Dataset: Middle Valley Chimney Sulfate Reduction Rates
Deployment: AT15-67

Sampling and Analytical Methodology: Once on board ship, samples were directly transferred to sterile anaerobic
seawater and handled/processed using appropriate sterile microbiological techniques. Subsamples were immediately transferred to gastight jars (Freund Container Inc.), filled with sterile anaerobic seawater containing 2 mM sodium sulfide at pH 6, and stored at 4 degrees C. Upon return to the laboratory, all samples were maintained in anaerobic seawater
(0.2 um filter-sterilized prior to use) supplemented with 2mM ΣH2S (defined as the sum of H2S, HS- and S2-) and adjusted to pH 6. The vent-like media was replenished every 8 to 12 weeks, and all samples were kept in the dark and 4 degrees C prior to incubation. Hydrothermal deposits were homogenized in a commercial blender (Xtreme™
blender, Waring Inc.) under a nitrogen atmosphere. Anaerobic homogenization was designed to minimize fine-scale geochemical and microbial heterogeneity and facilitate more accurate experimental replication. Hydrothermal homogenate (made up of both mineral deposit and interstitial fluid)) was aliquoted volumetrically (7.5 mL, ca. 29 g wet weight and ca. 20 g dry weight) into Balch tubes in an anaerobic chamber. The tubes were supplemented with 15 mL of sterile artificial vent fluid media designed to mimic the geochemical composition of fluids within the pores of a sulfide deposit (pH 6, 14 mM SO42-, 2.3 mM NaHCO3, 1 mM H2S, and 10 uM each of pyruvate, citrate, formate, acetate, lactate). Organic acid concentrations are comparable to those measured in situ along the Juan de Fuca ridge (Lang et al. 2006). Sufficient 35SO42- was added to achieve 555 kBq (15 uCi) of activity. Due to technical difficulties with post processing methodology, shipboard incubations using fresh material were not successful. The data presented here were generated using samples that had been maintained in sulfidic ventlike effluent (as described above) for one year. Samples were incubated anaerobically for 7 days at 4, 30, 40, 50, 60, 80 and 90 degrees C. Controls for sulfate reduction consisted of samples amended with 28 mM molybdate, a competitive inhibitor of sulfate reduction (Saleh et al. 1964; Newport & Nedwell, 1988). Six biological replicates were run for each treatment, and three biological replicates for each control. Upon completion, reactions were quenched with the injection of 5 mL 25% zinc acetate (which is ~20-fold more Zinc than the maximum sulfide concentration), and all samples were frozen at -20 degrees C to enable further analysis.

1 gram (wet weight) of crushed mineral (about 60% mineral, 30% interstitial fluid) was added to 10 mL of a 1:1 ethanol to water solution in the chromium distillation apparatus, and then degassed with nitrogen for 15 minutes to achieve anaerobicity. 8 mL of 12 N HCl and 16 mL of 1 M reduced chromium chloride was added anaerobically to the chamber and gently heated to a slow boil for 3 hours to evolve hydrogen sulfide gas. The resulting sulfide gas was carried via nitrogen gas through a condenser to remove any ethanol or water vapor, and was then trapped as zinc sulfide in a 25% zinc acetate solution. The radioactivity of the resulting sulfide (Zn35S) and the remaining sulfate from the supernatant (35SO42-) were measured via liquid scintillation counter in Ultima Gold scintillation cocktail (ThermoFisher Inc., Waltham, MA).