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ThesisItem Open Access Bioprocessing of Indian gooseberry for the production of lactic acid beverage and pickles(Punjab Agricultural University, Ludhiana, 2019) Kirandeep Kaur; Sahota, Param PalThe plant-based fermented beverages have received considerable interest for their rich nutrition, high content of soluble and insoluble fibers and for their fermentability by lactic acid bacteria (LAB). A set of ten allochthonous lactic acid bacterial strains phenotypically and genotypically characterized for their probiotic potential were used as starter culture for the optimization and production of Indian gooseberry-based functional beverage and lacto-pickle. The optimized bioprocess comprised of gooseberry juice blend [gooseberry juice 2: ginger extract 1: guava juice 2], dilution ratio (1:3), condiment concentration (0.7%), pasteurized at 82ºC for 10 seconds and 5.0% (v/v) (106 CFU/mL) active starter culture for fermentation at 37ºC for 24 hr. The microbiological and physicochemical parameters of stored beverage (90 days) were; 1ºBrix, Titrable acidity 0.30-0.89 %, pH 6.0 -2.6, Total Sugars 44.3-35.3 mg/100mL, Reducing sugars 5.4-10.7 mg/100mL and antioxidant activity (85-67%), Total polyphenolic content (42.5-32.6 mg GAE/100mL),Total flavanoids (14.5-28.7 mg/100mL) with LAB capable of maintaining cell counts of 7.39 log CFU/mL. The bioprocess optimized for the Indian gooseberry lacto pickle as 5.0% (v/v) (106 CFU/mL) active starter culture and 5% brine solution for fermentation at 37ºC for 24 hr. The microbiological and physicochemical parameters of stored lacto-pickle (90 days) were; LAB count-6.22-7.27 log CFU/mL, 1ºBrix, Titrable acidity 0.24-0.64 %, pH 7.0-2.8, Total Sugars 20.1-11.6 mg/100g, Reducing sugars 2.60-14.6 mg/100g, viz. nutraceuticals Total antioxidant activity 86.1- 90.2%, Total polyphenolic content 16.6-37.8 mg GAE/100g and Total flavanoids 4.007-55.7 mg/100g. The overall acceptability of beverage and pickle was 7±0.2 on nine point hedonic scale. The bio-interventions: Gooseberry beverage and pickle endowed with organic acids, metabolites, antioxidants, polyphenols and flavonoids showed antimicrobial activity against food borne pathogens Staphylococcus aureus MTCC3906, Listeria monocytogenes MTCC657, Klebsiella pneumoniae MTCC109, Escherichia coli MTCC443, Aeromonas hydrophila MTCC173 as well as arrested the initial phase of Molt-4 cancer cell lines, down regulating the expression of proto-oncogenes and up regulating the tumor suppressor gene exhibiting the antitumorigenic effect.ThesisItem Restricted Bioprocessing of Turmeric (Curcuma longa Linn.) for the production of lactic acid beverage and pickles(Punjab Agricultural University, Ludhiana, 2019) Makkar, Avneet Kaur; Sahota, Param PalThe plant-based fermented beverages have received considerable interest for their rich nutrition, high content of soluble and insoluble fibers and for their fermentability by lactic acid bacteria (LAB). A set of ten allochthonous lactic acid bacterial strains phenotypically and genotypically characterized for their probiotic potential were used as starter culture for the optimization and production of Turmeric-based functional beverage and lacto-pickle. The optimized bioprocess comprised of turmeric juice blend [Turmeric juice 100mL: ginger extract 1.5mL: lemon juice 5mL], dilution ratio (1:3), condiment concentration (1.0%), pasteurized at 52ºC for 15 seconds and 5.0% (w/v) (106 CFU/mL) active starter culture for fermentation at 37ºC for 24 hrs. The microbiological and physico-chemical parameters of stored beverage (90 days) were; 1ºBrix, titrable acidity 0.30-0.80 %, pH 4.8-2.5, total sugars 48.56-13.0 mg/100mL, reducing sugars 11.54-4.70 mg/100mL and antioxidant activity (70.37-68.58%), total polyphenol content (44.20-40.00 mg GAE/100mL), total flavonoids (38.06-48.11 mg/100mL) with LAB capable of maintaining cell counts of 6.99 log CFU/mL. In addition, the beverage prepared from: 2% turmeric powder (var. Punjab Haldi-1) showed a significant difference in total polyphenol (37.12 mg GAE/100mL) and flavonoid content (43.78 mg/100mL). The bioprocess optimized for the Turmeric-lacto pickle as 5% (w/v) (106 CFU/mL) active starter culture and 5% brine solution for fermentation at 37ºC for 24 hrs. The microbiological and physico-chemical parameters of stored lacto-pickle (90 days) were; LAB count 7.05-5.82 log CFU/mL, 1ºBrix, titrable acidity 0.28-1.12 %, pH 4.6-2.2, total sugars 10.21-7.26 mg/100g, reducing sugars 6.54-7.50 mg/100g, total antioxidants 53.04-72.17%, total polyphenolic content 45.35-57.12 mg GAE/100g and total flavonoids 39.25-45.15 mg/100g. The overall acceptability of beverage and pickle was 8±0.2 on nine point hedonic scale. The bio-interventions; Turmeric beverage and pickle endowed with organic acids, metabolites, antioxidants, polyphenols and flavonoids showed antimicrobial activity against food borne pathogens Staphylococcus aureus MTCC3906, Listeria monocytogenes MTCC657, Klebsiella pneumoniae MTCC109, Escherichia coli MTCC443, Aeromonas hydrophila MTCC173 as well as arrested the initial phase of Molt-4 cancer cell lines, down regulating the expression of proto-oncogenes and up regulating the tumor suppressor gene exhibiting the antitumorigenic effect.ThesisItem Open Access Characterization of stress tolerant microalgae strains for biogas production(Punjab Agricultural University, Ludhiana, 2019) Sharma, Nishu; Phutela, Urmila GuptaThe present research work was aimed at characterization of stress tolerant microalgae strains for biogas production. A total of six strains, five stress tolerant microalgae strains viz. BGLR4, BGLR7, BGLR10, BGLR18, BGLRS and one standard strain Spirulina sp. NCIM 5143 were used in the present study. These strains were screened for maximum biomass production, chlorophyll content, protein, carbohydrate and lipids on four different media viz. Blue green-11(BG-11), Bold’s basal medium (BBM), Algal culture medium (ACM) and Zarrouk’s medium (ZM) and on different concentrations of unsterilized dairy wastewater (20%, 40%, 60%, 80% and 100%). Among all the six strains, BGLR18 and BGLRS were found to produce maximum biomass BBM and ACM respectively while Spirulina sp. NCIM 5143 produced maximum biomass on ZM. BGLR18 and BGLRS showed maximum growth on 60% concentration of dairy wastewater whereas Spirulina sp. NCIM 5143 on 100% concentration of dairy wastewater. Maximum percent reduction in various physico-chemical parameters of dairy wastewater was shown by BGLR18 and BGLRS at 60% dairy wastewater while Spirulina sp. NCIM 5143 at 100%. Further, studies were carried out with strains BGLR18 and BGLRS and compared with standard strain Spirulina sp. NCIM 5143. Cultural conditions of BGLR18, BGLRS and Spirulina sp. NCIM 5143 were optimized using response surface methodology (RSM) based on central composite design. The optimized and most desirable cultural conditions of pH, temperature, light intensity, growth period and inoculum concentration for BGRL18 were 10.02, 20.00°C, 7599.41 Lux, 21.75 days and 1.00% ; for BGLRS were 11.17, 26.03 °C, 4000.02 Lux, 39.37 days and 10.00% and for Spirulina sp. NCIM 5143 were 10.35, 24.38 °C, 8000.00 Lux, 25.13 days and 1.00% respectively. On molecular identification, BGLR18 showed 96% resemblance to Chlorosarcinopsis eremi and BGLRS showed 99% resemblance with Scenedesmus sp. MKB. These three strains showed wide variation in their total nitrogen, total protein,total phenols, antioxidant activity, DPPH radical scavenging activity. ICP-AES analysis showed that heavy metals were negligible in all the three strains while essential elements like Ca, Fe, P and Mg were present in higher concentrations