Thank you, Referee #1, for your thorough review and insightful comments on our study. 

We appreciate your understanding of the importance of the research topic, acknowledgment of the scientific soundness of the manuscript, recognition of the appropriateness of the experimental procedures and data, and positive evaluation of the literature citation. We have also noted your point about the lack of mention of technical replications in the geochemical analysis. This is an important aspect to ensure the reliability and reproducibility of the results. Therefore, we have described this information in the revised manuscript as below, and also summarized it in the Supplementary Table S3: 

“The number of replication and the standard deviation value for each measurement are provided in the Supplementary Table S3.” 

“Each isotopic ratio was measured 6 times per sample, with the standard deviations of δD and δ18O less than 2.0‰ and 0.5‰, respectively.” 

“Each sulfur isotope ratio represents the average of duplicate measurements, with deviations less than 0.4‰.” 

Q. In the deepest sample (1373 mbgs), no mcrA gene could be quantified. However, if we consider the acetoclastic methanogenesis in that sample, it is the highest. Why? Discuss.

Response: As you indicated, mcrA gene copy numbers could not be quantified despite the high potential activity of acetoclastic methanogenesis in the deepest sample. We believe that this is due to the limitation of the mcrA gene sequencing analysis because we did not obtain the mcrA gene sequences of Archaeoglobaceae, which may be involved in acetoclastic methanogenesis in the deepest sample. In the revised manuscript, we have addressed this limitation of the mcrA gene sequencing analysis as follows:  

“The absence of mcrA gene sequences of Archaeoglobaceae, which can participate in acetoclastic methanoenesis in deeper samples (as described above), indicates the bias and limitation in the mcrA gene analysis, which may also explain the discrepancy between the highest 14C acetoclastic activity and the lowest mcrA gene copy numbers in the deeper samples at 1115 and 1373 mbgs.”

Q. Line 138: Is it murA gene or mcrA gene?

Response: It is mcrA gene. We have corrected it in the revised manuscript. 

Q. Line 198: Remove ‘of sulfur’

Response: We have modified the sentence accordingly. 

Thank you, Referee #2, for your insightful comments on our manuscript. We appreciate your interest in our work and acknowledge that there are areas requiring improvement. We are committed to addressing the issues identified to enhance the quality and clarity of our publication.

Q1.    The objective of this study is not clear, and the introduction section must be substantially reorganized.

Response: As you suggested, we have thoroughly revised the introduction section (please also see the responses to the comments #2, 3, 4 & 5) as follows:

“The deep subsurface environment harbors a substantial fraction of Earth's prokaryotes (Magnabosco et al. 2018; McMahon & Parnell 2014), constituting over 80% of the total prokaryotic biomass (Bar-On et al. 2018). The metabolic activities of these microorganisms play a pivotal role in global biogeochemical cycling, such as carbon, nitrogen and sulfur (Aloisi et al. 2006; Magnabosco et al. 2018). A prominent example is methane production. Much of the methane hydrate, the largest methane reservoir on Earth, is suggested to be generated by these subsurface microorganisms (Kvenvolden 1995). Additionally, coalbed methane and shale gas reservoirs also may contain a significant amount of microbially-derived methane (Vinson et al. 2017). 

In the absence of light energy, microorganisms inhibiting deep sedimentary environments rely on chemical energy derived from the oxidation of reduced substances in sediments with organic matter oxidation being particularly critical (Arndt et al. 2013; Jørgensen & Boetius 2007; Lovley & Chapelle 1995). The labile components are consumed during burial, limiting the availability of energy sources for microorganisms as sediment age increases (Middelburg 1989). However, substantial populations of active microbial cells have been observed even in deep buried sediments older than 16 million yr (Schippers et al. 2005). It has been hypothesized that temperature increase during burial stimulate thermal or biological degradation of recalcitrant organic matter, possibly sustaining microbial activities (Parkes et al. 2000). To address the fundamental questions of how microbial cells in the deep biosphere can survive with limited energy sources, this hypothesis has been examined through numerical simulations (Horsfield et al. 2006), laboratory incubation experiments (Parkes et al. 2007), and geochemical analysis (Malinverno & Martinez 2015) of subseafloor sediments. Thus, while temperature increase during sediment burial is posited to drive subsurface microbial activity, field observation-based microbiological research remains limited, and the mechanisms are not fully understood.

