中国科学院海洋研究所机构知识库

深海冷泉是由深部甲烷或其他还原性流体向海底渗漏或喷发而形成的流体系统，塑造了以化能合成细菌为主要初级生产力的特殊生态系统，是研究甲烷转换、深部地化循环的天然实验室。冷泉的地化环境在垂直方向和水平方向上均存在明显的梯度变化，而水岩界面是垂直方向上环境变化最为剧烈的区域。冷泉环境的异质性分布塑造了迥异的微生物群落，代谢功能也具有多样性，认知南海冷泉不同区域和不同水层微生物组合和功能对于理解冷泉喷发对区域海洋生态系统的影响至关重要。因此，本研究聚焦中国南海冷泉，通过沉积物原位保真取样系统、大体积原位水体过滤系统获取了南海冷泉区沉积物、水-岩/生物群落/沉积物界面上覆水体，以及冷泉渗漏/喷口上方不同水层水体样品，采用扩增子技术、宏基因组技术开展了冷泉区不同微环境下微生物的多样性与潜在代谢过程研究。

通过宏基因组分析技术对南海北部F冷泉区还原性沉积物中的微生物群落和代谢过程进行了分析。结果表明，在表层沉积物中，弯曲杆菌（Campylobacterota）、变形杆菌（Proteobacteria）、脱硫杆菌（Desulfobacterota）、拟杆菌（Bacteroidota）和甲烷八叠球菌（ANME-2/3）的相对丰度较高；随着深度增加，脱硫杆菌和ANME-1a/b占据主导。浅层沉积物中微生物主要通过CBB途径固碳，深层沉积物中主要为rTCA循环和WL途径；介导了硫化物氧化、硫代硫酸盐氧化及硝酸盐还原过程的基因集中在8cm以浅的表层沉积物中，固氮相关基因在50cm以深的沉积物相对丰度较高，可为营养贫乏的深海沉积物提供大量氮资源；甲烷厌氧氧化、硫酸盐还原基因的分布特征提示该冷泉区的硫酸盐-甲烷转换带（SMTZ）处于较浅层沉积物中。对可能参与硫代谢过程的微生物宏基因组组装基因组（MAGs）分析表明，冷泉表层沉积物中丰富的硫氧化菌参与了对硫化物及硫代硫酸盐的氧化过程，生成的硫酸盐可支持浅层SMTZ中的甲烷厌氧氧化过程，限制释放到底层水中的甲烷量，对于控制甲烷等全球变暖气体进入海洋至关重要。

界面层是连接上层水柱和底层沉积物的过渡区域。由于特殊的地质条件，冷泉区界面层表现为高度动态和非均匀的微环境，在这里，不同微区的微生物发生着不同的生物地球化学反应，这些过程也受到上覆水、喷发流体以及沉积物等多种环境的相互作用。而介导这些过程的微生物类群在沉积物和上层水柱之间的界面层的物质交换和能量流动中起着至关重要的作用。通过扩增子技术，以16S rRNA为目的基因，对南海陵水冷泉和F冷泉的底边界层上覆水中微生物进行初步的多样性分析，以及潜在功能预测，结果发现，处于早期发育阶段的陵水冷泉由于甲烷流体的大规模和持续性的喷发，界面区微生物多样性较F冷泉低，与甲烷消耗相关的微生物呈富集状态，甲烷的消耗主要是由于甲烷的好氧氧化和反硝化型甲烷厌氧氧化；在F冷泉的氧—缺氧波动的界面区，微生物群落多样性较高，相对缓慢渗漏的甲烷流体被甲烷氧化细菌消耗，碳、硫、氮化合物也在此发生强烈的氧化还原耦合反应。

冷泉羽流形成的上升流塑造了当地微生物群落组成，极大地提升了甲烷氧化菌的丰度，同时，水体环境也是一道天然的甲烷过滤屏障。本研究通过宏基因组技术，对F冷泉全水深水体微生物进行了相关研究，结果表明，F冷泉（全水深约1150米）喷口上方微生物群落具有明显的垂直差异。底层界面和表层界面中自养微生物的比例远高于中部水体，同时微生物间也存在更多的互作。特别是底层界面水体微生物合作更为密切，例如，界面层微生物可以通过合作进行羟胺和硫化氢的氧化。此外，界面层Pseudomonadales和Burkholderiales多个MAGs具有降解长链烷烃和芳香烃的通路和基因，因此可能是未来石油降解的重要目标类群。另外，我们发现了明显的硝酸盐还原偶联甲烷和硫化物氧化的证据，增强了深海微生物在有氧无氧周期变化环境的生存能力。甲烷氧化菌丰度随着距底距离快速递减，氨氧化古菌和异养菌如酸杆菌等成为丰度最高的类群，在600m深度甲烷氧化菌仍然普遍存在，但不同水深的甲烷氧化菌属于不同的分类单元，可能对甲烷浓度表现为不同的偏好。海面表层（5米）和亚表层（100-200米）水体中甲烷代谢相关的微生物群落丰度非常低，由此认为，表层与亚表层几乎不受冷泉喷发的影响。我们的研究结果展示了冷泉上方水体全水深中不同水层微生物组成和功能的异质性，特别是甲烷喷发导致的冷泉区全水深微生物介导的地球化学反应过程存在的差异。

