Dataset: Sulfur cycling and sulfur stable isotopes 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 between September and November of 2019. 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 U1545B is considered a reference site since it was free of sill intrusions and unaffected by active hydrothermal circulation. The geothermal gradient in hole U1545B was 227ºC/km. The geothermal gradient in hole U1546C, 221ºC/km, was similar to that measured in hole U1545B. Sites U1547 and U1548 were 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  (27º30.413’N, 111º40.734’W, water depth 1585.6m) exceeded 50°C in the upper 50 mbsf. The geothermal gradient in the U1547B hole was between 529ºC. The geothermal gradient in the U1548C hole was between 958ºC. 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. Site U1550 (27°15.170′N, 111°30.445′W, water depth 2001.2 ) 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 U1550B was 135ºC/km. These sites are described in more detail in Teske et al. (2021).

To preserve sediment for solid-phase sulfur analysis and incubation experiments, sectioned core samples were placed in N₂-filled gas-tight bags, and subsequently transferred to anoxic glass bottles and stored at 4℃ until further subsampling. Porewater concentrations of sulfate, methane and sulfide, and solid phase parameters, including total organic carbon and total sulfur, were analyzed onboard using established IODP protocols (Teske, 2021). These data were obtained from the IODP database at https://web.iodp.tamu.edu/OVERVIEW/ (Teske, 2021). Briefly, methane concentrations were determined using a gas chromatograph (Agilent 7890A), sulfate concentrations were analyzed by ion chromatography (Metrohm 850 Professional IC), and sulfide samples were fixed with zinc acetate solution and measured spectrophotometrically (Cline, 1969). For total carbon (TC) and total sulfur (TS) contents, samples were freeze-dried and powdered, and ~15 mg was accurately weighed. Analysis was conducted using an elemental analyzer (Thermo Finnigan FlashEA 1112 CHNS). Inorganic carbon (IC) content was measured using a Coulometrics 5012 CO₂ coulometer. Freeze-dried and powdered sediments was reacted with 6.5 mL of 2 mol L^–1 HCl in a glass vial to liberate CO₂. Total organic carbon (TOC) content was quantified by the subtraction of IC from TC content. The statistical ANOVA of TOC to TS (C/S) ratios at the six sites was performed using Origin 2025 software (OriginLab Corporation). Significance was determined at a threshold of p < 0.05.

For solid-phase sulfur species extraction, ~1.5 g subsamples were transferred to centrifuge tubes containing  5% zinc acetate solution. After centrifugation to remove the supernatant, elemental sulfur (ES) was first extracted via sustained agitation for 15 hours with 30 mL 100% methanol (Zopfi et al. 2004). Dissolved ES was quantified via reversed-phase liquid chromatography (Agilent, 1260 Infinity III) with a C-18 column (Agilent ZORBAX Eclipse XDB-C18), using 100% methanol as the eluent. The pump speed was set to 1 mL min^–1, and absorbance at 265 nm was measured via UV detection. The detection limit for this method is <1 µmol L^–1, and the analytical precision (relative standard deviation, RSD) was better than 0.7%.

The separated solid residue was subsequently treated with a cold chromium distillation procedure (Kallmeyer et al. 2004, Røy et al. 2014), where acid volatile sulfur (AVS) was released with 6 mol L^–1 HCl, and chromium reducible sulfur (CRS, mainly pyrite) was sequentially extracted with an acidified chromium chloride solution (CrCl₂). The sulfide liberated from these solid-phase sulfur species was trapped by 5% (w/v) zinc acetate to form ZnS precipitate and then the concentration of each species was determined spectrophotometrically (Cline, 1969).

Reactive iron was extracted using a citrate-dithionite buffer (Raiswell et al. 1994). 0.2 g sediment subsample was extracted with 50 mL 50 g L^–1 sodium dithionite in pH 4.8 buffer at room temperature for 2 hours. The concentration of iron released was determined spectrophotometrically using the ferrozine method (Stookey 1970). The degree of pyritization (DOP) was used to determine the effects of iron limitation on pyrite formation and the calculation formula was as follows (Berner 1970):

DOP = Pyrite Fe / (Pyrite Fe + Reactive Fe)

For porewater sulfate sulfur isotope analysis, a thawed pore fluid volume (give volume) was fixed with saturated barium chloride solution to precipitate sulfate as barium sulfate. The BaSO₄ was then rinsed with 6 mol L^–1 HCl and Milli-Q water. After determination, the precipitated zinc sulfide from CRS was subsequently converted to Ag₂S by addition of silver nitrate and ammonium hydroxide to the recovery vessel. Cleaned and dried BaSO₄ and Ag₂S was mixed with excess V₂O₅ and the stable sulfur isotopic ratio was measured on an isotope ratio monitoring mass spectrometer (IRMS; Thermo Delta V Plus) coupled to a Flash elemental analyzer. Standard deviations were better than 0.3‰ (n ≥3), estimated using one International Atomic Energy Agency (IAEA-3) standard (–32.30‰) and two national (China) first level sulfur isotope standards (–0.07‰ and 22.15‰), respectively. The stable sulfur isotope values, δ³⁴S, were calculated using the following formula:

δ³⁴S = [{(³⁴S/³²S)_sample/(³⁴S/³²S)_standard} - 1] × 1000

The units of δ³⁴S are per mil (‰) and are reported relative to the Vienna Canyon Diablo Troilite (VCDT) standard.  

