Humic substances as electron acceptors for anaerobic oxidation of methane driven by ANME-2d

Humic substances (humics) are ubiquitous in terrestrial and aquatic environments where they can serve as electron acceptors for anaerobic oxidation of organic compounds. Methane is a powerful greenhouse gas, as well as the least reactive organic molecule. Anaerobic oxidation of methane (AOM) coupled to microbial reduction of various electron acceptors plays a crucial role in mitigating methane emissions. Here, we reported that humics could serve as terminal electron acceptors for AOM using enriched nitrate-reducing AOM microorganisms. AOM coupled to the reduction of humics was demonstrated based on the production of ¹³C-labelled carbon dioxide, and AOM activity was evaluated with different methane partial pressures and electron acceptor concentrations. After three-cycle reduction, both AOM activity and copy numbers of the archaea 16S rRNA and mcrA genes were the highest when anthraquinone-2,6-disulfonic acid and anthraquinone-2-sulfonic acid were electron acceptors. The high-throughput sequencing results suggested that ANME-2d were the dominant methane oxidation archaea after humics reduction, although the partner bacteria NC10 trended downward, other reported humics reduction bacteria (Geobactor and Anammox) appeared. The potential electron transfer models from ANME-2d to humics were proposed. These results enable a better understanding of available electron acceptors for AOM in natural environments and broaden our insight into the significant role of ANME-2d.