Methylotrophic methanogenesis occurring within sulfate-rich zones of coastal and marine sediments is functionally linked to anaerobic methane oxidation (AOM), together constituting a cryptic methane cycle. This investigation presents data on such cryptic methane cycling across a land-to-sea transect comprising four sampling sites within the Carpinteria Salt Marsh Reserve (CSMR) in southern California, USA: two brackish, one marine, and one hypersaline. Surface sediments (upper 20 cm) were examined using geochemical profiling and radiotracer incubation experiments employing 35S-labeled sulfate, 14C-labeled monomethylamine, and 14C-labeled methane.   Methane concentrations in sediment porewater were generally low (3–28 µM) across all sites, except at the marine station, where levels increased with depth, reaching up to 665 µM. Methane production from monomethylamine was detected throughout the depth profiles at all stations, with estimated rates ranging from sub-nanomolar to nanomolar per cubic centimeter of sediment per day. AOM, quantified via 14C-CH₄ tracer, co-occurred with methylotrophic methanogenesis at each station, exhibiting activity levels between 0.03 and 19.4 nmol cm⁻³ d⁻¹.   Porewater chemistry revealed elevated concentrations of sulfate and dissolved iron across all sites. Sulfate levels (9–91 mM) remained sufficiently high to support sulfate reduction, which showed activity levels from 1.5 to 2,506 nmol cm⁻³ d⁻¹. Vertical profiles of sulfide and Fe(II) suggested a geochemical shift along the transect—from iron-dominated reduction at the brackish stations to sulfate-dominated reduction at the marine and hypersaline stations. AOM activity overlapped with zones of sulfate reduction and Fe(II) enrichment, indicating that methane oxidation may be coupled to both sulfate and iron reduction across all sampling locations.

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

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).

* Sheet 1 of submitted file "Krause et al., CSMR2019_datasets_BCO-DMO_Revised.xlsx" was imported into the BCO-DMO data system for this dataset. Values "NA" imported as missing data values. Table will appear as Data File: 965250_v1_cryptic-ch4-cycling.csv (along with other download format options).

Missing Data Identifiers:
* In the BCO-DMO data system missing data identifiers are displayed according to the format of data you access. For example, in csv files it will be blank (null) values. In Matlab .mat files it will be NaN values. When viewing data online at BCO-DMO, the missing value will be shown as blank (null) values.

* Column names adjusted to conform to BCO-DMO naming conventions designed to support broad re-use by a variety of research tools and scripting languages. [Only numbers, letters, and underscores. Can not start with a number]

* ISO DateTime with timezone (UTC) column added

* latitude and longitude rounded to 5 decimal places

Coverage: Carpinteria Salt Marsh Reserve, California, USA

NSF Award Abstract:
This research project investigates the close relationship between methane production and consumption in sediments by microbes in the oxygen-free zone of a coastal wetland in Southern California. The direct exchange of methane between those two closely related microbial groups, which has been named "cryptic methane cycling", has only recently been identified and little is known about its importance in reducing methane emissions from coastal wetlands. This research will reveal how carbon moves between the two types of microbes, and will identify the microbes involved. It will also reveal the important metabolic reactions responsible and the balance between methane production and consumption under different environmental conditions. Towards broader impacts, this project will provide training for two undergraduate and one graduate student in interdisciplinary wetland science and state of the art laboratory methods. A new freshmen course on global methane emissions will bring undergraduate students into the field to provide education on local coastal wetland environments. Results of the project will be disseminated to public and academic groups and will provide a better understanding of methane production and consumption in coastal wetlands.

Concentrations of atmospheric methane have more than doubled since the pre-industrial era, hence we urgently need to understand the mechanisms that control the emission of this potent greenhouse gas. Recent studies have provided the first evidence for the simultaneous microbial production and consumption of methane in the sulfate reduction zone of organic-rich sediments, a process named the "cryptic methane cycle." In this process, methane is proposed to be passed directly from methylotrophic methanogenesis to anaerobic oxidation of methane (AOM). However, little is known about the identity of the organisms involved, the trail of carbon from one metabolism to the other, or the environmental net result of the two processes. Without the details of this metabolic relationship, methane budgets of sediments remain incomplete. Coastal wetlands are of particular interest for the study of cryptic methane cycling, because their organic and sulfate rich sediments promote the production of methylated substrates for methylotrophic methanogenesis and provide electron acceptors for AOM. Yet, anaerobic microbial removal of methane from this ecosystem, particularly along the sulfate gradient between ocean and land, is still not well understood. Towards intellectual merit, this study elucidates the identity of methanogenic and methanotrophic archaea involved in cryptic methane cycling in a coastal wetland as well as the selection of electron acceptors that fuel methane removal in this metabolic relationship. The research provides new metabolic clues to unravel the versatility of the enzymatic machinery that drives methanogenesis and AOM. By capturing environmental factors that control the balance between the two processes working in close proximity, the results of this work further provide an enhanced understanding of methane dynamics in coastal wetland sediments. This information can be applied to biogeochemical models to improve the prediction of methane emissions from this ecosystem, which is found throughout the global coastal zone. Towards broader impacts, the research provides training in innovative analytical and experimental techniques to two undergraduate and one graduate student. The project further engages 20 undergraduates per year in a newly developed freshman seminar, "Methane - the Other Greenhouse Problem" including a field trip to the local wetland. The goal of the seminar will be to educate students, including those not entering STEM fields, about global sources and sinks of methane and its involvement in global warming now and in the future. Results of this study will be broadly disseminated to educational and public outreach platforms, such as high schools, community colleges, and environmental non-profits to teach scientific methodologies, concepts of biogeochemical cycling, and enhance the appreciation of this vital coastal environment.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.