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ThesisItem Open Access Effect of ACC (1-aminocyclopropane-1-carboxylate) utilizing rhizobia and Pseudomonas on nodulation and plant growth of clusterbean (Cyamopsis tetragonoloba (L.) Taub.)(CCSHAU, 2010) Khandelwal, Aakanksha; Sindhu, S. S.Clusterbean (Cyamopsis tetragonoloba (L.) Taub.) is an important commercially utilizable crop in arid zone of India. The nodulation status of this crop is poor under field conditions and therefore, its productivity is low. Recently, ACC deaminase containing rhizobia have been found to improve nodulation and plant growth of pea and alfalfa by lowering the level of ethylene, which acts as inhibitor of nodulation. Therefore, 95 isolates of Bradyrhizobium/Rhizobium and 55 isolates of Pseudomonas were obtained from the nodules and rhizosphere of clusterbean and screened for utilization of ACC on Dwarkin and Foster’s minimal medium. ACC utilization studies showed that 78% cultures of Bradyrhizobium/Rhizobium and 64% of Pseudomonas cultures had the ability to grow on ACC supplemented plates. Six Bradyrhizobium/Rhizobium isolates GRA11, GRA114, GRA115 and GRA6 caused significant root and shoot elongation of clusterbean at 5 and 10 days of growth on water agar plates. Most of the Pseudomonas isolates showed retardation effect on root growth but isolate CPA123 showed root and shoot elongation at 10 days of growth. Bradyrhizobium isolate GRA11 and two Rhizobium isolates GRA6 and GRA110 formed 35 to 47 nodules per plant and 136 – 255% increase in plant dry weight was observed at 45 days of growth in sterilized chillum jar assembly conditions. Coinoculation of ACC utilizing Bradyrhizobium isolate GRA11 with Pseudomonas isolate CPA123 (ACC +) formed 48 nodules per plant and caused 293% increase in plant dry weight whereas coinoculation of CPA123 (ACC +) isolate with Rhizobium culture GRA6 formed 40 nodules per plant and 274% increase in plant dry weight was observed at 60 days of growth. These three cultures having the ability to increase nodulation and plant growth under pot house conditions could be further used for inoculation of clusterbean under field conditions.ThesisItem Open Access Biogas production from cattle dung supplemented with paddy straw and evaluation of manurial value of spent slurry(CCSHAU, 2010) Narula, Amrita; Leela WatiThe basic problem we face today is that the earth's natural resources of energy are being depleted at a most alarming rate but our conventional energy resources are limited and already have been over exploited. It indicates that there is need to look up for alternate resources of energy. Motivated by the need to meet the ever-increasing energy demand and sustainability consciousness, many Governments have promoted renewable energy technologies such as biogas. Biogas (a mixture of approximately 60% methane and 40% carbon dioxide) is a wellestablished fuel that can supplement as an energy source for cooking and lighting in developing countries. Most of the biogas plants in India are being operated with diluted cattle dung slurry at 7-8% total solid concentration. Due to constraints in these conventional biogas systems, solid state fermentation system operated at about 15% total solid concentration using cattle dung as substrate has come into existence. Availability of animal waste is one of the major problem for successful operation of biogas digesters. So researchers are looking towards other alternative substrates. Agricultural residues represent the most important energy sources readily available for biogas production and paddy straw is one of such residue. Anaerobic digestion of paddy straw alone is inefficient because the nutrients and minerals required for bacterial growth are not present at sufficient level . Paddy straw in India is generated in huge amount and its management for farmers has become a big challenge so it is burnt. To reduce environmental pollution and to supply sufficient biogas production necessitated this study, with the ultimate goal of assessing the effect of co-digestion of cattle dung and paddy straw on biogas production. Cattle dung and paddy straw were mixed in ratios 1:00, 1:0.31, 1:0.46, 1:0.63 and 1:0.78 on dry matter basis and digested in semi continuous mode at two total solid (TS) concentrations i.e 12-13% and 14-15% for ten weeks. The maximum biogas production of 53.7 l/kg and TS degradation of 19.6% was obtained on supplementation of 5% paddy straw to cattle dung at 14-15% total solid concentration. The N, P and K content of effluent were 1.56, 0.54 and 1.55% respectively. On adjustment of C/N ratio of paddy straw and cattle dung to the level of cattle dung a constant rate of biogas i.e 0.311 l/l/day was obtained in all supplementation levels of paddy straw to cattle dung. Composting of spent substrate after biomethanation with Trichoderma reesei. inoculation resulted in further reduction of C/N ratio within one month producing quality manure.ThesisItem Open Access Utilization of xylose for bioethanol