) 25.00 (g/1), Sodium chloride 15.00 (g/1), CaS04 0.500 (g/1), KH2P04 0.01 (g/1), MgS04.7 H2O 0.01 (g/1), CuS04 0.0050 (g/1), FeSO4 0.005 (g/1)) without agar, autoclaved at 121°C /15 Ib for 15 min. The medium was inoculated with the spores of Trichoderma species with final concentration of Ix 106 conidia /ml from one week old slant eluted in pre-sterile Tween 80 (0.02 % v/v in water) solution and incubated on rotary shaker and agitated at 180 revolutions per minute and 2.5 cm throw at 30°C for 4 days. At the end of the fermentation time no vegetative cells or chlamydospores were seen under microscope. An average conidia count of 5x 10 /ml broth was obtained. The viability of spores as determined by colony forming units on standard Potato Dextrose Agar (PDA) was of the order of 92 to 100 %. In the FM1 medium (with higher concentrations of ingredients) spore count (5x o 10 /ml) is achieved which is also 100 times from the initial inoculum during the above incubation period as against the conventional medium (Molasses-yeast extract medium containing Molasses 7.50 (g/1), Glycerin 7.50 (g/1), Sodium Chloride 10.00 (g/1), Yeast Extract 5.00 (g/1), CaSO4 0.25 (g/1), KH2PO4 0.006 (g/1), MgS04.7 H2O 0.005 (g/1), CuS04 0.001 (g/1), and Trace Elements 0.0016 (g/1)). Only five-fold increase in the number of spores from the initial inoculum is achieved in the Molasses-yeast extract medium during the same incubation period. Besides, the number of chlamydospores in this medium is much higher at any given time as compared to the new medium. However, the spore concentration per ml and incubation period remained the same in both FM1 medium with higher concentrations of ingredients and FM1 medium with lesser concentration of ingredients (medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2P04 0.0060 (g/1), MgS04.7 H2O 0.0050 (g/1), CuS04 0.0010 (g/1), FeSO4 0.0016 (g/1). Example 3: Production of spores of Trichoderma species in 7 litre batch fermentor using the FMl medium. The seed was raised in Erlenmeyer flasks in FMl medium (medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2PO4 0.0060 (g/1), MgSO4.7 H2O 0.0050 (g/1), CuS04 0.0010 (g/1), FeSO4 0.0016 (g/1) as described in example 1. 7 litres of FMl medium (medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2PO4 0.0060 (g/1), MgS04.7 H2O 0.0050 (g/1), CuS04 0.0010 (g/1), FeSO4 0.0016 (g/1) along with 3ml of silicon oil as antifoam agent was sterilised in-situ in a 101 fermentor (New Brunswick, USA) at 121°C /15 Ib. for 15 min. The medium on cooling was inoculated with 100 ml of freshly grown seed culture with the final spores concentration of 2 -3 x 106. The fermentor was set with the following parameters: Agitation = 250 rpm Aeration rate = 0.5 vvm Temperature = 29°C ± I Pressure = 1.2 psig The fermentation was continued for 72 hours until the spore concentration of 1.0 x 109 was achieved (Table 1). Data of periodic sampling for spore counting at regular intervals of time Using both FMl and conventional medium (Molasses-yeast extract medium containing:Molasses 7.50 (g/1), Glycerin 7.50 (g/1), Sodium Chloride 10.00 (g/1), Yeast Extract 5.00 (g/1), CaSO4 0.25 (g/1), KH2PO4 0.006 (g/1), MgS04.7 H2O 0.005 (g/1), CuS04 0.001 (g/1), and Trace Elements 0.0016 (g/1)) media are shown in the Tables 1&2. Table 1: Growth and sporulation of Trichoderma species in 7L fermentor using novel FM1 medium (Table Removed) Table 2: Growth and sporulation of Trichoderma species in 7L fermentor using conventional (Molasses- yeast extract) medium (Table Removed) This novel medium (FM1) reduces fermentation period from 72 hrs to 48 hrs for sporulation. Moreover spore count (3.0x108 ) is achieved in this novel medium which is 100 times from the initial inoculum in 48 hrs whereas in case of conventional medium (Molasses-yeast) it is less than 10 times (l.OSxlO7) 72 hrs. Besides, number of chlamydospores in conventional medium is much higher as compared to the novel medium as any fixed time internal. Example 4: Production of spores of Trichoderma species in 7 litre batch fermentor using FM1 medium with higher concentration of ingredients. The seed was raised in Erlenmeyer flasks in FM1 medium (medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2PO4 0.0060 (g/1), MgSO4.7 H2O 0.0050 (g/1), CuSO4 0.0010 (g/1), FeSO4 0.0016 (g/1) as described in example 1. 