Isolation And Identification Of Thermohalophile Gen. Nov., Sp. Nov. And Diversity Of Microorganism In Sediments Of Hydrothermal Fluid Of Pacific, Atlantic And Indian Ocean

Microorganisms are one of the most valuable natural biology resources on the earth, and thermophile as a most important part of it, because of their extreme special living conditions, make themselves a unique enzyme system and defense mechanism to adapt the environment which is hard for most ordinary living organisms. Their enzymes and cells possess special molecular structure which is consistent with the heat resistance prosperity, this becomes one of the most important resources that appeals human beings. The new industry used enzymes and bio-activity materials play important parts in gene engineering, protein engineering, fermentation engineering and exploitation of mine resources. What is more important, thermophle have a special status in the evolution history of organisms and rich information of life evolution process. Research on the physical mechanisms of how they adapt to the extreme environment will show us clues which is related to the essence of life and the origin of living organisms.In the present study, we collected samples from hot springs near Xiamen and sediments and water samples from deep sea hypervent in three oceans, then incubated in different conditions, we got more than 200 strains of bacteria. Among which there are bacteria producing protease, agar consuming bacteria, flocculant which resist high temperature, some bacteria can produce metabolism substance which inhibit the growth of tumor cells, some can produce small molecules which can regulate the receptor of nerve peptide. All of which are valuable to be exploited.Due to fussy traditional method of separation and identification microorganisms, to get the process efficiently, we applied methods of molecular biology, including SDS-PAGE electrophoresis of whole bacterial cell protein, RAPD and amplification of 16S-23S rDNA spacer regions. By combining these three methods, we had isolated 7 strains of bacteria that belong to different genus among 60 strains. These 7 different strains were further confirmed by sequencing the 16S rDNA after amplification by PCR, these results provided a reliable basis for further study on the thermophilic bacteria.On the basis of previous study, we isolated one strain from sediment of hot spring dear Xiamen, which is a moderately thermohalophilic, facultatively anaerobic fermentative bacterium named HS1^T This strain is Gram negative, lives in the optional temperature between 37℃to 56℃, and grows better between 50-54℃, bears the highest salt concentration at 8 g/L. This strain can also deoxidize nitrate to N₂, sulphate and sulfite into H2S. C15:0 (21.27%), C15:1 ISO (21.11%) and C15:1 ANTEISO (16.03%) is the main three fat acid of this strain. The sequence of 16S rDNA indicates this strain belongs to Bacteroide phylum. 1448 of 16S rDNA sequence shows a 91.829 % homonology with Anaerophaga thermohalophila Fru22, according to the other factors such as phenotype, physical and biological characteristics, G-C content, we definite HS1^T (DSM 19012^T, CGMCCC 1.5071^T) as a new genus of thermohalophaga, name it Thermohalophaga xiamenensisas the model strain.In the study, we also carry out the comparison and investigation of micro diversity between some deep sea hypervent sites in three oceans by phylogenetic and multiple molecular biology methods. We extracted total DNA from sediments in different sites, constructed bacteria 16S rDNA cloning library, analyzed by PCR-RFLP and DGGE based method, we got information about the microorganisms in the sediment and its relationship with environments. Results showed, 16S rDNA sequences from different sites shared a little common of the known sequence, which also indicated that there are many genus of bacteria uncultured in deep sea hypervents. The characteristics of most bacteria related to the hypervent environment: symbiot bacteria, bacteria related to C,S,N metabolism, thermophile, anaerobe, iron metabolization and so on. The results illustrated that these sites possessed a typical deep sea hypervent ecosystem characteristic, metabolism of CH₄ and sulphur plays an important part in the energy circulation chain. Proteobacteria distributes abroad in all sites, and this is consistent with previous study. Anyway, the existence of numerous new extreme bacteria indicates that these regions have a big potential for microorganism exploitation.