99 Contrasting methane, sulfide and nitrogen regimes in coastal sediment bioreactors The first MBAE14/Pseudomonadales IMCC2047 MAG was described in 2019 in a similar ecosystem with oxygen-limitation and the presence of sulfide, methane, and ammonium (Mori et al., 2019). Based on the genomic potential, it was postulated to act as putative mixotrophic nitrifier-denitrifier. The MBAE14 MAG reconstructed here seems unlikely to be a functional nitrifier; instead, the substrate range of the unusual Cu-MMOs should be further investigated (e.g. by (meta)transcriptomics) as it could provide key insights into the ecological niche of this bacterium. Another insight from Mori et al. and our study is that the MAG has high AAI similarity to marine heterotrophs, including Oleiphilus spp. (> 90%), Marinobacter spp. (> 90%), and the denitrifier Pseudomonas stutzeri (> 88.4%), and heterotrophic nitrifier/ denitrifier Alcaligenes faecalis (> 78.9%) (Mori et al., 2019). The microorganisms are known to oxidize aliphatic (Oleiphilus spp. and Marinobacter spp) or aromatic (Pseudomonas stutzeri) hydrocarbons (Bowman & McMeekin, 2015; Lenferink et al., 2024; Singh & Tiwary, 2017; Yakimov et al., 2007). Alcaligenes faecalis can produce large amounts of hydroxylamine during heterotrophic nitrification (Lenferink et al., 2024). When building the Cu-MMO tree, we also included sequences belonging to the Azoarcus genus (Figure 5D), as both genomes from the genus Pseudomonas and Azoarcus have been co-enriched in a membrane-aerated nitrification-denitrification tank (Lan et al., 2023). Lastly, we describe a novel Rugosibacter MAG (Supplementary Figure 13) that was present in the biofilms of both eutrophic and oligotrophic bioreactors (0.91% enriched at month 15.5) (Figure 5B and Supplementary Table 1). This genus has only a single cultured isolate, which lacks a Cu-MMO but can degrade aromatic compounds (Corteselli et al., 2017). Interestingly, the PQQ-ADH of this MAG shows a high similarity (100% query, 94.72% alignment) to the one of an unpublished groundwater MAG of Rugosibacter, with cryptic sulfur and nitrogen cycling potential (Locus: MDO8347641; Bioproject: PRJNA700657). However, this putative groundwater Rugosibacter MAG lacks a Cu-MMO, which are often mis-binned and may be missing from the published Rugosibacter MAG. Salinity was increased from 1% to 2% over the course of one month, from month 14.5 to 15.5 (Supplementary Figure 1). We observed no salinity effect on the microbial community composition (Figure 2, Supplementary Figure 5 and 3
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