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ThesisItem Open Access Improvement in the nutritive value of groundnut shelly by fungal cultivation(Department of Microbiology College of Basic Sciences & Humanaties PAU, Ludhiana, 1986) Sukheja, Alka; Gupta, V. KThesisItem Open Access Effect of hydrogen uptake strains of rhizobium on moong (Vigna radiata L.)(Department of Microbiology College of Basic Sciences & Humanaties PAU, Ludhiana, 1985) Sharma, Poonam; Chahal, V. P. SThesisItem Open Access Studies on the effect of azotobacter on vegetable crops(Punjab Agricultural University, Ludhiana, 1975) Khullar, Subhash; Ghahal, V. P. SThesisItem Open Access Synergistic effect of Azotobacter, actinomycetes and phosphate solubilising bacteria on growth and yield of wheat (Triticum aestivum L.)(Punjab Agricultural University, Ludhiana, 2017) Kiranpreet Kaur; Gangwar, MadhuramaThe present investigation was undertaken to evaluate the synergistic effect of four isolates comprising Azotobacter, Sphingobacterium and two actinomycete isolates (Streptomyces badius and W9) for their functionality traits. All the isolates were able to produce Indoleacetic acid (IAA) and gibberellic acid (GA). Actinomycete isolate W9 showed maximum IAA (98.80 µg/ml) and GA (154.6 µg/ml). Similarly all the isolates were tested for siderophore production and phosphate solubilzation where Streptomyces badius exhibited maximum catechol type of siderophore (268 µg/ml) and hydroxamate type of siderophore (252.9 µg/ml). Sphingobacterium sp. isolate exhibited maximum phosphate solubilization activity (37.8±0.9mg/100ml). All the four isolates were screened for their hydrolytic enzymes production. Actinomycete W9 showed maximum amylolytic, chitinolytic, cellulolytic and proteolytic activity with dissolution factor 1.53 cm, 1.58cm, 1.33cm and 1.25 cm respectively. On the basis of the plant growth promoting traits, all the isolates were tested for growth promotion of wheat crop under field conditions. In both the trials, plant growth parameters like shoot and root length, fresh and dry weight of shoot and root, chlorophyll content were found to be significantly enhanced in inoculation with 100% RDF +Azotobacter + W9 over the control. In first trial (2015-16), maximum grain yield was showed by T11 (100% RDF +Azotobacter+ W9) i.e. 51.88 q/ha as compared to control (T2 & T1) was 45.41 & 40.13 (q/ha). Similar trend was observed in second trial (2016-17), where also T5 (100% RDF+ W9+ Azotobacter) showed maximum grain yield (49.5 q/ha) as compared to control (40.3 q/ha). Hence, these actinomycete isolate W9 and Azotobacter are potential candidates for enhancing plant growth that can help in the use of eco-friendly bio-fertilizers.ThesisItem Open Access Development of microbial consortia for pretreatment of paddy straw and its utilization for ethanol production(Punjab Agricultural University, Ludhiana, 2017) Lota, Pardeep; Kocher, Gurvinder SinghThe present study was conducted with the objective of standardization of a fungal consortium from selected ligninolytic cultures for biological pretreatment of paddy straw and its subsequent conversion to bioethnaol. The proximate analysis of paddy straw revealed that 30 mesh straw had highest proportion of cellulose (39.5%) besides, 23.3% hemicellulose and 11.5% lignin. Among different chemical pretreatment methods, acid (1.0% H2SO4) –autoclave (15 psi for 90 min) followed by alkali (2.0% NaOH) pretreatment resulted in 82.61% and 59.23% decrease in lignin and hemicellulose, respectively and 36.60% increase in relative proportion of cellulose. Four fungal strains (viz., LS1, LS2, LCu1 and LCu2) from a total of fifty nine strains isolated from diverse natural habitats, tested positive for the production of laccase (Lacc), lignin peroxidase (LiP) and manganese peroxidase (MnP). Out of three consortia developed on the basis of ligninolytic activities, consortium III comprising Pleurotus ostreatus and Phanerochaete chrysosporium, resulted in Lacc, LiP and MnP activities of 2.40, 37.92 and 62.50 nkat/gds, respectively, significantly higher than the activities of individual fungal strains. Biological pretreatment of paddy straw under Response surface methodology optimized conditions (moisture, 121.0 %; temperature, 31.3°C and log spore count, 8.0 spores/ml) resulted in cellulose, 43.0%; hemicellulose, 12.7%; lignin, 7.0%; total sugars,13.65 mg/gds and reducing sugars, 