52 Chapter 2 2019). Normalized mapped read (NMR) values for specific genes were calculated as follows: NMR = the number of mapped reads to the gene /(the length of the gene in bp*103)*(the total number of reads (mapped + unmapped) in the metagenome/106). See Supplementary Information for more details. Potential methane production rate measurements Sediments for incubations to measure potential methane production rates were sliced in a N2-filled portable glove bag and placed into sterile plastic bags (VWR International BV, Amsterdam). The resulting slices had a resolution of 4 cm and were stored anoxically into sealed aluminum bags (Gruber-Folien GmbH & Co. KG, Germany) in the dark at 4ºC for one month from sampling collection until bottles were assembled. For this, 5 g of wet sediments were placed into 60 mL-serum bottles, and 5 mL of sulfate-free artificial seawater (ASW) medium at pH 7.5 were added to create a 1:1 diluted slurry. Bottles were degassed and sealed under argon gas at atmospheric pressure (Linde Gas Benelux). The ASW medium was adapted from a previously published study (Kester et al., 1967) to achieve a salinity of 5.3 (van Helmond et al., 2020) and contained, per liter, 3.418 g NaCl, 1.54 g MgCl2.6H2O, 0.097 g KCl, 0.21 g CaCl2.2H2O, 0.014 g KBr, 0.0037 H3BO3, 0.003 g SrCl2.6H2O. 0.0004 g NaF, and 0.028 g NaHCO3. No trace elements or vitamins were added. From each site, six depths were incubated in triplicate at 4°C, resulting in 54 serum bottles. Methane production was monitored via injection of 100 µl-headspace samples into an HP 5890 gas chromatograph equipped with a Porapak Q column (80/100 mesh) and flame ionization detector (Hewlett-Packard, USA) with a detection limit of <1 ppm. Each gas sample was measured in triplicate and areas were averaged. Methane concentrations were calculated using the Henry’s law coefficient Hcp (Sander, 2015). Potential rates of methane production were calculated using linear regression of methane measurements obtained in the first 8-9 days of sediment incubation. More details are provided in the supplement and all calculations are provided in Supplementary Table 1. Sulfide toxicity experiment and AOM rate measurements Sediments for incubations to measure AOM rates under different sulfide concentrations were retrieved from previously stored, anoxically sealed aluminum bags kept in the dark at 4ºC for approximately two years after sample collection
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