兰州大学机构库 >地质科学与矿产资源学院
不同地质环境中微生物群落与极端微生物的研究
Alternative TitleStudy of Microbial Community and Extremophiles in Different Geological Environments
黄志勇
Thesis Advisor宋春晖
2019-03-15
Degree Grantor兰州大学矿产学院
Place of Conferral兰州
Degree Name博士
Degree Discipline矿物学、岩石学、矿床学
Keyword地质微生物 气候变化 微生物群落 极端微生物 基因组
Abstract地球上生命的出现和参与,深刻地影响和改变了地球的演化和历史进程,形成了地圈-生物圈-大气圈。生物作用作为活跃和强大的地质营力,无论是岩石圈物质的风化、搬运、沉积和成岩,或是对成矿元素的迁移富集,都发挥了巨大的作用,而且对地球表生系统的环境变化和元素循环产生深刻的影响。随着技术进步,学科交叉不断推动科学发展,形成许多综合性学科。在地学领域,地质微生物学就是地球科学与生物科学的交叉学科,可以全面系统的阐明生物地球化学作用、揭示地球环境变化规律,已经成为国际地学界关注的热点。微生物通过代谢活动完成地球元素循环,在全球或局部尺度上与许多重大环境变化和地质气候事件密切相关,特别是极端微生物不仅能帮助人们揭示地圈和生物圈起源的奥秘,阐明生物多样性形成机制和生命演化规律,同时能为探索全球环境变化提供新的认识,在环境保护、气候变化、资源开发、人类健康和生物技术等领域发挥巨大的作用。本文针对不同地质环境中的微生物,以新疆准噶尔盆地泥火山、青藏高原东北缘临夏盆地沉积剖面和酸性黄铁矿浸出液为研究对象,利用基因组测序、生物信息分析等分子微生物生态技术,研究不同环境下微生物代谢途径、群落结构、微生物多样性、以及微生物间相互作用,探索微生物地球化学作用及其对环境变化的指示作用,取得了如下主要成果和认识:1)新疆准噶尔盆地泥火山微生物群落研究泥火山是地下泥浆与气体喷发堆积而成,在地球碳循环中发挥着关键作用。以新疆准噶尔盆地两座泥火山(独山子和白杨沟)为研究对象,通过微生物组成差异分析,探索泥火山形成机制,并首次报道了独山子和白杨沟泥火山的微生物群落及多样性。研究表明,两座泥火山细菌和古菌的群落结构差异显著,其中白杨沟泥火山的细菌群落多样性高于独山子,而古菌群落多样性低于独山子。在独山子泥火山中发现的古菌为厌氧或极端嗜热菌,而白杨沟泥火山中发现的古菌为嗜盐古菌,与两座泥火山特殊的地理性质相似。此外,在两座泥火山中均发现大量的未知菌种。本研究成果不仅对揭示泥火山的形成机制和物种起源具有重要意义,也为今后准噶尔盆地油气探勘提供依据。2)青藏高原东北缘临夏盆地毛沟剖面沉积物中微生物记录的干旱化研究以青藏高原东北缘临夏盆地有年代控制的新生代毛沟剖面为研究对象,采用高通量测序技术对沉积样品的微生物群落结构进行解析,探索微生物在气候变化中的变化规律和指示作用。研究结果显示,毛沟剖面在26.47 Ma(30 m)、14.04 Ma(204 m)、8.03 Ma(314 m)和5.41 Ma(398 m)沉积序列的微生物群落结构简单、多样性指数较小,厚壁菌门(Firmicutes)相对丰度高;与之相反,其他沉积样品中的微生物群落结构复杂、多样性指数较高,变形菌门(Proteobacteria)相对丰度高。由于变形菌多数喜温湿环境,抗逆性较差,而厚壁菌更能适应干旱和极端环境,可以推断在26 Ma、14 Ma、8 Ma和5.4 Ma左右,毛沟剖面沉积序列所处的新生代时期发生过多个干旱化事件。3)嗜酸浸矿微生物的筛选、鉴定及功能解析嗜酸菌是一类适应酸性环境生长的极端微生物,对研究硫化矿的成因以及在地球铁硫元素循环中生物地球化学作用有重要意义。利用基因组、宏基因组和转录组等分子生物技术对嗜酸菌进行功能分析,探索嗜酸菌群的演化规律和生物地球化学作用。对实验室前期获得的嗜酸浸矿菌群5Biol进行驯化,发现驯化菌群可明显改变物种组成,Acidithiobacillus在驯化前后都是优势种,但驯化后在菌群中所占比例有79 %增加到86.77 %;边缘物种中Sulfobacillus的比例也有提升,由0.76 %上升至0.84 %。驯化后嗜酸浸矿菌群功能基因减少,但生物活性没变,其功能向简单化方向演化。从嗜酸浸矿菌群中筛选到一株极端嗜酸菌hq2,最适生长温度42 oC、最适pH 2,GC含量为52.44 %,可将Fe2+氧化为Fe3+,能利用Yeast但不能利用葡萄糖、蔗糖,甘露糖,海藻糖,苹果酸等有机碳源,也能利用S0和K2S4O6。基因组分析结果显示,hq2菌的基础代谢中有完整的卡尔文循环和磷酸戊糖途径(PPP),但糖酵解途径(EMP)和三羧酸循环不完整,而且不能通过还原硝酸盐/亚硝酸盐获得氮源。hq2菌株的硫代谢途径中没有发现编码Sox系统的基因,但有编码硫氧化还原酶(SOR)的基因,推测其利用Sor系统完成硫代谢。另外,菌株hq2的基因组中有完整的鞭毛合成基因,和抗砷、抗铜、抗汞等基因,具有很好的环境适应能力和趋化性。经系统分类鉴定,该菌有可能是一株新发现的混合营养型铁氧化菌,暂时命名为Sulfobacillussp. hq2。将两株从嗜酸浸矿菌群5Biol中筛选到的嗜酸微生物Sulfobacillus sp. hq2和Acidithiobacillus sp. AC进行混合培养,建立共培养体系,模拟菌群功能。通过比较转录组分析的结果显示,经过培养后Acidithiobacillus sp.AC在共培养体系中逐渐占据优势,与5Biol浸矿菌群的物种比例一致。Sulfobacillus sp. hq2不仅对Acidithiobacillus sp. AC细胞组分的合成、蛋白分子功能等细胞生物学过程起到激活作用,可显著促进Acidithiobacillus sp.AC的生长;还能增强Acidithiobacillus sp. AC氧化单质硫的能力,使整个共培养体系的铁氧化能力大幅提高。另外,与纯培养条件相比,Acidithiobacillus sp. AC在共培养体系中的硫代谢和氧化磷酸化基因上调,储存能量的能力变强;趋化调控基因和鞭毛合成基因也显著上调,表明细菌的趋化性增强,可以使得微生物细胞提高菌体对单质硫的获取能力,更好的利用环境,趋避有害环境。在嗜酸浸矿菌的共培养体系中Sulfobacillus sp. hq2对Acidithiobacillus sp. AC的全局基因转录产生巨大影响,从分子水平上揭示了双菌混合的共培养体系具备更强代谢能力。
Other AbstractThe emergence and participation of life on the earth profoundly influenced and changed the evolution and historical process of the earth, forming the geosphere-biosphere-atmosphere. Biological processes, as active and powerful geological forces, play an important role in weathering, transportation, sedimentation and diagenesis of lithospheric materials, as well as in migration and enrichment of ore-forming elements, and have a profound impact on environmental changes and element cycle of the Earth's supergene system. With the progress of technology, the intersection of disciplines promotes the development of science and forms many comprehensive disciplines. In the field of geoscience, geomicrobiology is an interdisciplinary subject between Geoscience and biological science. It can comprehensively and systematically clarify the biogeochemical action and reveal the law of the