We thank the referee very much for his/her comments.

10: I invite the authors to remove “and to explore natural gas deposits”, since this has a connotation of resource exploitation in a sector that already is a big emitter of GHG. The research should not highlight the use of methanogenesis ecological research as a survey tool for oil & gas industries, in particular when exploration will result in aquifer destruction.

Response: As suggested by the referee, we remove the sentence accordingly.

18-19: I suggest to change “Cultivation of the hydrogenotrophic methanogens that dominated in the….” To “Cultivation of the dominant hydrogenotrophic methanogens in the…” it makes better sense.

Response: As suggested by the referee, we modify the sentence accordingly.

25-26: It would be better to leave it as the reservoir of organic matter where microbial processes drive geochemical cycling. The reference to a large fraction of the microorganisms present on earth is out of context or quantifiable reality.

Response: As suggested by the referee, we modify the sentence as follows: 

Line 25. Terrestrial subsurface environments are massive reservoirs of water and organic matter where microbial processes drive geochemical cycling (Lovely and Chapelle, Reviews of Geophysics, 33, 365-381, 1995; McMahon and Parnell, 2014). Aquifers formed in…

31-32: The authors have to take care of story development, since one sentence states that only CO2 remains as electron acceptor, yet later introduce the 3 methanogenic pathways where acetate of methylated compounds are also acceptors. I may add, all those are mcrA dependent and authors did not make any reference to novel methanogenic pathways recently discovered. They may not be archaea, or methanogenic for the purpose of energy conservation but they exist. Else, the authors should start the paragraph stating this is exclusively about archaea methanogenesis.

Response: As suggested by the referee, we modify the relevant sentences as follows: 

Line 31. Methanogenesis, biological methane formation, is a terminal process involving the degradation of organic matter in anoxic environments (Lovely and Chapelle, Reviews of Geophysics, 33, 365-381, 1995), although the occurrence of non-methanogenic microbial methane production has recently been revealed as well (Liu et al., Science of the Total Environment, 806, 151362, 2022). Methanogens comprise a diverse group of archaea that produce methane from H2 and CO2 (hydrogenotrophic), methylated compounds (methylotrophic), acetate (acetoclastic), methoxylated aromatic compounds (Mayumi et al., Science, 354, 222-225, 2016) or alkanes (Zhou et al., Nature, 601, 257-262, 2021). 

35: loose sentence that does not integrate the story forming, it breaks narrative with the gas resources which to me is an energy-sector relevant sentence that should not mix with the science.

Response: As suggested by the referee, we remove the sentence accordingly.

54:55: the depths 100, 149, 476, 613, 715, 943, should have an indication for resolution of the soil/rock type which visually is hard to discern from the figure.

Response: High resolution of lithology of the study sediments has previously been demonstrated (Ikawa et al., 2014). In this manuscript, we have included the lithology of sampled aquifers as follows: 

Line 54. In this study, water samples were… Yuchi Formation [at 476 (fine sand), 613 (coarse sand), 715 (fine sand), 943 (fine sand) mbgs] and from the freshwater aquifers… Sarabetsu Formation [at 100 (coarse sand), and 149 (coarse sand) mbgs], as reference…

75: The volumes of the wellbore were extracted in different excess, why? This means you did not use the same criteria for extraction of “noise” water volume. Is 4 times sufficient to purge the water that is not representing the desired depth? In addition, how was this applied to all different depths? This is crucial for the soundness of your study.

Response: In this study, the criterion for water sample collection is based on the stabilization of specific chemical parameters, including water temperature, electrical conductivity, pH, and oxidation-reduction potential, as described Line 74-75. To ensure that the sampled groundwater is indeed from the aquifer itself, it is standard practice to pump 3-5 times the volume of water in the wellbore or the area sealed off by packers, as recommended by Kieft et al. in “Manual of Environmental Microbiology” (pp. 799-817, ASM Press, 2007). The water volume pumped from the well in our study also met this criterion.

79: filled with water to maintain anaerobic conditions? But the samples are…. Water? This is very confusing.

Response: The bottle was filled with “water sample” to raise the internal pressure of the bottle and prevent the penetration of air (oxygen). We modify the relevant sentences as follows:

Line 78. Water samples for microbial cultures were collected in sterilized glass bottles with butyl rubber stoppers and screw caps. The bottles were purged with N2 gas before and during the sample collection and then filled with water sample to raise the internal pressure of the bottles, effectively preventing the penetration of air. 

83-84: the gas separation during sampling should be explained in detail, not all readers are experts in these methods.

