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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 Studies on characterization and role of 2,4-Diacetylphloroglucinol and Pyoluteorin produced by fluorescent Pseudomonas species in replant site of apple orchard(YSPU, 2015) Rana, Sheetal; Kaur, MohinderThe present study focuses on characterization of secondary antifungal metabolites i.e. 2,4- diacetylphloroglucinol and Pyoluteorin produced by fluorescent Pseudomonas sp. isolated from the normal and replant sites of apple orcards. Six strains were isolated from the replant sites of Maggota, Sharontha and Siao of Shimla distt. and thirteen already isolated strains of fluorescent Pseudomonas sp. from replant sites of apple were used for screening of direct and indirect plant growth promoting activities like antifungal, siderophores, phosphate solubilization and production of HCN, ammonia, plant growth regulators (auxins, cytokinins and gibberellins) and lytic enzymes. Isolates Sh2r, Sh4r and B showed 99.7%, 98.4% and 99% similarity with Pseudomonas poae DSM14936 and Pseudomonas gessardii CIP105469 respectively. Six fungal pathogenic sp. isolated from replant sites of apple of Shimla distt. i.e. MgF1 and MgF2 showed 99.2% and 99.1% similarity with Rosellinia necatrix and Fusarium oxysporum, ShF1 and ShF2 showed 99.5% and 99.2% similarity with Fusarium oxysporum and Schizophyllum commune & SiF1 and SiF2 showed 99.2% and 100% similarity with Fusarium oxysporum and Pythium ultimum. Optimization of media, time of incubation, temperature, pH, carbon, nitrogen and mineral sources were done for production and extraction of antifungal metabolites i.e. 2,4- diacetylphloroglucinol & Pyoluteorin and for mass multiplication of inoculums for field trials. These metabolites were extracted with ethylacetate and identified through TLC at Rf 0.8 and 0.5 respectively. Antifungal activity, MIC and thermal stability of both these metabolites were evaluated. 2,4-diacetylphloroglucinol was further characterized through HPLC and NMR techniques. Antifungal activities of Pseudomonas sp. help in suppression of plant pathogens in soil. Antifungal activity along with other plant growth promoting activities may be the reasons for better establishment and growth promotion of replanted apple. Treatments with formulations of consortial strains were found to be more effective in growth and establishment of replanted apple. Therefore these strains can be exploited for the management of replant problem of apple.ThesisItem Open Access Purification and characterization of amylase produced from potential extremophiles for industrial application(YSPU, 2015) Gitanjali; Sharma, NiveditaMushroom compost being a highly probable source for amylogenic microorganisms was utilized as a source for isolation of amylolytic microorganisms. In total, 13 amylolytic bacteria have been screened from mushroom compost. Among them, alkalothermophilic hyperamylogenic strains GC6 and GV2 were selected and identified as B. aerius GC6 |KJ 775810.1| and B. sonorensis GV2 |KJ775811.1|. Cultural conditions and process parameters viz. media types, pH, temperature, inoculum size, incubation time, substrate concentration, divalent ions and surfactants etc. were optimized firstly through classical one variable at a time (OVAT) followed by statistical optimization by employing central composite design of response surface methodology. The enzymes obtained from both the strains were purified to homogeneity by following a sequential purification approach. B. aerius GC6 α-amylase was purified to a final purification fold of 17.86 and had a molecular weight of 43 kDa whereas B. sonorensis GV2 α-amylase was purified to a final purification fold of 14.52 and had a molecular weight of 45 kDa. α-amylase activity was found to be maximum at 50 oC and pH 9.0 for B. aerius GC6 and at 50 oC and pH 10.0 for B. sonorensis GV2. α-amylase from both the strains was quite thermostable with retention of more than 50% activity after incubation of 90 min at 45-60 oC. Kinetic