The key microbial group involved in anaerobic methane oxidation is anaerobic methanotrophic archaea (ANME). From a terrestrial mud volcano, we enriched a microbial community containing ANME-2a, using methane as an electron donor, Fe(III) oxide (ferrihydrite) as an electron acceptor, and anthraquinone-2,6-disulfonate as an electron shuttle. Ferrihydrite reduction led to the formation of a black, highly magnetic precipitate. A significant relative abundance of ANME-2a in batch cultures was observed over five subsequent transfers. Phylogenetic analysis revealed that, in addition to ANME-2a, two bacterial taxa belonging to uncultured Desulfobulbaceae and Anaerolineaceae were constantly present in all enrichments. Metagenome-assembled genomes (MAGs) of ANME-2a contained a complete set of genes for methanogenesis and numerous genes of multiheme c-type cytochromes (MHC), indicating the capability of methanotrophs to transfer electrons to metal oxides or to a bacterial partner. One of the ANME MAGs encoded respiratory arsenate reductase (Arr), suggesting the potential for a direct coupling of methane oxidation with As(V) reduction in the single microorganism. The same MAG also encoded uptake [NiFe] hydrogenase, which is uncommon for ANME-2. The MAG of uncultured Desulfobulbaceae contained genes of dissimilatory sulfate reduction, a Wood-Ljungdahl pathway for autotrophic CO2 fixation, hydrogenases, and 43 MHC. We hypothesize that uncultured Desulfobulbaceae is a bacterial partner of ANME-2a, which mediates extracellular electron transfer to Fe(III) oxide.
Keywords: ANME; AOM; Desulfobulbaceae; MAG; archaea; diversity; metagenome; methane; microbial communities; mud volcano.