139 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea INTRODUCTION Anthropogenic emissions of the greenhouse methane (CH₄) significantly impact climate change, driving global warming and altering climate patterns (IPCC, 2023; Saunois et al., 2024). Although carbon dioxide (CO₂) is the main greenhouse gas, methane has a much stronger short-term impact on global warming. Methane remains in the atmosphere for approximately 12 years; however, during that time, it is about 80 times more effective at trapping heat compared to CO₂ when considering its impact over a 20-year period (IPCC, 2014). Methane is produced in anoxic environments by methanogenic archaea that use a limited amount of substrates including H2, methanol, acetate and dimethyl sulfide (DMS) (Kurth et al., 2020). Fortunately, a significant proportion of the methane produced is subsequently removed by anaerobic and aerobic methanotrophic biofilter before reaching the atmosphere (Gao et al., 2022; Knittel & Boetius, 2009). The anaerobic oxidation of methane (AOM) is mediated by methanotrophic (ANME) archaea that can use a variety of electron acceptors: from the energetically less favorable sulfate in marine environments to the more favorable nitrate, humic substances, or metal oxides in brackish and freshwater systems (Glodowska et al., 2022; Zhao et al., 2024). The aerobic oxidation of methane is mediated by methane-oxidizing bacteria (MOB). Many MOB have the genomic potential for (partial) denitrification, fermentation, and occur in the anoxic zones of lakes (Kalyuzhnaya et al., 2019; Schorn et al., 2024a). Some reports also document the use of metal-oxides by MOB (Li et al., 2023; Zheng et al., 2020). Furthermore, methanotrophs of the NC10 phylum or “Candidatus (Ca.) Methylomirabilis” have the ability to dismutate nitric oxide into oxygen and nitrogen gas, using the produced oxygen most likely for the activation of methane via methane monoxygenase (Ettwig et al., 2010; Versantvoort et al., 2018). Understanding microbial methane oxidation is critical for managing methane emissions and their implications for climate change (Jones et al., 2023; Kirschke et al., 2013). Research on the role of ANME archaea in removing methane is gaining momentum and importance, especially in methane seeps, marine, and freshwater environments including engineered ecosystems such as wastewater treatment plants (Bhattarai et al., 2019; Haroon et al., 2013; Ruff et al., 2016; Segarra et al., 5
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