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.

Reviewer Note: I want to personally apologize to the authors for the delayed review. A family member experienced a significant medical emergency and required care, which impacted my ability to submit this review in a timely manner.

General Assessment: This manuscript analyzes sediment cores from Lake Joux to determine the long-term impact of eutrophication on methane (CH₄) cycling. By pairing a historical deposition record dating back to the 16th century with high-resolution geochemical profiles and 16S rRNA gene amplicon sequencing, the authors aim to link sediment history to modern benthic biogeochemistry. The study identifies 3 depth specific clusters, which correlate with sediment geochemistry and shifts in the methanogens/methanotrophs. Deep sediments with a geochemical record of eutrophic deposition exhibited a distinct niche for methylotrophic methanogenic taxa. Enrichment of cyanobacteria amplicon sequencing variants (ASVs) and increased sulfur compounds relative to the middle carbonate layer provided indirect evidence for methylated compounds in these deep eutrophic sediments. The authors conclude differences in historical sediment deposition influence modern biogeochemical cycling.

The manuscript provides robust data to support the conclusions. However, I have comments concerning the interpretation of the geochemical and 16S rRNA gene amplicon sequencing variant (ASV) data. Given the issues listed below, care should be taken to update the manuscript to improve clarity.

Major Comments:

1. Rewrite final paragraph of Introduction: The final paragraph of the Introduction currently includes an overview of carbon isotopes. While this background is relevant, placing it in the closing paragraph distracts from the study objectives and findings. Consider moving this text to the Discussion. Please revise the final paragraph to focus on the study objectives and the significance of linking the Lake Joux depositional record to modern methane cycling.
2. Statistical testing of depth specific clusters: The text describes three distinct depth-specific clusters derived from center-log ratio (CLR) transformed 16S rRNA data in Section 3.3.4 and supplemental figure 2. While distinct groupings are visually observed along the first principal component (explaining 95.1% of the variance), the differences between the clusters could be interpreted as subjective. Please justify the differences among clusters with appropriate statistical tests to confirm significant differences between groups.
3. Interspersed Results and Discussion sections: There are several instances where results are presented in the Discussion and findings are interpreted in the Results. For example, the correlation between methanotrophic taxa and nitrate/phosphate (Fig. 5) was presented in the Discussion, when it belongs in the Results section. Conversely, interpretative text and literature citations were included when describing the high abundance of Nanoarchaeota in lines 384-386 of the Results. I recommend the authors review the manuscript to separate the description of data in Results from interpretation in Discussion.
4. Describing Methanomassiliicoccales ASVs as strict methylotrophic methanogens: Methanomassiliicoccales taxa are dependent on methylated compounds for growth and methane production. However, the growth rate of Methanomassiliicoccus isolates on methanol increase when utilizing hydrogen (H₂) as an electron donor [1–3]. A recent metagenomic survey of Methanomassiliicoccales identified the genes required for the hydrogen dependent reduction of methanol to methane across all analyzed metagenome-assembled genomes (MAGs) [4]. Consider identifying Methanomassiliicoccales ASVs as hydrogen-dependent methylotrophic methanogens.

Minor Comment:

1. Functional Assignment of nitric oxide dismutase (NOD) from 16S rRNA data: The authors assign the presence of nitric oxide dismutase (NOD) to specific Bacteroidota taxa in line 581 of the Discussion. However, the NOD annotation method was not described. The distribution of NOD to specific taxa appears to be inferred by matching the identified ASVs with the NOD database presented in Ruff et al. 2024 [5]. Please describe the NOD annotation method with a reference to the database or remove the description of NOD containing taxa.

Line specific comments by section:

Methods

Line 152: The text cites Fig 1 for the site location, but the figure also contains an NMDS plot. Please redefine the citation as Fig 1A.

Line 199-206: The method for measuring δ¹³CDIC is described, but the method for determining the DIC concentration has been omitted. Please clarify how DIC concentrations were determined.

Line 236-239: Please specify the gas chromatography detector used to measure bulk CH4.

Line 270-273: The method for generating the geochemical NMDS (Fig 1B) is in the section describing 16S rRNA gene amplicon sequence analysis. I suggest the description of the method be moved to the end of Section 2.5 for clarity.

