83 Contrasting methane, sulfide and nitrogen regimes in coastal sediment bioreactors Genomes containing Cu-MMO were downloaded from JGI’s IMG/MER after identification of Pfam02461 using the pfam search of their “all isolate” database, noting that several genomes in the database appear to be MAGs. Several additional genomes were downloaded due to shared taxonomy or identification of the pfam motif in other automated mining efforts of large genomic databases (e.g., AnnoTree, Mendler et al., 2019). Reference genomes were annotated using DRAM (v1.2.2) (Shaffer et al., 2020) to homogenously call genes and identify Cu-MMOs. The amino acids of the A subunits of Cu-MMOs were aligned using MAFFT’s “ginsi” setting (1000 iterations, global alignment; v7.397) (Katoh & Standley, 2013), trimmed automatically using Trimal (v1.4.rev22) with default settings (Capella-Gutiérrez et al., 2009), and the alignment input into IQTree (1.6.12) with 1000 bootstraps and automatic model selection (Nguyen et al., 2015). Ammonia oxidizing bacteria references employed to analyze the operon architecture of amoCABEDcycAB and haoAB included: Nitrosococcus oceanii strain ATCC 19707, “Ca. Nitrosacidococcus tergens” sp. RJ19 (Picone et al., 2021) and Nitrosomonas europaea strain ATCC 19718. For the pyrroloquinoline quinone (PQQ)-ADH phylogenetic tree generation, reference (Keltjens et al., 2014) and study sequences were first aligned using with default MAFFT setting from EMBL-EBI web browser (Madeira et al., 2022), trimmed with (--gappy out) using Trimal v1.4.rev15 (Capella-Gutiérrez et al., 2009) and ran with IQ-TREE v2.0.3 using flags -st AA -m MFP -bb 1000 -nt AUTO (Nguyen et al., 2015). Fast amino acid identity matrixes were generated with the online tool of the Kostas lab with default settings (Rodriguez-R & Konstantinidis, 2016). The biogeography of “Ca. Methanoperedens BLZ2” and the Methylomonadaceae family was conducted using SingleM-resolved Sandpiper (April 2024) (https://sandpiper. qut.edu.au/). RESULTS Fluctuations in sulfide, nitrate, nitrite, and ammonium concentrations in between substrate loadings For the eutrophic bioreactor, sulfide was fully consumed during the monitoring period, whereas ammonium remained below 0.5 mM, and nitrate fluctuated between 2 to 7 mM (Supplementary Figure 3). Nitrite did not accumulate to more than 1-5 µM except after substrate loading when mM nitrite levels were reached for 3
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