本研究通过对南海两个冷泉区不同的微环境下微生物群落组成和代谢过程研究，揭示了冷泉环境中甲烷氧化耦联氮代谢、硫代谢的重要性以及微生物与其环境之间的相互依存关系，为解析冷泉独特生态系统的生物地球化学过程提供了新的视角。研究成果也将大大拓宽对冷泉微生物群落结构和生态功能的认知，有助于全面阐释冷泉微生物在地球重要元素的生物地球化学循环中的驱动机制。

The deep-sea cold seep is a fluid system formed by deep methane or other reducing compounds leaking or erupting into the sea floor, which has shaped a special ecosystem with chemosynthetic bacteria as the main primary productivity. This system is a natural laboratory for the study of methane conversion and deep geochemical cycle. The geochemical environment of cold seep has obvious gradient changes in both vertical and horizontal directions, and the water-rock interface is the area with the most intense environmental changes in the vertical direction. The heterogeneous distribution of cold seep environment has shaped diverse microbial communities and metabolic functions. Exploring the microbial assemblages and functions in different regions and water layers of cold seeps in the South China Sea is crucial for understanding the impact of cold spring eruption on regional marine ecosystems. Therefore, this study focused on the cold spring in the South China Sea. And samples of sediments, overlying water at the water-rock/biome/sediment interface, and water in different layers above the leakage/vent of the cold seeps in the South China Sea were obtained through in-situ fidelity sampling system of sediments and large-scale in-situ water filtration system. Amplicon and metagenomic techniques were used to study the diversity and potential metabolic processes of microorganisms in different microenvironments in cold seep area.

The microbial communities and metabolic processes in the reduced sediments of F cold seep in the northern South China Sea were analyzed by metagenomic analysis. It turned out that in the surface sediments, the relative abundances of Campylobacterota, Proteobacteria, Desulfobacterota, Bacteroidota and ANME-2/3 were high. As the depth increased, Desulfobacterota and ANME-1a/b gradually dominated. The microorganisms in shallow sediments mainly fixed carbon through CBB cycle, while in deep sediments they mainly adopted rTCA and WL cycles. The genes mediating sulfide oxidation, thiosulfate oxidation and nitrate reduction were concentrated in shallow sediments above 8cm below interface, and the nitrogen fixation genes were relatively abundant in deep sediments below 50cm, which can provide a large amount of nitrogen resources for nutrient-poor deep-sea sediments. The distribution of methane anaerobic oxidation (AOM) and sulfate reduction genes suggested that the sulfate-methane conversion zone (SMTZ) in the cold seep area was located in shallow sediments. The analysis of microbial metagenomic assembly genomes (MAGs) that may be involved in sulfur metabolism showed that the abundant sulfur-oxidizing bacteria in the surface sediments participated in the oxidation of sulfur and thiosulfate, and the generated sulfates could support the anaerobic oxidation of methane in the shallow SMTZ, which could limit the amount of methane released into the bottom water. Therefore, the methane oxidation coupled by sulfate reduction was crucial to controlling the flow of global warming gases like methane into the ocean.

The interface layer is a transition area that connects the upper water column with the underlying sediment. Due to the special geological conditions, the interface layer of the cold seep region showed a highly dynamic and non-uniform microenvironments, where microorganism in different microzones contained different biogeochemical reactions. These processes were also subject to the interaction of overlying water, eruptive fluids, sediments and other environments. The microbiome that mediates these processes played a crucial role in the exchange of matter and the flow of energy at the interface layer between the sediment and water column. By amplification technique with 16S rRNA as the target gene, the diversity of microorganisms in the bottom boundary layer of Lingshui and F cold seeps in the South China Sea was preliminarily analyzed and their potential functions were predicted. The results showed that the microbial diversity in the interface region of Lingshui cold seep in the early development stage was lower than that of F cold seep due to the large-scale and continuous eruption of methane fluid. The microorganisms related to methane consumption are enriched, and the methane consumption is mainly due to the aerobic oxidation and denitrifying anaerobic oxidation of methane. In the oxygen-anoxic fluctuation interface area of F cold seep, the diversity of microbial community was high, and the methane fluid that leaked slowly is consumed by methane-oxidizing bacteria. Significantly coupling redox reactions among the carbon, sulfur and nitrogen compounds also occurred here.

