| The environment of hot spring was quite similar with early Earth, and the ecology of microbial in hot spring was simple and stable. The exploration of microbial diversity in such ecosystem was important for the understanding of the microbial community structure, physiology and the relationship to the geochemical conditions. It also was helpful for understanding of the origin and the evolution of life.Thermophiles were the unique microbial source and rare material of scientific research. Hot spring had attracted broad interest because of the unique thermophilic properties of the organisms thriving in these biotopes and the description of an increasing number of new thermophilic species. Thermostable enzymes synthesized by thermophilic microorganisms were the important resources in many industrial processes and biochemistry. Cellulases played an important role in the biomass energy, especially thermostable cellulases.In the study, the bacterial and arechaeal diversity of the coastal hot spring in Xiamen were investigated by culture-independent molecular approaches. Three16S rDNA gene libraries of bacterial community from different temperature hot spring sediments were constructed and the analyses were performed by amplified ribosomal DNA restriction analysis (ARDRA) technique. The sequence similarities were analyzed by using the BLAST programs for searching the GenBank DNA databases. The phylogenetic analyses indicated that most of the bacterial16S rDNA sequences belonged to at least eight groups, including Proteobacteria, Aquificae, Bacteroidetes, Chloroflexi, Thermotogales, Firmicutes and Acidobacteria, and a few belonged to unknown groups. All bacterial libraries were dominated by Proteobacteria. In hot spring of90℃,Alphaproteobacteria was the predominant species (37.5%). While Betaproteobacteria was dominant in the hot spring of70℃(43.9%) and80℃(50.6%)。To describe arechaeal diversity, the arechaeal16S rDNA gene libraries of arechaeal community from different temperature (70℃and80℃) hot spring sediments were constructed. The phylogenetic analyses indicated that the arechaeal16S rDNA sequences belonged to Crenarchaeota and Euryarchaeota. Most of the archaeal phylotypes were related to sequences of yet-uncultivated microorganisms retrieved from terrestrial geothermal springs, deep-sea hydrothermal vents and the subsurface. All archaeal libraries were dominated by Crenarchaeota, and the proportion of Crenarchaeota in hot spring of80℃and70℃were89.2%发and91.3%resperctively. Clone SA-61belonged to unknown phylum of archaea. The archaeal phylotypes belonged to Thermoproteales and Desulfurococcales of Crenarchaeota phylum, and Thermococcales and Archaeoglobales of Euryarchaeota phylum, and a few belonged to unknown groups. Desulfurococcales were dominant in the both hot springs.Culture-based approaches were used in isolating thermophiles from Hot spring. In total, more than100thermophilic bacterial strains were isolated from hot springs in Nevada of USA and Yongtai country of China. Superior cellulose and hemicellulose decomposing strains were screened and identified by16S rDNA sequencing analysis. LY7and LY8which degraded cellulose were indentified as Alicyclobacillus sp. LY7nd Geobacillus sp. LY8. Geobacillus sp. LY8could be cultured at temperature range from40℃to70℃, while the optimal temperature was65℃. LY-1, LY-2, LY-3and LY-4which degraded hemicellulose, were assigned to Geobacillus sp. based on16S rDNA sequencing analysis. They could grow at temperature range from40℃to70℃, while the optimal temperature was65℃.The β-glucosidase was the rate-limiting enzyme in cellulose hydrolysis of many microorganisms and the resulting accumulation of cellobiose inhibits endoglucanase and cellobilhydrolase activities. To increase production of the enzyme and study the character of thermostable enzyme, the gene encoding a thermostable β-glucosidase from Geobacillus sp. LY8was cloned and sequenced. According to the amino sequence, β-glucosidase from Geobacillus sp. LY8belonged to glycosyl hydrolase family1. After PCR amplification, the β-glucosidase gene was cloned into the pGEX4T-2vector. The recombinant plasmid containing β-glucosidase gene was transformed into E. coli BL21.The recombinant β-glucosidase was heterologously overexpressed in the intracellular space. Then it was purified and the biochemical characteristics were also investigated. The results showed that:(1) The optimal temperature for the enzymatic reaction was65℃, and had good enzyme activity at30～90℃. The recombinant β-glucosidase worked well at90℃and was stable when treated at55～75℃for120min. All of these showed that the recombinant β-glucosidase was an excellent thermostable enzyme.(2) The recombinant β-glucosidase had good activity at broad pH range from pH4to pH8.(3) The recombinant β-glucosidase was unusually stable in organic solvents and detergents. In conclusion, the recombinant β-glucosidase was an excellent thermostable enzyme. It had good enzyme activity at high temperature and acid or natural pH.Fungi was the most important microorganism that degrading cellulose. Culture-based approaches were used in isolating thermophilic fungi from hot spring which degraded cellulose. In total,21thermophilic fungi strains were isolated and eight of them were identified by18S rDNA sequencing analysis. C1and WJM-4-WG belonged to Cladosporium sp.; W3W, CC-1, C2F1and C2F2belonged to Aspergillus sp.; CC-8belonged to Trichosporon sp. While WJM-1-G had low homology with sequences in NCBI, and maybe it belonged to a novel species. Fermentation conditions of three superior cellulose decomposing strains CC-1, CC-8and WJM-4-WG were further studied. The gene encoding a thermostable cellobilhydrolase from Aspergillus sp.CC-1was cloned and sequenced. According to the sequence, cellobilhydrolase from Aspergillus sp.CC-1belonged to glycosyl hydrolase family6. Cloning of cellobilhydrolase gene of themophilic fungi founded the base not only for constructing the engineering strain with high-yield cellulose, but also for the study of unusual structure and function of thermostable enzyme. |
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