53 Sulfide toxicity as key control on anaerobic oxidation of methane in eutrophic coastal sediments until bottles were assembled. For these incubations, Site 5 was selected due to highest methanotroph genome coverages, and samples were combined and homogenized, resulting in a mixture spanning the depth of 8 to 28 cm. Five grams of wet sediments were placed into 60 mL-serum bottles and mixed with 5 mL of a solution containing MgSO4 8 mM and 0.5% NaCl, to achieve a final concentration of 4 mM sulfate and a pH buffered to 7.05. Bottles were degassed with argon gas and a headspace at 0.5 bar of overpressure was created, containing also 0.5% of N2, 0.5% CO2), and 20% 13CH4. To one set of triplicate bottles, sulfide was added to a final concentration of 2 mM, while the other bottles did not receive sulfide. After 13 weeks of incubation in the dark at 4ºC, AOM was confirmed by detection of 13CO 2 production (data not shown). Then, sulfide was added to the remaining bottles, in triplicate incubations, targeting final concentrations of 0, 0.5, 1, 2 and 4 mM. To monitor 13CO 2 production, bottle pressure was monitored using a GHM 3111 Digital Pressure Meter with a GMSD 2 BR – K31 sensor, and 50 µl of headspace was injected into an Agilent 6890 series gas chromatograph coupled to a mass spectrometer equipped with a Porapak Q column heated at 80°C with helium as the carrier gas as previously described (Dalcin Martins et al., 2021). Liquiddissolved 13CO 2 was estimated with the equation ∑13CO 2 = 13CO 2(g) [1 + kRT Vliquid/ Vgas (1+ KZ/[H+])]60 and summed to headspace 13CO 2 derived from calibration curves for AOM rate calculations (see Supplementary Table 2 for calculations). Bottles were sampled for sulfide determination as described in the section collection of porewater and sediment samples for geochemical analysis. For that, approximately 1 mL of slurry was anoxically withdrawn and filtered through a 0.2µm Nylon syringe filter. Approximately 300 µL of filtrate was mixed with 1.2 mL of an anoxic 2% zinc acetate solution and stored at 4ºC until analysis. Removed liquid volumes were considered for AOM rate calculations. Finally, sulfide concentrations were measured as described in the section “porewater analyses”. 2
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