The upwelling from the cold seep plumes shaped the composition of the local microbial community, greatly increasing the abundance of methane oxidizing bacteria, and the water environment acted as a natural methane filtration barrier. In this study, metagenomic technology was used to study the microorganisms in the whole water depth of F cold seep. The results showed that the microbial communities of this site (the whole water depth is about 1150 meters) had obvious vertical differences. The proportion of autotrophic microorganisms in the bottom interface and surface water was much higher than that in the middle water body, and there were more interactions among microorganisms there, which was more closely in the bottom interface in particular. For example, microorganisms in the interface layer could oxidize hydroxylamine and hydrogen sulfide through interspecific cooperation. In addition, multiple MAGs belonging to Pseudomonadales and Burkholderiales at the interface layer contained pathways and genes for the degradation of long-chain alkanes and aromatics, so they might be important target groups for future petroleum degradation study. We also found clear evidence of nitric acid reduction coupled to methane and sulfide oxidation, which enhanced the survival ability of deep-sea microorganisms in an environment with varying aerobic and anaerobic cycles. The abundance of methane-oxidizing bacteria decreased rapidly with distancing from the bottom, and the ammonia-oxidizing archaea and heterotrophic bacteria such as Acidobacterium became the highest abundance group in the middle depth water. Methane-oxidizing bacteria were still common at the depth of 600m, which belonged to different taxa in different water depths and may show different preferences for methane concentration. The abundance of methane metabolism-related microbial communities in surface (5 m) and subsurface (100-200 m) waters was very low, suggesting that the surface and subsurface waters are almost unaffected by cold seep eruptions. Our results demonstrated the heterogeneity of the composition and function of microorganisms in different water layers in the whole water depth above the cold seep, especially the differences in the microbial geochemical reaction processes of the whole water depth affected by methane eruption.

By studying the microbial community composition and metabolic processes under different microenvironments in two cold seep regions of the South China Sea, this study revealed the importance of methane oxidation coupled by nitrogen and sulfur metabolism in the cold seep environment, as well as the interdependence between microorganisms and their environment, providing a new perspective for the analysis of biogeochemical processes in this special ecosystem. The research results will also greatly broaden the understanding of the community structure and ecological function of cold seep microorganisms, and help to fully explain the driving mechanism of cold seep microorganisms in the biogeochemical cycle of important elements on Earth.

第1章 绪论 1

1.1 深海冷泉生态系统简介 1

1.2 我国的冷泉系统 2

1.3 冷泉独特的环境特征 3

1.4 冷泉微生物多样性和功能 5

1.5 冷泉微生物参与广泛的地化过程 7

1.5.1 微生物介导的深海碳循环 8

1.5.2 微生物介导的海底氮循环 12

1.5.3 微生物驱动的海底硫循环 14

1.6 无法培养微生物常用的研究方法 20

1.7 本研究的站位简介 21

1.8 本研究的目的和意义 22

第2章 冷泉沉积物中的微生物多样性及其硫循环中的贡献 24

2.1 研究背景 24

2.2 材料与方法 25

2.2.1 样品采集 25

2.2.2 DNA提取 27

2.2.3 宏基因组测序 27

2.2.4 基因组分箱 27

2.2.5 系统进化分析 28

2.3 结果与讨论 28

2.3.1 冷泉还原性沉积物微生物组成 28

2.3.2 冷泉沉积物微生物介导的元素循环基本过程 31

2.3.3 冷泉硫代谢相关微生物筛选及代谢潜力分析 33

2.4 结论 37

第3章 冷泉水岩界面微生物多样性及地化循环贡献 39

3.1 研究背景 39

3.2 材料与方法 40

3.2.1 样品采集和地球化学测量 40

3.2.2 DNA提取及文库制备 44

3.2.3 生物信息学分析 44

3.3 结果 44

3.3.1 地球化学测量结果 44

3.3.2 微生物群落组成 46

3.3.3   两个冷泉区微生物群落功能预测 50

3.4 讨论 53

3.4.1 复杂多变的界面带微环境导致微生物空间异质性 53

3.4.2 界面微环境中DAMO/SAMO发挥重要作用 55

3.5 结论 58

第4章 冷泉羽流区微生物多样性特征、生态功能及甲烷过滤作用 60

4.1 材料与方法 61

4.1.1 样品采集和环境参数获取 61

4.1.2 DNA提取和测序 62

4.1.3 数据挖掘和生信分析 62

4.2 结果与讨论 64

4.2.1 水体的关键理化参数特征 64

4.2.2 不同深度微生物群落的层化特征 65

4.2.3 不同水层微生物的主要功能 71

4.2.4 MAG构建和代谢模型分析 80

4.2.5 F冷泉区域不同水层关键代谢过程 85

4.2.6 冷泉不同水层微生物竞争合作潜力差异 86

4.2.7 底层水岩界面CN过程的耦合 88

4.2.8 甲烷有氧氧化——冷泉的甲烷过滤器 89

4.2.9 Pseudomonadales、Rhodobacterales、Burkholderiales，BWI水层的全能异养菌 90

4.3 结论 91

第5章  总结与展望 92

参考文献 95

附录 116

致谢 155