Anaerobic oxidation of methane (AOM) is hypothesized to occur through reverse hydrogenotrophic methanogenesis in marine sediments because sulfate reducers pull hydrogen concentrations so low that reverse hydrogenotrophic methanogenesis is exergonic. If true, hydrogenotrophic methanogenesis can theoretically co-occur with sulfate reduction if the organic matter is so labile that fermenters produce more hydrogen than sulfate reducers can consume, causing hydrogen concentrations to rise. Finding accumulation of biologically-produced methane in sulfate-containing organic-rich sediments would therefore support the theory that AOM occurs through reverse hydrogenotrophic methanogenesis since it would signal the absence of net AOM in the presence of sulfate. Methods16S rRNA gene libraries were compared to geochemistry and incubations in high depth-resolution sediment cores collected from organic-rich Cape Lookout Bight, North Carolina. ResultsWe found that methane began to accumulate while sulfate is still abundant (6–8 mM). Methane-cycling archaeaANME-1,Methanosarciniales, andMethanomicrobialesalso increased at these depths. Incubations showed that methane production in the upper 16 cm in sulfate-rich sediments was biotic since it could be inhibited by 2-bromoethanosulfonoic acid (BES). DiscussionWe conclude that methanogens mediate biological methane production in these organic-rich sediments at sulfate concentrations that inhibit methanogenesis in sediments with less labile organic matter, and that methane accumulation and growth of methanogens can occur under these conditions as well. Our data supports the theory that H2concentrations, rather than the co-occurrence of sulfate and methane, control whether methanogenesis or AOM via reverse hydrogenotrophic methanogenesis occurs. We hypothesize that the high amount of labile organic matter at this site prevents AOM, allowing methane accumulation when sulfate is low but still present in mM concentrations. 
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                    This content will become publicly available on January 2, 2026
                            
                            Terrestrial Organic Matter Amplifies Methane Emissions Across Sediments of the Mississippi River Headwaters
                        
                    
    
            Abstract Terrestrial organic matter (tOM) plays a critical role in aquatic ecosystems, influencing carbon processes and greenhouse gas emissions. Here, we investigate the impact of tOM on methane production in littoral and pelagic sediments from the Mississippi River headwaters using a microcosm approach. Contrary to our expectations, tOM addition universally increased methane production across lentic sediments, with no significant difference between littoral and pelagic zones. Methane production was influenced by select sediment microorganisms, primarily methanogens and lignocellulose degrading bacteria, which responded similarly across different sediment habitats. The study highlights the role of cytochrome-containing methanogens and their syntrophic relationships with fermentative bacteria, emphasizing the significance of microbial community structure in sediment methane dynamics. Our findings suggest that increasing tOM loads to freshwater systems could have broader implications for methane emissions, driven by specific microbial interactions. Author Contribution StatementHMS and TLH conceived the study and obtained the funds. HMS led fieldwork and microcosm set-up. HMS and LAD analyzed gas samples and HMS performed the data analysis and graphical representation of the results. HMS wrote the first draft of the manuscript, and all authors contributed significantly to the preparation of the final draft. Scientific Significance StatementAs human activities and climate change increase the amount of organic material entering lakes and rivers, understanding the effects this has on greenhouse gas emissions is crucial. Our study reveals that adding terrestrial organic matter to freshwater sediments universally boosts methane production, a potent greenhouse gas. Through the exploration of microbial communities responsible for this process, our research highlights how changes in terrestrial organic matter export to aquatic systems could increase methane emissions from sediments. Data Availability StatementAdditional Supporting Information can be found in the online version of this article, including an extended version of methods and supplementary tables. Sequencing data associated with this paper is available on NCBI, BioProject PRJNA1164797. 
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                            - Award ID(s):
- 1948058
- PAR ID:
- 10583272
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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