DIVERSE MICROORGANISMS MEDIATING THE ANAEROBIC OXIDATION OF METHANE IN METHANE SEEPS REVEALED BY DIRECTLY COUPLED ISOTOPIC AND PHYLOGENIC ANALYSES

Microbially mediated anaerobic oxidation of methane (AOM) is a significant biogeochemical process, serving as the primary methane sink in the world’s oceans. The recognition of uncultured Archaea (ANME-1 and ANME-2), phylogenetically related to methanogens, as mediators of this globally important process has recently been established by coupled isotopic and molecular surveys of lipid biomarkers, 16S rRNA genes, and fluorescent in situ hybridization. In this study, we examined the diversity of Archaea and sulfate-reducing bacteria involved in AOM within methane seep sediments of the Eel River basin using directly coupled isotopic and phylogenetic analyses at the level of individual microorganisms. The combined application of secondary ion mass spectrometry and fluorescent in situ hybridization (FISH-SIMS) revealed extreme carbon-13 depletion (down to –96‰) in the biomass of the ANME-1 and ANME-2 cells, providing direct evidence of methane assimilation by both archaeal groups within anoxic seep sediments. Members of the ANME-2 and/or ANME-1 were abundant in methane-laden sediments underlying dense chemosynthetic clam beds, sulfide-oxidizing microbial mats, and bubbling vents. Both groups formed physical associations with Bacteria including, but not limited to, the sulfate-reducing Desulfosarcina/ Desulfococcus. In contrast to the tight syntrophic association observed between the ANME-2 and the Desulfosarcina group, the carbon-13 depleted ANME-1 archaea were frequently found without a bacterial partner, suggesting that this group may be capable of oxidizing methane autonomously by a currently unrecognized mechanism. Additionally, stable carbon isotope values for select microorganisms, not thought to be involved in AOM (e.g. Beggiatoa-like filamentous bacteria), also contained traces of methane carbon in their biomass. This indicates the potential for ecosystem-wide incorporation of methane-derived substrates by the greater microbial methane seep community. The results from these coupled fine-scale isotopic and phylogenetic analyses illustrate the complexity of the microbial communities and interactions within the seep environment and suggest the existence of multiple microbial groups and mechanisms involved in oxidizing methane anaerobically.