Dear aoxue Yin:

Thanks for taking the time to review our manuscript "Dissolved organic matter fosters core mercury-methylating microbiome for methylmercury production in paddy soils". We deeply appreciate your insightful and constructive feedback, which has significantly improved the clarity and overall quality of our work. We have carefully addressed all your comments and made the necessary revisions to the manuscript. For your convenience, the changes have been highlighted in yellow. Additionally, we provided a detailed point-by-point response to each of your comments below. We hope that the revised version meets your expectations and adequately addresses all the concerns raised in the review process.

With sincere appreciation,

Dr. Bo Meng, on behalf of all co-authors

General comments

This manuscript presents an insightful study investigating the relationship between dissolved organic matter (DOM) and methylmercury (MeHg) production in paddy soils, particularly focusing on the core microbiome responsible for mercury methylation. The study employs advanced genomic and metagenomic techniques to identify key microbial taxa involved in mercury methylation and explores the impact of DOM on the activity of these microorganisms. The authors highlight that DOM plays a pivotal role in shaping the composition and functional activity of the Hg-methylating core microbiome, which in turn regulates MeHg production. The conclusions are supported by extensive field sampling, genome-resolved metagenomic analysis, and controlled incubation experiments.

Response: Thank you for your valuable feedback on our manuscript. We appreciate your positive comments regarding the investigation of the relationship between dissolved organic matter (DOM) and methylmercury (MeHg) production in paddy soils, especially focusing on the core microbiome responsible for mercury methylation.

In response to your comments, we have carefully revised the manuscript. We hope the changes address all concerns and improve the overall quality of the work.

Comment 1: The discussion section could further expand on how the findings integrate with broader environmental mercury cycling processes. A more detailed comparison with existing literature on Hg-methylating microbes in non-paddy soils would enhance the paper’s contribution to microbial ecology.

Response: Thank you for your valuable comment. We have expanded the discussion section to better integrate our findings with broader environmental mercury cycling processes, as you suggested. Specifically, we highlight how dissolved organic matter (DOM) impacts mercury methylation not only in paddy soils but also in other soil environments, comparing our findings with existing literature on Hg-methylating microbes in non-paddy soils. To enhance clarity, the corresponding sentences were added in the revised manuscript as below:

"In addition to paddy soils, DOM's influence on microbial Hg methylation has been observed in other ecosystems, such as wetlands and sediments, where DOM enhances Hg bioavailability and shapes microbial community structures to promote methylmercury (MeHg) production. For instance, in wetlands, DOM-bound Hg has been found to change the community assembly of mercury for methylating microbes (Fagervold et al., 2014), similar to what we observed in paddy soils." (Main text, Lines 293-297).

Comment 2: The manuscript discusses DOM composition but lacks specific details on the types of organic compounds most relevant to stimulating Hg-methylating activity. Including additional chemical analyses of DOM could strengthen the conclusions.

Response: Thank you for your valuable comment. While we did not conduct additional chemical analyses, we did examine the composition of dissolved organic matter (DOM) across paddy soils with varying levels of Hg contamination (NMS, MMS, and HMS). Our findings revealed that low-molecular-weight organic acids such as oxalic acid, formic acid, tartaric acid, and acetate were highly abundant in these soils, with their concentrations being highest in NMS soils, followed by MMS, and lowest in HMS. These compounds are known to serve as key carbon sources for Hg-methylating microorganisms, particularly in enhancing their activity.

Although we do not have direct data on the influence of aromatic compounds and humic substances on Hg methylation, it is possible to hypothesize that their more complex structures may make them less bioavailable or more resistant to microbial degradation, potentially leading to a weaker stimulation of Hg-methylating activity compared to low-molecular-weight organic acids. This hypothesis warrants further investigation in future studies. To clarify, we have added the corresponding sentences to the revised manuscript as below:

"While we did not conduct additional chemical analyses, we examined the DOM composition across paddy soils with different Hg contamination levels (NMS, MMS, HMS). Our analysis found that low-molecular-weight organic acids, including oxalic acid, formic acid, tartaric acid, and acetate, were highly abundant, particularly in NMS soils, with decreasing concentrations in MMS and HMS. These organic acids serve as key carbon sources for Hg-methylating microorganisms. Although we do not have direct data on the effects of aromatic compounds and humic substances on Hg methylation, it is possible to hypothesize that their more complex structures might reduce their bioavailability or slow microbial degradation, potentially resulting in a weaker effect on Hg-methylation compared to low-molecular-weight organic acids. This hypothesis could be explored in future studies, offering a pathway for a more detailed understanding of how DOM composition influences Hg methylation." (Main text, Lines 328-336).

Comment 3: The authors hypothesize that DOM stimulates microbial activity through metabolic pathways but do not delve deeply into the mechanistic underpinnings. Further discussion on the microbial metabolic pathways involved, possibly supported by metabolomic data, would improve the mechanistic understanding.