For the determination of potential rates of sulfate reduction (SR) and anaerobic oxidation of methane (AOM), 10 g sediment subsamples were weighed in a N₂-filled glove box and transferred into 120 mL glass vials. Next, 40 mL artificial seawater medium was added and the vials were closed with blue butyl rubber stoppers and crimped. The seawater media for sediment slurry incubations were prepared as follows: 200 mg KH₂PO₄, 250 mg NH₄Cl, 25 g NaCl, 0.5 g MgCl₂ × 6H₂O, 0.5 g KCl and 150 mg CaCl₂ × 2H₂O, dissolved with 1 L Mill-Q water in a glass bottle (Heuer 2017). Sediment samples collected from above the sulfate-methane transition zone (SMTZ) were supplemented with 5 mmol L^–1 sulfate in the artificial seawater medium, whereas samples from below the SMTZ were maintained without the addition of Na₂SO₄. After autoclaving, 5 mL sterile-filtered 154 mM Na₂S and 1 M NaHCO₃ was added to the media to assure anoxia and to modulate the pH, respectively.

After pre-incubation, ~3 mL sediment slurry was introduced into a modified cut-end Hungate tube sealed with a gray chlorobutyl stopper and a screw cap. 100 µL of radiolabeled ³⁵S-Na₂SO₄ solution (~167 kBq) and ¹⁴C-CH₄ solution (~17 kBq) was injected into the quadruplicate sediment samples (one killed control and three live samples) for SR and AOM rate measurements, respectively. Samples were incubated concurrently under in situ temperature for 20 days. After incubation, microbial activities of SR and AOM were terminated by injecting 3 mL 20% (w/v) zinc acetate and 3 mL 2 mol L^–1 NaOH, respectively.

Zinc acetate-fixed slurry samples were transferred to falcon tubes and then centrifuged. The supernatant was obtained to count unreacted ³⁵S-SO₄²- radioactivity, and the pool of total reduced sulfur species in the remaining sediment was recovered by the cold chromium distillation (Røy et al. 2014). The extracted sulfide was trapped with 5% (w/v) zinc acetate solution to measure ³⁵S-H₂S radioactivity. The sulfate reduction rates were calculated from the following formula:

SRR = {(³⁵S-H₂S ) / [(³⁵S-H₂S ) + (³⁵S-SO₄^2- )]} ×  [SO₄^2- ] × 1.06 / t × 1,000,000

where SRR is the sulfate reduction rate presented in pmol cm^–3 day^–1; (³⁵S-H₂S) is the measured ³⁵S-H₂S radioactivity (decays per minute, dpm); (³⁵S-SO₄^2-) is the radioactivity of unreacted ³⁵S-SO₄^2- (dpm); [SO₄^2-] is the total sulfate concentration of slurry (i.e. sediment porewater sulfate plus added sulfate times the porosity); 1.06 is the correction factor for the expected isotopic fractionation; t is incubation time in days; 1,000,000 is the unit conversion factor. The units of sulfate reduction are pmol cm^-3 d^-1.

For AOM rate measurements, ¹⁴C-CO₂ production from ¹⁴C-CH₄ was quantified with the acid digestion method (Joye et al. 2004, Zhuang et al. 2019). Briefly, homogenized sediment slurries were transferred to a 250 mL glass bottle and purged with air to remove unreacted ¹⁴C-CH₄. Then 5 mL of 30% (v/v) sulfuric acid was added to the bottle and then liberated ¹⁴C-CO₂  was trapped with 3-methoxypropylamine. AOM rates were calculated using the following equation:

AOM= {(¹⁴C-CO₂) / [(¹⁴C-CO₂) + (¹⁴C-CH₄ )]} ×  [CH₄ ] × 1.06 / t × 1,000

where AOM is the anaerobic oxidation of methane calculated in pmol cm^–3 day^–1; (¹⁴C-CO₂) is the ¹⁴C-CO₂ counts (dpm); (¹⁴C-CH₄) is the total injected ¹⁴C-CH₄ (dpm); [CH₄] is the methane concentration of sediment slurry (µmol L^–1); 1.01 is the carbon isotopic fractionation correction factor; t is incubation duration in days; 1,000 is the unit conversion factor. The units of anaerobic oxidation of methane are pmol cm^-3 d^-1.

The reported microbial activity rates reflected the potential metabolic capacity of the in situ microbial community within the sediment, constrained by ambient geochemical conditions and substrate availability.