| Management of N in agricultural ecosystem is one of the most essential factors for agricultural sustainability. Injudicious use of nitrogen fertilizer has induced a crucial environmental problem, which includes volatilization of NOX to the atmosphere, acid rain, induced leaching of soil nutrients, changes in the global N cycle, and nitrate pollution of groundwater. Heavy use of fertilizer N to maximize crop production is only likely to aggravate these problems. However, to exploit and utilize Biological Nitrogen-Fixation is one of the important ways to solve those problems.It has been 40 years since Dobereiner and Ruschel isolated the nitrogen-fixing bacteria from the rhizosphere of sugarcance plants and demonstrated the potential of diazotrophs to associate with graminaceous plants. More recent evidence of significant biological nitrogen fixation in economical important Graminous species, particularly sugar cane, rice and forage grasses, has induced tremendous interest in NI fixation by non-legumes. Compared with fertilizer nitrogen, biological fixed N2 is less susceptible to volatilization, denitrifiaction and leaching because N from this source is used directly by the plant. It is promising to alleviate those problems through exploring and utilizing associative nitrogen fixation properly. In this thesis, a strain of associative nitrogen fixing bacteria with high nitrogenase activity was selected and the further studies for it were conducted, the results obtained are as follows:Selection and identification of associative nitrogen fixing bacteria with high nitrogenase activity. The nitrogenase activities detected by means of the acetylene reduction method and growth rates tested by optical density of germ suspension under the X 400nm (OD4oo) of all the strains tested except for the strain W12 reduced gradually due to frequent subculture during a year. However, the strain W12 has been showing high and stable nitrogenase activity and growth rate since it was isolated five years ago. In the nitrogen-free medium, it grew well and the acetylene reduction activity (ARA) reached up to 1800 nmol C2H2 per ml culture in 48 hours. The results of rice inoculation experiment in a spermosphere model system with various nitrogen-fixing bacteria suspensions showed that the strain W12 represent highest ARA amongst all the strains tested, the differences between it and the other44strains were highly significant. Moreover, the high ARA of the strain W12 associated with different rice genotypes were less varied. In nitrogen-free medium, the logarithmic phase of the strain W12 was about 13-48 h. In logarithmic period its doubling time was 2-4 h in nitrogen-free medium, 0.9-lh when enough NK? was supplied. Its essential characteristics are as follows: short rods, 2.0x1.6 ^m in diameter, arranged singly, Gram negative, facultatively anaerobic, having both a respiratory and a fermentative type of metabolism, oxidase negative and catalase positive. To combine the results of morphological and physiological identification and the result of BIOLOG system test, the strain W12 was recognized as Klebsiella pneumoniae ss pneumoniae, belonging to Klebsiella, Enterobacteriaceae. The result of pathogenic examination showed that it was nontoxic and nonpathogenic.Applicability of the strain W12 to diverse environmental factors. Effects of diverse environmental factors on the growth rate (OD4oo) and nitrogenase activity (ARA) of the strain W12 hi nitrogen-free culture were investigated in our experiments. The results implied that the strain W12 could easily adapt to different cultural conditions: it could use various carbon sources(especially glucose, sucrose, malic acid, mannitol), propagate quickly and fix nitrogen at a temperature range of 15癈 to 40癈 and at 25-35 癈 for optimum, at a pH range of 4 to 8.5, at a saline concentration range of 0.01% to 1.5%; Low NlV" concentration had little effect on its Nitrogenase activity. ARA could also be detected when it grow in the culture media with 5mmol/L NtV".The effects of inocul...
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