Dataset: Rates of methanogenesis in subsurface sediments from R/V JOIDES Resolution IODP-385 drilling expedition in the Guaymas Basin between September and November, 2019

Subsurface sediment samples were collected from four drilling sites in the Guaymas Basin, Gulf of California, during IODP Expedition 385 “Guaymas Basin Tectonics and Biosphere” using the research vessel R/V JOIDES Resolution. Rates of methanogenesis were determined at four sites. Sites 1545 (27º38.230’N, 111º53.329’W) and 1546 (27º37.884’N, 111º52.781’W) were located roughly 52 km and 51 km, respectively, northwest of the axial graben of the northern spreading segment. Both sites are highly sedimented and a 75-meter thick inactive (~thermally equilibrated) basaltic/doleritic/gabbroic sill was present at site 1546 between ~355 to 431 meters below the seafloor (mbsf). Site U1545 is considered a reference site since it was free of sill intrusions and unaffected by active hydrothermal circulation. Samples to determine methanogenesis rates were collected from the "B" hole at every site (during IODP expeditions, several, closely-spaced holes are drilled at each site so that sufficient sediment is available for all of the proposed work.

The geothermal gradient at hole U1545B was 227ºC/km. The geothermal gradient at site U1546B was similar to that measured in hole U1545B, 221ºC/km, since the sill at 1546B was thermally equilibrated. Sites U1547 (27º30.413’N, 111º40.734’W) was located inside the periphery of an active, sill-associated hydrothermal mound located about 27 km northwest of the axial graben of the northern spreading segment. Temperatures in hole U1547B exceeded 50°C in the upper 50 mbsf. The geothermal gradient at this site was between 511ºC to 960ºC/km. Site U1549 was located near a cold seep sustained by a deeply buried, thermally equilibrated sill intrusion at several hundred meters depth. The geothermal gradient at hole U1549B was 194ºC/km.

Sediment samples were collected using an advanced piston coring system (APC) and a half-length APC (Teske et al. 2021). After retrieval on deck, sediment cores were sectioned in a designated core cutting area. Approximately 0.5 cm of the outer sediment layer was carefully removed to minimize potential contamination. For analysis of hydrocarbon gases, ~ 5 mL sediment was transferred into 21.5 mL clean, pre-combusted glass vials using a cut-off syringe. The vials were sealed immediately with polytetrafluoroethylene septa and aluminum caps and were incubated for 30 min at 70°C to allow dissolved hydrocarbon gases to equilibrate with the headspace before analysis, as described in Teske et al. (2021). For molecular hydrogen analysis, duplicate 3 mL sediment samples were collected immediately after core recovery using a cut-off syringe. The subsample was transferred into 21.5 mL glass vials and incubated and processed according to the procedures of Lin et al. (2012).

Whole round core (WRC) segments for methanogenic activity measurements were cut from cores retrieved from hole B at each site and stored temporarily in a nitrogen-filled glove bags until processing in the radioisotope tracer isolation space on the R/V JOIDES Resolution. Manipulation of samples for rate measurements were carried out in an anaerobic chamber (95:5 [V/V] N2:H2) to prevent oxygen contamination. Methanogenesis rate assays were carried using modifications of our previous methods (Bowles et al. 2011, Zhuang et al. 2018, Zhuang et al. 2019); the modifications increased the sensitivity of the rate measurements. Replicates for each sample were prepared in quadruplicate (three live and one killed control) for each substrate by transferring 5 cm³ of sediment from the inner-most part of a whole round core into a modified cut-end Hungate tube (Bellco Glass, 2407-00125) using a sterile cut-end syringe inside an anaerobic chamber (95%:5%, N₂:H₂). The headspace was removed by inserting a modified butyl rubber stopper (Bellco Glass, 2048-11800A) into the cut-end of the tube, and then a septum (Bellco Glass, 2047-11600) and screw cap (Bellco Glass, 2047-16000) were placed on top of the tube to seal the sample. All vials, stoppers, and septa were autoclaved and stored anoxically prior to use as described by Zhuang et al. (2019).

Replicate and control samples were injected with 100 μL of ¹⁴C -labeled substrate (American Radiolabeled Chemicals, Inc.). Radiotracer stocks were diluted into pH-adjusted (pH-7) milliQ water to achieve the target activity in a 100 µL injection, which was 390 to 427 kBq for ^2-14C-acetate, 637 to 1286 for ¹⁴C-bicarbonate, 427 to 451 kBq for ¹⁴C-formate, 417 to 3294 kBq for ¹⁴C-methanol, or 155 to 375 kBq for ¹⁴C-methylamine. Prior to the addition of radiotracer, the killed control samples were amended with 4 mL of helium-purged 2M NaOH and homogenized using a vortex. Live and killed samples were incubated for 2-4 weeks at 1 atmosphere and temperatures near in situ values (within 10°C of in situ temperature). The live incubations were terminated by adding 4 mL of helium-purged 2M NaOH and homogenizing samples using a vortex. All samples were stored at room temperature for shore-based processing and analysis in the University of Georgia laboratory.

