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Dr. Y. S. Parmar University of Horticulture & Forestry, Solan
Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, was established on 1st December, 1985 with the objective to promote education, research and extension education in the fields of Horticulture, Forestry and allied disciplines. Late Dr. Yashwant Singh Parmar, the first Chief Minister and the architect of Himachal Pradesh perceived the importance of Horticulture and Forestry to develop and improve the State economy which led to the establishment of this University. Its history lies in erstwhile Himachal Agricultural College, Solan, established in 1962 and affiliated to the Panjab University. It became one of the campuses of Agriculture Complex of Himachal Pradesh University on its formation in 1970. Consequent upon the establishment of Himachal Pradesh Krishi Vishvavidyalaya in 1978, this campus became its Horticulture Complex and finally in 1985, assumed the status of a State University, being the only University in the country engaged exclusively in teaching, research and extension in Horticulture and Forestry.
The University is located at Nauni in Solan District of Himachal Pradesh, 13 km from Solan on Solan-Rajgarh Road, at an elevation of 1300 metres above mean sea level. Solan town is situated on national highway (NH-22) and is well connected by train and bus services.
The University has four constituent colleges, out of which, two are located at the main campus Nauni, one for horticulture and the other for forestry, having 9 and 7 departments, respectively. The third College i.e., College of Horticulture & Forestry is located at Neri in Hamirpur District on Nadaun-Hamirpur state highway, about 6 Km from Hamirpur town and is well connected with bus service. The college offers three Undergraduate Degree Programmes i.e. BSc (Hons.) Horticulture, BSc (Hons.) Forestry and B. Tech. Biotechnology and MSc degree programme in a few subjects. The fourth college i.e. College of Horticulture and Forestry, Thunag (Mandi) is located at Thunag District Mandi. This college offer BSc (Hons.) Horticulture and BSc (Hons.) Forestry degree programme. In addition, there are five Regional Research Stations, 12 Satellite Stations and five Krishi Vigyan Kendras (KVKs) situated in different zones of the State.
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ThesisItem Open Access Characterization of plant growth regulators produced by fluorescent Pseudomonas species and their role in control of replant problem of apple and pear(DYSPU, 2013) Sharma, Shweta; Mohinder KaurIsolation, identification and characterization of native fluorescent Pseudomonas isolates for multifarious plant growth activities from normal and replant site of apple and pear orchard was done for selection of best plant growth regulators producing strains. These were screened out for plant growth promoting activities like production of plant growth regulators, phosphate solubilization, antifungal, siderophores, HCN, ammonia and lytic enzymes. Plant growth regulators are important secondary metabolites produced by fluorescent Pseudomonas sp. such as auxins, gibberellic acid and cytokinins which play significant role in increasing the root surface and length that may help in early establishment of plant in replant site. On the basis of PGPR activities, ten isolates were genotypically characterized. Two best isolates (An-1-kul and An-13-Kul) were selected for 16S rRNA sequencing. The sequence of the 16S rRNA gene is used as a molecular clock to estimate relationships among bacteria and to identify an unknown bacterium to the genus or species level). RAPD studies were also carried out with ten selected isolates which showed the best relatedness among isolates which were isolated from same location. Three regulators auxins, gibberellins and cytokinins were extracted and purified from twobest selected strains of Pseudomonas aeruginosaAn-1-kul and An-13-kul and were purified and characterized by TLC, Sephadex G-25, Dowex-50 column chromatography, high performance liquid chromatography (HPLC) and their specific bioassays. Partial purified cytokinins and auxins extracted from these two strains An-1-kul and An-13-kul were also evaluated by tissue culture bioassay on callus formation and shoot regeneration in broccoli explant and root regeneration in in vitrodeveloped shootlets of cabbage respectively. Three strains of Pseudomonas aeruginosaAn-1-kul and An-13-kul, An-4-shr