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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 AN INNOVATIVE GREEN TECHNOLOGY FOR AGRICULTURAL WASTE UTILIZATION(NAUNI,UHF, 2020-10) SHARMA, KANIKA; SHARMA, NIVEDITAABSTRACT In the present investigation, different agricultural wastes were collected from Himachal Pradesh and their adjoining states i.e. Punjab and Haryana and an attempt had been made to utilize this waste as substrate for its enzymatic degradation to enhance their saccharification and bioconversion to ethanol by co-fermentation with ethanologens. Based upon the chemical composition of mixed agricultural waste and large availability of rice straw, these were finally selected for further study. Different physico-chemical pretreatment methods applied to rice straw/mixed waste, where maximum reducing sugars i.e. 46.98 and 44.45 mg/g from rice straw/mixed waste respectively were obtained in microwave pretreatment. After optimization of different process parameters by one factor at a time (OFAT) approach for enzymatic saccharification under SmF, highest reducing sugars reported from untreated and pretreated rice straw/mixed waste were 34.40 and 56.70 mg/g; 38.0 and 63.50 mg/grespectively, at enzymatic ratio of 3.5:1.5, 50oC temperature, 5.5 pH, incubation period 72 h and 7.5% biomass loading followed by CCD of RSM by varying temperature, pH and incubation time. SEM analysis revealed that the surface structure of rice straw/mixed waste was significantly changed after pretreatment and enzymatic hydrolysis. The reducing sugars so obtained were analyzed using HPLC technique. In 2nd mode i.e. SSF (biological degradation), two fungal strains selected P. chrysoporium and P. ostreatus fungal strains had showed maximum amount of reducing sugars produced i.e. 55.70 and 49.05 mg/g by using microwave pretreated rice straw and mixed waste respectively. After optimization of process parameters through OFAT and RSM maximum reducing sugars from untreated and pretreated rice straw (450 watt) /mixed waste (600 watt) had the critical values as 51.12 and 37.98 mg/g; 71.99 and 73.10 mg/g respectively, at 30 and 25oC temperature and incubation period 10 days respectively. Among two processes of ethanol fermentation evaluated in the present study, SHF was found to be the best in terms of highest ethanol productivity with S. cerevisiae I+ P. stipitis as best fermenting microorganism from enzymatic saccharified and fungal degraded sugary syrup. Estimation of bioethanol production after enzymatic saccharfied/ biological degrdaded microwave pretreated rice straw/mixed waste hydrolysate fermented by co-culture of S. cerevisae I and P. stipitis was done with the help of GC-MS. Scale up of ethanol fermentation using 7.5 l stirred tank bioreactor, batch conversion of rice straw/mixed waste enzymatic saccharified hydrolysate to ethanol was carried out by co-culture of S. cerevisiae I + P. stipitis under SHF. The highest ethanol yield of 52.14 and 46.89 g/l with 88.12 and 80.01 % of fermentation efficiency was achieved after 48 and 36 h of fermentation with an agitation speed of 200 rpm and aeration rate of 0.05vv, pH 6 at 25 ±2°C and inoculating the bioreactor with 24 h old co-culture of yeasts (S. cerevisiae I + P. stipitis). Also to combat the pollution problem, value addition of agricultural waste for cultivation of mushroom supplemented with apple pomace had emerged as an ecofriendly technique with higher yield and better utilization substrate.ThesisItem Open Access Evaluation of different physicochemical and biological pretreatments for enzymatic hydrolysis of hardwood for ethanol production(YSPU, 2014) Kaushal, Richa; Sharma, NiveditaIn 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 production.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 Detection and characterization of gene encoding bacteriocin in lactic acid bacteria and to study preservative potential of purified bacteriocin(YSPU, 2012) Gautam, Neha; Sharma, NiveditaThe 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 breviswhile G2 identified as Lactobacillus spicheriby 16S r RNA gene technique and registered in NCBI under accession no. JX46150 and JX48191 respectively. Bacteriocin productionwas optimized through classical one variable at a time method. Both the isolates showed maximum bacteriocin production at early stationary phase, at pH4.0, temperature 35 0 C 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 2×10 3 to 8×10 3 AU/ml in both cases. Purified bacteriocin titers of L. brevis UN increased by 87.5 % against L. monocytogenes, 66.6 % againstS.aureus and 75 % against C. perfringens. In case of L. spicheriG2 bacteriocin titers increased by 75 % , against L. monocytogenes, S. aureusand 15 % against C. perfringensrespectively. 