characteristics of α-amylase from both the strains showed that the enzyme was very efficient qualitatively as well as quantitatively. Raw starch adsorption and hydrolysis ability shown by α-amylase from both the strains is a rare feature of bacterial α-amylase making it a potential candidate for starch processing industry. Immobilization of purified α-amylase from both the strains onto natural hydrogels showed a considerable retention upto 8 cycles however immobilization using iron oxide nano particles proved to be quite stable with apparently no loss in activity even after 20 cycles. Applicability of purified α-amylase from B. aerius GC6 was assessed by utilizing it for the hydrolysis of Hydrodictyon biomass for the production of bioethanol. Mixture of α-amylase, β-amylase, cellulase and pectinase from potential inhouse microorganisms was used for saccharification of biomass followed by its fermentation to ethanol using S. cerevisiae-I (MTCC3089). A final ethanol concentration of 13.93 g/l and 16.67 g/l with a fermentation efficiency of 64.84% and 66.68% was obtained in a stirred tank bioreactor under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) mode respectively. This is the first report on α- amylase from B. aerius and B. sonorensis. Thermal and alkaline stability of this enzyme makes them a potential candidate for use in starch processing industry.ThesisItem Open Access Characterization of alkaline protease from plant growth promoting rhizobacteria and their potential applications(YSPU, 2015) Shiwani; Shirkot, C.K.Rhizosphere of apple trees of Trans Himalayas is great niche of proteolytic bacteria which may have potential for use in industries. A total of 76 proteolytic bacteria were obtained from rhizosphere soil and root endosphere of apple trees from different sites of four locations viz., Chamba, Shimla, Kinnaur and Kullu of Himachal Pradesh, India, which were screened for plant growth promoting traits including phosphate solubilization, IAA, hydrogen cyanide production and production of antifungal metabolite. Among them, 17 isolates that exhibited maximum alkaline protease activity were selected. The diversity elucidation and differentiation of isolates was achieved through their biochemical characterization and amplified ribosomal DNA restriction analysis with three tetra-cutter restriction enzymes (Hpa II, Hinf I and TaqI), the representative cluster groups were identified by 16S rDNA sequencing. This brings to light the predominance of Bacillus sp. and presence of remarkable intraspecies functional and genotypic diversity in apple rhizosphere. Proteolytic acitivity among selected isolates ranged between 50 to 1610 μg/ml/min at pH 8.0 and maximum was observed for Bacillus amyloliquefaciens SP1 (1610 μg/ml/min). Maximum enzyme production for B. amyloliquefaciens SP1 was observed, when fermentation medium, supplemented with 1.0% gelatin at pH 8.0 was incubated at 35°C for 48 h with 1.0% inoculun size and maltose as carbon source. Further optimization by Plackett Burman design and Response surface methodology resulted 2.08-fold increase in alkaline protease production (3350 μg/ml/min). The enzyme was purified by gel permeation and cation exchange chromatography and had a molecular mass of 43 kDa. Protease activity was optimum at pH 8.0 and 60°C. The half-life of the enzyme at 50, 60 and 65⁰C was 77, 19.8 and 13.33 min. The protease had Km, V max and Ea values of 0.125 mg/ml, 12820 μg min-1 and 34.52 kJ/mol in casein hydrolysis, respectively. All metal ions except Hg2+ and Cu2+ were well tolerated. The deduced internal amino acid sequence of B. amyloliquefaciens SP1 protease by MALDI-TOF MS resembled the sequence with D-alanyl-D-alanine carboxypeptidase of Bacillus subtilis strain 168. An alkaline protease gene was amplified from genomic DNA, cloned and successfully expressed in competent cells of E. coli DH5 . Most reliable three dimensional structure for protease was predicted by using Phyre2 server. This study reported the shortest time of 1:30 min at 3630 μg/ml/min