Response: As suggested by the referee, we modify the relevant sentences as follow: 

Line 78. The gases that were associated with the formation water in aquifers (i.e., under high pressure conditions) were naturally separated from the water when it was sampled under atmospheric pressure. These separated gases were collected using the water displacement method.

90-92: details on the column could be useful to include.

Response: The description of the gas composition and isotope ratio measurement methods has been revised to include information about the analysis columns, as follows:

“The gas composition was analyzed using two gas chromatographs: a Shimadzu GC-8A equipped with a thermal conductivity detector and a Molecular Sieve 5A column, and an Agilent 6890 equipped with a flame ionization detector and a PoraPLOT Q column. Stable carbon (δ¹³C) and hydrogen (δD) isotope ratios of methane, along with δ¹³C of carbon dioxide, were measured using a Trace Ultra gas chromatograph connected to a DELTA V plus isotope ratio mass spectrometer (IRMS) through a GC IsoLink combustion/pyrolysis interface (Thermo Fisher Scientific), with the column being PoraPLOT Q”. 

115: perhaps a more expert opinion is needed, but I do NOT think the primer selection and references for microbial analysis are up to date. What about the former Rice ClusterI? Now to be Methanomassiliicocci? Also the use of mcrA gene markers as a stand-alone method to classify has been flagged as non sufficient by the recent Proposed minimal standards for description of methanogenic archaea. I suggest the authors review this recommendation and describe the fulfilment with discussion around their findings according to emerging needs to harmonize the way methanogens are described and reported: Prakash et al., Int. J. Syst. Evol. Microbiol. 2023;73:005500 DOI 10.1099/ijsem.0.005500

Response: Firstly, we would like to confirm Referee #1 that our analysis includes not only mcrA gene sequences but also 16S rRNA gene sequences. For further details, please refer to the section “3.3 Microbial community compositions in the water samples”. Secondly, we acknowledge that the paper by Prakash et al. presents the minimal standards for taxonomic description and classification of methanogens, but it falls outside the scope of this study. Thirdly, it is worth noting that the former Rice Cluster I has now been reclassified as Methanocella. Nevertheless, our study did not detect Methanocella, formerly known as Rice Cluster I.

154: not methylated compounds? Like methylamines?

Response: In this study, we did not attempt to culture methylotrophic methanogens using methylated compounds as methanogenic substrates, as our primary focus was not on this pathway of methanogenesis. As supported by the 16S rRNA gene sequencing analysis, methylotrophic methanogens were found to be present in only minor proportions (Fig. 3c). 

230: The authors should make a discussion around other methanogens in reference to their gene primers dating almost 2 decades ago and also on new markers used in more recent research. What about Bathyarchaeota? All your findings come from Euryarchaeota and this seems biased to your methods. What about the Asgard superphylum? To give a more holistic view of carbon cycling in these environments? This can be regardless of methanogenic potential but a complete view of the diversity likely missed by your gene markers of choice.

Response: As already mentioned, we analyzed 16S rRNA gene amplicon sequences targeting both bacteria and archaea for the water samples (Fig. 3). However, potential methanogens possessing the Mcr gene but not classified within Euryarchaeota, such as Methanomethylicales, Nezhaarchaeales, Korarchaeia and Methanodesulfokores, were not detected in 16S rRNA gene sequencing reads. Additionally, the 16S rRNA gene sequences that were closely related to those of Bathyarchaeota and Asgard, known to possess Acr but not Mcr (for more information, refer to Mei et al., PNAS Nexus, 2, pgad023, 2023), were not detected in this study either. Consequently, to investigate whether the diversity of methanogens observed in the 16S rRNA gene analysis was also present in the mcrA gene analysis, we conducted traditional mcrA gene sequencing. Regarding carbon cycling, we have detected Ca. Methanoperedens that can oxidized methane by coupling to nitrate reduction and discussed the possibility of methane oxidation in that system (in the Line 252-255). We add the relevant sentences as follows:

Line 245. … were detected. However, no other potential candidates for methanogens or hydrocarbon-oxidizing archaea, known to possess Mcr or Acr (alkyl-S-CoM reductase) gene in their genomes, such as Methanomethylicales, Nezhaarchaeales, Korarchaeia, Methanodesulfokores, Bathyarchaeota and Helarchaeales (Mei et al., PNAS Nexus, 2, pgad023, 2023), were not detected in any of the samples.

