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ThesisItem Open Access STRAIN IMPROVEMENT OF Calocybe indica THROUGH MUTAGENESIS AND ITS AGRONOMIC OPTIMIZATION(PAU Ludhiana, 2011) JATINDER KAUR; H. S. SodhiTremendous revolution in the mushroom technology in recent years with respect to types and strains of cultivated mushrooms has made Calocybe indica, a specialty mushroom, the third most popular and commercially grown mushroom in India. A lot of work has been done as far its cultivation technology is concerned but no systematic work has yet been undertaken for its genetic improvement. During present investigation nine strains of C. indica were cultivated on wheat straw using recommended technology. Strain Ci- 3 gave maximum biological efficiency of 81.28%. Basidiospores from all the nine strains were collected and subjected to germination trial on different media but no germination was observed. Improvement of high yielding strain, Ci-3 using mutagenic treatment on protoplasts was conducted. Four mutagenic treatments (one physical and three chemical) yielded 30 putative mutants. Growth studies of putative mutants indicated maximum growth rate of CMB-4 on wheat straw while CMN-11 gave maximum biomass on complete yeast extract medium. Mutant CMN-9 gave maximum endoglucanase (0.345 μg/min/ml) and xylanase (0.540 μg/min/ml) activity. Seven mutants were identified on the basis of growth and enzymes studies. A set of 14 primers were used for molecular characterization of mutants along with the parent using RAPD-PCR. Out of these only 6 primers resulted gave distinct amplification products. Six primers yielded 68 scorable bands ranging from 40bp to 280bp. Similarity coefficient of Ci-3 ranged from 0.706 for CMN-3 to 0.794 for CMU-2. Three mutants (CMU-2, CMU- 5 and CME-2) showed 4 bands each having similar relative mobility values (0.2, 0.35, 0.5, 0.65) during protein profiling using SDS-PAGE. Three bands were obtained for CMN-11, CMB-4 and Ci-3 and 2 bands for CMN-9. Only one band was obtained in case of CMN-3 with relative mobility of 0.2. Cultivation of the mutants was carried out on wheat straw following hot water treatment. Four mutants (CMN-3, CMN-9, CMN-11 and CMB-4) were found to give significantly higher yield than the parent while only three mutants (CMN-3, CMN-9 and CMN-11) gave better results on mixture of wheat straw and paddy straw and on steam sterilized wheat straw. During nutritional studies mutant CMN-9 was found to be nutritionally better with high protein, tocopherol, β- carotene and lycopene content. Postharvest treatment of mutants indicated cabinet drying as a better option in terms of colour and texture retention.ThesisItem Open Access STUDY ON ERGOSTEROL CONTENT AND PROTEIN PROFILE OF MEDICINAL MUSHROOM, Ganoderma lucidum(PAU Ludhiana, 2013) Anna Goyal; H. S., SODHIGanoderma 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.ThesisItem Open Access “BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ALKALINE PROTEASE OF BACILLUS CIRCULANS MTCC 7906”(PAU Ludhiana, 2011) Inderjeet Kaur; Gurvinder Singh, KocherThe present study was conducted to characterize alkaline protease of Bacillus circulans MTCC 7906. Cotton deoiled meal (2.1%) supported optimum enzyme production in 144 hours of incubation. Among the agricultural byproducts used to improve alkaline protease production, the combination of Soybean meal and Glucose supported maximum enzyme production in 96 hours of incubation which was comparable with the control Reese medium (Casein + Glucose) in terms of alkaline protease activity as well as incubation time required for enzyme production. The alkaline protease was purified (16.2 fold) to homogeneity from the culture supernatant by the combination of ammonium sulphate precipitation (30-60%) and DEAE-cellulose anion exchange chromatography. The biochemical characterization of partially purified alkaline protease displayed maximum activity at a pH of 9.0, temperature of 60°C, an enzyme concentration of 0.1 ml/3.0 ml and substrate concentration of 12 mg/ml. The Km and Vmax of partially purified alkaline protease were found to be 4.5 mg/ml