and 4:30 min at74 μg/ml/min of enzyme activity for hydrolysis of gelatin layers of used X-ray film. It has revealed strain removal property and dehairing activity for animal hide without chemical assistance and without hydrolyzing fibrous proteins. This study also reported significant role of protease in antagonistic activity against C. michiganensis, R. solanacearum and F. oxysporium.ThesisItem Open Access CHARACTERIZATION OF POTENTIALLY ACTIVE NEW PROBIOTIC STRAINSISOLATEDFROM DIFFERENT SOURCES AND TO STUDY THEIR PROSPECTS AS NUTRACEUTICAL AGENTS(2015) GUPTA, ANUPAMA; SHARMA, NIVEDITAABSTRACT The present investigation was carried out to isolate most efficient potential probiotic lactic acid bacteria from rare and unexplored food sources, their screening, identification, safety assessment, evaluationof probiotic attributes, metabolic fingerprinting as well the development of novel nutraceutical products. In total 27 bacterial isolates were obtained from fermented/non fermented food sources. Out of all, 6 bacterial isolates were screened by bit/disc as well as well diffusion method on the basis of their wide inhibitory spectrum against tested pathogens. Isolates C-1, LB-CC, LB-WC, Ch-1, Ch-2 and 107 were selected for further studies having widest antagonistic potential, which were isolated fromkodo millet flour, lasoda bari, chuli and salori, respectively that are traditional food products of Himachal Pradesh. Isolates C-1, LB-CC and LB-WC were identifies as Pediococcus pentosaceus, Ch-1 as Enterococcus faecium, Ch-2 as Pediococcus acidilactici while 107 identified as Pediococcus damnosus by 16S rRNA gene technique and registered in NCBI under accession no. KM251461, KM251460, KJ489415, KJ541885, KJ541886 and KJ577588, respectively. Safety assessment of isolates was done by evaluating antibiotic susceptibility, haemolytic, DNase and gelatinase activities. All the isolates exhibited 80-100% antibiotic sensitivity, non-heamolytic, non-DNase and non-gelatinase activities, thereby proving their safe status. These screened isolates were further examined for their probiotic potential viz. acidity tolerance, bile tolerance, simulated gastrointestinal transit, auto-, co-aggregation, hydrophobicity, adhesion to gastric as well as mammalian cell lines, competitive exclusion of pathogens, antimicrobial potential, exopolysaccharides and β-galactosidase production, compatibility and cumulative probiotic potential. All the six screened isolates were found to be highly acidity tolerant strains with 93.65-99.41 % survival rate at pH 3 for 3 h. These six isolates were able to resist high bile salt concentration i.e. 2.0% with 93.12-98.22 % survival rate for 8 h. Resistance to gastrointestinal transit as found to be in a range from 43.09-90.12 %. All the six isolates exhibited good autoaggregation capacity i.e. greater than 40% after 5 h and showed string hydrophobicity towards xylene i.e. > 40%. All the isolates were able to adhere to gastric mucin with 14.7-49.5% adhesion and all showed adhesion potential to mammalian epithelial cell lines. These screened probiotic isolates showed broad and strong inhibitory spectrum against both gram positive as well as gram negative pathogenic bacteria through secretion of antimicrobial metabolites viz. lactic acid (7.18-8.46 mg/l), H2O2 (0.24-0.38g/l) and bacteriocins (77.33-100.0 AU/ml). Metabolic fingerprinting was done to elucidate the total metabolic profile of isolates and P. pentosaceus LB-CC and P. acidilactici Ch-2 were being first lactic acid bacteria to be reported for the production of squalene with anticancerous properties. The entire screened six isolates were highly qualified with cumulative score of 95.83% and are being used to prepare probiotic nutraceutical products with high antioxidant content viz. novel cereal based probiotic acidulant, probiotic enriched mango fortified shrikhand and probiotic enriched pulse fortified yogurtand are successfully accepted in their sensory evaluation. Hence, this study affirms the use of P. pentosaceus