As sediment compaction progresses with burial, pore size and permeability decrease, reducing the living space, available water, and nutrients, thereby inhibiting microbial growth (Fredrickson et al. 1997). To investigate the impact of temperature increase on subsurface microbial ecology, it is essential to target subsurface environments where these inhibitory factors are minimized. Therefore, we focused on aquifers, which, even at great depths, maintain high porosity and permeability, providing ample living space and water for microorganisms (Fredrickson et al. 1997; Krumholz et al. 1997; Lovley & Chapelle 1995; Mcmahon & Chapelle 1991).

In this study, we targeted a gas field in central Japan, where aquifers exhibit a wide temperature range, spanning approximately 35 to 80 °C, due to a steep geothermal gradient (5 °C per 100 m) (Kato 2018). We collected formation water (FW) from each aquifer and employed a comprehensive approach, including radiotracer experiments, molecular sequencing, microbial analysis, and geochemical analysis, to evaluate microbial diversity, community structure, and potential metabolic processes. Furthermore, in this field, high-temperature oil layers are situated deeper than the series of aquifers. We, therefore, also examined the impact of oil components on microorganisms in the upper aquifers. The aim of this study is to elucidate the effects of temperature rise and associated geochemical processes, such as the decomposition of sedimentary organic matter and petroleum formation, on microbial diversity, community structure, and metabolic processes, including the conversion of carbon and sulfur compounds.”

Q2.    L30-31: The first sentence does not link to any parts of this study.

Response: The first sentence and related sentences in the introduction section has been modified so that it links to the aim of this study as follows: 

“The deep subsurface environment harbors a substantial fraction of Earth's prokaryotes (Magnabosco et al. 2018; McMahon & Parnell 2014), constituting over 80% of the total prokaryotic biomass (Bar-On et al. 2018). The metabolic activities of these microorganisms play a pivotal role in global biogeochemical cycling, such as carbon, nitrogen and sulfur (Aloisi et al. 2006; Magnabosco et al. 2018). A prominent example is methane production. Much of the methane hydrate, the largest methane reservoir on Earth, is suggested to be generated by these subsurface microorganisms…” 

Q3.    L41-61: The paragraph of the site description should be moved to the result section. Most of the contents are not suitable for a part of Introduction section.

Response: As you suggested, the site description has been moved to the Materials & Methods and Results sections in the revised manuscript as follows:

“The chemical and isotopic compositions of natural gases (Kaneko and Igari, 2020) indicate that methane dissolved in upper aquifers is primarily of microbial origin, whereas that from lower oil deposits primarily originates from oil-associated thermogenic processes (Fig. S1) based on the classification by Milkov and Etiope (Milkov and Etiope, 2018). In this gas field, gases are dissolved in FW and produced for commercial purposes by pumping gas-associated FW from upper aquifers. Crude oil and gases are also collected from lower oil deposits.” 

“The water temperatures ranged from 38 °C to 81°C in the FW samples from upper aquifers (Fig.1c) and from 67 to 96 °C in the samples from lower oil deposits (Supplementary Table S1).” 

Q4.    L50: The temperature boundary described in the previous study is not a rational why the authors did not analyze a sample of 90 ˚C or higher temperature in this study.

Response: Within the upper aquifers where biogenic natural gas is deposited, there are no gas production wells with water temperatures above 81 °C. Therefore, we collected oil-associated formation water sample for microbial cell counts from lower oil deposits (1733 mbgs), in which water temperature was measured to be 96 °C. As a result, microbial cells were not observed in this sample, and molecular gene sequencing analysis was not conducted. Although these results were described in the original manuscript, we have modified the relevant sentences to clarify this point as follows: 

“No microbial cells were observed in the oil-associated FW sample (1733 mbgs), in which water temperature was measured to be 96 °C (Table S3). Accordingly, molecular gene sequencing analysis was not conducted on oil-associated FW samples from lower oil deposits.” 

We also note that, with the revision of the introduction section, references to the temperature boundary in both the main text and the title have been deleted.

Q5.    L63: It is not clear why the objective is important.

Response: As described above, we have thoroughly revised the introduction section to clarify why the objective of this study is important. Our research investigates the potential metabolic activities of microorganisms in the deep subsurface environment, which play a significant role in global biogeochemical cycles. By focusing on aquifers with varying temperatures, this study aims to understand how microbial communities adapt and survive with limited energy sources, particularly in high-temperature environments. Additionally, we examine the impact of oil components on microbial activity, contributing to our knowledge of subsurface microbial ecology and its implications for methane production and other biogeochemical processes.

Q6.    The mcrA gene analysis was highly biased, and probably novel lineages of methanogen and anaerobic menthane oxidizers cannot be detected with the primer set. The authors should mention the limitation.

