Core collection:
AT50-14: Gravity core material was sectioned with a tubing cutter, capped, and stored at 4 degrees Celsius until incubation processing. Sediment depths 50-75 centimeters (cm), 110-125 cm, and 220-230 cm were selected for incubations based on sulfate zonation.

AT50-29b: Push core material was sectioned through extrusion out the top at 5 cm resolution. Sediment depths 0-5 cm, 5-10 cm, and 10-15 cm were stored in three separate glass jars (Ball) and pooled with those depth intervals from two other cores at 4 degrees Celsius until incubation processing.

Incubation setup: Headspace-free bag incubations were conducted with sediment from each study site. The overall idea was to fill foil bags with a sediment slurry that had been equilibrated with differing concentrations of CH4 and SO4 to mimic in situ geochemical conditions characteristic of each site. Three geochemical zones were targeted for each incubation to reflect the vertical zonation observed in the in situ data: the sulfate zone (SZ; high SO4, low CH4), the transition zone (TZ; low SO4, low CH4), and the methanogenic zone (low SO4, high CH4). First, in situ SO4 data was used (AC) to inform the zonation of the collected cores prior to incubation. However, SO4 data was not available for SHR when sectioning; therefore, sediments were sectioned into 5 cm intervals based on SO4 data previously reported for SHR bacterial mat sites (Boetius et al., 2000; Boetius & Suess, 2004; Torres et al., 2002). Next, incubations were set up with experimental and control treatments. For all incubations, a slurry was created in a 2:1 sediment to anoxic MQ ratio. Slurries were sieved with a mesh strainer (steel single weave #20 mesh) to remove shell fragments that could puncture the incubation bags or clog the stopcock fitted to the incubation bag. Prior to starting incubations, storage tests were conducted to ensure the bags were gas-tight and didn’t cause isotopic fractionation. The slurries were then transferred to 500 milliliter (mL) syringes without a headspace. All slurry manipulations were conducted in an anaerobic chamber (Coy). A mixture of 100% CH4, 99% 13C-CH4 (Cambridge Isotope Laboratories), and ultra-high purity (UHP) nitrogen gas (N2) was introduced to the syringe and equilibrated with the slurry by rotating for 90 minutes to achieve CH4 concentrations targeted to in situ concentrations. After equilibration, the headspace was removed and the slurry was injected into a 1 liter (L) N2-flushed foil sampling bag equipped with a 3-way stopcock (Calibrated Instruments).

Sediment/porewater sampling:
CH4, CO2, δ13C-CH4, δ13C-CO2: 10 mL glass serum vials containing 3 mL sediment slurry were subsampled using the headspace equilibration method following Magen et al. (2014). Briefly, 8 mL of ultra-zero purity air (UZA) was injected into the sediment vial with a plastic syringe equipped with a 22-gauge needle. Vials were shaken and the headspace was mixed for 2 minutes, after which 8 mL of headspace gas sample was removed. Headspace samples were subsequently diluted with UZA to fall within the instrumental range for CH4 and CO2 measurements. Because the sulfide in the incubation subsamples was not bound like in the characterization cores, a copper trap was used at the intake of the CRDS to remove sulfide before entering the machine (Malowany et al., 2015).

SO4 and Cl: 2 mL microcentrifuge tubes containing 40 microliters (uL) porewater sample acidified to pH 2 with 0.1M H3PO4 were diluted (135x) with Milli-Q water prior to analysis. IAPSO certified seawater standards (Ocean Scientific International Ltd.) were used for all samples and precision is +/- 2%.

DOC and δ13C-DOC: 40 mL amber VOA vials containing 1 mL porewater sample acidified to pH 2 with trace metal clean HCl were measured with the wet oxidation method, similarly to the setup detailed in Lalonde et al. (2014). Samples are acidified and sparged to remove inorganic carbon then reacted with Sodium persulfate as wet oxidation. Data were normalized using two different internal organic standards (precision +/- 0.5 ppm for concentrations and 0.2‰ for isotopes).

DIC and δ13C-DIC: 10 mL glass serum vials containing 1 mL porewater sample acidified to pH 2 with H3PO4 were measured at CBL CRDS was used as detailed for the CH4 and CO2 concentrations and stable carbon isotopes.

POC and δ13C-POC: After frozen sediment samples were sampled for CH4 and CO2 concentrations and stable carbon isotope ratios, one of the duplicate samples was uncapped, dried at 60 degrees Celsius (°C) for three days, ground, and acidified with vapor acidification.