二次离子质谱表征地杆菌共生团聚体外表面特征分子

	二次离子质谱表征地杆菌共生团聚体外表面特征分子
	魏文超
学位类型	硕士
导师	刘芳华研究员
	2017-12
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	红粘层地杆菌团聚体微生物种间电子传递飞行时间二次离子质谱仪
其他摘要	微生物种间电子传递（Microbial Interspecies Electron Transfer, MIET）作为厌氧能量代谢过程中的重要环节，已经成为国际研究热点，然而在滨海湿地生态系统中的研究相对较少。前期研究表明黄河三角洲滨海湿地广泛存在一种特殊红粘层，该层含有丰富的铁氧化物，是研究MIET的理想场所。本研究选取该区域含有红粘层的不同深度的土壤样品作为研究对象，对其理化性质进行了测定，并对其微生物多样性进行了高通量分析。同时，通过实验室富集培养，探究其潜在的微生物种间电子传递机制。理化性质测试结果表明红粘层土壤含有较高含量的无定形铁和游离氧化铁；高通量测序结果表明红粘层土壤微生物群落结构明显区别于其它层，且富含铁还原菌（Geobacter等）和产甲烷菌；富集培养实验过程中，发现甲烷的产生总要滞后于无定形铁的还原过程，暗示红粘层土壤中存在的铁氧化物作为电子受体与二氧化碳竞争电子，抑制了产甲烷过程；且随着富集代数的增加，铁还原能力逐渐下降，产甲烷能力逐渐增强且启动时间缩短，暗示着微生物群落发生变化且其中的电子流向可能发生改变。本研究首次对黄河三角洲滨海湿地红粘层土壤中微生物多样性及其潜在的种间电子传递机制进行了探究，对初步揭示红粘层中微生物在滨海湿地碳、铁循环中的重要作用提供了基础数据。为了深入探究黄河三角洲滨海湿地红粘层中微生物潜在的种间电子传递机制，本研究选取了2株已证实存在直接种间电子传递（Direct Interspecies Electron Transfer, DIET）的异化铁还原菌Geobacter metallireducens和Geobacter sulfurreducens作为模式菌株，并建立二者共生体系。已有研究结果表明，地杆菌共生团聚体的形成有利于共生体系中G. metallireducens 和G. sulfurreducens进行DIET，然而其重要界面——团聚体外表面，其分子组成仍不清楚。为了进一步揭示团聚体外表面的分子组成及其形成机制，本研究首次使用表面灵敏度极高的飞行时间二次离子质谱仪（Time of Flight Secondary Ion Mass Spectrometry, ToF-SIMS）对G. metallireducens和G. sulfurreducens形成的共生团聚体样品以及一系列对照样品进行了分子表征。主成分分析结果表明地杆菌共生团聚体与地杆菌共生浮游细菌样品具有明显差异，意味着团聚体表面分子组成及结构具有特异性。结合主成分分析及质谱峰鉴定结果，本研究揭示了引起团聚体表面特异性组成的一系列物质：氨基酸、脂肪酸、脂质等。其中，氨基酸组成方面的差异最大，表明蛋白质在共生体系中的重要性。群体感应信号分子N-butyryl-L-homoserine lactone在地杆菌共生团聚体样品中的丰度明显高于共生浮游样品，表明其在共生团聚体形成过程中起到了重要作用。接受电子的微生物G. sulfurreducens为共生体系中的优势菌种，在共生体系的形成方面起到主导作用。本论文利用ToF-SIMS技术，从表面分子表征角度，探究了地杆菌共生团聚体的形成及其互营机制，证明了ToF-SIMS在研究生物膜表面方面的应用潜力，同时也为深入揭示滨海湿地MIET机制提供可能性。; As an important step of the anaerobic energy conversion, microbial interspecies electron transfer (MIET) has become a hot issue in the world. However, the researches focused on coastal wetland ecosystem are insufficient. Previous studies shown that a special red clay horizon, which contains considerable amount of iron oxides and is an ideal place to study MIET, widely existed in the Yellow River Delta. In this study, soil samples of different depths were collected, which contains the red clay horizon. In order to investigate the potential mechanism of the MIET within the red clay horizon, soil physicochemical properties and microbial diversities were measured, designed enrichment experiment of soil microbes was performed. The results showed that the red clay horizon soil samples contained higher amount of amorphous and free iron oxides than its upper and lower layers. Besides, the microbial community structures of the red clay horizon were unique from other layers, which had higher abundance of iron-reducing bacteria (such as Geobacter) and methanogens. The enrichment experiment results showed that the production of methane was lagging behind the reduction of amorphous iron oxide, suggesting the competition for electron acceptors between the iron oxides and carbon dioxides would inhibit the production of methane. After transferring for several generations, the iron reducing ability of the enriched microbes decreased, accompanied with less start time and the increased ability of methane production, suggesting the changes of the microbial communities and the flow direction of electrons. This is the first exploration for the microbial diversity and potential mechanisms of the microbial interspecies electron transfer in the red clay horizon, which located in the Yellow River Delta. This study provided basic data and would be helpful for revealing the important role of the red clay horizon in the carbon and iron cycling of the coastal wetlands. In order to further investigate the potential mechanisms of the microbial interspecies electron transfer in the red clay horizon of the Yellow River Delta, two kinds of dissimilatory iron reducing bacteria Geobacter metallireducens and Geobacter sulfurreducens were selected and co-cultured together in this study. Previous studies showed that, the formation of the aggregates was beneficial for the direct interspecies electron transfer (DIET) within the co-cultured aggregates. However, the composition of the outermost surface, which is the important biointerface of the co-cultured aggregates, remains unclear. In order to further reveal the composition of the outermost surface of the co-cultured aggregates and its formation mechanisms, a series of samples associated with syntrophic G. metallireducens and G. sulfurreducens were characterized by surface sensitive time of flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis was used in spectral analysis. Our results show that the syntrophic Geobacter aggregates are significantly different from their planktonic cells, indicating a distinct chemical composition (i.e., amino acids, fatty acids, and lipids) and structure formed on their surface. Among these characteristic components, amino acid fragments dominated in the variance, suggesting the importance of proteins in the co-culture. Additionally, the quorum sensing signal molecule N-butyryl-Lhomoserine lactone was observed in co-cultured Geobacter aggregates, implying its role in syntrophic growth and aggregate formation. Furthermore, the electron acceptor organism G. sulfurreducens was shown to be the dominant species in syntrophic communities that drove the syntrophic growth. In this study, the mechanism of the co-cultured aggregates formation and its syntrophic growth was explored by using ToF-SIMS, which suggests that ToF-SIMS may be a promising tool to understand the complex biofilms systems and provides the possibility of further investigation of the mechanism of MIET in the coastal wetlands.
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/23475
专题	中国科学院烟台海岸带研究所知识产出_学位论文海岸带生物学与生物资源利用重点实验室_海岸带生物学与生物资源保护实验室
作者单位	1.中国科学院烟台海岸带研究所 2.中国科学院大学
推荐引用方式 GB/T 7714	魏文超. 二次离子质谱表征地杆菌共生团聚体外表面特征分子[D]. 北京. 中国科学院大学,2017.