than Spirulina sp. NCIM 5143. GC-MS analysis of methanolic extract showed that many bioactive compounds were present in all the strains. These strains were evaluated for their biogas production potential alone and in co-digestion with paddy straw. The highest biogas was produced by BGLRS (Scenedesmus sp. MKB) (28.80 Litres) with highest biogas production potential (P) of 30.85 mLg-1 VS showing maximum biogas production rate (Rm) of 0.58 mLg-1d -1 with a lag phase (λ) of 1.30 days. While in co-digestion experiment, the highest biogas production was recorded by BGLR18 (Chlorosarcinopsis eremi) (168.46 Litres) with maximum biogas production potential (P) of 214.30 mLg-1 VS showing maximum biogas production rate (Rm) of 8.76 mLg-1 d -1 with a lag phase (λ) of 12.10 days. Hence, the stress tolerant microalgae strains can be co-digested with paddy straw for enhancing biogas production.ThesisItem Open Access Isolation and characterization of Rhizobium spp. for its plant growth promoting potential in Lucerne (Medicago sativa L.)(Punjab Agricultural University, Ludhiana, 2019) Jotshana Kumari; Khanna, VeenaThe present investigation was undertaken to access the effect of rhizobial isolates on plant growth and symbiotic parameters in Lucerne (Medicago sativa L.).Forty rhizobial isolates were identified and characterized culturally, morphologically, biochemically and found belonging to genera Rhizobium. The isolates were functionally characterized on the basis of phosphate solubilization, Indole acetic acid, siderophore, cyanide and gibberellic acid production. Indole acetic acid production ranged from 2.6 -77.7 μg/ml in presence of tryptophan after 3 days of incubation and increased to 2.9-101.7 μg/ml on 5th day.The range of PSI (Phosphate solubilization index) was 13-17 mm with highest solubilizing index shown by LNR-6, LNR-20, LNR-23 and LNR-39 (17mm).Twenty eight of the forty isolates were able to produce siderophore as evidenced on Chrom-azurol-S-agar, maximum catecholate type siderophore produced by LNR-39 (43.4 μg/ml). 57.5% isolates produced HCN out of which 13 % were strong and 39.1% were moderate producers. The gibberellic acid production ranged from 41.2 -96.6 μg/ml with LNR-39 (96.6 μg/ml) being the potent gibberellic acid producer. The study carried out showed that isolates LNR-2,LNR-4, LNR-6, LNR-22, LNR- 32 and LNR-39 exhibited growth and functionality traits which were at par, hence they were selected for field trail. The field experiment revealed enhanced stem and root length,dry weight of nodules, nitrogen content, leghemoglobin and chlorophyll content , green fodder yield under Rhizobium treatment. Thus, the present study revealed that treatment with Rhizobium LNR-39 and treatment LNR-4 were effective for plant growth promotion and enhanced symbiotic effeicency in Lucerne.ThesisItem Open Access Biosorption of electronic waste through microbial cultures: a bioremediation technology(Punjab Agricultural University, Ludhiana, 2019) Preetiman Kaur; Sharma, ShivaniSeven microbial cultures Lentinus edodes, Pleurotus florida, Ganoderma lucidum, Aspergillus niger, Trametes versicolor, Streptomyces spp. and Pseudomonas spp. were evaluated for biosorption and bioleaching from electronic-waste (e-waste) under submerged conditions. Pleurotus florida and Pseudomonas spp. showed the maximum specific activity of laccase enzyme (6.98 U/mg and 5.98 U/mg respectively) on 20th day of incubation in comparison to other microbial cultures. Maximum biosorption and bioleaching of copper was observed in Pleurotus florida (97.26 mg/g and 44.30 mg/l respectively) followed by Pseudomonas spp. (96.20 mg/g and 42.06 mg/l respectively). However, Pseudomonas spp. showed maximum leaching (46.33 mg/l) and absorption (98.00 mg/g) for iron followed by Pleurotus florida (43.13 mg/l and 94.13 mg/g). Presence of large halo orange