167 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress INTRODUCTION Coastal freshwater wetlands are dynamic ecosystems and hotspots for methane cycling, a greenhouse gas of growing concern (Rosentreter et al., 2021; Saunois et al., 2020). Biological methane production in these systems is performed by marine methanogenic archaea present in deeper sediment layers (Treude et al., 2014). Methanotrophs constitute a natural methane biofilter that mitigates part of the coastal methane emissions (Knittel & Boetius, 2009; Wallenius et al., 2021). In the deeper anoxic layers of the sediment, anaerobic methanotrophic archaea (ANME) couple methane oxidation to the reduction of a range of electron acceptors including nitrate, sulfate, metal oxides, and natural organic matter (Chadwick et al., 2022; Egger et al., 2018; Glodowska et al., 2022; Knittel & Boetius, 2009). In cases where nitrate is the most competitive electron acceptor, members of the archaeal family Methanoperedenaceae (formerly ANME-2d) are thought to dominate the removal of methane (Wallenius et al., 2021). In the top sediment layer and water column of coastal systems, methane oxidation occurs mostly under oxic conditions by methanotrophic bacteria (Venetz et al., 2023; Żygadłowska et al., 2023). Coastal freshwater regions are currently facing a plethora of anthropogenic challenges, including intensive agricultural land use and climate change-driven sea level rise (IPCC, 2023; Kaushal et al., 2021). Sea water intrusion to coastal ecosystems poses hyperosmotic pressure on freshwater microbial communities, including bacterial methanotrophic communities (Ho et al., 2018; Osudar et al., 2017). This osmotic pressure makes the Methanoperedenaceae ANME a relevant group of microorganisms to investigate regarding their stress response to salt concentration increase; salinity-derived methane emission reduction has been described for aquatic inland waters (Soued et al., 2024) but the effect on anaerobic methanotrophs has to be further unraveled. Investigations on osmoregulation are strongly biased towards bacteria or halophilic archaea with a few exceptions in methanogens such as Methanosarcina mazei (Jurdzinski et al., 2023; Mukhtar et al., 2020; Spanheimer & Müller, 2008) thereby leaving the comprehensive physiological response of ANME to salt-stress largely unexplored (Li et al., 2022). 6
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