海岸带生物学与生物资源保护实验室知识库

甲烷是一种重要的温室气体，可以由产甲烷菌产生，滨海河流沉积物是其重要的潜在来源。传统观念认为，铁氧化物能够与产甲烷过程竞争电子，从而抑制甲烷产生。本研究团队前期的工作显示，在异化铁还原菌存在并适合胞外电子传递发生的条件下，可以种间通过直接电子传递的方式促进甲烷产生。因此，揭示该区域内铁还原菌多样性及其电活性、产甲烷菌多样性及其产甲烷类型，并尝试解析铁氧化物转化过程对甲烷产生过程的影响，对于评估河海交互区域温室气体排放潜力，阐明生物地球化学碳、铁耦合循环具有至关重要的科学意义。本研究以受人为活动影响明显的渤海及其入海河流沉积物为研究对象，分析了滨海河流不同区域的沉积物中铁还原菌的多样性，并分离得到一株新的具有电活性的铁还原菌；揭示了铁氧化物在厌氧产甲烷条件下的转化规律；解析了渤海不同断面沉积物中产甲烷菌的空间分布规律，以及由河到海空间与盐度梯度条件下产甲烷菌多样性与产甲烷类型的变化规律。在研究过程中获得的主要结论如下：

1、通过对滨海河流沉积物中电活性铁还原微生物的研究，结果发现界河沉积物Jh2、Jh3样品具有明显的铁还原活性；克隆文库表明两个位点具有特征性的铁还原细菌群落结构，Jh2样品中铁还原菌主要以Geobacter sp.为主；而Jh3样品中铁还原菌主要以Clostridium sp.为主；并在滨海河流沉积物中，分离得到了一株具有电活性的铁还原菌（JhA）。以上结果拓展了对于Anaerosinus sp.这一新菌种电活性的认识；同时，环境中新分离得到具有电活性的铁还原微生物，将为重金属环境修复提供菌株来源，具有潜在的应用价值。

2、通过对磁铁矿在滨海河流沉积物产甲烷体系中产生与转化的研究，发现了该体系内含有铁还原微生物与产甲烷菌。其中，铁还原微生物在无定形铁存在情况下，与产甲烷菌竞争电子，从而抑制甲烷产生；但随着无定形铁转化为磁铁矿，产甲烷体系中具备了磁铁矿介导的胞外电子传递条件，从而促进了甲烷产生；而磁铁矿本身也可以作为电子受体，被铁还原菌还原竞争电子，抑制了前期甲烷产生。研究结果揭示了产甲烷菌群中磁铁矿的产生和转化对其中铁还原菌和产甲烷古菌间电子传递过程的影响。

3、通过对渤海不同海域沉积物中微生物群落多样性与产甲烷潜力的研究表明，海底沉积物中微生物T-RFs片段相近，但其群落在空间分布上存在差异；沉积物中可提取态总铁对细菌群落结构具有显著影响，但未有显著影响古菌群落的环境因子；样品中脱硫弧菌（Desulfovibrio）和硫杆菌属（Thiobacillus）是主要优势细菌，甲烷杆菌属（Methanobacterium）和甲烷八叠球菌属（Methanosarcina）是主要优势古菌；富集过程中不同区域沉积物样品产甲烷量不同，表现为近岸样品产甲烷量较高。由以上结果推测，渤海沉积物中细菌尤其是与铁循环相关细菌可能是影响产甲烷潜力的重要因素；同时，人为活动，如水产养殖，污水排放等造成的有机物输入，可能增加了近海区域甲烷排放潜力。

4、通过对渤海及其入海河流沉积物中产甲烷菌多样性的研究，结果发现由河到海过程中产甲烷菌多样性降低，且营养类型由复杂到单一；河海环境中甲基营养型产甲烷菌存在差异性，渤海中以甲烷类球菌属（Methanococcoides）为主，而黄河以甲烷八叠球菌属（Methanosarcina）为主；在海水富集培养过程中海洋样品仍以Methanosarcina为主，而河流样品却以未在原位样品中检测到的甲烷叶菌属（Methanolobus）为主，该结果说明，渤海沉积物中产甲烷菌并非主要来源于黄河。同时，在河海环境中均分离得到梅氏甲烷八叠球菌（Methanosarcina mazei），说明该菌种具有良好的环境适应性，而且可能是滨海环境中重要的产甲烷菌与碳循环参与者。

