Imprint of eutrophication on methane-cycling microbes in freshwater sediment

Abstract. Eutrophication can alter methane (CH₄) cycling in lakes, yet its long-term effect on sediment microbial communities remains unclear. To elucidate these effects, we analyzed a 400-year-old sediment record from the historically eutrophied Lake Joux, Switzerland, combining porewater and solid-phase geochemistry with 16S rRNA gene amplicon analyses. Lithological and chemical stratification defined three intervals (deep eutrophic, middle carbonate, upper eutrophic) that were correlated with changes in organic matter sources. Methanogens were clearly depth-partitioned: methylotrophic Methanomassiliicoccales dominated deep eutrophic sediments, whereas hydrogenotrophic Methanomicrobiales and Methanobacteriales increased upward in shallower, more recent sediments with fresher organic matter. Paired isotopic data support this substrate-driven shift in CH₄ production. Although O₂ was not detected below ~0.4 cm, sequences of aerobic gammaproteobacterial methanotrophs (Crenothrix and Methylobacter) were abundant in surface sediments down to ~20 cm sediment depth, correlating with NO₃^- and PO₄^3- concentrations. The absence of anaerobic methanotrophs and C-isotopic evidence for ongoing methane oxidation suggests that these O₂-requiring, methane monooxygenase-utilizing Methylococcales constitute the dominant CH₄ sink in these surface sediments. These findings reveal that eutrophication can cause a stratification of methane-cycling microbial communities, highlighting the role of sedimentary legacies in regulating benthic CH₄ emissions from freshwater ecosystems.