Abstract Anaerobic oxidation of methane (AOM) and the environmental conditions supporting AOM on continental margins is an essential component to global methane budgets. Diagnostic lipid biomarkers and their compound specific isotope analysis preserved in authigenic carbonates at cold seeps can serve as “fingerprints” to archaeal−bacterial consortia involved in AOM. However, despite the discovery of several hundreds of seeps along the United States Atlantic Margin (USAM), there are relatively few biomarker investigations of cold seep carbonates along this passive margin. A lipid biomarker, carbon isotope, and DNA marker gene study was therefore undertaken to determine the microbial origins of authigenic carbonates from two USAM seeps, Norfolk and the Baltimore Canyon seep fields. Results from this study capture a distinct archaeal lipid signature from putative methanotrophic archaea, including archaeol (I), sn-2-hydroxyarchaeol, 2,6,10,15,19-pentamethylicosane (PMI), and crocetane. The 13C-depleted AOM-related archaeal lipid samples (i.e., archaeol: −91.6‰, sn-2-hydroxyarchaeol: −129.2‰, PMI −92.8‰, and crocetane: −70.9‰) confirm the dominance of methane assimilation and isotope fractionation during AOM. These results are consistent with the detection of archaeal anaerobic methanotrophs (ANMEs) based on 16S rRNA gene sequencing. The Norfolk authigenic carbonate contained ANME-1a, -1b, 2a-2b, and 2c whereas only the ANME-2 clade was detected at Baltimore and present as the subclusters 2a-2b and -2b. The ANME-2d clade may also be present, particularly at the Baltimore seep site, given the high abundance of Candidatus Methanoperedens nitroreducens detected in the mcrA gene sequencing. The presence of terminally branched fatty acids, antesio- and iso-C15:0 components, as well as C16:1ω7 with δ13C values as low as −107.6‰, are indicative of sulfate-reducing bacteria (SRB) at the Norfolk seep site and supports syntrophy of SRB with methane-oxidizing archaea. In contrast, nitrate-driven AOM in syntrophy M. nitroreducens at the Baltimore seep site is consistent with elevated fatty acid δ13C values and lack of Deltaproteobacteria at the Baltimore seep site. Taken together, the range in lipid composition, distribution, and carbon isotopic composition observed at the Norfolk and Baltimore seep sites suggests AOM is performed by multiple archaea instead of a single species and may be paired with either or both nitrate- and sulfate-reduction. Given the heterogeneous nature of cold seep ecosystems, this study fills a critical spatial gap in our knowledge of AOM activity at two seep sites along a passive margin.

中文翻译：

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美国大西洋边缘冷渗流处甲烷代谢的分子指标

摘要 甲烷厌氧氧化 (AOM) 和大陆边缘支持 AOM 的环境条件是全球甲烷收支的重要组成部分。在冷渗漏处保存在自生碳酸盐中的诊断性脂质生物标志物及其化合物特异性同位素分析可以作为参与 AOM 的古细菌菌群的“指纹”。然而，尽管沿美国大西洋边缘 (USAM) 发现了数百处渗流，但对沿该被动边缘的冷渗碳酸盐岩的生物标志物研究相对较少。因此，进行了脂质生物标志物、碳同位素和 DNA 标记基因研究，以确定来自两个 USAM 渗流、诺福克和巴尔的摩峡谷渗流场的自生碳酸盐的微生物来源。这项研究的结果从假定的甲烷营养古细菌中捕获了独特的古细菌脂质特征，包括古细菌 (I)、sn-2-羟基古细菌、2,6,10,15,19-五甲基二十烷 (PMI) 和克罗辛烷。耗尽 13C 的 AOM 相关古菌脂质样品（即，古菌醇：-91.6‰、sn-2-羟基古菌醇：-129.2‰、PMI -92.8‰和crocetane：-70.9‰）证实了甲烷同化和同位素分馏的主导地位在 AOM 期间。这些结果与基于 16S rRNA 基因测序的古细菌厌氧甲烷氧化菌 (ANMEs) 的检测一致。诺福克自生碳酸盐包含 ANME-1a、-1b、2a-2b 和 2c，而在巴尔的摩仅检测到 ANME-2 分支，并作为子簇 2a-2b 和 -2b 存在。ANME-2d 进化枝也可能存在，特别是在巴尔的摩渗漏点，考虑到在 mcrA 基因测序中检测到的高丰度的 Methanoperedens nitroreducens。存在末端支化脂肪酸、反 C15:0 和异 C15:0 成分，以及 C16:1ω7 的 δ13C 值低至 -107.6‰，表明诺福克渗漏点存在硫酸盐还原菌 (SRB) 和支持 SRB 与甲烷氧化古菌的共养。相比之下，巴尔的摩渗漏部位的同养型硝基还原菌中硝酸盐驱动的 AOM 与巴尔的摩渗漏部位脂肪酸 δ13C 值升高和缺乏 Deltaproteobacteria 一致。总之，脂质组成、分布的范围，在诺福克和巴尔的摩渗漏点观察到的碳同位素组成表明，AOM 是由多个古菌而不是单个物种进行的，并且可能与硝酸盐和硫酸盐还原中的一个或两个配对。鉴于冷渗流生态系统的异质性，本研究填补了我们对沿被动边缘的两个渗流点的 AOM 活动知识的关键空间空白。