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Open Access
DEVELOPMENT OF HYBRIDS OF PLEUROTUS FLORIDA AND VOLVARIELLA VOLVACEA THROUGH PROTOPLAST FUSION
(2013) Zenebe Teka Mengesha
Protoplast fusion using Polyethylene glycol (PEG) and electrofusion was carried out between strains of Pleurotus florida and Volvariella volvacea to develop improved hybrids. Preliminary screening was carried out based on responses to growth at different temperature regimes, colony radial growth, morphology and growth patterns of colony, hyphal size, presence or absence of clamp connections, and other unique characters like production of reddish exudates. Following the rigorous screening, eight intergeneric hybrids (Pv14, Pv19, Pv21, Pv27, Pv45, Pv66, Pv102 and Pv127) were selected and further evaluated for fruit body formation. Pv45, Pv66 and Pv102 were unable to give primordia after 25 days of growth and were considered sterile. Among those which developed fruit bodies, Pv21 and Pv127 had brownish circular caps with regular margins and central stipes. Other strains showed cap morphologies closely related to PF5. Isozyme (alcohol dehydrogenase, malate dehydrogenase, esterase and super oxide dismutase) pattern analyses indicated presence of bands in all the strains which verified hybridization between the two parents. Besides, two bands observed neither in the parents nor in the other strains were noticed in pv27 and pv127. Amplifications of genomic DNA from all the strains using 14 arbitrary decamer RAPD primers resulted in 98 bands, with size range of 160bps to 2500bps, of which 57.14 per cent were polymorphic. The bands were obtained only from 8 primers. A combined clustering analysis of these bands using NTSYSpc software indicated that Pv127 exhibited marked divergence from the other strains in the phylogenetic clusters and had better performance. Therefore, the salient investigation of the current work was screening of a hybrid strain (Pv127) with high BE (52.71%) and low temperature tolerance compared to V. volvacea
Open Access
Characterization of potential Bradyrhizobium/ Ensifer strains for improving biological nitrogen fixation and yield in soybean [Glycine max (L.) Merrill]
(PAU Ludhiana, 2013) Davinderdeep Kaur; Poonam Sharma
he present investigation was carried out to study the characterization of potential Bradyrhizobium/Ensifer strains for improving biological nitrogen fixation and yield in soybean [Glycine max (L.) Merrill]. Nine authentic strains of rhizobia were collected from different AICRP centres of soybean in India. Out of nine strains, 5 were identified as slow grower Bradyrhizobium strains (IND1, LSBR3, PANT1, SB271 & DS1) and 4 strains (IND2, LSER7, LSER8 & PANT2) as fast grower on the basis of their morphological and biochemical characteristics. Further, These strains were investigated for their functionality traits viz. indole acetic acid (IAA), phosphate (P) solubilization hydrogenase activity and intrinsic antibiotic spectra. Significantly high IAA was recorded with LSER8 (25.75µg ml-1) in the presence of L-tryptophan (0.01%). Significantly high P solubilization of TCP (100 mg) in Pikovaskaya’s medium was observed with IND2 (5.80 mg 100 ml-1) at 12th day. In qualitative screening of hydrogenase activity 2 Bradyrhizobium strains (LSBR3 and DS1) and 3 Ensifer strains (LSER7, LSER8 and PANT2) showed red coloration on YEMA media amended with 0.01% TTC dye. Roots from soybean seeds bacterized with Bradyrhizobium strain LSBR3 produced significantly high amount of flavonoid like compounds. Potential of different rhizobial strains were further evaluated for symbiotic efficiency, growth improvement and grain yield in soybean (SL744) under field conditions. Significantly high growth parameters, symbiotic traits and grain yield was observed with two Bradyrhizobium strains (LSBR3 and PANT1) and Ensifer strain (LSER8). These rhizobial strains can be explored as future biofertilizer to promote growth and yield in soybean.
Open 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.