zones in ewaste treated sample of Pseudomonas spp. confirmed the production of siderophores. The conditions for laccase production using best screened microbial cultures were optimized by Response Surface Methodology using Design Expert 12.0 and regression equations were developed with R2 value ranging between 0.7817 to 0.9257 indicated high degree of fitness. The protein profiling of these optimized cultures by SDS-PAGE revealed the presence of more number of protein bands in e-waste containing samples in comparison to untreated cultures. The topographical studies of samples treated with e-waste through SEM showed the etching of surfaces along with deposition of certain vegetative and spores cells, extracellular material secreted by these microbial cultures and heavy granular pits. Further EDX studies showed the decrease of metal ions from their respective initial concentrations present in untreated samples which confirmed the bioleaching followed by biosorption.ThesisItem Open Access Calocybe indica strain evaluation for lignocellulolytic enzymes in relation to mushroom yield(Punjab Agricultural University, Ludhiana, 2019) Karanpreet Singh; Sodhi, Harpreet SinghCalocybe indica, milky mushroom is a tropical mushroom of Indian origin that grows well at 28-35°C. C. indica strains were evaluated for their mycelial growth, extracellular enzymes producing capability and their yield potential. Different characteristic of nine strains of Calocybe indica Ci-01 to Ci-09 and a Macrocybe were grown on Potato Dextrose (PDA), Complete Yeast Extract (CYM), Mushroom Minimal Media (MMM) and their broth at 30±2°C. After 10 days, maximum radial growth was observed on PDA in Ci-06 (87.6 mm) and Ci-09 (87 mm) strain of C. indica. The maximum biomass was recorded in CYM in Ci-06 (16.1g/l) and Ci-07 (15.5 g/l). The maximum linear growth on wheat straw after 10 days was observed in Ci-07 (63mm) and Ci-09 (66mm). In wheat straw, the maximum biological efficiency estimated from the harvested yield (kg/q dry straw) was observed in the strain Ci-06 (57.3%). Two strains Ci-07 (48.5%) and Ci-09 (45.7%) were found to give yield at par with each other. Maximum number of fruiting bodies were recorded in the strain Ci-06 (1552 no./q dry straw). In culture filtrate endoglucanase enzyme activity range between 1.91-4.42 U/mg, endoxylanase activity ranged between 2.73-4.76 U/mg, laccase activity ranged between 6.94-10.2 U/mg. During spawn run, endoglucanase and endoxylanase enzyme activity ranged between 0.58-1.03 U/mg and 0.74 -2.11 U/mg, laccase activity was ranged between 1.51-4.54 U/mg. During pinhead, endoglucanase and endoxylanase activity ranged between 0.61-1.59 U/mg and 1.57-2.96 U/mg, whereas laccase activity range between 6.53-10.6 U/mg. In fruiting bodies, endoglucanase and endoxylanase activity of the strains ranged between 0.94-1.88 U/mg and 0.93-2.36 U/mg, laccase activity ranged between 5.43-9.21 U/mg. A positive correlation of yield with biomass and endoglucanase has been observed in C. indica strains Ci-03, Ci-06, Ci-07 and Ci-09.ThesisItem Restricted Pretreatment of paddy straw for cultivation of Vplvariella (Bull.ex Fr.) Singer(Punjab Agricultural University, Ludhiana, 2018) Swarthi K.; Sodhi, Harpreet SinghTwo strains of Volvariella volvacea, VV3 and VV6 were evaluated for their mycelia growth rate and yield potential on pretreated paddy straw as substrate under Punjab conditions. Mycelial growth studies on Nutrient agar (NA) and Potato Dextrose agar (PDA) inoculated with Bacillus sp. and Delftia sp. separately revealed that bacteria had no antagonistic effect on the growth of V. volvacea. The mycelia growth rate of V. volvacea on PDA inoculated with bacterium was statistically at par with control and covered plate in 5 days whereas on NA mycelia growth covered the plate in 8 days. The effect of pretreatment of paddy straw on its chemical composition was studied with 6 different pretreatments: urea 1% (w/w), urea 0.5% (w/w), urea 0.5% (w/w)+Delftia sp. 0.5% (v/w), urea 0.5% (w/w) +Bacillus 0.5% (v/w), Delftia sp. 0.5%, Bacillus sp. 0.5% (v/w) and water 0.5% (v/w) as control. The pretreated paddy straw was analysed for proximate principles at a periodic interval of 5 days upto 20 days. Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), Cellulose, hemicelluloses, Acid Detergent Lignin (ADL), Ash content and crude protein content were analysed. A significant decline in NDF value from 75.7% to 46 % and 34.4% was observed when urea 0.5% + Bacillus 0.5% + Delftia 0.5% were used as pretreatment of paddy straw respectively.The ADF value was significantly reduced to 23% and 33% when urea 0.5% +Delftia 0.5% and urea 0.5% + Bacillus 0.5% were used to treat paddy straw. A significant reduction in cellulose content was observed from 40% to 25% to 20% with urea 0.5%+ Delftia 0.5% and urea 0.5% + Bacillus 0.5% respectively. The hemicelluloses content recorded maximum reduction to 11.45% from 23.4% with urea 0.5% + Delftia 0.5%. There was a rapid decline in ADL with urea 0.5% + Delftia 0.5% and urea 0.5% + Bacillus 0.5% from 12.3% to 4.3% and 7% respectively. The ash and crude protein content increased with all the pretreatments. Studies on mycelia growth rate on pretreated paddy straw revealed that the addition of bacteria along with urea had improved the growth rate of both the strains of V. volvacea indicating the significant role of bacterial pretreatment in the mycelia growth of V.volvacea. Cultivation of V.volvacea strains on pretreated paddy straw had shown the maximum yield was obtained from pretreated paddy straw from VV6 (14.84 kg/ q dry straw) followed to strain VV3 (13.82 kg/q dry straw) on pretreated paddy straw. The yield from untreated (control) paddy straw was 10.5 kg/q dry straw from VV3, 12.5 kg/q dry straw from VV6. Present study revealed that pretreatment of paddy straw with urea 0.5%+Delftia sp. 0.5% gave better yield than untreated paddy straw.ThesisItem Open Access Cellulose degrading bacteria, Decomposition, Green manure, Maize, Microbial dynamics, Nutrient mineralization, Yield(Punjab Agricultural University, Ludhiana, 2019) Charnjeet Kaur; Khanna, VeenaThirty four rhizobacterial isolates were isolated from mungbean rhizosphere soil collected from different locations of the Punjab. The isolates were evaluated for their PGP traits at 35°Cand 40°C but, it was found that rhizobacteria were more acclimatized to 35°C as compared to 40°C. All the isolates showed IAA production at 35°C (0.13- 56.44μg/ml) and (0.22-36.80μg/ml) at 40°C. The isolates were effective P and Zn solubilizers with P-solubilisation index ranging from 6.9-15.5 at 35°C and from 5-13.8 at 40°C. Zn solubilisation index ranging from 3.8-24.0 at 35°C and 4.6-15.3 at 40°C, most potent isolates being G-2, G-3, G-33 and G-34. Isolates G-2, G-3, G-33 and G-34 were HCN and ammonia producers as well. The biocidal impact of the rhizobacterial isolates against F.oxysporum and R.solani was studied and eight isolates showed antagonistic activity against F. oxysporum and R. solani. Isolates G-2, G-3, G-33 and G-34 proved to be strong antagonistics with 27.0-37.5% inhibition of the phytopathogens. The field experiment conducted revealed that co-inoculation of Rhizobium and G-33 proved to be the most beneficial, resulting in the improvement of symbiotic traits viz nodule number per plant (66.6), nodule dry weight (22.2 mg/plant), root (0.29g/plant) and shoot (11.84g/plant) dry weight and the yield (1134 kg/ha) of mung bean (ML2056) as compared to Rhizobium treatment alone (1093 kg/ha).ThesisItem Open Access Development of bacterial consortium to improve green manure degradation and nutrient availability in kharif maize(Punjab Agricultural University, Ludhiana, 