were used individually and their consortia for treatment of apple and pear seedlings before plantation in replant field at Bajaura and Sharontha respectively. The performance of apple and pear seedlings was much better in terms of plant establishment, growth promotion in terms of plant height, number of nodesand number of branches, chlorophyll content of leaves and NPK of rhizospheric soil over their respective control after nine and twelve months of plantation. These strains can be further exploited and recommended for the management of replant problem of apple and pear after conducting more field trials in replant sites . These characterized strains of Pseudomonas aeruginosa can have great importance in the field of horticulture, especially in the treatment of replant problem of apple and pear especially in Himachal Pradesh.ThesisItem Open Access Production, purification and characterization of cellulase free xylanase from Cellulosimicrobium cellulans in solid state fermentation of apple pomace and its application in pulp biobleaching(YSPU, 2013) Walia, Abhishek; Shirkot, C.K.Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is first report on actinomycete from Cellulomonas genera to produce cellulase-free xylanase, which is an important industrial enzyme used in the pulp and paper industry. Strain CKMX1 was characterized by metabolic fingerprinting, whole-cell fatty acids methyl ester analysis and 16Sr DNA and found to be Cellulosimicrobium cellulans CKMX1. Cultural conditions and process parameters i.e. type of medium, particle size of carbon source, incubation period, temperature, initial pH, inoculum size, yeast extract, NH4NO3, urea, peptone, CMC, MgSO4 and CaCO3 were optimized using one factor at a time approach and xylanase activity was increased to 570.0 U/g DBP. CMCase, avicelase, FPase and -glucosidase activities were not detected, highlighting the novelty of the xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using central composite design following response surface methodology with eight independent variables which resulted in very high levels of xylanase (1027.65 U/g DBP). The optimization resulted in 3.1-fold increase of xylanase production, compared with the lowest xylanase production of 331.50 U/g DBP. The enzyme was purified by gel permeation and anion exchange chromatography and had a molecular mass of 29 kDa. Xylanase activity was maximum at pH 8.0 and 60ºC. The enzyme was somewhat thermostable, retaining 50% of the original activity after incubation at 50ºC for 30 min. The xylanase had Km and Vmax values of 26.4 mM and 2,000 μmol/min/mg protein, respectively. All metal ions except HgCl2, CoCl2 as well as CdCl2 were well tolerated and did not adversely affect xylanase activity. The deduced internal amino acid sequence of C. cellulans CKMX1 xylanase by MALDI-TOF MS resembled the sequence of acetyl xylan esterase of the Thermotoga sp. EMP (Accession. no. WP_008192031). Molecular cloning was done by using pGEM-T easy vector and sequence of xylanase gene of C. cellulans CKMX1 showed maximum homology (98%) with xylanase gene of Cellulosimicrobium sp. (Accession no. FJ859907.1). The hydrolytic products of the insoluble oat spelt xylan incubated with xylanase were identified by xylose standards using HPLC. Xylanase effectively hydrolyzed xylan and the hydrolysis by xylanase produced mainly xylose as the main product after 5 min incubation time. Cellulase-free xylanase from C. cellulans CKMX1 under C-EP-D sequence has been shown to bring about a 12.5% reduction of chlorine, decrease of 0.8 kappa points (40%) and gain in brightness was 1.42 % ISO points in 0.5% enzyme treated pulp as compared to control where no enzyme pre-treatment was given, when enzymatically prebleached pulp was charged with 7.4% of total chlorine. From the present studies it is clear that C. cellulans CKMX1 xylanase is having the characteristic suited for an industrial enzyme (xylanases that are active and stable at elevated temperatures and alkaline pH).ThesisItem Open Access Bioconversion of cellulosic waste into bioethanol as biofuel(DYSPU, 2013) Sharma, Nisha; Sharma, NiveditaThe present investigation was carried out to isolate, screen and identify the most efficient cellulolytic and xylanolytic microorganisms from soil. Mutation of hypercellulase and xylanase producers, enzyme production, optimization, partial purification, bioconversion of cellulosic waste into bioethanol and scale up studies were performed with selected