Response: Thank you for your insightful comment. We fully acknowledge the importance of understanding the microbial metabolic pathways involved in DOM-stimulated Hg methylation, as it is crucial for elucidating the underlying mechanisms of this process. Although our current study primarily focused on identifying core Hg-methylating microorganisms and analyzing DOM composition, we agree that a deeper exploration of the metabolic pathways involved would significantly enhance the mechanistic understanding of how DOM influences these microorganisms.

In our study, we observed that different DOM compounds, particularly low-molecular-weight organic acids such as oxalic acid, formic acid, and acetate, were utilized by key Hg-methylating microorganisms (e.g., Geobacter and Desulfovibrio species). These microorganisms are known to employ key metabolic pathways such as the TCA cycle (tricarboxylic acid cycle) and acetate metabolism, which could be directly linked to the utilization of DOM components. For instance, Geobacter sulfurreducens and Desulfovibrio desulfuricans preferentially use acetate, which feeds into the TCA cycle, providing electrons for anaerobic respiration and potentially influencing Hg methylation rates (Hu et al., 2013; Liu et al., 2018b). Additionally, Methanoregula and Methanosarcina species involved in methanogenesis could be utilizing formate and acetate through the methanogenesis pathway, further contributing to Hg methylation (Sakai et al., 2010; Schöne et al., 2022).

While we did not include metabolomic data in this study, we recognize the need for future research in this area. Incorporating metabolomic profiling would be highly beneficial for identifying key metabolites and providing more detailed insights into the specific metabolic pathways influenced by DOM, such as acetate fermentation, methanogenesis, and electron transfer processes involved in Hg methylation. To enhance clarity, the corresponding sentences were added in the revised manuscript as below:

"Although metabolomic data were not included in this study, future research incorporating such analyses could provide valuable insights into how specific DOM components influence microbial metabolism and Hg methylation, revealing key metabolites and pathways such as acetate fermentation, methanogenesis, and electron transfer processes." (Main text, Lines 318-320).

Minor Comments

Comment 4: The introduction is well-written but could benefit from a clearer explanation of the broader ecological importance of Hg methylation in paddy fields versus other environments.

Response: Thank you for your constructive comment. We appreciate your suggestion to provide a clearer explanation of the broader ecological significance of Hg methylation in paddy fields compared to other environments. We have expanded the introduction to address this point:

"Compared to other environments such as wetlands and aquatic sediments, paddy fields present unique ecological conditions that make them significant hotspots for Hg methylation. The frequent flooding and draining cycles, high organic matter content, and dynamic redox conditions in paddy soils create an environment that supports high levels of microbial activity, particularly Hg-methylating microorganisms (Yin et al., 2013). These conditions not only enhance MeHg production but also increase the likelihood of MeHg entering the food web through rice consumption, posing significant health risks (Zhang et al., 2010). Understanding Hg methylation in paddy fields is therefore crucial, as rice is a critical exposure route for MeHg in humans." (Main text, Lines 43-49).

Comment 5: Line16 suggest “remains”

Response: Thank you for your suggestion. We have revised the sentence as recommended.

Comment 6: Line 64 “…other factors also play roles in MeHg production…”

Response: Thank you for your comment. We have revised the sentence as suggested.

Comment 7: Line 62, Line 237, Line 299, Lne 321 suggest unify the description of "low-molecular-weight DOMs" throughout the maus.

Response: Thank you for your comment. We have unified the description of "low-molecular-weight DOMs" throughout the manuscript for consistency.

Comment 8: Line 255 Whether the results support the argument that "MeHg concentration was strongly linked to hgcA gene abundance even compared to abiotic factors", it seems to be no description of the relevant results

Response: Thank you for your comment. Our results demonstrated that the contribution of hgcA gene abundance to MeHg concentration was stronger compared to the contributions of Hg bioavailability and redox conditions. Specifically, alteration of the core Hg-methylating microbiome composition, which is closely linked to hgcA gene abundance, significantly regulated soil MeHg concentration (λ = 0.84, p < 0.001) (Fig. 4). In comparison, the contributions of Hg bioavailability (10%) and redox conditions (25%) were much lower than that of DOM (65%) (Fig. 4), further supporting the link between hgcA gene abundance and MeHg concentration, even in the presence of varying abiotic factors. To enhance clarity, the corresponding sentences were re-organized in the revised manuscript as below:

"Additionally, SEM result showed that the core Hg-methylating microbiome composition, which is closely linked to hgcA gene abundance, significantly regulated soil MeHg concentration (λ = 0.84, p < 0.001) (Fig. 4). In comparison, the contributions of Hg bioavailability and redox conditions to the core Hg-methylating microbiome composition are 10% and 25%, respectively, which are much lower than that of DOM (65%) (Fig. 4)." (Main text, Lines 242-246).

Dear Reviewer:

We sincerely thank you for taking the time to review our manuscript titled "Dissolved organic matter fosters core mercury-methylating microbiome for methylmercury production in paddy soils". Your constructive feedback has been invaluable in improving the quality and clarity of our work. In response to your comments, we have carefully revised the manuscript and highlighted all changes in yellow for your convenience. Additionally, we have provided a detailed, point-by-point response to address each of your suggestions and concerns. We deeply appreciate your thoughtful review and believe that the revisions have significantly enhanced the manuscript. We hope that the revised version meets your expectations and satisfactorily addresses all the points raised during the review process.