C-1, P. pentosaceus LB-CC, P. pentosaceus LB-WC, E. faecium Ch-1, P. acidilactici Ch-2 and P. damnosus 107 in the development of new functional foods and nutraceutical preparations to impart betterment to the public health as these six strains isolated in the present study have been proved safe as well as highly effective probiotic candidatesThesisItem Open Access EVALUATION OF DIFFERENT PHYSICO-CHEMICAL AND BIOLOGICAL PRETREATMENTS FOR ENZYMATIC HYDROLYSIS OF HARDWOOD FOR ETHANOL PRODUCTION(2015) KAUSHAL, RICHA; SHARMA, NIVEDITAABSTRACT In the present investigation, an attempt was made to utilize hardwood as substrate for its degradation by potential microorganisms and evaluated different pretreatments to enhance their rate of hydrolysis - a key step for its bioconversion to ethanol. In total 20 microorganisms including 17 bacteria and 3 fungi were isolated. Among them, SD5 and RS2 were screened for cellulase and SD8 for xylanase production and were identified as B. simplex SD5, B. subtilis RS2, B. subtilis SD8 by 16S rRNA PCR technique and registered with NCBI under accession no KF844070, KF844069 and KF844068, respectively. Among fungi, WF5 and RF1 were selected for enzyme production and were identified using ITS 5.8S rRNA technique as T. harzianum WF5 and R. oryzae RF1 and registered under accession no. KF844067 and KJ1921199, respectively. Cellulase and xylanase enzymes were optimized through classical approach one factor at a time (OFAT) and Response surface methodology (RSM) varying medium, pH, temperature, inoculum size, incubation time, and substrate concentration. The partial purification of hydrolytic enzymes was done by ammonium sulphate precipitation. Full length gene sequences of BsSD8-xylanase of B. subtilis SD8 and four GHs namely three subunits of cellulase, Endoglucanase (ThWF5-Endo-glucanase), Exo-glucanase (ThWF5-Exo-glucanase) and -glucosidase (ThWF5- Glucosidase), and xylanase (ThWF5-Xylanase) of T. harzianum WF5 were pulled out and characterized. To reduce the production cost of ethanol, cheap untreated and pretreated lignocellulosic forest biomass i.e. hardwood were used as a substrate for sugar production. Among different hardwood species used, Eucalyptus and P. deltoides wood were selected for saccharification by bacterial and fungal hydrolytic enzymes, respectively. Among different physical, chemical and biological pretreatments, H2SO4 + H2O2 + steam pretreatment was found best for sugar production. Bioconversion of H2SO4 + H2O2 + steam pretreated E. teretecornis and P. deltoides wood to ethanol was studied under two different fermentation processes i.e separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). Different protocols had been designed to delimit the constraints of fermentation process. SHF was evaluated by modifying four different sub-processes of non-detoxification and detoxification as well as nonpooling and pooling of pretreated liquor. Maximum ethanol was achieved in protocol IV i.e. 7.02 g/l by co-culture of S. cerevisiae I + P. stipitis in E. teretecornis wood and 15.62 g/l in P. deltoides wood with fermentation efficiency of 61.05%. Scale up of SHF with P. deltoides wood using fungal enzymes and co-culture of S. cerevisiae I + P. stipitis was performed in 7.5 L bioreactor, achieving highest ethanol production after 52 h of fermentation. Among SHF and SSF, SHF in protocol IV i.e. pooled sample followed by detoxification was found to be the best and in case of strains used for fermentation, co-culture of S. cerevisiae I + P. stipitis was observed the best combination for highest bioethanol productionThesisItem Open Access CHARACTERIZATION OF POTENTIALLY ACTIVE NEW PROBIOTIC STRAINS ISOLATED FROM DIFFERENT SOURCES AND TO STUDY THEIR PROSPECTS AS NUTRACEUTICAL AGENTS(2015) GUPTA, ANUPAMA; SHARMA, NIVEDITAABSTRACT The present investigation was carried out to isolate most efficient potential probiotic lactic acid bacteria from rare and unexplored food sources, their screening, identification, safety assessment, evaluation of probiotic