滨海河流沉积物中产甲烷分离物的获得及其电化学特征

	滨海河流沉积物中产甲烷分离物的获得及其电化学特征
	李莹
学位类型	硕士
导师	刘芳华
	2016-05-21
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	生物工程
关键词	产甲烷分离物种间直接电子传递甲烷八叠球菌铁还原菌电活性
摘要	滨海河流是是海岸带区域重要的组成部分，连接陆地和海洋两大碳库，受到全球气候变化和人类活动的双重影响，滨海河流沉积物中二价铁含量丰富，其中富含铁还原微生物。本研究目的在于从富含铁的环境中获得有电活性的铁还原微生物，研究其与产甲烷菌之间的直接电子传递，为将来应用于沉积物微生物燃料电池进行污染物原位修复打下基础。本论文以界河流域为研究对象，分析不同样点理化性质，选取二价铁含量相对较高的 Jh3 样点进行铁还原富集培养，通过 T-RFLP 及构建克隆文库分析 Jh3 原位及富集样品的微生物群落多样性；采用 Hungate 厌氧滚管法从 Jh3 铁还原富集样品中分离获得具有潜在种间直接电子传递的产甲烷分离物，分析其微生物群落多样性，比较铁还原及产甲烷能力。构建微生物燃料电池研究产甲烷分离物S6是否具有电活性，进一步验证其存在的直接电子传递途径，是否具有应用于沉积物燃料电池的潜力；对 S6 进行厌氧滚管分离纯化，获得产甲烷菌及铁还原菌纯菌。主要研究结论如下： (1) 铁还原富集培养过程中Jh3具有较好的铁还原能力，比较Jh3原位样品及富集样品的微生物群落多样性，发现Jh3原位样品细菌群落中含有地杆菌、梭菌等铁还原菌，富集培养后异化铁还原菌地杆菌属 (Geobacter) 成为优势菌，拟杆菌的丰度显著增加。Jh3古菌群落中的优势菌主要为产甲烷菌，富集培养后以甲烷杆菌属/甲烷球形菌属为主。铁还原富集培养使样品富含铁还原菌及产甲烷菌，为研究产甲烷菌与铁还原菌间的直接电子传递提供了基础。 (2) 以乙醇为唯一电子供体从铁还原富集培养样品中分离获得了产甲烷分离物 (S6)。通过T-RFLP及克隆文库分析微生物群落多样性，发现梭菌 (Clostridium spp.，与C. tunisiense 同源性最高) 和甲烷八叠球菌 (Methanosarcina barkeri) 是S6 中的优势菌群。同时实验表明，S6 与 Geobacter metallireducens 共培养后铁还原和产甲烷能力未明显增加，因此推测 Clostridium spp. 可能与 G. metallireducens 类似，将电子直接传递给产甲烷菌 M. barkeri 产甲烷。此外，微生物燃料电池电化学分析发现，透析袋包裹电极后阻碍微生物与电极表面直接接触形成生物膜，其电流密度显著降低，并且循环伏安扫描无明显氧化还原峰，表明 S6 中存在直接电子传递途径，即产甲烷分离物中占优势的革兰氏阳性菌 Clostridium spp. 和 M. barkeri 之间可能存在种间直接电子传递。 (3) 进一步分离纯化产甲烷分离物，获得一株能够同时利用乙酸、甲醇、三甲胺底物的甲烷八叠球菌 (M. barkeri) ，命名为 Jh-Ar，其电镜形态为八叠球状的团聚状态，具有较高的产甲烷能力。同时分离到一株能够利用乙酸、乙醇、甲醇和三甲胺生长的梭菌目韦荣球菌科铁还原菌，命名为F9，该菌电镜下形态为长杆状，与 Selenomonadaceae str. SB90 同源性最高，序列相似度达到98.82%。F9不仅能够还原柠檬酸铁，还具有产电活性。 Clostridium不仅能够进行胞外电子传递，还可能与 M. barkeri 通过种间直接电子传递互营产甲烷。这一发现将以前限定在革兰氏阴性菌介导直接电子传递的认识推广到革兰氏阳性菌。
其他摘要	Coastal riverine is an important composition of coastal zone area, connect the two carbon library of terrestrial ecosystem and ocean, which dual influenced by global climate change and human activities. This work is object to acquire iron(III) reducing bacteria with electroactivity, investigating the direct electron transfer with methanogens and lay a basis for in situ bioremediation by sediment microbial fuel cells. We selected JieHe riverine as the research object, and enrichment the Jh3 sediment sample with iron (III)-reducing medium. Apply Terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis based on 16S rRNA genes revealed the microbial community of in situ and enrichment sample of Jh3. We further employed roll-tube (Hungate) anaerobic method to get syntrophic methanogenic isolation (S6) with ethanol as the sole electron donor. Then analyze the microbial community and compare the capacity of iron(III) reduction and methane production between methanogenic isolation and co-cultured system with Geobacter metallireducens. For further study, we construct microbial fuel cells to investigate the electroactivity and verify whether existence of direct electron transfer or not in S6. At last, we further separate and purified the methanogen and iron(III) reducing bateria from S6. The main conclusions are as follows: (1) Site Jh3 has the capability of iron (III) reduction which contain iron (III) reducing bacterias such as Geobacter and Clostridium based on T-RFLP and clone libraries. After enrichment of iron reduction medium, Geobacter became the predominant bacteria of Jh3 and the relative abundance of Bacteroides was increased. Methanogens were dominant in archaea community of Jh3 and Methanobacterium/Methanosphaera became predominant archaea in enrichment samples. (2) Acquire methanogenic isolation (S6) from Jh3 iron-reducing enrichment with ethanol as the sole electron donor. Clone library analysis of 16S rRNA gene showed that Clostridium spp. (close to C. tunisiense) and Methanosarcina barkeri was predominant in the bacterial and archaeal community, respectively. Interestingly, addition of G. metallireducens into S6 did not increase the ability of both iron (III) reduction and methanogenesis, indicating that Clostridium spp. may play a similar role in direct interspecies electron transfer from G. metallireducens to M. barkeri. Furthermore, current generation of the S6 suspension dramatically decreased when contact between the organisms and the electrodes was prevented by dialysis bag, and CV showed no obvious redox peaks. These results suggested that there was direct electron transfer in the methanogenic isolation, and the dominant Gram-positive Clostridium spp. can potentially directly transfer electron to M. barkeri in a methanogenic isolation. (3) Further separate and purify the methanogenic isolation. Gain a strain of methanosarcina barkeri which can utilize acetate, methanol, trimethylamine to produce amounts of methane, named Jh-Ar. This strain presents a conglobate and general form aggregate under observation by SEM. Separate another iron reduction bacteria named F9 which can utilize acetate, ethanol, methanol and trimethylamine, belonging to Clostridiales Veillonellaceae (maximum homology of 98.82% with Selenomonadaceae str. SB90). This strain presents a long rod-shaped under observation by SEM and can not only reduces the ferric citrate but also shows electrical activity. Clostridium has the potential capabilities of extracellular electron transfer and syntrophic methane production with methanogens by interspecies direct electron transfer. This discovery expands the gram-negative microorganism to gram-positive microorganism which can participate in the interspecies direct electron transfer.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/13842
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	中国科学院烟台海岸带研究所
第一作者单位	中国科学院烟台海岸带研究所
推荐引用方式 GB/T 7714	李莹. 滨海河流沉积物中产甲烷分离物的获得及其电化学特征[D]. 北京. 中国科学院大学,2016.

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