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 beenreported for the first time in present study. Both isolates were tested for their probiotic attributes and werefound 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.spicheriG2 showed resistance to the spoilage causing microorganisms in milk and apple juice. L. brevisUN and L. spicheriG2 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 Purification of cellulase and xylanase enzymes produced from potential microorganisms isolated from compost for industrial application(YSPU, 2012) Sanjeev Kumar; Sharma, NiveditaThe present investigation was carried out to isolate, screen and identify the most efficient cellulolytic and xylanolytic microorganisms from compost. Enzyme production, optimization, purification, characterization, kinetics, cellulase/xylanase gene encoding and scale up studies were performed with selected strains to recommend their use for industries. In total 102 microorganisms including 93 bacteria and 9 fungi were isolated. Among them, three bacteria SH8, SH0 and BM-1 and two fungal isolates i.e. T2 and W2were screened for cellulase and xylanase enzyme production studies. The bacterial isolates were identified as B. amyloliquefaciens, B. tequilensis and B. subtilisrespectively by 16S rRNA PCR technique and registered in NCBI under accession no. JX129360.1, JX129359.1 and JX129361.1 while fungiwere identified as T. longibracheatum and R. oryzae by ITS 5.8S rRNA technique and being assigned accession no. JX213811.1 and JX213812.1. in NCBI. Cellulase and xylanase enzymes were optimized through classical approach one factor at a time (OFAT) as well as response surface methodology (RSM) under submerged fermentation varying medium, pH, temperature, inoculum size, carbon source, substrate concentration and incubation time and in solid state fermentation using different pretreatments i.e. pH, temperature, moisture ratioand incubation time. The percent increase in enzyme activity obtained after optimization of different process parameters was 204.51% for cellulase of B. subtilis BM-1, 222.91% and 210.52% for xylanase of B. amyloliquefaciensSH8 and B. tequilensisSH0, respectively in SmF. The increase in cellulase and xylanase of T. longibracheatumT2 was recorded 63.65% and 95.96% while in R. oryzaeW2 it was 107.38% and 125.64%, respectively under SSF. The purification of hydrolytic enzymes was proceeded following multistep purification technique using ion exchange chromatography and gel exclusion chromatography and the molecular weight of partially purified xylanase enzymes of B. amyloliquefaciensSH8, B. tequilensisSH0 and T. longibracheatumT2 were found in the range of 14 kDa to 93.4 kDa, respectively. The partially purified xylanase of both bacterial and fungal origin were further characterized by studying, the effect of various parameters viz. the effect of pH, temperature, metal ions, substrate specificity, substrate concentrations and their kinetic parameters were derived. The partially purified thermostable xylanaseof bacterial origin was active at 90 o C, pH 6.0, showed high activity on xylan containingsubstrates and depicted cellulase free nature. The K m and V max of B. amyloliquefaciens SH8 xylanase for birch wood xylan were 166.67 µmol/mg/min and 5.83 mg/ml, while for B. tequilensisSH0 were 166.66 µmol/mg/min and 10.99 mg/ml. Similarly, partially purified enzyme of T. longibracheatumT2 was optimally active at 60 o C, pH 5.0 and showed high activity on xylan containing substrates. The K m and V max of T. longibracheatum T2 xylanase for birch wood xylan were 125.0 µmol/mg/min and 1.55 mg/ml. Cellulase and xylanase abilities of the isolates were targeted by amplification of eglS and Xyn genes. Optimum conditions i.e. inoculum age, inoculum size, aeration rate and agitation rate were explored at pilot scale for xylanase enzyme by B. amyloliquefaciens SH8. The inoculum age of 4h, inoculum size @ 10% concentration, 1.0vvm and agitation rate of 200 rpm were found best for production of xylanase enzyme in 7.5 L bioreactor. Mathematical model based on experimental results for xylanase production was proposed. The production of xylanasewas found growth associated. The model consists ofa set of ordinary differential equations taking into account the bacterial growth, substrate utilization and xylanase production with time.ThesisItem Open Access Preparation of bioactive phosphocompost and its effect on soil properties and crop yield(YSPU, 2014) Thakur, Deepshikha; Kaushal, RajeshA total of twenty four purified bacterial, seventeen actinomycetes and twenty three fungal isolates were isolated from various samples collected from different niches and locations of District Shimla and Solan of Himachal Pradesh. These isolates were screened for their cellulolytic abilities and multifarious plant growth promoting traits. A compatible consortium consisiting of efficient isolates of bacteria (DB1), actinomycetes (DA7) and fungi (DF14) was developed for faster decomposition