Dataset: Sediment geochemistry and microbial metabolic activity data on cryptic methane cycling in the Carpinteria Salt Marsh Reserve, California from sampling in July of 2019

The top 15 to 20 cm of sediment at each station was collected in large (10 cm i.d.) and small (2.6 cm i.d.) polycarbonate push cores. Push cores were carefully inserted into sediment by hand including approximately 5 cm overlying water. Large push cores were inserted approximately 20 cm from each other, while small push cores were inserted approximately 15 cm from each other to provide sufficient space for push core extraction. Sediment surrounding the push cores was carefully removed to place a metal plate under the bottom to safely extract the push cores. Any air headspace within the push cores were filled bubble-free with overlying-water from the station location along the creek or hypersaline pool and sealed with rubber stoppers and electrical tape. Sediment push cores were transported to the home laboratory on the same day, stored in the dark at room temperature and processed within 1 d to 1 week of collection, depending on the analysis type.

One day after collection, one large push core from each station was selected for porewater geochemistry analysis. At all stations the top layer of the sediment was sliced at 1.5 cm followed by 1 cm increments due to natural slopes found at the sediment surfaces during the time of sampling. All sediment was sliced under a constant flow of argon gas to minimize oxidation of oxygen-sensitive substrates. The sediment sections were transferred to pre-argon flushed 50 mL centrifuge vials and centrifuged at 4300 g for 20 mins. Immediately after centrifugation, the separated porewater was analyzed spectrographically for dissolved sulfide according to (Cline, 1969) and iron (II) according to (Grasshoff et al., 1999) using a Shimadzu UV-Spectrophotometer (UV-1800) equipped with a sipper unit. The remaining porewater was frozen (-30 °C) and later measured for dissolved porewater sulfate and chloride concentrations. 

Porewater sulfate and chloride was determined using an ion chromatograph (Metrohm 761) (Dale et al., 2015). Analytical precision of these measurements was <1% based on repeated analysis of IAPSO seawater standards. Absolute detection limit of sulfate was 1 mM, which corresponds to 30 mM in the undiluted sample. Porewater salinity at each station was calculated from chlorinity using Knudson’s equation (Salinity = 1.805 * Chlorinity) assuming that the major ionic ratios in the porewater and in seawater are similar (Knudsen, 1901). 

For methane concentrations, porosity/density, solid-phase carbon/nitrogen, and molecular analysis, a separate large push core from each station, the top 1.5 cm was sliced followed by 1 cm increments because of natural slopes at the sediment surface found at the time of sampling. For methane concentrations, 2 mL of sediment at each interval was subsampled using a 3 mL cut-off plastic syringe and transferred to a 12 mL glass serum vial filled with 5 mL of 5% NaOH and sealed with grey butyl rubber stoppers. Headspace methane concentrations were later determined using a Shimadzu gas chromatograph (GC-2014) equipped with a packed Haysep D and flame ionizer detector. The column was heated to 80 °C and ultra-high pure helium was used as the carrier gas, set to 12 mL per minute. A methane standard (Scotty Analyzed Gases) was used to calibrate for methane concentrations with a ± 5% precision.

For porosity and density, 8 mL of sediment was collected from each 1 cm layer using a 10 mL plastic cut-off syringe, transferred to pre-weighed plastic 10 mL vials (Wheaton). The wet samples were then weighed and then stored at 4 °C. The samples were later dried at 75 °C for 72 hrs and then reweighed. Sediment porosity was determined by subtracting the dry sediment weight from the wet sediment weight and dividing by the total volume. Sediment density was determined by dividing the wet weight by the total volume of the sample.