Methane is an important greenhouse gas, which is produced by methanogens, and the coastal area sediments are important potential sources. In conventional opinion, the iron oxides can compete with methanogens for electrons and thereby inhibiting methane production. Our previous research has showed that it would be different for iron-reducing bacteria with the ability of extracellular electron transfer. Therefore, how to reveal the diversity of iron-reducing bacteria with electronchemical activity and methanogens with different metabolic pathways in this region, as well the effect of iron oxide transformation processes on the methanogenesis, which are of crucial scientific significance for assessing the greenhouse gas emissions in the river-sea interaction region and elucidating the biogeochemical carbon and iron coupling cycle. In this study, the sediments from the Bohai Sea and its inlets to the sea were studied. Firstly, the diversity of iron-reducing bacteria in different regions of the coastal river was analyzed and a new iron-reducing bacterium with electroactivity was isolated. Secondly, the iron oxides transformation under anaerobic methanogenesis condition was revealed. Lastly, the spatial distribution of methanogens in sediments of different sections in Bohai Sea, with the diversity and production of methanogens under different gradients, including spatial and salinity, across river to sea were analyzed. The main conclusions obtained are as follows:

With the study of electrochemically active iron (III)-reducing bacteria in coastal riverine sediments, the results showed that the Jh2 and Jh3 samples of the JieHe river sediments had iron reducibility. And the clone library showed that the two sediments had the characteristics of the iron reducing bacteria community structure. The iron reducing bacteria in the Jh2 samples were mainly dominated by Geobacter sp., while the Jh3 samples was Clostridium sp. And the electroactivity bacteria (JhA) was isolated from coastal river sediments. These results expand the understanding of the electrical activity of the new strain of Anaerosinus sp. At the same time, the electroactive isolates from the environment will provide the source of strain for the bioremediation of heavy metal environment with important application value.

With the study of the production and transformation of magnetite in the methanogenic consortia from coastal riverine sediments, it has been found that iron-reducing bacterial and methanogens were contained in it. In the presence of amorphous iron(III) oxides, the iron-reducing bacteria would reduce the ferric iron that compete the electrons with methanogens, thereby inhibiting the methan production. But with the conversion of amorphous iron to magnetite, the extracellular electron transfer occurred mediated by magnetite, so the methane production was promoted. Beyond our consideration, the magnetite itself could also act as an electron acceptor. As a result, the magnetite was reduced by iron-reducing bacteria with competing electrons and inhibiting methane production. These results revealed that the effect of magnetite production and conversion in methanogens consortia on the electron flow between iron-reducing bacteriaand methanogens.

Studies on microbial community diversity and methanogenic potential in sediments from different areas of the Bohai Sea showed that there were similar main microbial T-RFs in the in situ samples with spatially different. Total iron extracted from sediments has significant effects on bacterial community structures, but not for archaeal community. Desulfovibrio and Thiobacillus were the dominanted bacteria in the sample, and Methanobacterium and Methanosarcina were the main dominanted archaea. The methane production of regional sediment samples varies in the enrichment process, showing a higher methane yield in near-shore samples. It is speculated that bacteria in the sediments of the Bohai Sea, especially those related to the iron cycle, might be an important factor affecting the potential for methanogenesis. It was deserved to mention that human activities might increase the potential for methane emission in the offshore regions.

In order to explore more detail information about the changes of methanogens across Yellow River to Bohai Sea gradient, it has been found that the diversity of methanogens in the sediments varied from multi-trophic communities to specific methylotrophic communities. Methylotrophic methanogens were in both in situ sediments. Methanococcoides was the main methanogens in the Bohai Sea, while that of the Yellow River samples were dominated by Methanosarcina. After seawater enrichment culture, it was revealed that active Methanococcoides dominated in the BS enrichment cultures with trimethylamine as the substrate, methylotrophic Methanolobus dominated in the YR enrichment cultures, which was limitedly detected in in situ sediment samples. Methanosarcina were also detected in this gradient samples as well. These results indicated that the methanogens in Bohai Sea were not mainly from the Yellow River. The same species of Methanosarcina mazei were isolated from the area across a river to sea by the culture-dependent method, which were wide distributed. Our results showed the distribution of diverse methanogens across a river to sea gradient may shed light on adaption strategies and survival mechanisms of methanogens.