Responses to Referee #2

GENERAL COMMMENTS

The manuscript by Katayama et al., entitled "Microbial methane formation in deep aquifers associated with the sediment burial history at a coastal site," presents an interesting study that analyzes the origin of methane in a natural gas-bearing sedimentary aquifer, located in a coastal area of northern Japan. The authors have used an elegant approach: They have investigated a special geological setting in which two aquifers of different geochemical composition are efficiently isolated by a clay aquitard. They have built their research on the careful characterization of the site in a previous study (Ikawa et al., 2014), and conducted a new sampling campaign to collect high-quality, large-volume water samples from six carefully selected target depths, up to 943 m deep below ground surface and with in situ temperatures up to 35°C. They have investigated these samples with state-of-the art methods, using stable isotope analyses of methane and water, DNA-based analysis of microbial communities in the pristine water samples, and additional cultivation experiments. 

The outstanding strength of this study is the selection of an ideal study site in a geological setting, which exhibits a distinct depth profile for the hydrogen isotopic composition of water. Thanks to this fact, the authors cannot only identify microbial CO2 reduction as the predominant pathway of methanogenesis, but also delineate the depth of the methane source. They show convincingly that methanogenesis is highest in the deepest aquifer, where water temperature, salinity, and total organic carbon contents of the adjacent mud sediments were highest. 

The scientific questions are relevant and within the scope of BG. The concept and data are novel, and the conclusion is substantial. The results are discussed in an appropriate and balanced way. Overall, the manuscript is well organized, reads well and I couldn't detect major scientific flaws. The topic is of interest to scientists both in the CH4 community and in the field of deep biosphere research. My recommendation is: Essentially, the paper can be published with some minor revisions as suggested below.

Response: We thank the referee very much for his/her thoughtful and positive evaluation of our manuscript. The careful characterization of the study site and the utilization of stable isotope analyses, DNA-based microbial community analysis, and cultivation experiments were integral to our research, and we're glad to see these aspects recognized. We also greatly appreciate the comments highlighting the distinct depth profile for the hydrogen isotopic composition (methane vs. water) as a key factor contributing to the strength of our study. It's rewarding to know that this factor enabled us to not only identify the dominant pathway of methanogenesis but also pinpoint the depth of the methane source. Furthermore, we extend our sincere gratitude for the suggestion that the paper can be published with minor revisions. We have carefully incorporated the recommended revisions into the manuscript, as outlined below.

MINOR COMMENTS

L. 37/Fig. S1: Location of the investigated site. To improve readability, please add another map with a larger scale to show the location of the investigated site in the northern part of Hokkaido/within Japan.

Response: As suggested by the referee, we have added another map with a larger scale showing the entire Hokkaido region in the Fig. S1.

L. 47: “Throughout the burial history of the Yuchi Formation, water salinity decreased while temperature increased along the geothermal gradient (Fig. 1d).” – This sentence is misleading. I don’t think that salinity decreased with the geothermal gradient. The opposite is true. Salinity increases with depth. Please clarify this statement. Two sentences might be better than one.

Response: Certainly, the statement in question appears to contain a potential misinterpretation. Considering that the alteration in salinity has been addressed earlier  (Lines 38-46 in the original manuscript), we focus solely on the change in temperature with depth in this sentence as follows:

Line 47. “Throughout the burial history of the Yuchi Formation, temperature increased along the geothermal gradient (Fig. 1d).”

L. 63/64: Please add information about the location of the site on Hokkaido, Japan.

Response: As suggested by the referee, we have modified the relevant sentences as follows:

Line 63. The study site was located on a sand dune 300 m from the coastline of Teshio Plain located in northern Hokkaido (44.9948° N, 141.6882° E) at an elevation of 5.2 m above sea level (Fig. S1).

L. 198: “In all water samples, CH4 accounted for approximately >75% of the total dissolved gas (Table 2).” This is the analytical result for the recovered gas phase. Please add information on the concentration of dissolved methane in the aqueous phase (in text and table). The conversion requires a mass balance calculation (total amount of released methane (moles) divided by water volume from which the gas was released). 

Response: The concentration of dissolved methane in the aqueous phase serves as an indicator reflecting the history of biogenic methane formation within each sediment. We agree with the perspective of Referee 2, acknowledging that incorporating such information could have strengthened the validity of the discussion presented in this paper. Regrettably, due to several constraints during field operations and the limited anticipation of its significance at the time of sample collection, measurements of dissolved methane concentration were not conducted. Consequently, we are unable to provide additional data of the concentration of dissolved methane in the aqueous phase. We appreciate Referee 2's insightful observation and understanding the circumstances that hinder our ability to address this point.

