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20133
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Subject: Microbiology × End date: 2013 × Start date: 2013 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    STUDY ON ERGOSTEROL CONTENT AND PROTEIN PROFILE OF MEDICINAL MUSHROOM, Ganoderma lucidum
    (PAU Ludhiana, 2013) Anna Goyal; H. S., SODHI
    Ganoderma lucidum, a specie belonging to the class basidiomycetes, family polyporaceae of the order aphyllophorales has been widely used as a source of potent nutracuetical products. Present study was planned to identify and characterize the role of proteins and ergosterol in the developmental process of Ganoderma lucidum. Four strains of Ganoderma lucidum (GL I - IV) showed a gradual increase in biomass to give 25.52g to 31.72g of biomass after three weeks of growth in mushroom complete medium broth with maximum in strain GL-III. Ganoderma lucidum strains were grown on wheat straw supplemented with 5% wheat bran with maximum biological efficiency for GL-I strain (31.27%) followed by GL-II (26.76%) and number of fruit bodies were 927 and 693 each weighing 33.7g and 38.6g, respectively. Ganoderma lucidum strain GL-I showed maximum ergosterol content (4601μg/g) whereas strain GL-III showed only 32μg/g. From spawn run, maximum ergosterol was obtained from GL-IV strain followed by strain GL-II while at pin head formation and fruit body formation ergosterol content was better for GL-II. Ergosterol content of fruit body of GL-1 was observed maximum (7009μg/g). Overall observation indicated that the ergosterol content increased with each stage of cultivation process i.e. from spawn run to pinhead and finally to fruit body formation. The intracellular and extracellular enzymatic studies have indicated enhanced activity during spawn run on solid substrate in comparison to that grown in the broth. The esterase and peroxidase activity significantly increased during the pinning of the cultures thus, indicating a positive role of these enzymes in fructification process. The FTIR analysis of proteins made during different stages of cultivation namely spawn run, pin head formation and fruiting indicated that the fruiting strains (GL-I and GL-II) have an ordered protein structure with hydrophobic amino acids. In case of GL-IV, unordered structure was obtained that could be related to the role of hydrophobin proteins in mushroom fructification process. Another observation on GL-IV indicated the presence of basic amino acids and aromatic amino acids with very low amount of acidic amino acids like aspartic acid and glutamic acid. The observation recorded during present study indicated a positive role of hydrophobic amino acids and hydrophobin proteins in mushroom fructification process. Ganoderma lucidum strain GL-II was also grown on selenium fortified mushroom minimal medium at different concentrations of 5ppm to 25ppm of sodium selenate. Scanning electron micrographs exhibited gradual decrease in hyphal diameter, spore number and spore diameter with increase in selenium concentration and the spore structures were altered. A significant decrease in spore diameter is observed in concentration of 20ppm and 25ppm (5.60 and 1.26 μm, respectively) as compared to control (10.04 μm). The SEM-EDS studies showed no selenium traces on the hyphal surface, however, on the contrary, SEM-EDS studies of crushate samples revealed selenium traces indicating selenium absorption as the cytosolic moieties as selenoproteins. Atomic absorption spectroscopy indicated an increasing trend in the uptake by the hyphal biomass as the concentration of sodium selenate increased with maximum absorption at concentrations of 15 ppm and 25 ppm (9.9%). It was concluded that fortification till 15 ppm can be used as stress was not that prominent and culture could grow rapidly without significant alteration in structure and morphology to enhance its biomedicinal properties. Present study has indicated that during the mushroom development process, ergosterol content increases with a positive role of proteins like peroxidases and hydrophobins at each stage of morphogenesis.
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    ThesisItemOpen Access
    Elucidation of phorate metabolism by bacterial isolates from agricultural soils for bioremediation
    (PAU, 2013) Jariyal, Monu; Gupta, V. K.
    Fifteen phorate metabolizing bacterial species isolated from sugarcane field soils were identified using 16SrDNA sequence homology. Based upon relative phorate degradation, Brevibacterium frigoritolerans, Bacillus aerophilus and Pseudomonas fulva were found to cause more than 98 per cent phorate reduction. These bacterial species could grow over a wide range of pH (4.0-11.0) and temperature (25-37°C), but optimally at pH of 6.0-6.5 and 37°C, in shaking cultures. Whereas, B. frigoritolerans was salt sensitive, P. fulva & B. aerophilus grew optimally in 3.0 and 4.0 per cent NaCl, respectively. All the three bacterial species grew optimally in the presence of glucose and peptone (1.0 % each). Only B. aerophilus carried a plasmid of around 4 kbp, but curing of this plasmid did not affect the phorate degradation establishing that phorate degradation genes are borne on chromosomal DNA. Induction by phorate resulted in only nondifferentiating protein profiles in all the three bacterial species establishing that phorate degradation is a constitutive character. In soils amended with upto 300 mg kg -1 phorate, these bacterial species within 42 days actively metabolized phorate by between 89.81 and 95.62 per cent with maximum capacity shown by P. fulva. This phorate degradation was further improved to 98.31 per cent, by mixed cultures of all the three bacterial species which constituted most effective bioremediation consortia for significantly relieving soils from phorate residues. The investigations in this study has isolated three bacterial species and established their potential for active bioremediation of phorate both in liquid cultures and agricultural soils.
