| In order to accelerate corn-stalk's rotting returned in the fields in Northern China, the complex microbial system CSS-1 with high efficiency and stable degrading ability for corn stalks was constructed and its microbial composition was analyzed. The CSS-1 was domesticated from the soils which had a long term of corn-stalk-returning into the fields in Shanxi Province, Shandong Province and Beijing using the methods of nutrition-restricted cultivation combining with gradient-temperature induction. Its decomposition ability was determined by the value of weight-loss percentage, C/N, CMC enzyme activity of the treated corn stalks. The microbial composition of CSS-1 was analyzed by cloning library method. The Stability of CSS-1 was confirmed by comparing the curves of weight-loss percentage and CMC enzyme activity, as well as the DGGE (denaturing gradient gel electrophoresis) analisis for the floras for the treated corn-stalk by CSS-1's different generations. The biological characteristic of CSS-1 for decomposition of corn stalk was determined by the growth curve of the microbial community, changes of CMC enzyme activity, xylanxylanohydrolase enzyme activity and pH in the decomposing process.The CSS-1 with high efficiency and stable degrading ability for corn stalks was domesticated finally from the soils which had a long term of corn-stalk-returning into the fields in Shanxi Province.The results indicated that the average of CMC enzyme activity and the maximum CMC enzyme activity produced by CSS-1 were respectively higher of 79% and 82% than those of the widely used commercial inoculant A in corn-stalk decomposition experiments. The weight-loss of the corn stalks treated with CSS-1 increased 62.62% and 173.65 % compared with that of blank control and the inoculant A, respectively. The microbial composition stability of CSS-1 was demonstried by valuating the DGGE profiles of bacteria and fungus community of the subcultures of the 22 generations. By the analysis of safety of the complex microbial system was demonstrated that it had better biosafety.The results of cloning library method showed that the dominant bacteria of CSS-1 belonged to Enterobacter sp., Cellulomonas sp., Streptomyces sp., Bacillaceae sp., Pantoea sp. and Cellvibrio sp. and the dominant fungi were Trichoderma sp. and Fusarium sp.. We also get some floras which have important function in cellulose degradation complex microbial system, such as Pseudoxanthomonas and Rhizobium sp., more species deversity than those by pure culture dependent method. Compared with the result of that clone library method had nicer application perspectives in analying the composition of microbial samples than the pure culture dependent method.The characteristics of CSS-1 for decomposition of corn stalk indicated that during the whole period of decomposition, the growth curve of the microbial community and changes of cellulose activity and pH in the system were identical. Growth curve of the CSS-1 included typical three periods,they were logarithmic phase(0-4 d), stationary phase(4-10 d) and decline phase(10-16 d), respectively. In the first four days of the decomposing process, the count of microorganism, pH, xylanxylanohydrolase enzyme activity increased sharply, meanwhile CMC enzyme activity decreased slowly; in the flowing days(4-10 d) the count of microorganism showed a trend of increase after initial decrease, CMC enzyme activity, xylanxylanohydrolase enzyme activity and pH reached maximum value; in the end phase (10-16 d) the indicators of decomposition characteristics tended to stabilize.The community composition and development were investigated by the means of DGGE, and the results showed that CSS-1 was constituted of 18 kinds of bacteria, 9 kinds of Fungi and presented fluctuation in some degree with decomposition.Furthmore, result of cluster analysis of DGGE profiles was accordant with the growth curve, and the degradation process could be divided into three stages:initial stage (0-2 d), intermediate stage(2-10 d)and end stage(10-16 d). It was in accordance with the growth curve of the CSS-1.In initial stage, every species grow fast; in intermediate stage some flora which have the function of cellulose degradation were dominant, such as Streptomyces sp., Micrococcus sp., Bacillus megaterium strain, Pseudomonas sp., Trichoderma sp., Fusarium sp.. After entering end stage, part of the functional species group declined for the reason of gradual depletion of the nutrients in substrate, such as Streptomyces sp. Actinomycetales bacterium; some of anaerobic microbes shows the contary trend, and gradually develop to grow, such as Uncultured Clostridium sp., Rumen bacterium. Meanwhile some species have always been to maintain arelatively high number, such as Trichoderma sp., Fusarium sp..The study also demonstrated that the method of Bead-beader was suitable for large quantity samples of DNA extraction from soils samples; clone library method and DGGE can be used well in the analyzing bacterial composition of complex microbial samples with good results. |
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