Assessing the Effect of Humic Substances and Fe(III) as Potential Electron Acceptors for Anaerobic Methane Oxidation in a Marine Anoxic System

Marine anaerobic methane oxidation (AOM) is generally assumed to be coupled to sulfate reduction, via a consortium of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). ANME-1 are, however, often found as single cells, or only loosely aggregated with SRB, suggesting they perform a form of AOM independent of sulfate reduction. Oxidized metals and humic substances have been suggested as potential electron acceptors for ANME, but up to now, AOM linked to reduction of these compounds has only been shown for the ANME-2 and ANME-3 clades. Here, the effect of the electron acceptors anthraquinone-disulfonate (AQDS), a humic acids analog, and Fe(3+) on anaerobic methane oxidation were assessed by incubation experiments with anoxic Black Sea water containing ANME-1b. Incubation experiments with (13)C-methane and AQDS showed a stimulating effect of AQDS on methane oxidation. Fe(3+) enhanced the ANME-1b abundance but did not substantially increase methane oxidation. Sodium molybdate, which was added as an inhibitor of sulfate reduction, surprisingly enhanced methane oxidation, possibly related to the dominant abundance of Sulfurospirillum in those incubations. The presented data suggest the potential involvement of ANME-1b in AQDS-enhanced anaerobic methane oxidation, possibly via electron shuttling to AQDS or via interaction with other members of the microbial community.