Open Access
CHARACTERIZATION OF NARINGINASE ENZYME FOR PRODUCTION OF DEBITTERED LOW ALCOHOLIC NATURALLY CARBONATED KINNOW BEVERAGE
(PAU Ludhiana, 2013) Navjot Kaur; Param Pal, Sahota
Naringin, the bitter flavonone glycoside and primary bitter component in citrus fruit juices can be hydrolysed by naringinase enzyme into tasteless component, naringenin. A rapid detection test for naringinase producing microorganisms using FeCl3 has been developed. Naringin an inducer in kinnow juice, mutated yeast Clavispora lusitaniae and induced the production of crude enzyme naringinase with the activity of 0.0135 IU/ml. The parameters optimized for naringinase production were pH (4), temperature (50°C), naringin (0.8%) and rhamnose concentration (0.6%) with high substrate activity Km value of 1.00 mM and Vmax value of 28.56 mM. Divalent cations Cu2+ and Ca2+ competitively inhibited enzyme activity where as Mn2+ and Zn2+ stimulated enzyme activity of 0.024 IU/ml and 0.0079 IU/ml, respectively. The debittering of kinnow juice by Clavispora lusitaniae mutant prompted for utilization of kinnow juice for the production of low alcoholic naturally carbonated fermented kinnow beverage. Technology for the production of low alcoholic naturally carbonated fermented kinnow beverage with yeast Clavispora lusitaniae under optimized fermentation conditions were developed. Microbiological, physiochemical and sensory evaluation of kinnow beverage with 40 percent juice revealed pH 4.2, TSS 10.5°B, acidity 0.53%, ascorbic acid 9.2 mg/100ml, reducing sugar 8.83 percent, total sugars 9.4 percent, limonin 1.7 ppm, naringin 120.8 ppm, β-carotene 0.26μg/L, alcohol 0.28% (w/v), CO2 1.09 bar and plate count 9.3x108 cfu/ml, ranked highest for taste 7.9, aroma 8.5, colour 7.8, astringency 8.25 and overall acceptability 7.8 during storage period of 70 days under refrigerated conditions (4°C). The percentage decrease in limonin and naringin on storage was 54 and 64.8 percent, below the threshold level of limonin (6 ppm) and naringin (600 ppm) respectively. The pasteurization conditions standardized for kinnow beverage were 85°C for 3 sec.
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.
Restricted
PRODUCTION OF RICE VINEGAR AND DNA FINGERPRINTING OF FERMENTING MICROORGANISMS
(2013) Sandeep Kaur
The present study envisaged to isolate ethanol producing yeast and acetic acid producing bacteria from natural sources for vinegar production from two rice varieties, Basmati (PUSA 1121) and parmal (PR 116). The saccharification of rice starch was optimised through response surface methodology. Using crude enzyme at 60°C, maximum sugars were obtained in the wort of 20% substrate i.e. 110 and 95 mg/ml glucose from rice varieties PUSA 1121 and PR 116 respectively. With commercial enzyme too, maximum sugars were obtained with 20% substrate i.e. 172 mg/ml glucose with PUSA 1121 at 54.5°C and 133 mg/ml glucose with PR 116 at 60°C, hence giving saccharification efficiency of 95% and 90% respectively. PUSA 1121 was validated to have saccharified optimally at 20% substrate, pH 5.3, temperature 54°C and 30 IU/g alpha amylase and 50 IU/g glucoamylase. Similarly PR 116 was best saccharified at 20% substrate, pH 5.6, temperature 60°C and 30 IU each of alpha amylase and glucoamylase. Out of 20 yeast isolates screened, the isolate Y-4 gave maximum 79.25% fermentation efficiency, hence was used to produce ethanol by two techniques, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). In SHF, Y-4 produced 7.64% (w/v) and 6.82% (w/v) ethanol from PUSA 1121 and PR 116 worts having initial sugar concentration of 17.2% and 14% respectively. However, SSF led to decreased ethanol production i.e. 5.88% (w/v) from parmal rice and 6.82% (w/v) from basmati rice with the same initial sugar levels in the wort. Characteristically Y-4 utilized fructose, dextrose, galactose and mannose, reproduced by lateral budding, sensitive to cycloheximidine, lacked lipolytic activity and unable to produce starch and was identified as Issatchenkia orientalis F701 (Pichia kudriavzeii). It stands deposited in GenBank with accession no. JX537791. On the other hand, standard culture of Acetobacter acetii NRRL 746 from the Dept. of Microbiology, P.A.U, Ludhiana was used for acetic acid production from ethanol. An 8% ethanol wort produced 6.51% (w/v) volatile acids in PUSA 1121 and 5.08% (w/v) volatile acids in PR 116 in the lab studies. In the scale up production of vinegar to 5 litre, the volatile acidity levels were 5.10 and 4.86% (w/v) for respective rice varieties. The sensory evaluation of vinegar samples scored 7.59 and 8.27 points on 10 point scale for basmati and parmal varieties of rice respectively. Both types of rice vinegar were analyzed to have elements such as Fe, Na, K, Mg, Ca, Zn within acceptable limits according to Codex Alimentarius (2000).