4.00 mg/gds. The pretreatment of paddy straw with concentrated (10x) ligninolytic enzyme under shake flask conditions using optimized physico-chemical parameters viz. paddy straw concentration, 2.5 g; enzyme volume, 8.0 ml; Ca2+ ions (10 mM) and incubation temperature, 45°C in 72 h of enzymatic action resulted in 80.87% and 47.64% decrease in lignin and hemicellulose, respectively and 29.96% increase in relative proportion of cellulose. For saccharification studies, from a total of forty seven fungal strains isolated from diverse natural sources, two strains (Aspergillus sp. CTS1 and Aspergillus sp. CTS2) tested positive for thermophilic and cellulolytic nature. The two strains were used for the development of fungal consortium which revealed filter paper, carboxymethyl cellulase and cellobiase activities of 10.2, 30.0 and 7.9 U/gds, respectively, significantly higher than the activities of individual fungal strains. Under the RSM optimized conditions [temperature, 55°C; pH,4.8 and substrate concentration, 5% (w/v)] and 30FPU/g dry substrate Arrowzyme yielded 476.0 mg/gds reducing sugars from biological pretreated paddy straw which were further increased to 492.0 mg/gds by the addition of Tween 20 at 0.2% (v/v). Enzymatic saccharification of biological pretreated straw by in-house concentrated cellulase (CC1) from thermophilic fungal consortium resulted in 229.0 mg/gds reducing sugars. The saccharification of chemical pretreated straw with Arrowzyme and CC1 resulted in 566.0 mg/gds and 250.0 mg/gds reducing sugar, respectively. Optimization of fermentation under separate hydrolysis and fermentation (SHF) conditions using RSM [S.cerevisiae inoculum size 10.0 % (v/v) and DAHP concentration, 0.2% (w/v)] resulted in 0.172 g/g and 0.193g/g ethanol from biological and chemical pretreated hydrolysate, respectively. Similarly, the CC1 saccharified hydrolysate resulted in an ethanol content of 0.076 g/g (biological pretreated) and 0.085 g/g (chemical pretreated). Fermentation of non-enzymatically saccharified biological and chemical hydrolysate by S.cerevisiae produced 0.013 g/g and 0.041 g/g ethanol, respectively, whereas sequential fermentation of chemical hydrolysate with S.cerevisiae and Pachysolen tannophilus produced 0.047g/g ethanol. The combined ethanol production from enzymatically and non-enzymatically saccharified biological and chemical pretreated paddy straw hydrolyaste was observed to be 0.185 g/g and 0.240 g/g, respectively.ThesisItem Open Access Production of extracellular proteases from Pleurotus florida (Mont.) Sing. and Volvariella volvacea (Bul. Fr.) Sing.(Punjab Agricultural University, Ludhiana, 2017) Prabhjot Kaur; Sodhi, H.S.Pleurotus florida and Volvariella volvacea are grown widely in subtropical and tropical areas. Proteases are a group of fundamentally hydrolytic enzymes that acylate and deacylate to peptide bonds. P.florida showed higher mycelial dry weight (13.4g/L) and wheat straw gave maximum yield for P.florida (72.5kg/q). In qualitative estimation fruit body of V.volvacea showed maximum halo diameter (1.17 cm). In quantitative analysis, for serine proteases in crude and partially purified extract, mycelium of P. florida (28.1 nmol/min/mg) and fruit body of P.florida (148.3 nmol/min/mg), showed maximum specific activity. The maximum specific activity of cysteine proteases in crude and partially purified extract was observed in fruit body of V.volvacea (28.1 nmol/min/mg) and fruit body of P.florida (145.2 nmol/min/mg). The maximum specific activity of aspartic proteases in crude and partially purified extract was observed in mycelium of P.florida (42.2 nmol/min/mg) and fruit body of P.florida (318.5 nmol/min/mg). Maximum molecular weight of protein was 50.2 kDa recorded in all the samples. The FTIR (Fourier transform infrared spectroscopy) assay indicated that the spent substrate of V.volvacea was devoid of Amide A bond, aromatic amino acids and β-sheet conformation. Arginine, lysine and histidine were present in all the samples, whereas sulfur containing amino acids were present in some of the samples. The proteases production potential is more in P.florida than V.volvacea.ThesisItem Open Access Enzymatic debittering and aroma enhancement of kinnow juice(Punjab Agricultural University, Ludhiana, 2017) Manmeet Kaur; Sahota, Param PalThe processing of acidic fruits to beverages has encountered commercial restrictions due to development