2019) Jaspreet Kaur; Gosal, S.K.The present investigation was conducted to isolate cellulose degrading bacteria with plant growth promoting traits to enhance green manure (Crotolaria juncea) decomposition as well as nutrient mineralization in maize crop. A total of 75 different bacteria were isolated from soil samples collected from various locations. Only 20 isolates were selected on their ability to degrade cellulose. Cultural and morphological characterization showed that most of these bacterial isolates were gram positive, non-motile and non-endospore forming cocci. Biochemical characterization of the isolates revealed that most of these isolates were positive for oxidase, catalase, methyl red, starch and citrate utilization test. Only two bacterial isolates (CD4 and CD6) were urease positive, one (CD11) was indole positive and three (CD1, CD11 and CD15) were found to HCN positive. Functional characterization of these isolates showed that the isolates CD2 and CD9 had highest cellulase (27.543 mg/ml) and β-glucosidase activity (15.46 μg pNP released/ml), respectively. Highest IAA production (42.25 μg/ml), phosphorous solubilization index (1.80) and quantitative P- solubilization (25.39 μg/ml) were recorded for CD9 bacterial isolate. All the isolates were found to have nitrogen fixing ability. The maximum amount of ammonia (6.659 μM/ml) was excreted by isolate CD18 on N-free Jensen‟s medium. On the basis of functional characteristics, three best isolates (CD2, CD9 and CD14) were evaluated for green manure decomposition and nutrient mineralization studies, under pot conditions under different soil (sterilized and unsterilized) and moisture regimes (aerobic and flooded). The application of isolates as consortium was found best for increasing green manure decomposition, nutrient mineralization and soil biological activities relative to uninoculated control treatments. These effects of consortium inoculation were found more pronounced in aerobic conditions as compared to flooded conditions. A field experiment was conducted for two consecutive years (2016 and 2017). A significant decomposition of green manure was observed with application of CDBC biofertilizer within the period of seven days in field condition. A total of eight treatments having different combinations of biofertilizer (CDBC), green manure and inorganic nitrogen fertilizers (75% and 100%) were used to study their effect on soil microbial dynamics, soil chemical properties and yield of maize crops (var. PMH1 and var. PMH4). Microbial population (total bacteria, fungi, P-solubilizer, amylolytic and cellulolytic bacteria) and enzymatic activities (dehydrogenase, alkaline phosphatase, urease, cellulase, amylase and invertase) in maize rhizosphere significantly increased in treatments having integrated application of CDBC, green manure and 100% whereas, soil actinomycetes and diazotrophic population were found highest in treatments with sole application of inorganic nitrogen fertilizer. Higher soil microbial populations and enzymatic activities were observed during second year of field study relative to first year. Soil chemical properties viz., electrical conductivity, organic carbon, available nitrogen, available phosphorous and potassium content were found highest under integrated application of CDBC with green manure and recommended dose of nitrogen fertilizer whereas soil pH varied non-significantly among the treatments. Maximum plant height, yield attributes and yield were observed in same treatment. The results indicated that application of CDBC biofertilizer not only decreased the fallow period of green manure decomposition but also enhanced soil health, fertility and yield of maize crop. So, application of CDBC along with green manure and inorganic nitrogen fertilizer can serve as sustainable agricultural practice for maize cultivation.