Open Access
EPIDEMIOLOGICAL SURVEY OF WATER BORNE PROTOZOA IN DRINKING WATER AND THE CORRELATION WITH FAECAL COLIFORMS AND CLOSTRIDIUM SP.
(Punjab Agricultural University, Ludhiana, 2013) Gupta, Anchal; Kocher, D.K.
Safe drinking water is a basic human right and its adequate supply is one of the major prerequisites for a healthy life, but waterborne diseases are a cause of deaths in many parts of the world. Three main types of micro-organisms responsible for water borne gastrointestinal illness are bacteria, viruses and protozoa. These pathogens enter the drinking water sources either by contamination of water by sewerage, unhygienic habits or by person to person contact or food borne. Giardia and Cyptosporidium protozoans act as indicators of water contamination, also the coliforms and Clostridium bacteria are world widely used as indicators of faecal contamination. A survey was conducted in rural and urban areas of Ludhiana district to find out the current status of water borne protozoans and their incidence in relation to microbial agents i.e coliforms and Clostridium sp. in water. In total, 100 water samples from various sources like submersible, handpumps, municipal corporation taps and filter were collected. Incidence of Giardia was not detected in any of the collected water samples. Results revealed that high incidence of Cryptosporidium, coliforms and Clostridium was found in handpump water followed by submersible and municipal corporation, while filter water showed their lowest incidence. Correlation of Cryptosporidium with Clostridium was also higher in handpump water (100%) followed by submersible (90%), while correlation of Cryptosporidium with coliforms was higher in submersible pumps (72.7%) and similar i.e 66.7% in the water samples collected from other sources.
Open Access
Submitted to the Punjab Agricultural University in partial fulfilment of the requirements for the degree of
(Punjab Agricultural University, Ludhiana, 2013) Kaur, Jatminder; Gupta Phutela, Urmila
Ethanol is the most consumed biofuel in the world. Sweet sorghum [Sorghum bicolor (L.) Moench], a C4 Graminaceous crop which has sugar rich stalks having good potential as an alternative feed stock for ethanol production. In present study the sweet sorghum juice of variety CSV19SS was procured and was being analyzed for storage stability and its fermentation potential. Sweet sorghum juice was found to be stable under refrigeration conditions for about 75 days without any significant changes in the sugar profile and acidity of the juice. Two Saccharomyces cerevisiae strains namely S. cerevisiae strain-35 and S. cerevisiae strain NRRL Y-2034 were used for fermentation of sweet sorghum juice. S. cerevisiae strain-35 was found to be more efficient yielding 8.6% ethanol with fermentation efficiency of 86.69% as compared to S. cerevisiae strain NRRL Y-2034 yielding 8.4% ethanol with fermentation efficiency of 86.34%. Optimization of cultural parameters for maximum ethanol production and fermentation efficiency was done with the help of statistical software Statgraphics Centurian XVI.I using Response Surface Methodology. Various optimizing parameters were inoculum concentration, agitation rate and temperature and the resultant responses were ethanol content, total acidity and pH, using sugarcane and sweet sorghum juice inoculated separately by Saccharomyces cerevisiae strain NRRL Y-2034 and Saccharomyces cerevisiae strain-35. Production of ethanol from sweet sorghum juice and sugarcane juice (COJ-89) was compared under optimized conditions. Sweet sorghum juice inoculated with S. cerevisiae NRRL Y-2034 yielded 8.85% (v/v) ethanol with fermentation efficiency of 87.35% under optimized conditions of temperature (30ºC), agitation rate (50 rpm) and inoculum size (7.5% v/v). The sugarcane juice