attributes, metabolic fingerprinting as well the development of novel nutraceutical products. In total 27 bacterial isolates were obtained from fermented/non fermented food sources. Out of all, 6 bacterial isolates were screened by bit/disc as well as well diffusion method on the basis of their wide inhibitory spectrum against tested pathogens. Isolates C-1, LB-CC, LB-WC, Ch-1, Ch-2 and 107 were selected for further studies having widest antagonistic potential, which were isolated from kodo millet flour, lasoda bari, chuli and salori, respectively that are traditional food products of Himachal Pradesh. Isolates C-1, LB-CC and LB-WC were identifies as Pediococcus pentosaceus, Ch-1 as Enterococcus faecium, Ch-2 as Pediococcus acidilactici while 107 identified as Pediococcus damnosus by 16S rRNA gene technique and registered in NCBI under accession no. KM251461, KM251460, KJ489415, KJ541885, KJ541886 and KJ577588, respectively. Safety assessment of isolates was done by evaluating antibiotic susceptibility, haemolytic, DNase and gelatinase activities. All the isolates exhibited 80-100% antibiotic sensitivity, non-heamolytic, non-DNase and non-gelatinase activities, thereby proving their safe status. These screened isolates were further examined for their probiotic potential viz. acidity tolerance, bile tolerance, simulated gastrointestinal transit, auto-, co-aggregation, hydrophobicity, adhesion to gastric as well as mammalian cell lines, competitive exclusion of pathogens, antimicrobial potential, exopolysaccharides and β-galactosidase production, compatibility and cumulative probiotic potential. All the six screened isolates were found to be highly acidity tolerant strains with 93.65-99.41 % survival rate at pH 3 for 3 h. These six isolates were able to resist high bile salt concentration i.e. 2.0% with 93.12-98.22 % survival rate for 8 h. Resistance to gastrointestinal transit as found to be in a range from 43.09-90.12 %. All the six isolates exhibited good autoaggregation capacity i.e. greater than 40% after 5 h and showed string hydrophobicity towards xylene i.e. > 40%. All the isolates were able to adhere to gastric mucin with 14.7-49.5% adhesion and all showed adhesion potential to mammalian epithelial cell lines. These screened probiotic isolates showed broad and strong inhibitory spectrum against both gram positive as well as gram negative pathogenic bacteria through secretion of antimicrobial metabolites viz. lactic acid (7.18-8.46 mg/l), H2O2 (0.24-0.38g/l) and bacteriocins (77.33-100.0 AU/ml). Metabolic fingerprinting was done to elucidate the total metabolic profile of isolates and P. pentosaceus LB-CC and P. acidilactici Ch-2 were being first lactic acid bacteria to be reported for the production of squalene with anticancerous properties. The entire screened six isolates were highly qualified with cumulative score of 95.83% and are being used to prepare probiotic nutraceutical products with high antioxidant content viz. novel cereal based probiotic acidulant, probiotic enriched mango fortified shrikhand and probiotic enriched pulse fortified yogurt and are successfully accepted in their sensory evaluation. Hence, this study affirms the use of P. pentosaceus C-1, P. pentosaceus LB-CC, P. pentosaceus LB-WC, E. faecium Ch-1, P. acidilactici Ch-2 and P. damnosus 107 in the development of new functional foods and nutraceutical preparations to impart betterment to the public health as these six strains isolated in the present study have been proved safe as well as highly effective probiotic candidates.ThesisItem Open Access CHARACTERIZATION OF ALKALINE PROTEASE FROM PLANT GROWTH PROMOTING RHIZOBACTERIA AND THEIR POTENTIAL APPLICATIONS(2015) SHIWANI; SHIRKOT, C.K.ABSTRACT Rhizosphere of apple trees of Trans Himalayas is great niche of proteolytic bacteria which may have potential for use in industries. A total of 76 proteolytic bacteria were obtained from rhizosphere soil and root endosphere of apple trees from different sites of four locations viz., Chamba, Shimla, Kinnaur and Kullu of