of delayed and inherent bitterness by limonoids and flavonoids. The present study has been undertaken with purified debittering enzymes; limonin dehydrogenase, naringinase and β-glucosidase to meet the consumer palatability of citrus juices. The limonin dehydrogenase enzyme has been purified to 1.14- fold with 33.98% recovery, 29.25 IU activity and 0.525 IU/mg specific activity resulted in 86.43% degradation of limonin in juice by catalyzing oxidation of limonin –A-ring lactone to the non-bitter 17dehydroxylimonoate. The debittering naringinase enzyme 24.8IU activity,0.442 IU/mg specific activity, purified to1.68 fold could reduce 57.93% naringin which is hydrolysed by the enzyme into non-bitter bioactive compound naringenin.. The flavour enhancing βglucosidase with activity 14.68 IU, specific activity 0.344 IU/mg purified to 1.18 fold resulted in the increase in glucose content upto 4.25µg/ml in kinnow juice. The debittered fermented low alcoholic naturally carbonated (LANC) beverage has been developed by optimizing the bioprocess conditions as TSS 13ºB, yeast inoculum @ 0.75%(v/v), incubation temperature 28±2ºC and time 36hrs.The physicochemical changes were recorded as TSS 13ºB, acidity 0.313%, naringin 181.13 ppm, limonin 4.73 ppm, glucose 1.3µg/ml, total sugars 42.97 µg/ml, ascorbic acid 34.22 mg/ml in fermented kinnow beverage after 25 days of storage at 4ºC. In kinnow juice, maximum reduction in bitter component limonin 87.34%, naringin 58.41% whereas increase in glucose upto 4.38 µg/ml, acidity 30.13% and total sugar content 42.97 µg/ml were observed. The nutraceuticals recorded in fermented kinnow beverage were flavonoids (mg/L)- gallic acid 0.075, caffeic acid 0.002, rutin 0.001, ferulic acid 0.001; Organic acids (mg/L) – oxalic acid 0.243,tartaric acid 3.698, mallic acid 0.018, citric acid 0.432; Fat soluble vitamins (mg/L)-Vitamin A (Retinol) 0.060, cholecalciferol (D3) 0.038, α-tocopherol (Vitamin E) 0.001, Vitamin K 0.018 ; water soluble vitamins (mg/L)thiamine 0.782, pyridoxine hydrochloride 0.040, pantothenic acid 0.002, riboflavin 0.001, biotin 0.011; Protein 6.42g/100g,Carbohydrate 40.95g/100g.ThesisItem Open Access Bioprospects of microalgal isolates from water logged area of Punjab for biogas production(Punjab Agricultural University, Ludhiana, 2017) Dar, Rouf Ahmad; Phutela, Urmila GuptaThe present study was aimed at isolation, identification, screening and characterization of microalgae from the waterlogged area of south-west Punjab, India. Optimization of cultural conditions of potential microalgal strains, their biogas production potential and anaerobic co-digestion studies were also conducted. Nineteen microalgal cultures (BGLR1-BGLR19) were isolated and were screened using different culture media for their growth kinetics. Isolate BGLR6 followed by BGLR5 showed the highest growth and biomass production in Algae culture medium and BG-11 medium respectively. These isolates upon molecular identification were found to be Asterarcys quadricellulare BGLR5 (MF661929) and Spirulina subsalsa BGLR6 (MF191711). The cultivation conditions for the enhanced production of biomass and other functional components (chlorophyll, carbohydrate, lipid and protein) of BGLR5 and BGLR6 were first screened by Plackett–Burman design and then the significant factors were optimized by Central Composite design (CCD). The optimal cultural conditions for BGLR5 and BGLR6 as per the model were pH of 9.92 & 11.5; temperature of 21.84 & 20°C; light intensity of 80.99 & 81 μmol m-2 s-1; growth period of 25 days (both); 15.00 mM NH4Cl/ CaCl2; 12.28 & 5.00 mM NaNO3 and 7.09 & 2.00 mM K2HPO4 respectively. Under these conditions, the response variables generated a desirability index of 84.10 and 94.91% for BGLR5 and BGLR6 respectively. A 0.42 and 1.60-fold increase in dry cell biomass yield was achieved in BGLR5 and BGLR6, compared to the basal condition (0.8858 and 1.0890 g biomass L-1) respectively. The biogas potential of the microalgal biomass under controlled temperature (35 ± 2°C) conditions revealed that in case of BGLR5, the highest biogas yield (51.712 Lkg-1algal biomass) was obtained in the hydrothermally (100°C 30min) pretreated biomass whilst in BGLR6, the highest biogas yield (42.73 Lkg-1algal biomass) was obtained in enzymatically (10% 24hr) pretreated biomass. Further, co-digestion of 50% paddy straw and 50% BGLR5 biomass (replacement of paddy straw