Diagnostic lipid biomarker and stable carbon isotope signatures of microbial communities mediating the anaerobic oxidation of methane with sulphate

Microorganisms living in anoxic marine sediments consume more than 80% of the methane produced in the world's oceans. In addition to single-species aggregates, consortia of metabolically interdependent bacteria and archaea are found in methane-rich sediments. A combination of fluorescence in situ hybridization and secondary ion mass spectrometry shows that cells belonging to one specific archaeal group associated with the Methanosarcinales were all highly depleted in 13C (to values of -96 per thousand). This depletion indicates assimilation of isotopically light methane into specific archaeal cells. Additional microbial species apparently use other carbon sources, as indicated by significantly higher 13C/12C ratios in their cell carbon. Our results demonstrate the feasibility of simultaneous determination of the identity and the metabolic activity of naturally occurring microorganisms.

Microbial communities in hydrothermally active sediments of the Guaymas Basin (Gulf of California, Mexico) were studied by using 16S rRNA sequencing and carbon isotopic analysis of archaeal and bacterial lipids. The Guaymas sediments harbored uncultured euryarchaeota of two distinct phylogenetic lineages within the anaerobic methane oxidation 1 (ANME-1) group, ANME-1a and ANME-1b, and of the ANME-2c lineage within the Methanosarcinales, both previously assigned to the methanotrophic archaea. The archaeal lipids in the Guaymas Basin sediments included archaeol, diagnostic for nonthermophilic euryarchaeota, and sn-2-hydroxyarchaeol, with the latter compound being particularly abundant in cultured members of the Methanosarcinales. The concentrations of these compounds were among the highest observed so far in studies of methane seep environments. The delta-(13)C values of these lipids (delta-(13)C = -89 to -58 per thousand) indicate an origin from anaerobic methanotrophic archaea. This molecular-isotopic signature was found not only in samples that yielded predominantly ANME-2 clones but also in samples that yielded exclusively ANME-1 clones. ANME-1 archaea therefore remain strong candidates for mediation of the anaerobic oxidation of methane. Based on 16S rRNA data, the Guaymas sediments harbor phylogenetically diverse bacterial populations, which show considerable overlap with bacterial populations of geothermal habitats and natural or anthropogenic hydrocarbon-rich sites. Consistent with earlier observations, our combined evidence from bacterial phylogeny and molecular-isotopic data indicates an important role of some novel deeply branching bacteria in anaerobic methanotrophy. Anaerobic methane oxidation likely represents a significant and widely occurring process in the trophic ecology of methane-rich hydrothermal vents. This study stresses a high diversity among communities capable of anaerobic oxidation of methane.