Himachal Pradesh, India, which were screened for plant growth promoting traits including phosphate solubilization, IAA, hydrogen cyanide production and production of antifungal metabolite. Among them, 17 isolates that exhibited maximum alkaline protease activity were selected. The diversity elucidation and differentiation of isolates was achieved through their biochemical characterization and amplified ribosomal DNA restriction analysis with three tetra-cutter restriction enzymes (Hpa II, Hinf I and TaqI), the representative cluster groups were identified by 16S rDNA sequencing. This brings to light the predominance of Bacillus sp. and presence of remarkable intraspecies functional and genotypic diversity in apple rhizosphere. Proteolytic acitivity among selected isolates ranged between 50 to 1610 μg/ml/min at pH 8.0 and maximum was observed for Bacillus amyloliquefaciens SP1 (1610 μg/ml/min). Maximum enzyme production for B. amyloliquefaciens SP1 was observed, when fermentation medium, supplemented with 1.0% gelatin at pH 8.0 was incubated at 35°C for 48 h with 1.0% inoculun size and maltose as carbon source. Further optimization by Plackett Burman design and Response surface methodology resulted 2.08-fold increase in alkaline protease production (3350 μg/ml/min). The enzyme was purified by gel permeation and cation exchange chromatography and had a molecular mass of 43 kDa. Protease activity was optimum at pH 8.0 and 60°C. The half-life of the enzyme at 50, 60 and 65⁰C was 77, 19.8 and 13.33 min. The protease had Km, V max and Ea values of 0.125 mg/ml, 12820 μg min-1 and 34.52 kJ/mol in casein hydrolysis, respectively. All metal ions except Hg2+ and Cu2+ were well tolerated. The deduced internal amino acid sequence of B. amyloliquefaciens SP1 protease by MALDI-TOF MS resembled the sequence with D-alanyl-D-alanine carboxypeptidase of Bacillus subtilis strain 168. An alkaline protease gene was amplified from genomic DNA, cloned and successfully expressed in competent cells of E. coli DH5 . Most reliable three dimensional structure for protease was predicted by using Phyre2 server. This study reported the shortest time of 1:30 min at 3630 μg/ml/min and 4:30 min at74 μg/ml/min of enzyme activity for hydrolysis of gelatin layers of used X-ray film. It has revealed strain removal property and dehairing activity for animal hide without chemical assistance and without hydrolyzing fibrous proteins. This study also reported significant role of protease in antagonistic activity against C. michiganensis, R. solanacearum and F. oxysporium.ThesisItem Open Access IMPROVEMENT OF BIOETHANOL PRODUCTION FROM ENZYMATIC DEGRADATION OF SOFTWOOD BIOMASS BY STANDARDIZING FERMENTATION PROCESSES(2015) TONDON, DIVYA; SHARMA, NIVEDITAABSTRACT The present investigation was carried out to isolate, screen and identify the most efficient cellulolytic, xylanolytic and lignolytic microorganisms from diverse sources. 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 23 microorganisms including 20 bacteria and 3 fungi were isolated. Among bacteria R2, R4 were found to be potent cellulase producer and SD9 were screened as hyper xylanase producer while a fungal strain WF3 was screened as best hydrolytic enzymes producer so selected for further studies. These microbial isolates were identified as B. licheniformis R2, B. mojavensis R4 B. atropheaus SD and Amylomyces rouxii WF3 and registered with NCBI under accession no. |KJ588781|, |KJ588787| and |KJ588788|. Cellulase and xylanase enzymes were optimized through classical approach one factor at a time (OFAT) as well as response surface methodology under submerged fermentation varying medium, pH, temperature, incubation time, substrate concentration and inoculums size. The percent increase in enzyme activity obtained after optimization of different process parameters was 106.25%, 105.76% for cellulase of B. licheniformis R2 and B. mojavensis R4 while 26.46% increase was noticed for xylanase of B. atropheaus SD9. To reduce the production cost of enzymes, various softwood