and 5555 nmole tyrosine min-1 ml-1, respectively. The enzyme activity was activated by divalent ions whereas a metal chelator EDTA and ammonium hydroxide reduced the activity. The molecular weight of the enzyme was estimated to be 46 kDa on SDS-PAGE. For, molecular analysis, full length alkaline protease gene (BcAP, 1329 bp) was PCR amplified from B. circulans DNA and cloned into pTrcHisA vector. Sequencing and alignment of nucleotide and predicted amino acid sequences of alkaline protease region identified a number of substitutions (mutation) which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The multiple alignment of nucleotide and amino acid sequences of alkaline protease gene of B. circulans established major matches with three closely related subspecies of B. subtilis (B. subtilis subspecies subtilis strain 168, B. subtilis BSn5 and B. subtilis subspecies spizizenii strain W23). Molecular evolution of B. circulans strain was determined by phylogenetic analysis of alkaline protease gene and predicted amino acid sequences. This analysis also suggested that alkaline protease gene is novel gene, which has been accessioned in GenBank with accession number JN645176.1. Recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the protein with estimated molecular size of 46 kDa as observed earlier.ThesisItem Open 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.ThesisItem Open Access Characterization of cellulase producing thermophilic fungi for saccharification of sweet sorghum (Sorghum bicolor L. Moench) bagasse in order to produce ethanol(PAU, 2014) Reetika; Kochar, G.S.Diminishing fossil fuel reserves and increase in greenhouse gas emissions resulting in environment pollution problems have led to a search for alternate sources of fuel. The availability of agricultural lignocellulosic biomass such as bagasse, straw, dry leaves etc. in huge quantities render such a biomass an ideal substrate for bioethanol production. Sweet sorghum, because of its inherent characteristics, such as salinity and lodging tolerance, ability to grow under water and nutrient deficient soils, rapid growth, high sugar accumulation and high biomass production makes it a potential candidate for bioethanol production. The present study was envisaged with the major objective to isolate, thermophilic cellulolytic fungi capable of producing cellulases with improved functional characteristics which could be eventually used for effective hydrolysis of sweet sorghum bagasse (SSB) for bioconversion of sugars produced during hydrolysis to bioethanol. Out of the ten isolates screened for cellulase production through solid state fermentation, two isolates WAT-1 and RWY showed relatively higher filter paper cellulase and β-glucosidase activity at temperatures ranging from 45-55 °C. Therefore, WAT-1 and RWY were identified on the basis of microscopic, macroscopic and molecular characteristics as strains of Aspergillus fumigatus and A. terreus, respectively. Enzyme characterization studies for endoglucanase produced by A. fumigatus and A. terreus revealed that the optimum temperature and pH were 60 ºC and 4.0 and 50 ºC and 5.0, respectively. The kinetic studies of the cellulase produced by A. fumigatus revealed Km value of 19.46 mg/mL and vmax with 74.86 mg/mL/min. Whereas, for cellulase produced by A. terreus Km value was 16.09 mg/mL and vmax was 64.60 mg/mL/min. Alkali pretreatment was found to be more effective than the dilute acid pretreatment or biological treatment. Thus, enzymatic hydrolysis of the alkali pretreated SSB on optimized parameters using response surface methodology (4% sodium hydroxide, 140 ºC, 15 min and 800 micron particle size) led to production of 61.29, 16.28 and 3.33 g/L glucose, xylose and arabinose, respectively. Hydrolysis of alkali-treated SSB using optimized parameters for cellulase obtained from A. terreus (30 FPU/gds crude enzyme, 20 % substrate loading, 50 ºC for 33 h) resulted in 75 g/L glucose, 10 g/L xylose and 3 g/L arabinose. Hydrolysis of alkali-treated SSB with crude concentrated cellulase from A. fumigatus using optimized parameters (30 FPU/gds crude enzyme, 