of organic wastes and higher phosphate solubilization from insoluble sources (rock phosphate and bone meal). The inoculation of tri-culture consortium (DB1+DA7+DF14) to the composting pits stacked with pine needles (pretreated with hot water > 70 °C) + agriwates + rock phosphate @ 3 % + bone meal @ 3 % + urea @ 1 %, resulted in higher reduction of C/N ratio (from 60:1 to 18:1), phosphate solubilization and increased cation exchange capacity over uninoculated control. The conjoint use of 75 % recommended dose of chemical fertilizers and 25 % phosphocompost, increased the yield by 58 % and 62 % for pea and tomato, respectively, over control. Besides saving 6.3, 14.9 and 14.4 kg /ha N, P2O5 and K2O, respectively in pea crop and 37.5, 30.08 and 15.6 kg/ha N, P2O5 and K2O, respectively in tomato crop, the conjoint use of phosphocompost and chemical fertilizers also improved fruit quality (number of grains per pod, pod length, pod girth, TSS in pea and fruit weight and TSS in tomato), available nutrient status, cation exchange capacity, soil enzymes (phosphatase, dehydrogenase and urease) activity and other microbiological properties (total microbial count, microbial biomass and microbial activity) of soil.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 Diversity and functions of phosphate solubilizing rhizobacteria associated with apple seedlings(YSPU, 2012) Mehta, Preeti; Shirkot, C.K.The ability of a few soil microorganisms to convert insoluble forms of phosphorus to an accessible form is an important trait in plant growth-promoting bacteria for increasing plant yields. Little is known about the composition of phosphate solubilizing bacterial(PSB) diversity associated with rhizosphere of apple trees. Therefore, the objective of the present study was to investigate the diversity of PSB population associated with rhizosphere of soil and roots of apple and characterize them for multifarious plant growth promoting traits and to test the hypothesis, whether root exudates of apple trees exert a selective pressure on the proliferation of similar or different phosphate solubilizing plant growth promoting rhizobacterial population. Two hundred and six phosphate solubilizing bacteria were isolated by using culture dependent procedure from rhizosphere soil and endorhizosphere of apple trees of four locations viz., Chamba, Shimla, Kinnaur and Kullu of Himachal Pradesh. All the PSB isolates were screened for plant growth promoting traits (PGPT), including IAA production, siderophore, HCN, chitinase synthesis ability and antifungal activity against D. necatrix. IAA production was detected in 50 isolates (24.2%), siderophore synthesis in 53 isolates (25.7%), HCN in 40 isolates (19.4%), chitinase synthesis ability in 61 isolates(29.6%) and percent growth inhibition against D. necatrixin 61 isolates (29.6%). The diversity index (Shannnon-Weaver index) revealed that PGPT-possessing PSBs in rhizosphere soil were more diverse than root endosphere. Eighty five of total 206 PSB isolates possessed one or more than one plant growth promoting traits, hence these 85 PGPT- possessing PSBs were selected further for RAPD analysis. After RAPD- PCR analysis with primerRBa-4, 85 PSBs were divided in to five major groups. Sixteen representative PSB isolates from RAPD groups (3, 2, 8, 1, 2 isolates of Ist, 2 nd , 3 rd , 4 th and 5 th group, respectively.) were identified further by 16S rRNA analysis. The 16S rRNA analysis identified 16 PSBs as Bacillus subtilis, B. licheniformis, B. pumilus, B. altitudinis, B. methylotrophicus and B. firmus. Among 206 PSBs, a highly efficient P-solubilizing isolate was selected and presumptively identified as Bacillus subtilis CB8 A. The isolate solubilized tri-calcium phosphate and produced substantial amount of soluble phosphorus (823 mg/l) in Pikovskaya’s (PVK) broth and exhibited the production of IAA (16.2 µg/ml), siderophore (85.21%) and growth inhibition against D. necatrix (56.67%). P- solubilization of B. subtilis CB8A was inversely correlated with pH (r = – 0.78) and positively correlated siderophore production (r = 0.81), IAA (r = 0.58) and antifungal antibiotic activity against D. necatrix (r = 0.63). Gluconic acid (1.43%) and citric acid (0.67%) were detected as the major organic acids with small percentages of 2- ketogluconic acid (0.34%), formic acid (0.15%), oxalic acid (0.10%) and fumaric acid (0.03%) during TCP solubilization by the B. subtilis CB8 A. P- solubilization and nitrogen fixing abilities of the isolate CB 8 A were confirmed by amplification of gdh and nifH genes. Thus, the ability of performing multifarious plant growth promoting activities in tandem suggest B. subtilis CB8 A a novel PSB isolate for plant growth promotion and has significance to develop a costeffective ecofriendly multifunctional biofertilizer for use in apple orchards, the most important cash crop in Himachal Pradesh.