strains to recommend their use for industries. In total 89 microorganisms including 84 bacteria and 5 fungi were isolated. Among them, ten hypercellulase and xylanase producing bacteria were subjected to mutation for enhanced enzyme production. N 12 (M) and Kd1(M) were screened for cellulase and xylanase enzyme production studies. The wild and mutant bacterial isolates were identified as B. stratosphericus N12 (W), B. stratosphericus N12 (M), B. altitudinis Kd1 (W) and B. altitudinis Kd1 (M) respectively by 16S rRNA PCR technique and registered with NCBI under accession no. |KC995116|, |KC995118|, |KC995115| and |KC995117|. Cellulase and xylanase enzymes were optimized through classical approach one factor at a time (OFAT) under submerged fermentation varying medium, pH, temperature, inoculum size, incubation time, carbon source and substrate concentration. The percent increase in enzyme activity obtained after optimization of different process parameters was 85.23% for cellulase of B. stratosphericus N12(M) and 85.60% for xylanase of B. altitudinis Kd1(M). To reduce the production cost of enzymes, cheap untreated and pretreated lignocellulosic forest biomass i.e. hardwood and softwood wereused as a substrate under SmF by B. stratosphericusN12(M) and B. altitudinisKd1 (M), SSF by M. thermophilaSH1 and among them alkaline hydrogen peroxide pretreated P. deltoideswood was found the best for hypercellulase and xylanase production under SmF as well as SSF. The partial purification of hydrolytic enzymes was done by ammonium sulphate precipitation. Scale up of cellulase from B. stratosphericus N12(M) as well as xylanase from B. altitudinisKd1 (M) was performed in 7.5 L bioreactor at 200 rpm, 1 vvm and 30 0 C, achieving 2.443 IU cellulase and 11.10 IU xylanase respectively, afteronly 8 h of fermentation. Bioconversion of alkaline hydrogen peroxide pretreated P. deltoideswood to ethanol was studied under three different fermentation processes i.e separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF). Different strategies had been designed to delimit the constraints of fermentation process. SHF was evaluated by modifying four different sub-processes of detoxification and non-detoxification as well as pooling and nonpooling of pretreated liquor. Maximum ethanol was achieved in method –IV of SHF i.e. 18.47g/l by co-culture of S. cerevisiae II and P. stipitis with the fermentation efficiency of 72.46%. Among all the three processes of fermentation evaluated in the present study, SHF was found to be the best and in case of strains used for fermentation, co-culture of S. cerevisiaeII and P. stipitiswas observed the best combination for highest bioethanol production.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF PLANT GROWTH PROMOTING RHIZOBACTERIUM AND ITS EFFECT ON GROWTH OF APPLE PLANTS(2013) MAHAJAN, RISHI; SHIRKOT, C.K.Apple cultivation is today a prominent industry in Himachal Pradesh, India occupying an area of 1,03,644 hectares of land of the total 2,14,574 hectares of land under horticulture. High yielding modern varieties are being adopted, to increase the productivity, which are highly responsive to external inputs. Indiscriminate use of chemical inputs poses potential threat to human health and environment. It is in this context, that PGPR play a pivotal role in organic/sustainable crop production. Work presented in here, highlights the novelty of Bacillus licheniformis strain CKA1, isolated from apple roots, in being used as a single strain with multifunctional plant growth promoting traits. Microbial formulation developed has been comprehensively evaluated under field conditions for its effect on early growth and yield of apple. Bio-control potential of the isolate has been assessed in white root rot infested apple trees. An attempt has also been made to decipher molecular mechanisms underlying the multifunctional plant growth promoting potential of strain CKA1. Application of this microbial formulation resulted in 59.61 to 75.83 per cent increase in germination; 59.58 and 61.89 per cent increase in shoot and root length respectively in nursery and increased Nitrogen (32.53%), Phosphorus (320.37%) and Potassium (48.27%) in the whole shoot system over un-inoculated controls. PGPR inoculation increased fruit yield by 35 to 44% in a three year trial in farmer’s field and completely rejuvenated diseased apple trees infected with Dematophoranecatrix. Cloning and sequencing of genes from strain CKA1 has paved the way for