inoculated with S. cerevisiae NRRL Y-2034 yielded 10.14% (v/v) ethanol under optimized conditions of temperature (30ºC), agitation rate (100 rpm) and inoculum size (5% v/v) with fermentation efficiency of 97.67%. When sweet sorghum juice inoculated with S. cerevisiae Strain-35 8.47% (v/v) ethanol was produced under optimized conditions of temperature (30ºC), agitation rate (50 rpm) and inoculum size (5% v/v) with fermentation efficiency of 83.56%. However in sugarcane higher ethanol production was observed (11.23% v/v) with fermentation efficiency of 95.62% under optimized conditions of temperature (30ºC), agitation rate (100 rpm) and inoculum size (7.5% v/v). Sweet sorghum juice and sugarcane juices are good substrates for ethanol production. However, lifecycle assessment of both the crops has an edge towards the utilization of sweet sorghum juice for ethanol production
Open Access
PROCESS OPTIMIZATION FOR ETHANOL PRODUCTION FROM SWEET SORGHUM JUICE USING Saccharomyces cerevisiae
(Punjab Agricultural University, Ludhiana, 2013) Kaur, Jatminder; Gupta Phutela, Urmila
Ethanol is the most consumed biofuel in the world. Sweet sorghum [Sorghum bicolor (L.) Moench], a C4 Graminaceous crop which has sugar rich stalks having good potential as an alternative feed stock for ethanol production. In present study the sweet sorghum juice of variety CSV19SS was procured and was being analyzed for storage stability and its fermentation potential. Sweet sorghum juice was found to be stable under refrigeration conditions for about 75 days without any significant changes in the sugar profile and acidity of the juice. Two Saccharomyces cerevisiae strains namely S. cerevisiae strain-35 and S. cerevisiae strain NRRL Y-2034 were used for fermentation of sweet sorghum juice. S. cerevisiae strain-35 was found to be more efficient yielding 8.6% ethanol with fermentation efficiency of 86.69% as compared to S. cerevisiae strain NRRL Y-2034 yielding 8.4% ethanol with fermentation efficiency of 86.34%. Optimization of cultural parameters for maximum ethanol production and fermentation efficiency was done with the help of statistical software Statgraphics Centurian XVI.I using Response Surface Methodology. Various optimizing parameters were inoculum concentration, agitation rate and temperature and the resultant responses were ethanol content, total acidity and pH, using sugarcane and sweet sorghum juice inoculated separately by Saccharomyces cerevisiae strain NRRL Y-2034 and Saccharomyces cerevisiae strain-35. Production of ethanol from sweet sorghum juice and sugarcane juice (COJ-89) was compared under optimized conditions. Sweet sorghum juice inoculated with S. cerevisiae NRRL Y-2034 yielded 8.85% (v/v) ethanol with fermentation efficiency of 87.35% under optimized conditions of temperature (30ºC), agitation rate (50 rpm) and inoculum size (7.5% v/v). The sugarcane juice inoculated with S. cerevisiae NRRL Y-2034 yielded 10.14% (v/v) ethanol under optimized conditions of temperature (30ºC), agitation rate (100 rpm) and inoculum size (5% v/v) with fermentation efficiency of 97.67%. When sweet sorghum juice inoculated with S. cerevisiae Strain-35 8.47% (v/v) ethanol was produced under optimized conditions of temperature (30ºC), agitation rate (50 rpm) and inoculum size (5% v/v) with fermentation efficiency of 83.56%. However in sugarcane higher ethanol production was observed (11.23% v/v) with fermentation efficiency of 95.62% under optimized conditions of temperature (30ºC), agitation rate (100 rpm) and inoculum size (7.5% v/v). Sweet sorghum juice and sugarcane juices are good substrates for ethanol production. However, lifecycle assessment of both the crops has an edge towards the utilization of sweet sorghum juice for ethanol production.

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