Methanogenic activity was determined by converting the ¹⁴C-CH₄ produced within each sample to ¹⁴C-CO₂ via combustion at 850°C using a copper oxide catalyst and trapping the evolved ¹⁴C-CO₂. For each sample, both atmospheric air and 100 μL of ultra-high purity ¹²C-CH₄ were added to generate a headspace and serve as a carrier, respectively. The produced ¹⁴C-CH₄ was driven into the headspace by vortexing the samples and ¹⁴C-CH₄was removed from the sample by flushing the headspace with 5% O₂ in a balance of N₂ for 1 hour. Carry-over of ¹⁴C-labeled organic substrates or carbon dioxide during ¹⁴C-CH₄ recovery would inflate the calculated rates, the carrier gas stream from the sample was purified through a series of substrate-dependent traps between exiting the sample vial and entering the combustion oven. The two parent tracer traps for bicarbonate, acetate, and formate consisted of 2M NaOH in saturated NaCl (trap 1) and NaOH pellets (trap 2). The three parent tracer traps for methylamine consisted of 1M H₂SO₄ (trap 1), 2M NaOH in saturated NaCl (trap 2), and NaOH pellets (trap 3). The four parent tracer traps for methanol were immersed in an ice bath and consisted of 1M NaOH (trap 1), 2M NaOH in saturated NaCl (trap 2), NaOH pellets (trap 3), and silica gel (trap 4). Extra traps were required for methanol due to its inherent volatility.

Downstream of the combustion oven, the evolved ¹⁴C-CO₂ was collected in a trap of 1.5 mL of 3-methoxypropylamine (Thermo Scientific, 99% purity, catalog number #AAB24095AU) and 4 mL of scintillation cocktail (Research Products International, Bio-SafeII, catalog number 111195) after passing through an empty trap that was in place to eliminate sample backflow into the combustion column. After one hour, the content of the final trap was transferred to a 7 mL scintillation vial and the ¹⁴C-activity was quantified using a liquid scintillation counter. Recovery tests with ¹⁴C-CH₄ showed that we recovered > 90% of the known activity of added. Blanks (empty tubes) were included in each sample set served to identify any carryover or contamination between runs.

The rate of ¹⁴C-substrate turnover to ¹⁴C-methane, the turnover constant, k, was calculated using the activity of the produced methane (¹⁴C-CH₄; disintegrations per minute (dpm)), the activity of the substrate added to the sample (¹⁴C-substrate; dpm), and the incubation period (t; days) (Equation 1). Potential rates of methanogenesis (pmol cm^-3 day^-1) were calculated using the equation below, where the turnover constant k (day^-1) was multiplied by the porosity-corrected substrate concentration in nmol cm^-3 wet sediment which was derived from multiplying the substrate concentration, nmol mL^-1, by the water content, mL water cm^-3 wet sediment, assuming that the mass of water in grams is equivalent to its volume in mL or cm^-3, and the isotopic fractionation factor, α. The following fractionation factors were used: 1.02 for acetate; 1.04 for bicarbonate; 1.07 for methanol; 1.06 for methylamine; 1.02 for formate) were used (Krzycki et al. 1987, Summons et al. 1998, Whiticar 1999). A factor of 1000 was applied to convert the rate from nmol cm^-3 day^-1 to pmol cm^-3 day^-1.

Methanogenesis rate (pmol cm^-3 d^-1) =
turnover constant (d^-1) x [substrate] (nmol cm^-3) x α x 1000 (pmol nmol^-1)

Sample counts were corrected for the killed control by subtracting the minimum detectable dpm (average dpm of killed controls plus three times the standard deviation of the killed controls, dpm) from the measured ¹⁴C-CH₄ activity (dpm) of each live sample. Samples with ¹⁴C-CH₄ activity (dpm) less than the minimum detectable dpm were considered to be below the detection limit. 

The detection limits for the different methanogenesis rates were: 1.26 pmol cc⁻¹ day⁻¹ for H₂/CO₂, 0.05 pmol cc⁻¹ day⁻¹ for acetate, 0.01pmol cc⁻¹ day⁻¹ for formate, 0.03 pmol cc⁻¹ day⁻¹for methanol, and 0.01 pmol cc⁻¹ day⁻¹ for methylamine.