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    ThesisItemOpen Access
    STRAIN IMPROVEMENT OF Lentinus edodes (Berk.) Sing. BY PROTOPLAST FUSION TECHNIQUE
    (Punjab Agricultural University, 2013) naresh kumar; Johal, P.P
    Protoplast fusion is one of the techniques to combine genetic characters across species barriers. In the present studies, dikaryotic mycelium of Lentinus edodes strains was used for protoplast fusion using polyethylene glycol (PEG) to develop hybrids with improved desirable traits like total yield, temperature tolerance, sporelessness/less spore producing and shorter cropping cycle. Optimized protocol was developed for protoplast isolation of using parameters like enzyme: combinations and concentrations, age of mycelium, duration of incubation period, osmotic stabilizers type. It was found that for both LeS and LeC, 3 days old mycelial culture, 0.6M NaCl prepared in 0.01 M phosphate buffer gave the maximum protoplast yield (2.40 x 107 and 1.80 x 107 protoplasts/mL) after 3 hours of incubation when enzyme combinations from group I of chitinase 3, β-1, 3-glucanase 4, driselase 7 and lyticase 5 concentrations (mg/mL) were used. The protoplasts subjected to polyethylene glycol (PEG) mediated fusion was regenerated on three regeneration media with four osmotic stabilizers. Maximum regeneration efficiency of putative hybrid fusants was obtained on SMY (starch-malt-yeast extract) medium (3.0%) where 0.6M sucrose was used. All together 124 intraspecific putative hybrid fusants were obtained and five hybrid fusants (L4, L13, L31, L58 and L76) were selected on the basis of radial growth. All the hybrid fusants shared almost similar colony morphology with some variation in growth pattern. Some unique characteristics like production of red exudate, softening of agar in hybrid fusants were also seen. Selected hybrid fusants were further characterized for genetic variability by high enzyme production, isozyme analysis and RAPD-PCR.Qualitative estimation of endoglucanase activity of parents and hybrid fusants indicated highest enzyme production by hybrid fusant L58 with 1.27 z/c value. Quantitative endoglucanase activity indicated highest enzyme production by hybrid fusant L58 (8.85 μmol/min/mL) on saw dust supplementation.Highest xylanase activity showed by hybrid fusant L58 (18.85 μmol/min/mL) on wheat straw supplementation. Highest laccase activity was observed in L4 (8.33 μmol/mL of culture filtrate) on saw dust supplementation. Isozyme analysis was done using Native-PAGE. Protein content found maximum in L58 hybrid fusant (182.5µg/mL). Isozyme analysis of parents and hybrid fusants revealed a maximum of 4 malate dehydrogenase isozyme, with maximum by hybrid fusant L58 at the Rm value of 0.33, 0.43 and 0.56. Peroxidase produced two isozyme at Rm value of 0.62 and 0.70 while superoxide dismutase produced only one isoform. A set of 10 primers were used for molecular characterization of hybrids along with the parents using RAPD-PCR. Out of these 4 primers gave distinct amplification products. These 4 primers yielded 69 scorable bands ranging from less than 100 bp to more than 1000 bp for all the genotypes. Maximum similarity coefficient of value 0.952 was obtained between hybrid fusants L58 and L13. Similarity coefficient of parent LeC with parent LeS was 0.416 which could be compared with hybrid fusants L13 and L58. A combined dendrogram was obtained from the RAPD data of all the four primers together and it was found that three hybrid fusants i.e. L58, L13 and L31 were closer to LeS parent strain. Cultivation of all the five hybrid fusants developed by protoplast fusion along with the parent strains LeS and LeC was carried out on combination of sawdust with wood chips and wheat straw as substrate. Spawn run period varied from 47 days (L31) to 65 days (LeS). Hybrid fusant L58 was found to give significantly higher yield (57.2 % and 56.9 % with wheat straw and sawdust and wood chips of red meranti tree respectively) than