change of the earth's environment. It has become a hot topic of international geoscience circles. Microorganisms complete the earth's element cycle through metabolic activities, and are closely related to many major environmental changes and geological and climatic events at global or local scales. In particular, extremophiles can not only help people to reveal the mysteries of the origin of the geosphere and biosphere, clarify the formation mechanism of biodiversity and the law of life evolution, but also provide new insights into global environmental change, and play a great role in environmental protection, climate change, resource development, human health, industrial biotechnology et al. In this study, aiming at microorganisms in different geological environment, such as mud volcanoes in Junggar Basin, sedimentary profiles in Linxia Basin in northeastern margin of Tibetan Plateau and acid pyrite leaching solution, we studied microbial community, metabolic pathway, diversity and interactions by using molecular techniques and bioinformatics analysis. The exploration of microbial geochemistry and its indicative role in environmental change has yielded the following achievements. 1) Microbial community of mud volcanoes in Junggar Basin, Xinjiang Mud volcanoes are formed by the eruption and accumulation of underground mud and gas, and play a key role in the earth's carbon cycle. Taking two mud volcanoes (Dushanzi and Baiyanggou) in Junggar Basin of Xinjiang as research objects, the formation mechanism of mud volcanoes was explored according to their microbial composition analysis. The results showed that there were significant differences in bacterial community structure and archaeal community structure between Dushanzi and Baiyanggou. The diversity of bacterial community in Baiyanggou mud volcano was higher than that in Dushanzi, while the diversity of archaeal community was lower than that in Dushanzi. The Archaea found in Dushanzi mud volcano were anaerobic or extremely thermophilic, while those found in Baiyanggou mud volcano were halophilic, which were similar to the unique geographical characteristics of the two mud volcanoes. In addition, a large number of unidentified microorganisms were discovered in both mud volcanoes. The results of this study are of great importance not only to reveal the formation mechanism and origin of mud volcanoes, but also to provide a useful basis for oil and gas exploration in Junggar Basin in the future. 2) Droughting of Microbial Records in Sediments of Maogou Section in Linxia Basin, Tibetan Plateau Taking the Cenozoic Maogou profile in Linxia Basin, northeastern margin of Tibetan Plateau as the research object, we utilized high-throughput sequencing and organic geochemical techniques to study the microbial community of sediment, and explore its action in climate change and indicative roles. The results show that the microbial communities in Maogou section at 26.47 Ma (30 m), 14.04 Ma (204 m), 8.03 Ma (314 m) and 5.41 Ma (398 m) were simple, the diversities were low, and the relative abundance of Firmicutes were high. On the contrary, the microbial communities in other depositional samples were complex in structure, high in diversity index, and Proteobacteriawere abundance. Because Proteobacteriamostly prefers warm and humid environment and has poor resistance to stress, while Firmicutesis more adaptable to drought and extreme environment, it can be inferred that there were several drought events in the Cenozoic period of Maogou section sedimentary sequence at about 26 Ma, 14 Ma, 8 Ma and 5.4 Ma.  3) Isolation, identification and functional analysis of acidophilic leaching microorganismsAcidophile