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ThesisItem Open Access Isolation and identification of bacteria having pathogenic interactions with termites (Isoptera)(CCSHAU, 2007) Yuvraj singh; Sindhu, S.S.In the present study, the population of bacteria in termite nest soil varied from 1.2 x 106 to 90.0 x 106 colony forming units/g soil on nutrient agar medium. On soil extract agar medium, the bacterial population varied from 28.3 x 106 to 103.2 x 106 cfu/g soil and it varied between 2.6 x 106 to 256.0 x 106 cfu/g soil on King’s B medium. Based on colony morphology, gum production, colony characteristics and pigment production, bacterial isolates NNY 1-NNY 84 were selected from nutrient agar medium, isolates NSY 1-NSY 40 from soil extract agar medium and isolates NKY 1-NKY 96 were selected from King’s B medium. From the gut of termites, bacterial isolates GNY 1-GNY 20 were selected from nutrient agar medium plates. Isolates GSY 1-GSY 10 were obtained from soil extract agar medium and isolates GKY 1-GKY 20 were obtained from King’s B medium. A total of 270 bacterial isolates, i.e. 220 isolates from termite nest soil and 50 isolates from termite gut, were screened for the potential to produce different enzymes, i.e. lipase, protease and chitinase on specific media. In the preliminary screening, only 83 isolates were found to express one or more of the enzyme activities. Seven bacterial isolates without any enzyme activity were also selected. For lipolytic activity, the clearance zone to colony growth ratio varied from 1.24 to 4.20 in different bacterial isolates. Bacterial isolates NNY 73 and NSY 19 showed very little lipase activity and eight isolates did not show any lipase activity. Three isolates NNY 19, NNY 39 and NSY 20 showed 3.50, 4.20 and 4.00 enzyme production efficiency, respectively. Proteolytic enzyme production in different cultures varied from 1.24 to 2.29. The bacterial isolates NNY 24, NNY 50 and NNY 62 showed highest ratio of clearance zone to colony size i.e. 2.17, 2.14 and 2.29, respectively. Fourteen bacterial isolates did not show any proteolytic activity. Chitinolytic activity varied from 1.24 to 2.67 in different bacterial isolates. Isolates NNY 18, NNY 52, NNY 73 and GKY 10 showed very little chitinolytic activity. Four isolates, NNY 23, NNY 43, NNY 34 and NKY 91 showed 2.67, 2.50, 2.40 and 2.50 enzyme production efficiency, respectively. The bacterial isolates NNY 58, NKY 48 and NKY 69 expressed only chitinolytic activity. Isolate NKY 66 expressed only proteolytic activity whereas isolates NKY 17, NKY 62 and NKY 79 expressed only lipolytic activity. Twelve bacterial isolates were found to express all three enzyme activities. During studies of pathogenic interactions, only 47 bacterial isolates were found to kill the termites at 5 day of observation. The killing frequency of different bacterial isolates varied from 5.7 to 100 per cent at 5 day. Bacterial isolates NNY 23, NSY 19 and NKY 83 caused 100 per cent killing of the termites whereas 14 other isolates caused more than 82 per cent killing at 5 day. At 7 days of observation, 100 per cent killing of the termites was observed with four more bacterial isolates NNY 19, NNY 43, NSY 3 and NKY 91. Bacterial isolate NNY 23 possessed all the three enzyme activities and caused 100 per cent killing of the termites at 5 day of observation. Another bacterial isolate NKY 64 which did not show any of the enzyme activity but it caused 57.8 per cent killing of termites at 5 day. These results indicated that besides the production of three enzymes, some other metabolites (toxin or siderophore) could also be contributing to the killing of termites. Reisolation of bacteria from dead termites showed that seven bacterial isolates viz. NNY 52, NNY 62, NSY 3, NSY 10, NKY 25, NKY 27 and NKY 31 killed even the gut bacteria of the termites and only colonies of inoculated pathogenic bacteria appeared on nutrient agar, soil extract agar and KB medium plates. In the study of other termites inoculated with other 16 bacterial isolates, more than two types of colonies were observed on medium plates indicating that gut bacteria were not killed after ingestion/infection of antagonistic bacteria. Twenty-one bacterial isolates having more than 80 per cent killing of termites at 7 day of observation were identified by standard procedures up to genus level as described in Bergey’s Manual of Determinative Bacteriology. Gram-positive staining was observed only in bacterial isolates NNY 10, NNY 34, NNY 50, NNY 52, NSY 2, NSY 3, NSY 19, NKY 83, NKY 91 and GNY 17. Based on the comparative analysis of various morphological (colony morphology, pigment production and sporulation) and biochemical characteristics (such as indole production, methyl red test, Voges-Proskauer reaction, citrate utilization, oxidase, catalase and hydrolysis of starch and cellulose), the antagonistic bacteria were found to belong to the genera of Arthrobacter, Bacillus, Chromobacterium, Enterobacter, Micrococcus, Neisseria, Pseudomonas and