Anaerobic methane oxidation by ANME-2a at two molar chloride in Orca Basin

Abstract Anaerobic methane oxidation, typically mediated by consortia of archaea and bacteria, is a key process in the global methane cycle, but little is known about its upper salinity limits. We characterized the microbial methane cycle in the anoxic, hypersaline Orca Basin using metagenomics, metatranscriptomics, fluorescence in situ hybridization, and geochemical measurements at sub-meter resolution. In the brine, we detected transcriptional activity of the halophilic methylotrophic methanogen Methanohalophilus , consistent with a biological source for Orca Basin methane. In the particle-rich halocline (∼2 M Cl - ; ∼2235 meters depth), high mcrA transcription by a novel ANME-2a species was co-located with a positive shift in δ 13 C-CH 4 indicative of anaerobic oxidation of methane. ANME-2a also transcribed genes for biosynthesis of the osmolyte N(ε)-acetyl-β-L-lysine, supporting adaptation for hypersaline conditions. At the same depth, consortia of sarcina-like archaea, likely ANME-2a, were observed in association with vibrioid and filamentous bacteria, potentially members of a halotolerant genus in the order Desulfobulbales (family SURF-16, which includes the previously identified ANME partner Seep-DBB) that were active at the same depth. At and above the oxic-anoxic interface, aerobic methane oxidation appears to be mediated by three genera of uncultivated Methylococcales bacteria. Our results double the upper salinity range of ANME-2a to ∼2 M Cl - and reveal the key microbial players in the methane bio-filter between the Orca Basin brine and overlying seawater.