119 Unraveling nitrogen, sulfur and carbon microbial bioreactor responses to stress MAG coverages revealed shifts in microbial community structure during experimental conditions Normalized genome coverage for each MAG was used as a proxy for organism abundances across time points. Under regular operation conditions (Figure 1, G0), the most abundant microorganism in the reactor was MAG 39 Nitrobium versatile, with a genome coverage of approximately 245x (Figure 3). MAG 37 Methylomirabilis lanthanidiphila had normalized genome coverage of 89x, MAG 60 Thermoanaerobaculia 1 of 26x, MAG 57 Sulfuricella denitrificans of 26x, and MAG 55 Scalindua rubra of 25x. However, large shifts in microbial community structure occurred after ammonium was removed from the medium and subsequently re-introduced (G1). MAG 36 Methanoperedens nitroreducens, which previously had a normalized genome coverage of 5x, increased to 278x, and MAG 38 Methylomirabilis tolerans, which had a coverage of 19x before, increased to 84x., while MAG 37 Methylomirabilis lanthanidiphila decreased to 4x. Other abundant genomes at G1 were MAG 44 Plantomycetes 1 (50x), MAG 22 Desulfobacterota (39x), MAG 32 Kuenenia stuttgartiensis (37x), and MAG 56 Sulfuricella (37x). These genomes remained the most abundant in the bioreactor during the recovery period (G2), with MAG 36 Methanoperedens nitroreducens reaching 366x coverage, and MAG 38 Methylomirabilis tolerans, 110x, indicating that the community reached another but distinct stable structure. During the preparatory phase for the sulfide and NO toxicity experiment (G3), MAG 38 Methylomirabilis tolerans became dominant (292x), followed by MAG 36 Methanoperedens nitroreducens (126x), MAG 60 Thermoanaerobaculia 1 (71x), MAG 44 Plantomycetes 1 (33x), and MAG 32 Kuenenia stuttgartiensis (18x). These MAGs remained, in this order, the most abundant genomes in the reactor until the sulfide and NO toxicity experiment started (G4). Finally, at the end of this experiment (G5), again shifts in community structure were detected: MAG 36 Methanoperedens nitroreducens returned as the dominant genome (207x), followed by MAG 38 Methylomirabilis tolerans (198x), MAG 65 Thiohalobacteraceae (52x), MAG 44 Plantomycetes 1 (36x), MAG 60 Thermoanaerobaculia 1 (29x), and MAG 32 Kuenenia stuttgartiensis (19x). Metatranscriptomic analyses reveal active metabolic pathways in key microbial community members cycling methane, nitrogen and sulfur. To investigate transcriptional responses of the bioreactor microbial community to experimental 4
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