65 Sulfide toxicity as key control on anaerobic oxidation of methane in eutrophic coastal sediments reduction, as previously observed in estuarine systems, potentially due to the abundance of substrates (Sela-Adler et al., 2017). This is also supported by our data, given the relative abundances of sequences affiliated to Methanoregulaceae and Methanosaetaceae. An anaerobic methane-oxidizing archaeon constitutes the benthic methane biofilter Based on phylogenetic analyses, MAG 011, affiliated to the archaeal order Methanosarcinales, was identified as representative of an ANME-2 archaeon (Supplementary Figure 3). This was the only detected genome representing a methane-oxidizing microorganism in our sequencing datasets, indicating that the benthic methane biofilter (the biological process of methane removal in sediments) was potentially constituted of a single organism. MAG 11 had highest ngCOV at Site 5 (9.5x at 22 cm) below the SMTZ (Figure 4), but reached only 0.3x at Site 3 at 2 cm and 0.06x at Site 7 at 2 cm. This genome had 66% average amino acid identity to genome MZXQ01 (NCBI accession number), which represents an ANME-2b archaeon obtained from sediment of the Hydrate Ridge North methane seep (Yu et al., 2018) classified as “Ca. Methanomarinus sp. nov. 1” (Chadwick et al., 2022) (Supplementary Figure 4).* MAG 011 was 98.4% complete and 2% contaminated and had potential for a full reverse methanogenesis pathway, as well as acetate production or assimilation via an acetyl-CoA synthase (acss) gene (Figure 5, Supplementary Table 4). MAG 011 was further analyzed in detail to elucidate its metabolic potential. Genes encoding proteins involved in the reverse methanogenesis pathway were mostly present as single copy (Supplementary Table 4), with a few exceptions: (i) hdrABC were present in three copies, (ii) a second copy of mtrAH was downstream of mtrX while mtrEDCBAFGH subunits were present in a separate contig, (iii) three copies of the gene encoding the formylmethanofurantetrahydromethanopterin N-formyltransferase (ftr) were present, and (iv) both molybdenum- and tungsten-dependent formylmethanofuran dehydrogenases were present (fmdCABDE and fwdGBDC), with fwdC present as a separate single subunit and also as a two-copy fwdC gene fusion (Supplementary Table 4). 2
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