Results

Line 303-304: Data supporting the correlation with previously dated (137Cs/210Pb) cores was not presented. Consider showing or explaining the stratigraphic alignment.

Line 320-322: sulfate and sulfide exhibit clear inverse gradients in the upper eutrophic sediment (< 7.5 cm), suggesting active sulfate reduction. This trend is described in lines 485-487 of the Discussion, but not presented in the Results. Please update the Results to describe these coupled profiles.

Line 327: The citation to Fig. 2E refers to the DIC concentration profile, but the text suggests a reference to the δ¹³CDIC profile in Fig 2F. Please update the text or reference accordingly.

Line 381-383: The text states diversity is lowest in the deep eutrophic layer. However, Figure 3C shows the upper eutrophic layer has lower species richness and evenness. Please verify or correct the text.

Line 438-440: The text discusses Methylomirabilota (NC10) abundance, but the abundance data is not presented in Figure 4B. Please add NC10 abundance to Fig 4B or remove the discussion of their abundance from the Results. 

Line 443: The text discusses methanotroph abundance but cites Fig 3C, which refers to the alpha diversity metrics of the sediment layers. Please correct the figure reference.

Discussion

Lines 474-475: A transition from methylotrophic to hydrogenotrophic methanogenesis is a point in the Discussion. However, families are introduced only by taxonomic name in the Results. Please explicitly assign the inferred substrate preferences of taxa when introduced in the Results section to improve clarity.

Lines 475-476: The link between the taxonomic shift of methanogens and the δ¹³CDIC discontinuity at 32 cm is correct, but the explaination is complex. I suggest adding a brief explanation to aid readers unfamiliar with stable isotopes. Consider including information from the last paragraph of the introduction here.

Line 528: The decline of methylotrophic methanogens is attributed to a decrease in the availability of methylated compounds. However, these compounds were not measured. Please revise the text to clarify that the decrease was inferred.

Line 550: This statement implies anaerobic methane oxidation (AOM) is dominant CH4 oxidation pathway across all sediment types. However, aerobic methane oxidation can be the dominant oxidation pathway in aerobic terrestrial sediments. Please update the text to reflect the specific environmental context of AOM.

Line 559: This text discusses Methylococcales abundance but cites a figure correlating abundance with nutrient availability (Figure 5). Please correct the reference.

Technical Corrections

Abbreviations: Define 'ISIC', 'TOC', and 'GCC-IRMS' at first mention. Explicitly define delta notation (e.g., δ¹³C-DIC) upon first use.

Figures: Increase font size of subplot legends in Figure 2 (i.e., increase size of A-P)

Line 241-242: Complete the sentence "Carbon isotopic fraction factors ... were as".

Line 434-435: Close the parenthesis

Line 560: The phrase "CH4-oxidation zone suggests that serve as the dominant" is incomplete.

Line 583: A transition is needed to connect oxygen production via NOD to denitrification by methanotrophic bacteria.

References

1.     Paul K et al. “Methanoplasmatales,” Thermoplasmatales-Related Archaea in Termite Guts and Other Environments, Are the Seventh Order of Methanogens. Appl Environ Microbiol 2012;78:8245–8253. https://doi.org/10.1128/AEM.02193-12

2.     Dridi B et al. Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. Int J Syst Evol Microbiol 2012;62:1902–1907. https://doi.org/10.1099/ijs.0.033712-0

3.     Iino T et al. Candidatus Methanogranum caenicola: a Novel Methanogen from the Anaerobic Digested Sludge, and Proposal of Methanomassiliicoccaceae fam. nov. and Methanomassiliicoccales ord. nov., for a Methanogenic Lineage of the Class Thermoplasmata. Microbes Environ 2013;28:244–250. https://doi.org/10.1264/jsme2.ME12189

4.     Speth DR, Orphan VJ. Metabolic marker gene mining provides insight in global mcrA diversity and, coupled with targeted genome reconstruction, sheds further light on metabolic potential of the Methanomassiliicoccales. PeerJ 2018;6:e5614. https://doi.org/10.7717/peerj.5614

5.     Ruff SE et al. Widespread occurrence of dissolved oxygen anomalies, aerobic microbes, and oxygen-producing metabolic pathways in apparently anoxic environments. FEMS Microbiol Ecol 2024;100. https://doi.org/10.1093/femsec/fiae132