20% substrate loading, 55 ºC for 24 h) yielded 85 g/L glucose, 13 g/L xylose and 4 g/L arabinose. Saccharification using cellulases in presence of 0.2% Tween 20 increased glucose production by 15-20%. Simultaneous saccharification and fermentation at 35 ºC, 20% substrate loading, 10% inoculum concentration for 24 h resulted in 19 g/L, 15 g/L and 23 g/L ethanol, respectively using cellulases from A. fumigatus and A. terreus and commercial enzymes and Pichia kudriavzevii for fermentation. Separate hydrolysis and fermentation (SHF) using statistically optimized parameters (35 ºC, 10% inoculum concentration for 16 h) resulted in 40 g/L , 49 g/Land 51 g/L ethanol while using sugars produced by crude cellulases obtained from A. terreus, A. fumigatus and commercial enzymes. The present study thus revealed that sweet sorghum bagasse (SSB) could be ideally used as a substrate for bioethanol production using the optimized pretreatment, hydrolysis and fermentation parameters.ThesisItem Open Access STRAIN IMPROVEMENT OF Lentinus edodes (Berk.) Sing. BY PROTOPLAST FUSION TECHNIQUE(Punjab Agricultural University, 2013) naresh kumar; Johal, P.PProtoplast 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) thanThesisItem Restricted Strain development of yeasts for efficient alcoholic fermentation utilizing xylose(Punjab Agricultural University Ludhiana, 2010) Sushma Gurumayum; Kalra, K. L.Two strains of yeasts, Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC1077 were used to develop improved strains of yeasts for efficient alcoholic fermentation utilizing xylose as carbon source by means of mutagenesis and protoplast fusion. Mutagenesis of xylose fermenting yeast, P. tannophilus MTCC1077 using physical mutagen, UV rays gave rise to 103 ethanol negative variants with the inability to use ethanol as carbon source for growth. The parent P tannophilus MTCC1077 was found to give a maximum ethanol yield of 0.339g/g with a corresponding fermentation efficiency of 66.47% from 20g/l xylose when fermentation was carried out at 30ºC at a pH of 5.0 with inoculum of 6% and an aeration of 100rpm. Two ethanol negative variants UVmut 62 and UVmut 41 gave ethanol yields significantly higher than the parent strain. UVmut 41 showed highest yield of ethanol of 0.350g/g with a fermentation efficiency of 68.53% while UVmut 62 gave a yield of 0.347 g/g with a fermentation efficiency of 67.92%. Mutagenesis of xylose fermenting yeast, P. tannophilus MTCC1077 using chemical mutagen, ethylmethanesulfonate (EMS) gave rise to 102 ethanol negative variants with the inability to use ethanol as carbon source for growth. Three ethanol negative variants EMSmut14, EMSmut39 and EMSmut26 showed significantly higher ethanol producing capacity than the parent strain. EMSmut26 gave the highest ethanol yield of 0.364g/g with a fermentation efficiency of 71.32% followed by EMSmut39 and EMSmut14 giving a yield of 0.350g/g and 0.346g/g respectively. Protoplast isolation rates of 99% for S. cerevisiae and 97% for P. tannophilus achieved with action of lyticase enzyme (0.08mg/ml) on cells for 60min at 30°C with 0.8M sorbitol, 1mM EDTA and 10mM Tris buffer at pH 7.5. Protoplast fusion was obtained when cells were treated for 30min with chemical fusogen, polyethyleneglycol (PEG mol wt. 6000) and highest protoplast regeneration frequency of 21% was obtained with 35% PEG and 0.8M sorbitol at 30ºC. Two fusants were able to produce ethanol from a mixture of glucose and xylose. Parent S. cerevisiae gave an ethanol yield of 0.430g/g with a fermentation efficiency of 84.21%. FUS1 giving a yield of 0.244g/g with a fermentation efficiency of 47.82% while FUS2 gave a yield of 0.273g/g with fermentation efficiency of ethanol at 72hr of fermentation. Scanning electron microscopy (SEM) study of parent strains and fusants revealed differences in the cell surface and also in budding pattern as compared to that seen with either of the individual parent strains. DNA contents of the two fusants were larger than that of any single parental strains, suggesting that these fusants have inherited genetic material from both parental strains.
