162 Chapter 5 indicated a drop from 20% to 11% of “Ca. Methanoperedens” (from T0 to T2), with no major changes from T2 to T3 (16-18%). We observed a minimal fraction of “Ca. Methanoperedens” in the planktonic fraction of T2 and T3, constituting about 0.50.9% of the total reads recovered (Supplementary Figure 3). We detected different RNA-seq trends on the granular “Ca. Methanoperedens”- specific cell division transcripts (ftsZ) and archaeal flagellin (flaF/flaH/flaI/flaJ) between control (T0) and T1 vs control (T0) and T2 or T3 (Supplementary Tables 3 and 7). Moreover, the general morphotype shift gene marker expression increased upon the first sulfide spike (Supplementary Table 7). This distinct morphotype gene marker expression shift in the different time points analyzed (Supplementary Table 7) showed a degree of similarity with the increase in markers for morphotype shift in “Ca. Methanoperedens” under oxygen and salt stress (Echeveste Medrano et al., 2024b, Chapter 6; Guerrero Cruz et al., 2018). Implications and future work This work presents the first physiological study on freshwater ANME “Ca. Methanoperedens” exposed to sulfide stress. We highlight a marked methane oxidation activity drop at 0.5 mM sulfide together with an increase in expression of “Ca. Methanoperedens” sulfite reductases (Group III Dsr-LP) as putative sulfite detoxification mechanism upon long-term exposure. In addition, the potential usage of PHAs together with sulfide scavenging community members appear to help enable “Ca. Methanoperedens” survival under sulfide stress. Future work on nitrite, salt, and sulfide stressors on anaerobic methanotrophs could benefit from the analysis of the response of distinct sulfite reductases to such stressors. Our study contributes to improved understanding of stress response in ANME archaea, particularly relevant in the context of eutrophic natural or engineered ecosystems. Data availability Presented 16S rRNA gene amplicon and metagenomics data is available under European Nucleotide Archive project number PRJEB81701.
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