| 24 Sep 2025
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 PO43- 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.
This manuscript explores whether past and present eutrophication affects microbial community structure in lake sediments, with a special emphasis on methane-cycling microbial communities. The authors characterized the sediment geochemistry and performed 16S rRNA gene amplicon sequencing at high resolution in sediment cores collected from the Swiss lake Lake Joux, which has a well documented history of human activity and the resulting effects on nutrient inputs and ecological changes. This context is very well outlined in the manuscript. In general, I think this manuscript is well-written and methodologically sound.
My main comment concerns the relatively superficial nature of the authors analyses of their amplicon data. In line 444, the authors state that aerobic methanotrophs were mainly represented by the genera Methylobacter and Crenothrix, both showing notable abundances. However, there is no information on how many ASVs were affiliated with these genera and whether there were depth-related differences in their abundance that could point to niche differentiation such as observed in other lacustrine systems. Such analyses would provide more detailed insights into community structure, especially within the upper sediment layers where chemical gradients are steepest. Integrating a phylogenetic analysis of the ASVs affiliated with the Methylobacter and Crenothrix could help to better resolve their niche partitioning and environmental roles. This would also strengthen the statement in line 591.
Secondly, the authors attribute the predominance of Methanomassiliicoccales in the deepest, eutrophic sediment layers to selection by past eutrophic conditions (see lines 493 and following). However, the prevailing understanding is that Methanomassiliicoccales are hydrogen-dependent methanogens. I wonder whether their distribution is influenced not solely by eutrophic conditions, but also by competition for hydrogen between them and hydrogenotrophic methanogens. I believe this aspect warrants further discussion and a more nuanced interpretation of the data.
Line-specific comments:
Line 184: Please add information on when sampling was conducted.
Line 197: Is there a reason why nitrite was not analyzed or was it not detected? Knowing where nitrite accumulates would help to define where conditions become denitrifying, information that could then be linked to the presence of specific MOB ASVs.
Line 246: Could you add here information on how relative abundances were calculated and does it refer to relative abundance of bacteria and archaea together?
Line 498: Methanol is also a common substrate for them and could be produced during the breakdown of organics.
Line 525: Again here, could competition for hydrogen influence the depth distribution?
Figures:
Fig 1. Please add information/description on panel B.
Fig 2 and supplementary table 2: The different oxygen profiles, are these repeated measurements of the same core or are these obtained from different cores?
Fig 4. Is there a reason to not show the distribution of NC10 in figure 4? I suggest to show NC10 here as well, either combined with the Methylococcales or in a separate panel.