production(CCSHAU, 2010) Singla, Ankit; Dhamija, S.S.The present investigation was undertaken to isolate xylose fermenting yeasts and examining their ability to ferment xylose in the presence of glucose. Seventeen yeast isolates were retrieved from different ecological niches. Grapes were found to be the most suitable source offering 5 isolates. Based on absorbance values various constituents of xylose- containing inoculum medium were optimized. The optimum xylose concentrations of inoculum and production media were found to be 1 and 10 %, respectively. All the isolates were subsequently screened for ethanol production on xylose medium. While seven isolates produced no alcohol at all on xylose, ten isolates produced 1-2 % (v/v) ethanol on xylose and 3-5 % (v/v) ethanol on dextrose. All the six yeast isolates which produced 5 % (v/v) ethanol on dextrose were screened for ethanol production on a medium containing both dextrose and xylose (4:3 ratio). Isolate 11 produced the maximum (4.5 %) ethanol from mixed carbon sources leaving behind only 0.5 % residual sugars, which seemed to represent xylose rather than dextrose. It was further examined for ethanol production on media containing either dextrose or both dextrose and xylose at 12 and 15 % sugar levels. In the latter, the two sugars were always in a ratio of 4 parts dextrose and 3 parts xylose. In case of the fermentation medium containing dextrose, a maximum of 5.5 % (v/v) ethanol was achieved at an initial sugar concentration of 12 % at 72 h. Similarly, on the medium containing both dextrose and xylose, a maximum of 4.7 % (v/v) ethanol was obtained at an initial sugar concentration of 12 % at 72 h, Based on the morphological and biochemical properties of isolate 11, the results were found to conform well with the characteristic properties of Candida.ThesisItem Open Access Molecular diversity of rhizobia Infecting broad bean (Vicia faba L.) in arid and Semi-arid regions of Haryana(CCSHAU, 2011) Kirti; Gera, RajeshVicia faba is a legume and forms symbiosis relationship with soil bacteria of the genus Rhizobium. Strains of Rhizobium infecting broad bean are generally assumed to be classified as R. leguminosarum bv. viciae. The diversity of V. faba which was carried out in earlier studies were mainly for rhizobial populations isolated from same region or for comparison with R. leguminosarum strains isolated from other legume species and no information is available about the diversity of these rhizobia in the soil under extreme environment such as soils of arid and semiarid regions. Therefore, the present investigation was started by collecting 107 soil samples from different arid and semi arid zones of Haryana to study the diversity of rhizobia nodulating Vicia faba. These soil samples were analyzed for pH, EC, organic C and total N. Soil pH ranged from 6.34 to 7.55 ,EC was in the range of 0.09 to 5.54 dSm-1. The organic C and total N was in the range of 0.05 to 0.61% and 0.044- 0.061%,, respectively. 184 rhizobial isolates were isolated from various soil samples using trap plant Vicia faba. Out of them 64 were selected on the basis Gram’ staining and peptone water test. The genomic DNA of 64 isolates was isolated and amplified for nodC gene using nod CI and nod CF primers and hence authenticated as rhizobia. The genomic DNA of these isolates was also amplified for nifH gene using 19F and 407R nifH gene primers, out of which 50 isolates showed nifH gene amplification. Genomic DNA of all the nodC +ve isolates was also amplified with 16S rDNA gene primers using 27F and 1378R primers. The amplified product was subjected to RFLP analysis with MspI and HaeIII restriction enzymes. RFLP analysis of 16S rDNA of rhizobial isolates from Vicia faba nodules using MspI and HaeIII showed wide diversity among themselves. These were distributed into two major groups with different subgroups and the divergence among these started at 66 and 54 % similarity with MspI and HaeIII, respectively. Three rhizobial isolates, Vf- 63, Vf -77 and Vf -103 which formed separate major group after RFLP analysis with MspI and HaeIII individually or in combination.ThesisItem Open Access Processing of spoilt wheat into an additive for maximization of ethanol production(CCSHAU, 2011) Swami, Urvashi; Dhamija, S.S.It is no secret that ethanol is an important chemical with uses such as a feedstock for chemical and beverage industries and an additive to fuel for automobiles. Over the years, its demand in general, has been on an increase and India is no exception. Clearly, maximization of ethanol production becomes imperative and could be achieved by considering, among others, the addition of yeast nutrients to the fermentation liquor. In the Indian alcohol industry, N and P, the commonly used nutrients are sourced from chemical fertilizers, which do have a certain carbon footprint. Ideally, organic/ renewable yeast nutrients would be more suitable, as they have a low net green house gas emission. However, so far, no dedicated formulation is available