L. 204-207: “Methane dissolved in water from the Koetoi Formation, which underlies the Yuchi Formation (Fig. S1), plotted near the boundary between a biogenic and a thermogenic origin (Tamamura et al., 2014) (Fig. 2a). The lack of thermogenic methane produced at great depth in the upper Sarabetsu and Yuchi Formation samples implies that methanogenesis occurred within these formations.” – This part could be refined. I understand that methane in the Koetoi Fromation has a distinctly different isotopic composition. The isotopic difference implies, that methane in the Yuchi Formation is not derived from thermogenic sources in the Koetoi formation. Please include some information on the line of arguments, if it is correct. L. 209-214/Fig. 2: Please use symbols instead of the words “squares”, “circles” and “triangels” to increase (quick) readability.

Response: As suggested by the referee, we have modified the relevant sentences as follows: Line 204. “Methane dissolved in water from the Koetoi Formation, which underlies the Yuchi Formation (Fig. S1), plotted near the boundary between a biogenic and a thermogenic origin (Tamamura et al., 2014) (Fig. 2a). The isotopic difference indicates that the proportion of methane in the upper Sarabetsu and Yuchi Formations derived from the lower Koetoi Formation was minimal.”

In accordance with the suggestion, we have now substituted the terms "squares," "circles," and "triangles" with their corresponding symbols in the caption of Figure 2.

L. 306: “A remarkably high methanogen population […]” – Why was the finding remarkable? What was the expectation?

Response: Based on this comment, we have recognized that the term “remarkably” might lead to confusion, and as a result, we have replaced it with “distinctly”.

L. 315: “Previous laboratory heating experiments simulating the burial of marine sediments have shown an increase in acetate, which may potentially sustain the deep subseafloor biosphere (Wellsbury et al., 1997).” – For your information: The effect of heating on acetate concentrations, a substantial stimulation of microbial activity, and the role of SAO have also been shown in deeply buried marine sediments with the help of pore-water depth profiles and radiotracer experiments, e.g. Heuer et al. (2020) and Beulig et al., (2022).

•    Heuer, V.B., Inagaki, F., Morono, Y., Kubo, Y., Spivack, A.J., Viehweger, B., Treude, T., Beulig, F., Schubotz, F., Tonai, S., Bowden, S.A., Cramm, M., Henkel, S., Hirose, T., Homola, K., Hoshino, T., Ijiri, A., Imachi, H., Kamiya, N., Kaneko, M., Lagostina, L., Manners, H., McClelland, H.L., Metcalfe, K., Okutsu, N., Pan, D., Raudsepp, M.J., Sauvage, J., Tsang, M.Y., Wang, D.T., Whitaker, E., Yamamoto, Y., Yang, K.H., Maeda, L., Adhikari, R.R., Glombitza, C., Hamada, Y., Kallmeyer, J., Wendt, J., Wormer, L., Yamada, Y., Kinoshita, M. and Hinrichs, K.U. (2020) Temperature limits to deep subseafloor life in the Nankai Trough subduction zone. Science 370, 1230-1234. doi: 10.1126/science.abd7934

•    Beulig, F., Schubert, F., Adhikari, R.R., Glombitza, C., Heuer, V.B., Hinrichs, K.U., Homola, K.L., Inagaki, F., Jorgensen, B.B., Kallmeyer, J., Krause, S.J.E., Morono, Y., Sauvage, J., Spivack, A.J. and Treude, T. (2022) Rapid metabolism fosters microbial survival in the deep, hot subseafloor biosphere. Nature Communications 13. doi: 10.1038/s41467-021-27802-7

Response: Thank you for bringing up this valuable point. Your references to the pore-water depth profiles and radiotracer experiments conducted by Heuer et al. (2020) and Beulig et al. (2022) indeed provide further insight into the influence of heating on acetate concentrations and the potential role of SAO on microbial activity in deeply buried sediments. We therefore add these references and revised the relevant sentences as follows:

Line 312. “Previous laboratory heating experiments simulating the burial of marine sediments have shown an increase in acetate, which may potentially sustain the deep subseafloor biosphere (Wellsbury et al., 1997). Indeed, porewater acetate concentration in deep subseafloor sediments increased with depth, which stimulates potential methanogenic activities (Heuer et al., 2020; Beulig et al., 2022).”

Line 322. “We further demonstrate the SAO activity coupled with hydrogenotrophic methanogenesis to convert acetate to methane in the deep part of this formation sample. Previous study also suggests the potential for SAO with methanogenesis in deep subseafloor sediments at high temperatures (Beulig et al., 2022). Investigating the diversity…”

L. 534/Table 2: For the data on isotopic compositions, please include information on the reference standards against which the delta-values are reported.

Response: As suggested by the referee, we have added information on the reference standards (VPDB and VSMOW for carbon and hydrogen isotopic compositions, respectively) in Tables 1 and 2.