understanding molecular mechanisms involved in (a) nutrient mobilization of essential nutrients and their subsequent enhanced uptake by plants (b) phytohormone production and (c) antifungal metabolite synthesis. Apple orchard rejuvenation projects are being ambitiously launched by State agencies for restoring six decade old planted apple orchards. Integrated nutrient management systems comprising biological systems especially, single strain multifunctional PGPR such as strain CKA1 opens up new avenues not only for improving crop yield but also in sustaining soil health.ThesisItem Open Access CHARACTERIZATION OF PLANT GROWTH REGULATORS PRODUCED BY FLUORESCENT Pseudomonas SPECIES AND THEIR ROLE IN CONTROL OF REPLANT PROBLEM OF APPLE AND PEAR(2013) SHARMA, SHWETA; KAUR, MOHINDERABSTRACT Isolation, identification and characterization of native fluorescent Pseudomonas isolates for multifarious plant growth activities from normal and replant site of apple and pear orchard was done for selection of best plant growth regulators producing strains. These were screened out for plant growth promoting activities like production of plant growth regulators, phosphate solubilization, antifungal, siderophores, HCN, ammonia and lytic enzymes. Plant growth regulators are important secondary metabolites produced by fluorescent Pseudomonas sp. such as auxins, gibberellic acid and cytokinins which play significant role in increasing the root surface and length that may help in early establishment of plant in replant site. On the basis of PGPR activities, ten isolates were genotypically characterized. Two best isolates (An-1-kul and An-13-Kul) were selected for 16S rRNA sequencing. The sequence of the 16S rRNA gene is used as a molecular clock to estimate relationships among bacteria and to identify an unknown bacterium to the genus or species level). RAPD studies were also carried out with ten selected isolates which showed the best relatedness among isolates which were isolated from same location. Three regulators auxins, gibberellins and cytokinins were extracted and purified from two best selected strains of Pseudomonas aeruginosa An-1-kul and An-13-kul and were purified and characterized by TLC, Sephadex G-25, Dowex-50 column chromatography, high performance liquid chromatography (HPLC) and their specific bioassays. Partial purified cytokinins and auxins extracted from these two strains An-1-kul and An-13-kul were also evaluated by tissue culture bioassay on callus formation and shoot regeneration in broccoli explant and root regeneration in in vitro developed shootlets of cabbage respectively. Three strains of Pseudomonas aeruginosa An-1-kul and An-13-kul, An-4-shr were used individually and their consortia for treatment of apple and pear seedlings before plantation in replant field at Bajaura and Sharontha respectively. The performance of apple and pear seedlings was much better in terms of plant establishment, growth promotion in terms of plant height, number of nodes and number of branches, chlorophyll content of leaves and NPK of rhizospheric soil over their respective control after nine and twelve months of plantation. These strains can be further exploited and recommended for the management of replant problem of apple and pear after conducting more field trials in replant sites . These characterized strains of Pseudomonas aeruginosa can have great importance in the field of horticulture, especially in the treatment of replant problem of apple and pear especially in Himachal Pradesh.ThesisItem Open Access DETECTION AND CHARACTERIZATION OF GENE ENCODING BACTERIOCIN IN LACTIC ACID BACTERIA AND TO STUDY PRESERVATIVE POTENTIAL OF PURIFIED BACTERIOCIN(2013) GAUTAM, NEHA; SHARMA, NIVEDITAABSTRACT The present investigation was carried out to isolate most efficient bacteriocin producing potential probiotic lactic acid bacteria from rare and unexplored food sources, their screening, identification, optimization to enhance the maximum bacteriocin producing potential, purification, characterization of purified bacteriocin on biochemical as well as on molecular level and application of bacteriocin as food biopreservatives. In addition, probiotic attributes of bacteriocin producing lactic acid bacteria and its role in cell mediated preservation was studied. In total 53 bacterial isolates were isolated from fermented/ non fermented food sources. out of all, 41 bacterial isolates which were catalase –ve were screened against ten spoilage causing food borne