biomass were used as a substrate under SmF by B. licheniformis R2 and B. mojavensis R4 and B. atropheaus SD9 and SSF by A.rouxii WF3and among them P. roxburghii 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. Selected biomass was subjected to various pretreatment for enhanced saccharification. Among all the pretreatments, NaOH+urea was found as the best pretreatment and was selected for further studies. Bioconversion of pretreated P. roxburghii wood to ethanol was studied under two different fermentation processes i.e separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). Different protocols 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 non pooling of pretreated liquor. Maximum ethanol was achieved in protocol–II of SHF i.e. 11.08g/l by co-culture of S. cerevisiae I and P. stipitis with the fermentation efficiency of 41.09%. Among all the three processes of fermentation evaluated in the present study, SSF was found to be the best and in case of strains used for fermentation, co-culture of S. cerevisiae I and P. stipitis was observed the best combination for highest bioethanol production. Scale up of ethanol production was performed in 7.5 L bioreactor under different modes of fermentation. Simultaneous saccharification produced maximum ethanol with efficiency of 70.12%. Mathematical model based on experimental results for ethanol production was proposed. The production of ethanol was found as mixed growth associated. The model consists of a set of ordinary differential equations taking into account the growth, substrate utilization and ethanol production with time.ThesisItem Open Access STUDIES ON CHARACTERIZATION AND ROLE OF 2,4- DIACETYLPHLOROGLUCINOL AND PYOLUTEORIN PRODUCED BY FLUORESCENT Pseudomonas SPECIES IN REPLANT SITE OF APPLE ORCHARD(2015) RANA, SHEETAL; KAUR, MOHINDERABSTRACT The present study focuses on characterization of secondary antifungal metabolites i.e. 2,4- diacetylphloroglucinol and Pyoluteorin produced by fluorescent Pseudomonas sp. isolated from the normal and replant sites of apple orcards. Six strains were isolated from the replant sites of Maggota, Sharontha and Siao of Shimla distt. and thirteen already isolated strains of fluorescent Pseudomonas sp. from replant sites of apple were used for screening of direct and indirect plant growth promoting activities like antifungal, siderophores, phosphate solubilization and production of HCN, ammonia, plant growth regulators (auxins, cytokinins and gibberellins) and lytic enzymes. Isolates Sh2r, Sh4r and B showed 99.7%, 98.4% and 99% similarity with Pseudomonas poae DSM14936 and Pseudomonas gessardii CIP105469 respectively. Six fungal pathogenic sp. isolated from replant sites of apple of Shimla distt. i.e. MgF1 and MgF2 showed 99.2% and 99.1% similarity with Rosellinia necatrix and Fusarium oxysporum, ShF1 and ShF2 showed 99.5% and 99.2% similarity with Fusarium oxysporum and Schizophyllum commune & SiF1 and SiF2 showed 99.2% and 100% similarity with Fusarium oxysporum and Pythium ultimum. Optimization of media, time of incubation, temperature, pH, carbon, nitrogen and mineral sources were done for production and extraction of antifungal metabolites i.e. 2,4- diacetylphloroglucinol & Pyoluteorin and for mass multiplication of inoculums for field trials. These metabolites were extracted with ethylacetate and identified through TLC at Rf 0.8 and 0.5 respectively. Antifungal activity, MIC and thermal stability of both these metabolites were evaluated. 2,4-diacetylphloroglucinol was further characterized through HPLC and NMR techniques. Antifungal activities of Pseudomonas sp. help in suppression of plant pathogens in soil. Antifungal activity along with other plant growth promoting activities may be the reasons for better establishment and growth promotion of replanted apple. Treatments with formulations of consortial strains were found to be more effective in growth and establishment of replanted apple. Therefore these strains can be exploited for the management of replant problem of apple.