69 Sulfide toxicity as key control on anaerobic oxidation of methane in eutrophic coastal sediments Figure 6. Average anaerobic oxidation of methane (AOM) rates measured in Site 5 sediments incubated with varying added sulfide concentrations (0, 0.5, 1, 2 or 4 mM). While black circles indicate that samples were pre-incubated with 13C-methane only, the grey circle indicates that samples were pre-incubated with 13C-methane and 2 mM of sulfide. Error bars provide the standard deviation across three biological replicates. Results from this experiment corroborate our key microbiological observation, the differential distribution of potential ANME-2 abundances across samples based on MAG 11 normalized genome coverages. Low coverages of MAG 11 were estimated in sediments of Site 3, in agreement with the low methane concentrations measured in situ and low potential methane production rates measured (Figure 4). At Site 5, MAG 11 coverages were the highest, within and below the SMTZ, matching abundant methane and sulfate substrates for S-AOM activity (Figure 4) and their calculated fluxes into the SMTZ (Table 3), as well as highest potential methane production rates. However, at Site 7, where methane and sulfate were also abundant and had similar high fluxes into the SMTZ (Table 3), putative ANME-2 abundances were near zero, based both on 16S analyses and MAG 11 coverages. We acknowledge that shallowing of the SMTZ and methanogenesis in surface sediments likely contributed to higher methane escape from Sites 5 and 7 relative to Site 3 (Wallenius et al., 2021). Such high benthic fluxes of methane are in accordance with previously reported values for the Stockholm Archipelago (Sawicka & Brüchert, 2017). However, differences in the SMTZ or in methanogenesis 2
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