on the Indian market. On the other hand, spoilt wheat which ferments faster than its normal version, due to the presence of amino acids, minerals, small molecular weight peptides etc, is able to expedite the fermentation of other raw materials as well. Obviously, it offers the potential for retrieval of its ethanol-expediting yeast nutrients which are water-soluble, renewable and less polluting. Efforts were, therefore, made to recover this ethanol-expediting nutrient activity from liberally available spoilt wheat grains and concentrate the same so as to transform it into a potentially marketable additive. Spoilt wheat sample tested positive for ethanol-expediting activity as addition of its aqueous extracts (20ml) from 40% slurry of grains and powder to 30% normal wheat powder produced 10.5%, 12.2% ethanol (v/v), respectively. Partial sterilization of extracts at 85-90oCfor 30 minutes seemed to have little effect on the thermostability of the activity, as 10.2% and 11.9% (v/v) ethanol, respectively, were still produced. As against this, the complete sterilization at 15lbs for 15 minutes reduced the activity correspondingly to 9.4% and 10.4% (v/v) ethanol. To explore the ability of the extract to concentrate its ethanol-expediting activity, 20% extract was loaded into the slightly modified domestic R.O. system. Analysis of the retentaie (reject) ought to be containing the concentrated activity, which was successively reloaded into the R.O. system, revealed that the incremental concentration of TDS over various rounds wasn’t as effective as expected and reached a maximum value of 3600 at the end of 6th round from an initial value of 3000 ppm. Correspondingly, ethanol-expediting activity measured in terms of ethanol production also didn’t register an appreciable increase, achieving a value of 12.3% (v/v) ethanol at the end of 6th round as compared to 11.0% ethanol initially. However, this finding signals that domestic R.O. system in its present form does concentrate the activity, but in a manner which is economically non-viable. It would, therefore, be reasonable to presume that the domestic R.O. system with larger R.O. columns which would obviously have larger membrane surface area, if used in series might produce larger volumes of the penneate and thereby lead to the concentration of the retentaie. Partial sterilization at 85-90oCfor 30 min improved the shelf life of retentaie which failed to detect any mycotoxins otherwise present in spoilt wheat.ThesisItem Open Access Isolation of potassium solubilizing bacteria and their inoculation effect on growth of wheat (Triticum aestivum L. em. thell.)(CCSHAU, 2011) Parmar, Priyanka; Sindhu, S.S.In the present study, seventy bacterial isolates obtained from wheat rhizosphere and 67 reference strains from the department were tested for potassium solubilization ability on Aleksandrov medium supplemented with mica as potassium source. Twenty isolates/strains solubilized potassium from mica and the amount of K released by the isolates/strains ranged from 15 mg/L to 48 mg/L. Maximum K solubilization occurred with glucose as carbon source, at 25°C temperature of incubation, 7.0 pH of the medium and with KCl as potassium source followed by K2SO4. Isolate HWP47 caused solubilization of potassium in mica only by acid production, isolates HWP28 and HWP69 by production of CPS and EPS, HWP38 by production of acid and CPS whereas isolates HWP15, HWP17, HWP53, HWP 57, HWP61 and HWP63 caused solubilization by production of acid, CPS and EPS. Inoculation of K solubilizing isolate HWP47 in wheat (Triticum aestivum L.) variety WH711 caused 51.46% increase in RDW in soil and 60.19% increase when rock material was added in pots at 60 days after sowing. Similarly, 44.28% increase in SDW was found in HWP47 inoculated plants. At 90 days after sowing, the inoculation of rhizobacterial isolate HWP15 caused 15.29% and 27.19% increase in RDW and SDW, respectively. The plant dry weight gain was further enhanced to 20.59% and 71.92% in RDW and SDW, respectively with amendment of rock material. Inoculation with isolate HWP47 showed 22.35% increase in RDW and 73.68% increase in SDW on addition of rock material. Isolates HWP15 and HWP47 also caused significant K uptake in the shoot tissues. Thus, potassium solubilizing bacteria HWP15 and HWP47 could be further exploited for plant growth improvement under field conditions.ThesisItem Open Access Characterization and molecular diversity of endophytic bacteria isolated from chickpea (Cicer arietinum) nodules(CCSHAU, 2011) Saini, Ranjana; Dudeja, S.S.Endophytic bacteria exert several beneficial effects on host plants, such as stimulation of plant growth, nitrogen fixation and induction of resistance to plant pathogens. Elsewhere, endophytic bacteria have been isolated from roots and nodules of several legume plants. In the present investigation, a total of 76 endophytic bacterial isolates were isolated from the nodules of chickpea plants. Gram positive isolates were more than the Gram negative isolates. Among the Gram positive bacterial isolates, 61 were spore formers having central to terminal spores. A total of 74.3% endophytic bacteria in chickpea root growth promotion assay showed growth promotion and 73.7% were phosphate solubilizer. A total of 80.3% endophytic bacteria were producing ammonia while only 18.4% were organic acid producers. A total of 55 isolates were selected for molecular diversity studies. 