【作者】 王红蕾；

【导师】 冯雁；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 微生物生态学是研究微生物与环境相互作用的基础学科，反映出了微生物群落与环境因素在物质、能量以及信息等方面的交互作用，体现出了生态系统的平衡机制。热泉是地下水经地热加热后的产物，与其特定的地质及环境因素相互作用，形成了特殊的生态系统。该系统中的微生物群落结构以及丰富的微生物资源一方面反映了热泉特殊生态系统的平衡机制和生物进化机制，另一方面为特定嗜热微生物的分离与利用提供了可能。本论文选择了长白山热泉群中三个代表性热泉（83oC，74oC，53oC）作为研究对象，通过16S rRNA基因文库及末端限制性片段长度多态性（T-RFLP）分析相结合的方法对长白山热泉微生物群落结构及其多样性进行了研究。综合两种方法所得结果表明：共检测出47个16S rRNA细菌序列，在系统进化树上可聚类为7大类群，包括17个属和一些未确定种属的细菌。微生物群落结构的物种组成、种群密度及相对丰度分析，发现栖热袍菌门（Thermotogae）、嗜热菌属（Thermus）、梭菌属（Clostridium Prazmowski）、嘉利翁氏菌科（Gallionellaceae）、产液菌门（Aquificae）和假单胞菌科（Pseudomonadaceae）在三个温度热泉中均存在，推测这些微生物是长白山热泉微生物群落的固有微生物。三个不同温度热泉中微生物群落分布有明显差异，处于生物进化最古老位置的栖热袍菌门（Thermotogae）和产液菌门（Aquificae）在两个高温热泉中（83oC，74oC）所占比例较大，鞘氨醇杆菌（Sphingobacteria）仅出现在2个高温热泉样品中。83oC热泉中的微生物群落多样性最丰富（R=10），74oC群落均匀性更好（E=0.924）。根据T-RFLP结果，高温热泉的微生物群落表现出更加多样化的特征。由于三个样品的来源环境接近，热泉泉水化学成分类似，因此我们认为三个热泉的温度差异是导致其微生物生态结构差异的主要因子。通过对三个样品的代谢类型进行分析，三个环境均富含化能自养型细菌，高温热泉中光能自养型细菌所占比重较大。对嗜热微生物的研究不仅在探讨生命起源、进化以及其高温适应机制方面有重要意义，更有实用价值的是能够获得具有工业应用潜力的嗜热酶资源。通过对基因文库的分析，以产纤维素酶微生物和产酯酶微生物为筛选目标。从长白山热泉分离得到具有纤维素降解能力的嗜热厌氧微生物CBS-ZT。经PCR扩增和16S rRNA全序列比较，CBS-ZT属于厚壁菌门，与其相似性最高的菌为Caldicellulosiruptor saccharolyticus DSM8903，相似度为97%。CBS-ZT的最适生长温度为74oC，最适pH为弱碱性（7.8左右），可利用纤维素作为其生长碳源，能产氢气；脂肪酸成分为C16:0（34.2%），iso-C16:0（18%），C18:0（12.8%），iso-C17:0（11.1%），DNA G+C含量比为36.8%，根据结果可确定其为Caldicellulosiruptor的新种。该菌具有产氢和可降解纤维素两大优势，是一株在生物能源领域很有应用潜力的新菌。根据嗜热微生物产酯酶能力分析，分离得到了嗜热菌CBS28，16S rRNA分析表明它属于地芽孢杆菌属成员，与Geobacillus属中成员Geobacillusstearothermophilus序列相似性97%。CBS28最适生长温度为65oC，但可以在80oC的高温下生存；最适pH值为8，但在较强碱性和酸性条件下均可以存活。生理生化特征的实验表明，CBS28还具有淀粉酶、酪素酶等胞外酶活性。这为开发在食品酿造工业、医药工业和环境保护等领域具有应用价值的嗜热酶提供了新基因资源。更多还原

【Abstract】 Microbial ecology, the basic subject considering the interaction betweenmicroorganism and environment, could reflect the communication on substance,energy, or information between microbial community and circumstance, whichilluminate the balance of the ecosystem. Hot springs are formed by geothermal energyheating the underground water. The specific geological property and environmentalfactors make the hot springs become some distinctive ecosystems on our planet.Research on the microbial community structures of these ecosystems as well asnumerous microorganism resources would help us understand the balance mechanismof the hot springs special ecosystems and microorganism evolution mechanism. Alsoit may provide more opportunities for exploring and utilizing novel thermophilicmicroorganisms.In this study, the microbial community structure of three Changbai Mountain HotSprings (83oC,74oC,53oC respectively) were analysed by16S rRNA gene librarysequencing and terminal restriction fragment length polymorphism (T-RFLP).47bacterial16S rRNA sequences were determined in the phylogenetic tree, which can beclassified into7groups,17genus and some unassigned bacteria. The speciescomposition, population density, and relative abundance analysis of three hot springsmicrobial community structure indicated that Thermotogae, Thermus, Clostridium,Gallionellaceae, Aquificales and Pseudomonas existed in three hot springs. Wespeculated that they were the endogenous populations in three hot springs. On theother hand, microbial community distribution were different among three hot springs.The oldest Thermotogae and Aquificales in the community structure in twohigh-temperature hotsprings in the dominant flora. And Sphingobacteriia appears only in the samples of the two high-temperature hotsprings. The74oC hot spring in themost abundant microbial community diversity and evenness better. The dominant taxain these three samples indicated that the structure of74oC hot spring exhibit morediversity but most bacteria are mesophilic bacteria compare to83oC. The differenceevolution of groups is cased by temperature when the other factors are similar. Toverify the phylogenitic relationships between phylotyes in74oC and83oC samples, theaverage taxonomic distinctness were also take into account. Three samples of thesource environment close to the hotsprings springs chemical composition similar，sowe believe that the three hotsprings temperature differences lead to the bacterialecological structural differences only selection pressure. Through the analysis of threesamples of the metabolic type, three environments are rich autotrophic bacteria，hightemperature hotsprings in the light energy is dominant autotrophic bacteria.Speculated that this related to the physical and chemical factors of the ChangbaiMountain Hotsprings.On the Basis of16S rRNA sequence similarity, the thermophilic anaerobicCBS-ZTwhich was isolated from Changbai Mountain was shown to belong toFirmicutes, being most closely related to Caldicellulosiruptor saccharolyticus DSM8903(97%). The optimum temperature and pH of CBS-ZTwas74oC and7.8withcellulose as a carbon source, moreover, CBS-ZTwas capable of producing hydrogen.The fatty acid of CBS-ZTincluded C16:0(34.2%)，iso-C16:0(18%), C18:0(12.8%)，andiso-C17:0(11.1%). The DNA G+C content of CBS-ZTwas36.8%. Based on these data,CBS-ZTrepresents a novel species, meanwhile, it was a promising bacteria which canbe used to produce hydrogen and degradate cellulose.According to the16S rRNA sequence similarity, CBS28was shown to be themember of the Bacillus, with high similarity (>97%) to the Geobacillusstearothermophilus. Optimal growth of CBS28occurs at65oC and pH8.0. It can alsosurvive at80oC or in strong alkaline and acidic conditions. On the basis of thephysiological and biochemical characteristics, the extracellular enzymes of CBS28displayed activity of amylase, esterase and so on, which make it good candidate for ne w genes applied in food industry, medicine and envir onmental protecti on.更多还原