149 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea stable during the experiment, compared to our incubations where the sulfide was quickly depleted (Supplementary Figure 1B). Considering the sulfide inhibition after repeated long-term sulfide exposure in the “Ca. Methanoperedens” enriched culture (0.25 mM/day), the tolerance reduces to ~ 20% (24-h rate) or ~ 30% (5-day recovery rate) after the second 0.5 mM sulfide addition. Overall, this suggests that the freshwater “Ca. Methanoperedens” is less tolerant to sulfide exposure compared to brackish/marine ANME. Such differential sulfide tolerance has also been observed within marine ANME species, with ANME-1 remained active under higher sulfide concentrations while ANME-2ab activity was negatively correlated with sulfide (Biddle et al., 2012; Timmers et al., 2015). The presence and transcriptional activity of “Ca. Methanoperedens” are not negatively impacted by sulfide exposure To follow changes in the microbial community composition and expression patterns, we sequenced both DNA and RNA at T0, T1, T2, and T3 (Figure 3). “Ca. Methanoperedens” dominated the overall community in both DNA and RNA fractions throughout the reactor run (T0, T2, and T3) (Figure 3). The rare-biosphere (“Others” in Figure 3) were hardly represented in the transcriptome, although they made up a significant portion (between 25% and 40%) of the DNA reads, indicating slow mineralization of decaying biomass, usage of exudates or, just dormant yet not dead. Regarding the general microbial community composition change, we observed that MAGs “Desulfobacillus 2”, “Thermoanaerobaculia g JACTMI01 2”, “Aquamicrobium A”, “Aggregatilineales g SB15 & g CFX10” and “Brocadia” anammox bacteria, among others, showed a general upward trend in both metagenomics and metatranscriptomics (Figure 3 and Supplementary Tables 1-2). In contrast, the methane-oxidizing partner and nitrite-scavenger “Ca. Methylomirabilis oxyfera” reduced its overall transcription after long-term exposure and sulfide spikes (Figure 3). 5
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