7 liters of FM1 medium with following ingredients Molasses 20 (g/1), ;Corn Steep Liquor (CSL) 25.00 (g/1), Sodium chloride 15.00 (g/1), CaSO4 0.500 (g/1), KH2P04 0.01 (g/1), MgSO4.7 H2O 0.01 (g/1), CuS04 0.0050 (g/1), FeSO4 0.005 (g/1) without agar, along with 3ml of silicon oil as antifoam was sterilised in-situ in a 10 1 fermentor (New Brunswick, USA) at 121 °C /15 Ib. for 15 min. The medium on cooling was inoculated with 100 ml of seed culture with the final spores concentration of 2-3 x 106, The fermentor was set with the following parameters: Agitation = 250 rpm Aeration rate = 0.5 vvm Temperature = 29°C + I Pressure = 1.2 psig The fermentation was continued for 72 hours until the spore concentration of 1.0 x 10 was achieved (Table 3). Data of periodic sampling for spore counting at regular intervals of time using both FM1 and conventional (Molasses-yeast extract medium containing Molasses 7.50 (g/1), Glycerin 7.50 (g/1), Sodium Chloride 10.00 (g/1), Yeast Extract 5.00 (g/1), CaSO4 0.25 (g/1), KH2PO4 0.006 (g/1), MgSO4.7 H2O 0.005 (g/1), CuS04 0.001 (g/1), and Trace Elements 0.0016 (g/1) media are shown in the Tables 3&4. Table 3: Growth and sporulation of Trichoderma species in 7L fermentor using novel FM1 medium with higher concentration of ingredients (Table Removed) Table 4: Growth and sporulation of Trichoderma species in 7L fermentor using conventional (Molasses- yeast extract) medium (Table Removed) Perusal of the fermentation data show that the spore concentration/ml and incubation period remained the same as in the FM1 medium with lesser concentration of ingredients (medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2PO4 0.0060 (g/1), MgSO4.7 H2O 0.0050 (g/1), CuSO4 0.0010 (g/1), FeS04 0.0016 (g/1). Thus the novel medium (FM1) with lower concentrations of ingredients is more economical & reduces fermentation period from 72 hrs to 48 hrs for sporulation as compared to the conventional medium (Molasses-yeast extract). Example 5: Production of spores of Trichoderma species in 50 litre pilot fermentor using the FM1 medium. Spores of Trichoderma species raised in Roux bottles containing solid FM1 medium of the composition as described in Examples 1 &3 above were eluted in Tween 80 (0.02 % in water). 50 litres of FM1 medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2P04 0.0060 (g/1), MgSO4.7 H20 0.0050 (g/1), CuSO4 0.0010 (g/1), FeS04 0.0016 (g/1) as described in Examples 1 &3 along with 15-25ml of silicon oil as antifoam (the amount of silicon oil varied depending upon development of foam during fermentation) was sterilised in-situ in the 100 1 fermentor (Andel, India) at 121 °C /15 psig for 15 min. The medium on cooling was inoculated with 450-550 ml of spore suspension in Tween 80 solution with the final spores concentration of 1 x 106/ml. The fermentor was set with the following parameters: Agitation = 250 rpm (revolutions per min.) Aeration rate = 0.5 vvm (volume per volume per min.) Temperature = 29°C + I Pressure = 1 .2 psig (pounds per square inch x g) The fermentation was continued for 48 hours until the spore concentration of 2.3 x 108 was achieved (Table 5). Periodic sampling for spore counting was carried out at regular intervals of time as shown in the Table 5. Table 5: Growth and spornlation of Trichoderma species in 50-L fermentor using new FMl medium (Table Removed) Example 6: Production of spores of Trichoderma species in 50 litre pilot fermentor using FMl medium with higher concentration of ingredients. Spores of Trichoderma species raised in Roux bottles containing solid FMl medium of the Composition as described in Examples 1,3 &5 above were eluted in Tween 80 (0.02 % in water) solution as described in examples 1,3 and 5 above. 50 litres of FMl medium with following ingredients Molasses 20 (g/1), Corn Steep Liquor (CSL) 25.00 (g/1), Sodium chloride 15.00 (g/1), CaS04 0.500 (g/1), KH2PO4 0.01 (g/1), MgSO4.7 H2O 0.01 (g/1), CuSO4 0.0050 (g/1), FeSO4 0.005 (g/1) without agar, along with 15-25ml of silicon oil as antifoam (the amount of silicon oil varied depending upon development of foam during fermentation) was sterilised in-situ in the 100 1 fermentor (Andel, India) at 121°C 715 psig for 15 min. The medium on cooling was inoculated with 450-550 ml of spore suspension in Tween 80 solution with the final spores concentration of 1 x 10 /ml. The fermentor was set with the following parameters: Agitation =250rpm (revolutions per min.) Aeration rate = 0.5 vvm (volume per volume per min.) Temperature = 29°C + I Pressure = 1.2 psig (pounds per