Serratia.ThesisItem Open Access Utilization of pearl millet for ethanol production(CCSHAU, 2007) Poonia, Bijender; Leela WatiEthanol is a known feed stock for countless chemicals and potable purposes. Recently the use of ethanol for fuel purpose is catching up faster world over. It is produced by fermentation of molasses by Saccharomyces cerevisiae in Indian distilleries. Molasses has become more expensive due to its decontrol and deteriorated in quality. Therefore, an alternate substrate that would be both cheaper and readily available in quantities, need to be sought and examined. Pearl millet grains were considered as potential substrate for ethanol production because of high starch content (56.9%). xv The optimum conc. of pearl millet flour slurry for liquefaction by a-amylase was found to be 30% which was liquefied by 600ml of enzyme at 85ºC in 10 min under shaking conditions (140 rpm). Saccharification of liquefied starch of pearl millet hybrids by spirizyme (750ml) at 60ºC under shaking conditions resulted in production of maximum 15.9% (w/v) reducing sugars in 2h. Fermentation of hydrolysate of various pearl millet cultivars at 30±2ºC by S. cerevisiae at pitching rate of 0.5% (w/v) generated 10.6-11.4% (v/v) ethanol with in 36 hrs. under stationary conditions. No significant effect of supplementation of hydrolysate with urea, peptone and yeast extract was observed for ethanol production suggesting that the pearl millet flour has sufficient N and P required for ethanolic fermentation.ThesisItem Open Access Isolation and characterization of yeast for glucoamylase production(CCSHAU, 2007) Nisha; Kundu, B.S.Glucoamylase is the major enzyme required for complete hydrolysis of starch to glucose, which can be used as carbon source in fermentation for ethanol production. It also has large application in food industries and distilleries. The demand for glucoamylase has increased many folds due to utilization of non-conventional substrates (starchy raw material) for industrial ethanol production. The screening of organism that produce large amount of enzyme has been a major area to improve the efficiency of starch processing. Nineteen starch assimilating yeasts were isolated from various sources (ripened fruits, spoiled vegetables, bakery samples, soil, unfermented wort and honey etc). Screening of glucoamylase producing yeast was done by starch plate iodine test and estimating glucoamylase activity. NY-19 and NY-3 isolated from unfermented wort and banana produced 9.2 and 9.6 IU/ml glucoamylase and were finally selected along with Saccharomycopsis fibuligera MTCC-3816 (reference strain). The reaction of 400 ml of cultural filtrate with 1% starch substrate at pH of 4.8 and 30oC temperature for one minute was found optimum for enzyme assay. At optima temperature (30oC), pH (5.0) glucoamylase production by NY-19, NY-3 and MTCC-3816 were 14.40, 13.00 and 15.97 IU/ml, respectively after 48 h of incubation under shake condition. Starch (3%), yeast extract (1%) and peptone (2%) were best carbon and nitrogen source respectively. However MTCC-3816 gave higher activity at 2% starch. The glucoamylase produced from NY-19 was partially purified to 10 fold with specific activity of 43.33 IU/mg of protein using (NH4)2SO4 fractionation. The partially purified enzyme has optimum temperature of 500C and pH 4.8 and was thermostable up to 400C for 8 h. 87% saccharification of rice flour slurry (30%) attained under optimum conditions of temperature (500C) and pH (4.5) in 2 h using Palkozyme HT Plus (liquefying enzyme) and glucoamylase preparation form NY-19. On the basis of morphological and biochemical characteristics isolate NY-19 was identified as Endomycopsis sp.ThesisItem Open Access Development of microbial inoculant for the growth of Ashwagandha (Withania angustifolia)(CCSHAU, 2007) Dinesh Kumar; Yadav, K.S.A total of 104 isolates of rhizobacteria were obtained from rhizosphere and rhizoplane of Ashwagandha plants collected from three locations of Haryana. Of these 36 were from rhizosphere and 68 were from rhizoplane. All the isolates were screened for their growth promoting activities in terms of biomass production in sandy soil without addition of any organic or inorganic fertilizers. Inoculation with these isolates produced more plant biomass than control to a varying level. Only four isolates (HRP-7, RRP-8, and RRP-26 & YRP-11) produced plant biomass more than 10 g /plant. Highest plant biomass was produced by inoculation of isolate YRP-11 (16.49 g/plant) followed by RRP-26 (15.86 g/plant). Isolate HRP-7 produced least plant biomass. Isolates selected on the basis of higher plant biomass production showed useful traits like nitrogen fixation, and production of growth promoting substances like indole acetic acid. Inoculation with selected isolates increased plant biomass more in presence of farmyard manure than without farmyard manure. Mixture of these isolates produced biomass at par with mixed biofertilizer formulation containing Azotobacter and phosphorus solubilizing bacteria.