Our approach provided strong evidence for the taxa responsible for methane
oxidation. The Tonya Seep harboured several taxa potentially capable of methane oxidation under both aerobic and anaerobic conditions. click here This suggests that the sediment is a robust methane filter, where taxa presently dominating this important process could be replaced by less abundant taxa should the environmental conditions change. Methods Sampling site Tonya Seep (34°24.043′N; 119°52.841′W) is located in the Coal Oil Point seep field offshore Santa Barbara, California, USA. Tonya Seep is primarily a single 2 m diameter pit with many vents inside that rapidly coalesce into a single plume. There was a high content of hydrocarbons and Crenigacestat order tar in the sediments. Four sediment cores, two for methane oxidation
studies and two for metagenomic analysis, were collected at 25 m depth on July 16th 2008 by UC Santa Barbara Marine Operation divers. The polycarbonate liners used (30 cm length and 3.5 cm diameter) were treated with 70% ethanol and dried before sampling. The parallel cores (core I, II, III and IV) were sealed at the seafloor and kept on ice during transportation back to shore. Gas Sample Collection Two seep gas samples (Gas samples I and II) were collected in the surface waters above the seep. The samples were collected on two occasions from small vessels via an inverted funnel method in which seep gas bubbles were captured into 120 mL glass serum Sclareol vials after rising through the water column. Bottles were capped underwater after filling to avoid contamination with atmospheric gases. Seep gases were analyzed by gas chromatography as previously described . Error associated with the concentration measurements was ±4%. Methane oxidation rates Cores III and IV designated for methane
oxidation rate (MOR) measurements were injected with radiotracer 14C-CH4 (1 kBq 14CH4 dissolved in water, 20 μL injection volume) at 2 cm intervals and incubated at near in-situ temperature. After 18 hours the core was sub-sectioned and placed into vials with 1 M NaOH and quickly sealed, ending the incubation and trapping the CO2. A small sample of headspace (0.2 mL) was removed to determine CH4 concentration (which is not affected by the 14CH4 spike) by GC-FID (Shimadzu GC-4A, 6 ft length 80/100 mesh Molsieve 13X packed column run isothermally at 140°C with N2 carrier flow at 15 mL min-1). The remaining 14CH4 in the headspace of the vial was purged via a slow flow of air through a combustion tube filled with Cu(II)-oxide and maintained at 850°C. The resulting 14CO2 was trapped using a mixture of phenethylamine and 2-methoxyethanol. The remaining 14CO2, which was assumed to be microbially produced, was Selleck Duvelisib measured by first transferring the sediment into a 100 mL Erlenmeyer flask fitted with a small (7 mL) phenethylamine/NaOH-filled scintillation vial suspended beneath its rubber stopper.