pathogens by bit/disc method. Among all, 8 isolates were further secreened to test bacteriocin production potential on the basis of their wide inhibitory spectrum against tested pathogens. Finally two isolates UN and G2 were selected for further studies being hyperbacteriocin producers, which were isolated from Dhulliachar and Gundruck respectively which are traditional fermented food products of North east India. Isolate UN was identified as Lactobacillus brevis while G2 identified as Lactobacillus spicheri by 16S r RNA gene technique and registered in NCBI under accession no. JX46150 and JX48191 respectively. Bacteriocin production was optimized through classical one variable at a time method. Both the isolates showed maximum bacteriocin production at early stationary phase, at pH4.0, temperature 350C with an inoculum size of 1.5 OD @ 10 %. Bacteriocins from both the isolates were purified by single step gel exclusion chromatography and their molecular weights were found to be 14 kDa and 43 kDa respectively. Activity units increased from 2×103 to 8×103 AU/ml in both cases. Purified bacteriocin titers of L. brevis UN increased by 87.5 % against L. monocytogenes, 66.6 % against S.aureus and 75 % against C. perfringens. In case of L. spicheri G2 bacteriocin titers increased by 75 % , against L. monocytogenes, S. aureus and 15 % against C. perfringens respectively. Purified bacteriocins of both the isolates were characterized by studying the effect of temperature, pH , proteolytic enzymes and storage stability on them. Both purified bacteriocins were maximum active against all the tested pathogens at neutral pH, both were found to have moderate thermostability and were sensitive to proteolytic enzymes trypsin and proteinase k. Molecular determinants for bacteriocin production in L. brevis UN showed that gene for bacteriocin production was plasmid bound. 1H-NMR revealed the unique combinations of different amino acids in biochemical structure of purified bacteriocin which has been reported for the first time in present study. Both isolates were tested for their probiotic attributes and were found to have sound probiotic potential.The use of both the strains in bacteriocin mediated preservation and cell mediated preservation have been found quite satisfactory. The purified bacteriocins produced by L. brevis UN and L.spicheri G2 showed resistance to the spoilage causing microorganisms in milk and apple juice. L. brevis UN and L. spicheri G2 were used successfully to prepare healthy and refreshing probiotics drinks viz. bioyogurt I, II, III and nutritionally rich cereal based probiotic product.ThesisItem Open Access BIOCONVERSION OF CELLULOSIC WASTE INTO BIOETHANOL AS BIOFUEL(2013) SHARMA, NISHA; SHARMA, NIVEDITAABSTRACT The present investigation was carried out to isolate, screen and identify the most efficient cellulolytic and xylanolytic microorganisms from soil. Mutation of hypercellulase and xylanase producers, enzyme production, optimization, partial purification, bioconversion of cellulosic waste into bioethanol and scale up studies were performed with selected strains to recommend their use for industries. In total 89 microorganisms including 84 bacteria and 5 fungi were isolated. Among them, ten hypercellulase and xylanase producing bacteria were subjected to mutation for enhanced enzyme production. N12 (M) and Kd1 (M) were screened for cellulase and xylanase enzyme production studies. The wild and mutant bacterial isolates were identified as B. stratosphericus N12 (W), B. stratosphericus N12 (M), B. altitudinis Kd1 (W) and B. altitudinis Kd1 (M) respectively by 16S rRNA PCR technique and registered with NCBI under accession no. |KC995116|, |KC995118|, |KC995115| and |KC995117|. Cellulase and xylanase enzymes were optimized through classical approach one factor at a time (OFAT) under submerged fermentation varying medium, pH, temperature, inoculum size, incubation time, carbon source and substrate concentration. The percent increase in enzyme activity obtained after optimization of different process parameters was 85.23% for cellulase of B. stratosphericus N12 (M) and 85.60% for xylanase of B. altitudinis Kd1 (M). To reduce the production cost of enzymes, cheap untreated and pretreated lignocellulosic forest biomass i.e. hardwood and softwood were used as a substrate under SmF by B. stratosphericus N12 (M) and B. altitudinis Kd1 (M), SSF by M. thermophila SH1 and among them alkaline hydrogen peroxide pretreated P. deltoides