square inch x g) The fermentation was continued for 48 hours until the spore concentration of 2.3 x 108 was achieved (Table 6). Periodic sampling for spore counting was carried out at regular intervals of time as shown in the Table 6. Table 6: Growth and sporulation of Trichoderma species 50 L fermentor using FM1 medium with higher concentration of ingredients. (Table Removed) Perusal of the data show that the novel medium (FMl) with lower concentrations of ingredients is more economical and has the same spore concentration/ml and incubation period as the FMl medium with higher concentration of ingredients. Further the FMl medium is far superior than the conventional medium (Molasses-yeast extract medium containing Molasses 7.50 (g/1), Glycerin 7.50 (g/1), Sodium Chloride 10.00 (g/1), Yeast Extract 5.00 (g/1), CaSO4 0.25 (g/1), KH2PO4 0.006 (g/1), MgSO4.7 H2O 0.005 (g/1), CuSO4 0.001 (g/1), and Trace Elements 0.0016 (g/1) when spore concentration/ml and incubation period is taken into consideration. Advantages: The process has the following advantages: 1. The major constituents of the medium for the mass production of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and any other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions, are inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor and molasses. 2. The designed medium (FMl (medium containing Molasses 7.50 (g/1), Corn Steep Liquor (CSL) 20.00 (g/1), Sodium chloride 10.00 (g/1), CaSO4 0.2500 (g/1), KH2PO4 0.0060(g/l), MgSO4.7 H2O 0.0050 (g/1), CuSO4 0.0010 (g/1), FeSO4 0.0016 (g/1) is not only inexpensive but results in profuse sporulation of the order of 200-300 million coniodiospores (2-3 xlO8 spores/ml) of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and any other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions. Trichoderma species are used as efficient biocontrol agents against the soil-borne Wilt and Root-rot diseases of various crop plants. 200-300 million coniodiospores (2-3 xl08 spores/ml) are produced in 48 h under submerged fermentation conditions in large fermentors compared to 7 days using the conventional media (Molasses-yeast extract medium containing Molasses 7.50 (g/1), Glycerin 7.50 (g/1), Sodium Chloride 10.00 (g/1), Yeast Extract 5.00 (g/1), CaSO4 0.25 (g/1), KH2PO4 0.006 (g/1), MgSO4.7 H2O 0.005 (g/1), CuSO4 0.001 (g/1), and Trace Elements 0.0016 (g/1). This novel medium (FMl) reduces fermentation period from 72 hrs in conventional medium to 48 hrs for sporulation. Moreover spore count (3.0xl08) is achieved in this novel medium which is 100 times from the initial inoculum (4-5 x I O6) in 48 hrs whereas in case of conventional medium (Molasses-yeast) it is less than 10 times (l.OSxlO7) in 72 hrs. Besides, number of chlamydospores in conventional medium (Molasses-yeast) is much higher as compared to the novel medium (FM1) at any fixed time internal. The spores of this biocontrol agent can be directly blended with the carrier agent without any further processing and used directly in the fields. The present invention particularly relates to the development of a novel medium for the production of spores of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and any other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions. The above novel medium composition is based on inexpensive agro-based industrial byproducts like beetroot pulp, wheat bran etc., particularly corn-steep liquor and molasses useful for growth and sporulation of these fungi. We Claim: 1. A composition useful as growth medium for fungi, essentially comprising of carbon and nitrogen rich agro-based industrial by products selected from beet-root pulp, wheat bran, corn-steep liquor and molasses preferably corn-steep liquor and molasses, said composition comprising Molasses 5-20 (g/l), Corn Steep liquor (CSL) 10- 25 (g/l), Sodium chloride 5-15 (g/l), CaS04 0.1 - 0.5 (g/l), KH2PO4 0.001-0.01 (g/l), MgSO4.7H20 0.001-0.01 (g/l), CuSO4 0.001-0.0050 (g/l), FeSO4 0.0009 - 0.005 (g/l) and silicone oil 0.1 - 0.5 (g/l). 2. A composition useful as medium for mass production of fungi substantially as herein described with reference to the examples.

Documents:

988-del-2000-abstract.pdf

988-del-2000-claims.pdf

988-del-2000-correspondence-others.pdf

988-del-2000-correspondence-po.pdf

988-del-2000-description (complete).pdf

988-del-2000-form-1.pdf

988-del-2000-form-19.pdf

988-del-2000-form-2.pdf

988-del-2000-form-3.pdf