Within the one day of collection, one small sediment whole round push core from each station was used to determine sulfate-reduction rates (SRR)  at the home laboratory. Radioactive, carrier-free 35S- SO42- sulfate (35S-SO42-; dissolved in MilliQ water, injection volume 10 µL, activity 260 KBq, specific activity 1.59 TBq mg-1 ) was injected into the whole-round cores at 1 cm intervals and incubated at room temperature and in the dark following (Jørgensen, 1978) . The incubation was stopped after ~24 hours. Sediment samples (including controls) were transferred, preserved, and stored according to Krause and Treude (2021).  Samples were analyzed using the cold-chromium distillation method and the results from the analysis were used to calculate the sulfate reduction rates according to (Kallmeyer et al., 2004).

The present study aimed to follow the methane production by methanogenesis from mono-methylamine (hereafter abbreviated MG-MMA) and the subsequent oxidation of the methane to dissolved inorganic carbon (DIC) by anaerobic oxidation of methane (hereafter abbreviated AOM-MMA) (i.e., cryptic methane cycling) in salt marsh sediments across a salinity gradient. To find evidence of concurrent MG-MMA and AOM-MMA, one small whole round core from each station was injected with radiolabeled 14C-mono-methylamine (14C-MMA) (14C-mono-methylamine dissolved in 1 mL water, injection volume 10 µL, activity 220 KBq, specific activity 1.85-2.22 GBq mmol-1) at 1-cm intervals according to (Krause and Treude, 2021)  and stored at room temperature and in the dark for 24 hrs. Incubations were terminated by slicing the sediment at 1-cm intervals into 50 mL wide-mouth glass crimp vials filled with 20 mL of 5% NaOH. After transfer of the sample, vials were immediately sealed with a red butyl stopper and crimped with an aluminum crimp. Control samples were prepared by sectioning the top 5 cm of a separate whole round core from each station in 1-cm intervals into 50 mL wide mouth vials filled with 20 mL of 5% NaOH prior to radiotracer addition. Vials were shaken thoroughly for 1 min to ensure complete biological inactivity and stored upside down at room temperature till further processing. The residual 14C-MMA in the liquid, the 14C-CH4 in the headspace of the sample vials produced by MG-MMA, and the 14C-TIC in the sediments as a result of AOM-MMA samples were determined by the analysis according to (Krause and Treude, 2021). 

To account for the 14C-MMA binding to mineral surfaces (Wang and Lee, 1993, 1994; Xiao et al., 2022), we determined the recovery factor (RF) for the sediment from stations BL, BH and M following the procedure of Krause and Treude (2021). For the HP station, the RF factor previously determined by Krause and Treude (2021) was applied. 

Estimates of metabolic rates of MG-MMA and AOM-MMA were calculated from the results of the 14C-MMA incubations. Natural concentrations of mono-methylamine in the sediment porewater were detectable (> 3 µM) but were below the quantification limit (10 µM). To enable rate calculations for MG-MMA (Eq. 1), we assumed an MMA concentration of 3 µM for all samples, i.e., the detection limits of the NMR analysis. For calculation, please see Krause and Treude (2021). Results from the 14C-MMA incubations were also used to estimate the AOM-MMA rates according to Krause and Treude (2021).

AOM rates from 14C-CH4 (AOM-CH4) were determined by injecting radiolabeled 14C-CH4 (14C-CH4 dissolved in anoxic MilliQ, injection volume 10 µL, activity 5 KBq, Specific activity 1.852.22 GBq mmol-1) directly into a separate small whole round core from each station at 1-cm intervals. Incubations were stopped after ~24 hours and stored at room temperature until further processing, similar to section 2.6. Sediments were then analyzed in the laboratory using oven combustion (Treude et al., 2005) and acidification/shaking (Joye et al., 2004). The radioactivity captured after the headspace combustion and acidification and shaking analysis were determined by liquid scintillation counting. 

Metabolic data from radiotracer incubations were used to calculate metabolic rate constants (k) to compare relative turnover of MMA and CH4. We define the rate constants as the metabolic products divided by the sum of the metabolic reactants and products, divided by the incubation time (Krause et al., 2023).