97 Contrasting methane, sulfide and nitrogen regimes in coastal sediment bioreactors Verrucomicrobial methanotroph Methylacidiphilum fumariolicum SoIV (Gwak et al., 2022; Schmitz et al., 2023). Both the genus Methylovulum and Methylomonadaceae subgroup KS41 showed preferential enrichment under lower sulfide loading in the biofilm (Figure 4A, Supplementary Figure 9 and Supplementary Table 1). While for the genus Methylovulum there are cultured representatives (Iguchi et al., 2011; Oshkin et al., 2016) that could be tested, the genus Methylomonadaceae KS41, with the highest amount of reads, has no cultured representatives yet (Supplementary Figure 15 and Supplementary Table 7). Better resolving the activities and interaction of C, N, and S pathways in methanotrophs may have substantial implications for understanding and modeling biogeochemical cycling in these ecosystems Freshwater “Ca. Methanoperedens BLZ2” sp. was the only archaeal methanotroph that became enriched from 7 months onwards in the oligotrophic system, reaching as much as 21% of the total metagenomic reads in month 15.5 (Supplementary Figure 5 and 7 and Supplementary Table 1) despite the 2% brackish salinity in the medium. “Ca. Methanoperedens BLZ2” showed a considerable contribution to DNRA via nrfA under nitrate limiting and methane saturated conditions, reaching more than 80% of nrfA reads mapping to “Ca. Methanoperedens BLZ2” at month 15.5, similar to what was found in previous studies (Arshad et al., 2017; Dalcin Martins et al., 2022, Chapter 4) (Figure 3B and Supplementary Figure 5 and 7). The observed co-occurrence of “Ca. Methanoperedens” spp. with Methylomonadaceae type I methanotrophs was also described in a hypoxic marine eutrophic lake, indicating their potential prevalence under nitrate-limiting conditions (Venetz et al., 2023). Together with abundant MOBs, “Ca. Methanoperedens BLZ2” sp. and sulfur oxidizers, we co-enriched novel bacteria affiliated with Pseudomonadales IMCC2047 and Rugosibacter (Figure 5). Both corresponding MAGs contained unusual CuMMO-A sequences that clustered separately in an extended Cu-MMO tree (Figure 5D and Supplementary Figure 13). Both MAGs also include a complete (p/a) moCAB-like gene operon, followed downstream by a single putative amoD/pmoDlike ORF (Koo & Rosenzweig, 2020) (Figure 5C and Supplementary Table 6). There is yet no consensus in literature for describing canonical amoCAB associated ED 3
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