can survive in acidic environment. They are of great significance to study the genesis of sulfide ores and their biogeochemical cycles of iron and sulfur in the earth. Molecular biotechnology, such as genome, macrogenome and transcriptome, was preferred to analyze the function of acidophile, and to explore the evolution law and biogeochemical role. Acidithiobacilluswas the dominant species before and after domestication, but its proportion increased from 79% to 86.77% after domestication, and the proportion of Sulfobacillusin marginal species increased from 0.76% to 0.84%. After domestication, the functional genes of acidophilic leaching bacteria decreased, but their biological activities remained unchanged, and their functions evolved towards simplification. An extremely acidophilic bacterium hq2 was isolated from acidophilic leaching microflora 5Biol. The optimum growth temperature was 42 oC and the optimum pH was 2. Fe2+could be oxidized to Fe3+. It could grow with yeast, but could not use other organic carbon sources such as glucose, sucrose, mannose, trehalose and malic acid. S0and K2S4O6could also be utilized. The ratio of GC was 52.44%. Genome analysis showed that the Calvin cycle and pentose phosphate pathway (PPP) in the basal metabolism of the strain were complete, but the glycolysis pathway (EMP) and tricarboxylic acid cycle were incomplete, and the nitrogen source could not be obtained by reducing nitrate/nitrite. No gene encoding Sox system was found in the sulfur metabolism pathway of hq2 strain, but genes encoding sulfur oxidoreductase (SOR) were found. It indicated that hq2 could grow with ferrous oxide, elemental sulfur and sulfide in the presence of organic substances. Additionally, the genome of strain hq2 contained complete flagellum synthesis genes, arsenic, copper and mercury resistance genes, which have good environmental adaptability and chemotaxis. It revealed that the isolate may be a newly discovered mixed-nutrient iron oxidizing bacteria, tentatively named Sulfobacillussp. hq2. Two acidophilic bacteria, Sulfobacillussp. hq2 and Acidithiobacillussp. AC isolated from acidophilic leaching microflora 5Biol, were mixed cultured to establish a co-culture system to simulate the function of 5Biol. The results of comparative transcriptome analysis showed that Acidithiobacillussp. AC gradually occupied the dominant position in the co-culture system after culture, which was consistent with the species proportion of 5 Biol. Sulfobacillussp. hq2 not only activates the cell biological processes such as the synthesis of Acidithiobacillus sp. AC cell components and the function of protein, but also significantly promotes its growth. It also enhanced the ability of Acidithiobacillussp. AC to oxidize sulfur and improved the iron oxidation ability of the co-culture system. Moreover, compared with pure cultures, genes of the sulfur metabolism and oxidative phosphorylation inAcidithiobacillussp. AC were increased in the co-culture system, which enhanced the ability to store energy. Chemotaxis regulation genes and flagella synthesis genes were also significantly increased, indicating that bacterial chemotaxis was enhanced. Sulfobacillussp. hq2 has a great influence on the global gene transcription of Acidithiobacillus sp. AC, which revealed at molecular level that the co-culture system of two bacteria has a stronger metabolic capacity.
Pages189
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/338385
Collection地质科学与矿产资源学院
Affiliation地质科学与矿产资源学院
First Author AffilicationSchool of Earth Sciences
Recommended Citation
GB/T 7714
黄志勇. 不同地质环境中微生物群落与极端微生物的研究[D]. 兰州. 兰州大学矿产学院,2019.
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