Specialists instead of generalists oxidize alkanes in anoxic marine hydrocarbon seep sediments

The anaerobic oxidation of non-methane hydrocarbons mediated by sulfate-reducing bacteria (SRB) is a major process of organic matter degradation at marine hydrocarbon seeps. Several SRB have been successfully cultured, however, knowledge about in situ active organisms is still very limited. Here, we identified alkane-degrading key players from two contrasting seeps at the Mediterranean Amon Mud Volcano (Amon MV) and Guaymas Basin in the Gulf of California using complementary stable-isotope probing (SIP) techniques. Anoxic sediments were incubated with 13C-labeled butane or dodecane under close to in situ conditions. DNA- and RNA-based SIP in combination with 454-pyrosequencing (PYRO-SIP) allowed the identification of four phylogenetically distinct deltaproteobacterial groups of alkane-oxidizing SRB within the family Desulfobacteraceae. We named the groups degrading short-chain alkanes ‘SCA-SRB1’ and ‘SCA-SRB2’ and those degrading long-chain alkanes ‘LCA-SRB1’ and ‘LCA-SRB2’. CARD-FISH with newly developed specific probes revealed a high relative in situ abundance of SCA-SRB1 and SCA-SRB2 with 2% of the total community, while groups LCA-SRB1 and LCA-SRB2 were below 1% of total cells. Protein-based SIP (Protein-SIP), which enables to trace stable isotopes from substrate to protein, confirmed alkane-degrading key players of the family Desulfobacteraceae. In addition, Protein-SIP indicated additional carbon sources for 13C biosynthesis besides alkanes, and gave insights into possible metabolic pathways: (1-methylalkyl)succinylation as initial step of butane degradation and the oxidative Wood–Ljungdahl pathway as terminal point of alkane degradation.

硫酸盐还原菌（sulfate-reducing bacteria, SRB）介导的非甲烷烃厌氧氧化，是海底烃类渗漏区有机质降解的核心过程。目前已有多种硫酸盐还原菌实现纯培养，但对原位活性菌群的认知仍十分有限。本研究借助互补性稳定同位素探针（stable-isotope probing, SIP）技术，对地中海阿蒙泥火山（Amon Mud Volcano, Amon MV）与加利福尼亚湾瓜伊马斯盆地两处特征迥异的渗漏区开展烷烃降解关键菌群的识别研究。实验将缺氧沉积物置于接近原位的环境中，以¹³C标记的丁烷或十二烷进行孵育。结合454焦磷酸测序的DNA与RNA层面稳定同位素探针技术（DNA/RNA-SIP联合PYRO-SIP），成功在脱硫杆菌科（Desulfobacteraceae）内鉴定出四株系统发育独立的δ-变形菌纲烷烃氧化型硫酸盐还原菌类群。我们将降解短链烷烃的类群命名为"SCA-SRB1"与"SCA-SRB2"，降解长链烷烃的类群命名为"LCA-SRB1"与"LCA-SRB2"。通过全新开发的特异性探针开展的催化报告沉积荧光原位杂交（Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization, CARD-FISH）结果显示，"SCA-SRB1"与"SCA-SRB2"的原位相对丰度可达总菌群的2%，而"LCA-SRB1"与"LCA-SRB2"的丰度则低于总细胞数的1%。基于蛋白质组的稳定同位素探针技术（Protein-SIP）可追踪底物到蛋白质的稳定同位素流转，进一步验证了脱硫杆菌科内的烷烃降解关键菌群。此外，Protein-SIP结果还表明，除烷烃外，微生物还可利用其他碳源进行¹³C生物合成，并揭示了潜在的代谢通路：丁烷降解的初始步骤为（1-甲基烷基）琥珀酰化，而烷烃降解的终末步骤则为氧化性Wood-Ljungdahl途径。