16S rDNA was amplified and RFLP with three restriction endonucleases viz., HaeIII, MspI and HinfI showed wide diversity among the chickpea nodule endophytes and atleast 13 biotypes of bacterial endophytes were present in chickpea nodules. Inoculation of nodule endophytes promoted the plant growth and enhanced nitrogen fixing parameters of chickpea and most effective isolates were CNE1036, CNE215 and CNE217. The effectivity of isolate CNE1036 and CNE215 should be further confirmed under field conditions so as to select best inoculant strain.ThesisItem Open Access Isolation, characterization and molecular diversity of berseem (Trifolium alexandrinum L.) rhizobia from Haryana soils(CCSHAU, 2011) Varsha; Kukreja, KamleshSymbiotic nitrogen fixation is an important source of nitrogen and the microbial symbiosis is suggested to be an ideal solution for the improvement of soil fertility. Berseem (Trifolium alexandrinum L.) is most important winter season legume cultivated in around 2 million hectares in India and occupies predominant place in farm economy. Berseem is usually nodulated by R. leguminosarum bv. trifolii. In the present study, a total of 65 rhizobial isolates were obtained from nodule samples of berseem crop collected from farmer’s fields of 17 villages of Haryana state. All the rhizobial isolates were characterized for Gram’s reaction and it was found that all were Gram –ve small rods. Authenticity of rhizobia was done by plant infection test under sterilized conditions. All the rhizobial isolates formed nodules except BK41, BK45 and BH56 and number of nodules varied from 12 to 45 per plant. On the basis of nodulation and plant biomass, 40 berseem rhizobia were selected for further studies. All the 40 rhizobial isolates were characterized for IAA production, siderophore production, P-solubilization and temperature tolerance. IAA production ranged from 19.9 to176.4 μg mL-1. None of the isolate was able to produce siderophore and solubilize P. Majority of the isolates were able to grow at all the three temperatures (25, 30, 35oC) but few isolates showed diminished growth at 25oC. Genomic DNA of these rhizobial isolates was extracted by CTAB method and was amplified using 16S rDNA primers. The amplified product was of 1351bp long. The amplified product was digested with HaeIII restriction enzyme. The restriction digestion pattern was analyzed with NTSYS programe. Dendrogram based on 16S rDNA profiles showed that rhizobial isolates were distributed in two major groups with different subgroups and the divergence among them started at 60 percent similarity coefficient. Seven biotypes were formed out of which two were most prevalent. Considerable diversity was observed among berseem rhizobial isolates.ThesisItem Open Access Growth promotion activity and molecular diversity of root endophytic bacteria from legume and non-legume plants(CCSHAU, 2011) Aggarwal, Vishal Kumar; Pathak, D.V.Large numbers of bacterial endophytes have been reported from different plant tissues. These endophytes exhibit many beneficial characters for plant growth promotion and nitrogen fixation. These endophytes may perform similar or different functions in different plants and in different tissues of a plant. Therefore, a total of 90 root endophytic bacteria were isolated from roots of different crops chickpea (12), pea (15), wheat (14), lucerne (24) and oat (25) after proper surface sterilization. Studies of different morphological characters showed that 52 isolates were gram positive and 38 isolates were gram negative. Almost all the 50 gram positive isolates were spore formers with terminal or central spore. All the endophytes were screened for the presence of beneficial characters, and found that 62.3% endophytic bacteria were root growth promoter, 47.8% endophytic bacteria were phosphate solubilizers, 67.7% root endophytic bacteria were ammonia producers and 20% root endophytic bacteria were organic acid producers. Molecular studies on diversity of selected 43 endophytic isolates showed that in chickpea roots five biotypes of endophytic bacteria were present, whereas four in lucerne and wheat and three in pea and oat roots were present. Seven biotypes in legumes and six in non-legumes were present. Overall 10 biotypes of bacterial endophytes were present in roots of legumes and non-legumes being grown under CCS HAU farm. Selected 40 root endophytes were screened under pot culture conditions using chickpea as test host. Inoculation of root endophytes promoted the plant growth and nitrogen fixation of chickpea and best isolates were LRE3, ORE3A, CRE13 and CRE1.