| The nitrogen fixation of marine microorganisms not only is the main source of marine nitrogen, but also is an important factor which can affect the growth of marine organisms, and the change of marine ecological environment. With the research deepening of the resources, ecology, and mechanism of marine nitrogen fixing microbial, people found there are a lot of non-photosynthetic heterotrophic nitrogen-fixing bacteria in ocean which play an important role in marine nitrogen source.In this study,72 and 101 nitrogen-fixing bacterial strains isolated from the seawater and seabed sediments of Chinese yellow sea (32°19.7’N～36°19.9’N, 119°50’E～126°14’E) have be studied by the nifH-PCR. and the phylogenetic analysis of 16S rDNA sequence. Moreover, one suspicious new specie M90 strain has be identified by the polyphasic taxonomy which includ the characteristics of phenotype, physiology, biochemistry, chemistry and genetics. The aims of this research are to comprehensively understand the diversity and distribution of non-photosynthetic heterotrophic nitrogen-fixing bacteria in Chinese yellow sea, fill in the aspects of related research in China, rich the library of marine nitrogen-fixing bacteria, and develop the taxonomic system of international marine nitrogen-fixing bacteria. The results as following:(1) The phylogenetic analysis results showed that 72 strains isolated from the seawater were distributed in 2 phyla,4 classes, and 15 genera.101 strains isolated from the seabed sediments were distributed in 3 phyla,5 classes, and 23 genera. Most of strains of seawater and seabed sediments were mainly distributed in the Gammaproteobacteria (80.6% of seawater bacteria,65.3% of sediment bacteria). These suggest that the diversities of sediment bacteria are higher than the seawater bacteria.(2) The results of nifH-PCR showed that 42 stains of 72 seawater bacteria and 59 strains of 101 seabed sediment bacteria have positive reaction, and many strains not had nifH gene. It suggests that the nifH primers used whould not be specification for those isolated stains.(3) 11 strains (2C,3A,7B,9B,18B,18E,25B,30E,31D, M90,35C) isolated from seabed sediments were found as potential new species(the similarities of 16S rDNA sequence were 96.76% ～ 97.56% with their standard strains). One strain M4 was found as potential new genus (the similarities of 16S rDNA sequence was only 94.81% with its standard strains).(4) The polyphasic taxonomy results showed that the 16S rDNA sequence similarity of M90 strain was 97.65% comparing with the standard strain Pararhodobacter aggregans D1-19T, the DNA-DNA hybridization homologous rate of M90 strain and Pararhodobacter aggregans D1-19T was 22.56%, and the G+Cmol% content of M90 strain was 68.36% (in Pararhodobacter range). In addition, on the characteristics of cell and colony morphology, aerobic, exercise, temperature resistance, salt tolerance, acid and alkali resistance, antibiotic resistance, sole carbon source and nitrogen source utilization, enzyme and hydrolysis reaction, as well as the polarester, fatty acid composition, M90 strain was basically the same with standard strain Pararhodobacter aggregans D1-19T, and also showed some differences. Therefore, M90 strain was identified a new species of Pararhodobacter, and be named Pararhodobacter huanghaigudans M90T. |
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