by equal amount of microalgal biomass) produced 168.32 L biogas kg-1 feedstock with the ultimate biogas yield potential of 361.81 mLg-1 VS, reduced lag phase (λ:2.81 d) and increased rate of biogas production (Rm: 8.19 mLg-1d -1) which was higher not only in comparison to the anaerobic digestion of paddy straw and algal biomass individually but also to that of co-digestion of BGLR6 biomass with paddy straw. Likewise, 50 and 100% supplementation of BGLR5 biomass led to enhanced biogas production. Utilization of algal biomass cultivated in the open pond for co-digestion study with paddy straw in field scale digesters resulted in an increase of 17.27% biogas compared to control. The enhancement of methane content from 46.4% (control) to 66.5% (digester containing algal biomass) was also achieved. Hence, the present study signifies that the Asterarcys quadricellulare BGLR5 and Spirulina subsalsa BGLR6 biomass could be utilized as a cofeedstock with paddy straw for biogas production.ThesisItem Open Access Effect of biofertilizers and inorganic fertilizers on soil health and growth of Poplar under nursery conditions(Punjab Agricultural University, Ludhiana, 2017) Khipla, Neha; Gosal, S.K.A pot experiment was conducted to study the effect of biofertilizers and inorganic fertilizers on soil microbial dynamics, nutrient content of soil and plant and plant growth parameters. Significantly higher bacterial (195 x 108 CFU/g of soil), fungal (29 x 104 CFU/g of soil) and PGPR (186 x 105 CFU/g of soil) population was recorded in treatment having consortium biofertilizer + N100P100 whereas actinomycetes population (51 x 105 CFU/g of soil) in treatment having inorganic fertilizers only. Highest diazotroph (77 x 105 CFU/g of soil) and PSB (42 x 104 CFU/g of soil) population was recorded in treatment having Azotobacter and PSB inoculation respectively, with inorganic fertilizers. Maximum soil dehydrogenase (14.35 μg TPF formed/min/g of soil), alkaline phosphatase (2.95 μg PNP formed/ h /g of soil) and urease (394.3 μg urea hydrolysed/ h/ g of soil) activity was observed where consortium biofertilizer + N100P100 was applied. Soil organic carbon (0.33%), available N (171.5 Kg/ha), P (21.3 Kg/ha) and K (142.0Kg/ha) were found to be significantly higher in treatment with consortium biofertilizer + inorganic fertilizers. Plant N (0.74 g/plant) and K (0.68 g/plant) uptake was recorded highest under Azotobacter inoculation whereas highest P (0.16 g/plant) uptake was recorded under consortium inoculation at N100P100 respectively. Plant growth parameters viz shoot fresh and dry weight, shoot length, leaf number per plant were significantly higher in treatment having Azotobacter + N100P100 while stem collar diameter and root length were recorded highest under consortium + N100P100. Treatment having Azotobacter or consortium biofertilizer with N100P75 varied non significantly for highest root fresh and dry weight among the treatments. The effect of treatments having AM cultures and inorganic fertilizers resulted in significantly higher bacterial (132 x 108 CFU/g of soil), diazotroph (49 x 105 CFU/g of soil) population in treatment having AM1 + N100P75 while PSB (32 x 104 CFU/g of soil) and PGPR (142 x 105 CFU/g of soil) in treatment having AM1 + N75P75. Inoculated treatment having AM1 + N75P100 was recorded with maximum fungal population (24 x 104 CFU/g of soil) whereas actinomycetes population (51 x 105 CFU/g of soil) was found highest in control. Highest soil phosphatase (1.92 μg PNP formed/ h /g of soil) and urease (399.5 μg urea hydrolysed/h/g of soil) activity was observed under AM1 + N100P75 whereas dehydrogenase activity (14.78 μg TPF formed/min/g of soil) under AM1 + N75P75. Maximum soil organic carbon (0.34%), available N (157.1 Kg/ha) and P (17.0 Kg/ha) content was recorded in treatment having AM1 + N100P75 while available K content (135.5 Kg/ha) in treatment having AM1 + N75P75. Significantly higher plant N (0.70 g/plant), P (0.15 g/plant) and K (0.54 g/plant) uptake was recorded in treatments with AM1 inoculation and different doses of inorganic fertilizers. Plant growth parameters viz. root- shoot biomass and length, leaf number per plant, collar diameter were recorded highest in treatment having AM1 inoculation + recommended dose of N and P fertilizer. Hence the combined effect of biofertilizers and inorganic fertilizer boosts soil health and growth parameters of Poplar.