| There is large amount of energy in the interior of the earth, this energy is in the form of geothermal manifestation. China is at the Crossroad of the Circum-Pacific geothermal belt and the Mediterranean-Himalayan geothermal belt, a lot of well-developed geothermal area located here. Many thermophilic microorganisms with special metabolic and physiological characteristics may harbor there. Although geothermal environment is considered inhospitable, a high diversity of thermophilic microorganisms including Bacteria, Archaea and Fungi are present inside. At the beginning of this study, Biolog ECO microplate technology was used to study the metabolic characteristics of microbial communities of typical samples in Tagejia geothermal area, Culture depended method was carried out to study the diversity of thermophiles, from which a large number of potential candidates of new species were explored. In addition, in order to explore diversity and phylogenetic distribution of thermophiles that produce cellulose and hemicellulase, part of isolates were screened to test their cellulose and xylanse degradation ability. Finally a high-temperature cellulase was cloned from one candidate strain which showed both activities of cellulose and hemicellulose.Total six typical samples were selected to analyse their microbial community metabolic ability based on Biolog ECO microplate technology. The result showed that the metabolic characteristics of microorganisms under different type of samples are different from each other among the geothermal environments; less biomass in sediments resulted negative metabolic activity detection when incubated in microplates; while metabolisms of microbial communities in hot hole soil showed a high diversity of metabolic activity. Cluster analysis showed thermophiles in hot spring sediments are more similar in metabolisms, and community metabolisms in hot holes soil are significantly different from the hot springs sediments, while the difference between them was obvious. Then 15 representative samples with different temperature and pH were selected to investigate their biodiversity based on culture depended method, total 378 of isolates were obtained, and the 16S rRNA gene sequencing were completed. Analysis based on 16S rRNA gene showed that these strains were distributed in the 5 phyla of Domain Bacteria,6 classes,12 orders,17 families and 26 genera. Total 6 genera of actinobacteria were isolated and 20 genera of bacteria were obtained. Geobacillus, Fictibacillus, Anoxybacillus and Bacillus were the highest frequency genera, Meiothermus and Thermus are typical thermophiles.9 new candidates, in total 58 strains, accounting for 15.3% of the total isolates were obtained, Polyphasic taxonomic study of a new candidate belongs to the genus Brevibacillus was carried out and its taxonomic status was determined.150 of representative thermophiles were selected for cellulases and hemicellulases screening, of which 71 thermophiles can degrade plant cell wall polysaccharides (cellulose and hemicellulose), accounted 47.33% of the total candidates. Among these 41 stains were capable of xylan degrading,57 strains were capable of carboxymethylcellulose degradation and 9 strains were capable of avicil degrading, accounted 27.3%,38.0% and 6.0% of these candidates, respectively.Finally, a cellulase gene was cloned from one thermophilic bacteria YIM 78625 which could degrade carboxymethyl cellulose, avicil and xylan, an exo-cellulase gene was obtained and expressed in E. coli successfully, the other cellulose was also cloned from YIM 77570 successfully.This study expands our knowledge about the phylogenetic diversity of culturable thermophiles in Tagejia geothermal field, what’s more, potential application of ellulose, hemicellulose degradation bacteria was assessed. |
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