wood was found the best for hypercellulase and xylanase production under SmF as well as SSF. The partial purification of hydrolytic enzymes was done by ammonium sulphate precipitation. Scale up of cellulase from B. stratosphericus N12 (M) as well as xylanase from B. altitudinis Kd1 (M) was performed in 7.5 L bioreactor at 200 rpm, 1 vvm and 300C, achieving 2.443 IU cellulase and 11.10 IU xylanase respectively, after only 8 h of fermentation. Bioconversion of alkaline hydrogen peroxide pretreated P. deltoides wood to ethanol was studied under three different fermentation processes i.e separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF). Different strategies had been designed to delimit the constraints of fermentation process. SHF was evaluated by modifying four different sub-processes of detoxification and non-detoxification as well as pooling and nonpooling of pretreated liquor. Maximum ethanol was achieved in method –IV of SHF i.e. 18.47g/l by co-culture of S. cerevisiae II and P. stipitis with the fermentation efficiency of 72.46%. Among all the three processes of fermentation evaluated in the present study, SHF was found to be the best and in case of strains used for fermentation, co-culture of S. cerevisiae II and P. stipitis was observed the best combination for highest bioethanol production.ThesisItem Open Access PRODUCTION, PURIFICATION AND CHARACTERIZATION OF CELLULASE FREE XYLANASE FROM Cellulosimicrobium cellulans IN SOLID STATE FERMENTATION OF APPLE POMACE AND ITS APPLICATION IN PULP BIOBLEACHING(2013) WALIA, ABHISHEK; SHRIKOT, C.K.ABSTRACT Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is first report on actinomycete from Cellulomonas genera to produce cellulase-free xylanase, which is an important industrial enzyme used in the pulp and paper industry. Strain CKMX1 was characterized by metabolic fingerprinting, whole-cell fatty acids methyl ester analysis and 16Sr DNA and found to be Cellulosimicrobium cellulans CKMX1. Cultural conditions and process parameters i.e. type of medium, particle size of carbon source, incubation period, temperature, initial pH, inoculum size, yeast extract, NH4NO3, urea, peptone, CMC, MgSO4 and CaCO3 were optimized using one factor at a time approach and xylanase activity was increased to 570.0 U/g DBP. CMCase, avicelase, FPase and -glucosidase activities were not detected, highlighting the novelty of the xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using central composite design following response surface methodology with eight independent variables which resulted in very high levels of xylanase (1027.65 U/g DBP). The optimization resulted in 3.1-fold increase of xylanase production, compared with the lowest xylanase production of 331.50 U/g DBP. The enzyme was purified by gel permeation and anion exchange chromatography and had a molecular mass of 29 kDa. Xylanase activity was maximum at pH 8.0 and 60ºC. The enzyme was somewhat thermostable, retaining 50% of the original activity after incubation at 50ºC for 30 min. The xylanase had Km and Vmax values of 26.4 mM and 2,000 μmol/min/mg protein, respectively. All metal ions except HgCl2, CoCl2 as well as CdCl2 were well tolerated and did not adversely affect xylanase activity. The deduced internal amino acid sequence of C. cellulans CKMX1 xylanase by MALDI-TOF MS resembled the sequence of acetyl xylan esterase of the Thermotoga sp. EMP (Accession. no. WP_008192031). Molecular cloning was done by using pGEM-T easy vector and sequence of xylanase gene of C. cellulans CKMX1 showed maximum homology (98%) with xylanase gene of Cellulosimicrobium sp. (Accession no. FJ859907.1). The hydrolytic products of the insoluble oat spelt xylan incubated with xylanase were identified by xylose standards using HPLC. Xylanase effectively hydrolyzed xylan and the hydrolysis by xylanase produced mainly xylose as the main product after 5 min incubation time. Cellulase-free xylanase from C. cellulans CKMX1 under C-EP-D sequence has been shown to bring about a 12.5% reduction of chlorine, decrease of 0.8 kappa points (40%) and gain in brightness was 1.42 % ISO points in 0.5% enzyme treated pulp as compared to control where no enzyme pre-treatment was given, when enzymatically prebleached pulp was charged with 7.4% of total chlorine. From the present studies it is clear that C. cellulans CKMX1 xylanase is having the characteristic suited for an industrial enzyme (xylanases that are active and stable at elevated temperatures and alkaline pH