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Oceanic methane from global deep-sea sediment is largely consumed through microbially mediated sulfate-coupled oxidation, resulting in  13 C-depleted cell biomass of anaerobic methanotrophic archaea (ANME). The general ecological importance of subseafloor ANME has been well recognized in the last two decades. However, the crucial biochemical pathways for the overall anaerobic oxidation of methane (AOM) still remain enigmatic. Here, methanotrophic pathways were analyzed to trace  13 C-depleted amino acid biosynthesis in two clades of ANME (ANME-1 and ANME-2) from the Black Sea. Compound-specific analysis of ANME-dominated microbial mats showed a significant  13 C-depletion trend in association with increasing carbon numbers in protein-derived amino acid families ( e.g. , the pyruvate family in the order of alanine, valine, isoleucine and leucine was down to −114‰). This result indicates a stepwise elongation of  13 C-depleted carbon during amino acid biosynthesis. The overall results suggest that intracellular protein amino acids and the most  13 C-depleted signature of leucine, which has a specific branched-chain structure, are potentially propagated as isoprenoid precursor molecules into archaeal biosynthesis, resulting in the extremely  13 C- and  14 C-depleted nature of ANME cells in the deep microbial oasis.

Insight into anaerobic methanotrophy from 13C/12C- amino acids and 14C/12C-ANME cells in seafloor microbial ecology