Award: EAR-1852912

We investigated the presence of cryptic methane cycling, which is the simultaneous production and consumption of methane by microbes, in sediment of a coastal wetland (Carpinteria Salt Marsh Reserve (CSMR), southern California). According to our scientific hypothesis, methane is produced in the sediment by methanogens from non-competitive, methylated compounds in the zone of sulfate reduction; however, the emission of this potent greenhouse gas into the atmosphere is hampered by prompt anaerobic oxidation of methane (AOM). Major project goals included: (1) detection and quantification of cryptic methane cycling in surface sediment of the CSMR, (2) identification of electron acceptors for microbial AOM, (3) identification of microbial key players involved in methane cycling, and (4) environmental controls of methane emission into the atmosphere. For the detection and quantification of cryptic methane cycling we adapted a radiocarbon tracer method, which allowed us to follow the flow of carbon from a methylated substrate (methylamine) to methane via methanogenesis, and finally to inorganic carbon (CO2) via AOM. By applying this method, we demonstrated the presence of cryptic methane cycling in surface sediment along a salinity gradient (brackish, marine, hypersaline) in the CSMR. The process appears to prevent major buildups of methane in the sediment and mitigates eventual emission into the atmosphere. By combining analyses of intact sediment cores with in-vitro sediment experiments, we detected that AOM activity can be linked to both sulfate and iron reduction, depending on the availability of the terminal electron acceptor. Results from sequential iron and sulfur extractions indicate that poorly crystalline iron (III) minerals were highly available in the subsurface sediment of the hypersaline station, where AOM rates peaked with no detectable sulfate reduction rate. This finding suggests the activity of iron-dependent AOM. In-vitro experimentation with 14C and 35S radiotracers further confirmed that iron (III) acts as the electron acceptor for AOM in iron-rich sediments. In sulfidic sediments, however, sulfate was identified as the dominant electron acceptor for AOM. Across the salinity gradient, molecular analyses of sediment detected methylotrophic methanogenic archaea alongside groups of sulfate-reducing bacteria. Surprisingly, we found only low abundances of the classical anaerobic methanotrophic (ANME) archaea, suggesting that other microbial groups could contribute to methane consumption. Molecular analysis further revealed groups of bacteria that have been associated with methylotrophic activity and thus could play a role in methylamine consumption beside methanogens. The application of in-situ emission chamber incubations with a coupled greenhouse gas analyzer revealed consistent results over seasonally differing field campaigns: We detected highest methane fluxes into the atmosphere at the marine station, moderate fluxes at the brackish station, and lowest fluxes at the hypersaline station. We analyzed a series of environmental factors to illustrate the controls on methane emissions. Our preliminary results suggest that a combination of large grain size and high methanogenesis rate leads to elevated methane emissions at the marine station, whereas the cryptic methane cycling dominates at the hypersaline and brackish stations where porewater methane concentrations were near the detection limit. We also identified a salinity control on methane emissions at the hypersaline station, whose salinity fluctuates over the year depending on rainfall and evaporation, revealing higher methanogenesis rates under lower salinity conditions. Finally, we developed a simple method to extract adequate amounts of methane from large volumes (5 Liter) of methane-poor sediment for methane isotopologue analysis with UCLA's Panorama mass spectrometer. Our goal was to identify a methane isotopologue signature characteristic for cryptic methane cycling. We also incubated sediment slurries from replicate samples with methanogenic substrates and AOM inhibitors to study the (unaltered) methane isotopologue signature generated by the indigenous methanogenic community. Our results suggest that the isotopologue signatures of natural methanogenesis at the CMSR are like those produced from pure culture experiments with methylotrophic methanogens, while the in-situ methane gas was highly altered by AOM because of cryptic methane cycling. The clumped isotope signature of methane in coastal sediments is dictated by the balance between methanogenesis and AOM, and it provides a unique lens to track metabolic pathways of methane cycling when rate information is not readily available. In addition to our studies in the CSMR, we applied the newly adapted radiotracer method to track cryptic methane cycling in surface sediments of the nearby Santa Barbara Basin. We detected the presence of cryptic methane cycling at all investigated stations (440 to 590 m water depth), suggesting that the process is widespread from shallow to deep benthic environments providing that substrates are available for methanogenesis and electron acceptors for AOM. In summary, our project deciphered mechanisms of cryptic methane cycling in coastal and deep marine environments and highlighted the importance of this process for controlling methane emissions from surface sediment into the atmosphere. Last Modified: 09/07/2023 Submitted by: Tina Treude