Pyro-SIP with seep sediments
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.