Response: As you suggested, we have discussed the limitation of mcrA gene sequencing analysis in the revised manuscript as follows: 

“T The absence of mcrA gene sequences of Archaeoglobaceae, which can participate in acetoclastic methanoenesis in deeper samples (as described above), indicates the bias and limitation of the mcrA gene analysis, which may also explain the discrepancy between the highest 14C acetoclastic activity and the lowest mcrA gene copy numbers in the deeper samples at 1115 and 1373 mbgs.”

Q7.    L11: See above.

Response: We also revised the abstract section in line with the changes in introduction section as follows: 

“Deep subsurface microorganisms constitute over 80% of Earth's prokaryotic biomass and play an important role in global biogeochemical cycles. Geochemical processes driven by geothermal heating are key factors influencing their biomass and activities, yet their full breadth remains uncaptured.”

Q8.    L15: “molecular gene analyses” instead of “molecular gene sequencing analyses”

Response: We have modified the sentence accordingly. 

Q9.    L20, 26 and through the manuscript: Only 80˚C sample is a growth range of hyperthermophiles.

Response: In the revised manuscript, we have avoided the use of terms indicating temperature range (i.e., mesophilic, thermophilic and hyperthermophilic). This is because exact temperature ranges for these terms vary between literatures, leading to potential misunderstandings.

Q10.    L39: microbial cell abundance

Response: We have modified the sentence accordingly. 

Q11.    L44-46: Awkward sentence.

Response: We have removed the relevant sentences.

Q12.    L119-: Please provide number of replicates for each.

Response: To clarify the number of replicates for each, we have revised the relevant sentence as follows:

“The activity measurements were conducted in triplicate for each of the three cultivation periods and for each of the three radiotracers.” 

Q13.    L138: mcrA

Response: We have modified the sentence accordingly. 

Q14.    L171: Please provide the gas pressure of N2/CO2.

Response: The pressure of N2/CO2 was 0.1 MPa. We have added the information in page X, line YY.

Q15.    L176: enrichment instead of sample

Response: We have modified the sentence accordingly. 

Q16.    L234-: Please define the nomenclature used in this study; e.g. GTDB, NCBI, SILVA, or ICNP with any others.

Response: We used SILVA taxonomy because the taxonomic classification of 16S rRNA gene amplicon reads was performed based on the SILVA dataset in this study. (page X, line YY)

Q17.    L248: In ICNP, Methanobacterota is effective but not Euryarchaeota.

Response: Based on the SILVA taxonomy, Euryarchaeota was used.

Q18.    L255:  Deferribacter also uses iron or other electron acceptors that are likely available in the subsurface environments.

Response: We agree with this point. We have emphasized the “common” catabolic metabolisms of abundant taxa detected in deeper samples. In addition, the chemical analysis of formation water (shown in Table S3) indicated that electron acceptors (iron, nitrate, nitrite, manganese) were almost depleted in the studied aquifers. Therefore, we consider it is not necessarily important to mention the potential of Deferribacter to use iron or other electron acceptors.

Q19.    L257-259: Too speculative.

Response: As you suggested, we have removed the relevant sentences.

Q20.    L262: Nanobdellota instead of Ca. Nanoarchaeota.

Response: Because SILVA taxonomy still uses Nanoarchaeota, we have modified the sentence as follows: Nanobdellota (formerly Ca. Nanoarchaeota)  

Q21.    L268: Please clarify is this a result of a bias of methodology or not?

Response: We believe that this is a result of primer bias. We have modified the relevant sentences as follows:

“The absence of mcrA gene sequences of Archaeoglobaceae, which can participate in acetoclastic methanoenesis in deeper samples (as described above), is due to the limitation in the mcrA analysis because the primers used did not match the mcrA gene sequences of Archaeoglobaceae. This limitation may also explain the discrepancy between the highest 14C acetoclastic activity and the lowest mcrA gene copy numbers in the deeper samples at 1115 and 1373 mbgs.” 

Q22.    L282: observed in the enrichment cultures at

Response: We have modified the sentence accordingly. 

Q23.    L288-294: Appropriate tables and/or figures should be given for each sentence.

Response: As you suggested, we added the appropriate tables and/or figures in parentheses at the end of each sentence. 

Q24.    L294: “ranges” should be deleted because no experiments at 90˚C in this study.

Response: As described above, we have avoided the use of terms indicating temperature range.

Q25.    L339: Please provide accession numbers for mcrA gene sequences.

Response: The accession numbers for mcrA gene sequences were described in page 6, line 163 in the original manuscript and page X, line YY in the revised manuscript.