With great gratitude,

Dr. Bo Meng, on behalf of all co-authors

Comment 1: In the abstract, DOM was crucial in determining core Hg-methylating microbiome composition (65%), what does the 65% mean, please explain it in the text.

Response: Thank you for your insightful question. The "65%" mentioned in the abstract refers to the contribution of DOM to the composition of the core Hg-methylating microbiome. This value was calculated using structural equation modeling (SEM) results based on the method described by Tao et al. (2015). Specifically, in SEM, the contribution of a variable to its target variable was calculated as the square of its path coefficient divided by the sum of the squares of all path coefficients pointing to the same target variable. Using this formula, we calculated the contribution of DOM to the core Hg-methylating microbiome, and the result showed that DOM contributed 65%.

To ensure clarity, we have revised the Materials and Methods section to provide a clearer explanation of the contribution calculation method in the revised manuscript as below:

"We further calculated the contribution of ecological parameters, including DOM, to the core Hg-methylating microbiome, and the contribution of the core Hg-methylating microbiome to MeHg production, following the approach described by Tao et al. This calculation was performed by determining the proportion of the squared path coefficient of each parameter relative to the sum of the squared path coefficients of all parameters influencing the same target variable (Tao et al., 2015)." (Mian text, Lines 181-185).

Comment 2: In the core Hg-methylating microbiome, how much does the Geobacter account? Why the authors use the Geobacter for pure incubation experiment? why not use the soil for incubation experiment? What criteria were used to select the Geobacter?

Response: Thank you for these excellent questions. Below, we provide detailed responses to each point:

(1)    How much does the Geobacter account in the core Hg-methylating microbiome?

In the core Hg-methylating microbiome, Geobacter accounts for an average of 44.5% (36.2%, 40.5%, and 56.8% in NMS, MMS, and HMS soils, respectively) of the community, based on our analysis (Fig. 2c). This highlights its significant presence and potential role in Hg methylation within the studied system.

To explicitly include the microbial abundances of the three most abundant genera in each soil type, we have incorporated the specific values into the revised manuscript as follow:

"Although most microorganisms are not annotated, the three genera with the highest abundance in each soil type are as follows: In NMS, Geobacter (36.2%), Syntrophus (1.7%), and Desulfomonas (0.4%) dominate; in MMS, Geobacter (40.5%), Granulicella (2.9%), and Olavius (2.9%) are the most abundant; and in HMS, Geobacter (56.8%), Methanoregula (0.6%), and Granulicella (2.3%) prevail (Fig. 2c)." (Mian text, Lines 227-231).

(2)    Why did the authors use Geobacter for pure incubation experiments? Why not use soil for incubation experiments?

The primary reason for selecting Geobacter for pure incubation experiments is its well-documented and dominant role in Hg methylation in various environments, as supported by prior studies (Bravo et al., 2018; Zhong et al., 2024). By using Geobacter, we aimed to isolate and clearly demonstrate the direct effects of DOM on Hg-methylation rates and microbial activity, without the interference of complex soil matrix effects.

While soil incubation experiments could provide additional context for ecosystem-level processes, they would introduce confounding factors, such as competing microbial communities and varying physicochemical properties of the soil. Our approach using a pure culture system allows for a controlled study to elucidate specific microbial and biochemical mechanisms.

To explicitly clarify the rationale for using Geobacter sulfurreducens PCA in the incubation experiments, we have added the text in the Methods section as follows:

"Geobacter was selected for these pure incubation experiments due to its dominant role in mercury methylation and its ability to isolate the effects of DOM on methylation rates without the interference of soil matrix complexity." (Mian text, Lines 160-161).

(3)    What criteria were used to select Geobacter?

Geobacter was chosen based on several criteria: (i) Its established status as a key Hg-methylating microorganism in diverse environments, including paddy soils (Zhong et al., 2024); (ii) Its high abundance within the core Hg-methylating microbiome observed in our study (Fig. 2c); (iii) Its metabolic versatility, which makes it an ideal model organism for studying mercury methylation pathways under controlled conditions (Schaefer and Morel, 2009). These factors collectively make Geobacter an excellent candidate for exploring DOM-microbe interactions and their implications for methylmercury production.

We hope this explanation clarifies our rationale and methodology.

Comment 3: Why wasn’t Hg speciation measured or presented to confirm the hypothesis?

Response: Thank you for your insightful suggestion. While Hg speciation measurements would undoubtedly provide additional insights into the mechanisms of Hg methylation, the primary focus of this study was to investigate the role of DOM and microbial dynamics, particularly the core Hg-methylating microbiome, in driving MeHg production in paddy soils. This decision was made to prioritize a detailed examination of how DOM influences microbial Hg methylation through changes in community composition and activity.

Additionally, indirect evidence from random forest analysis and SEM highlighted the dominant influence of DOM on the core Hg-methylating microbiome compared to abiotic factors such as redox conditions and Hg bioavailability (Fig. S4 and Fig. 4). These results support the robustness of our conclusions regarding DOM's central role in Hg methylation.

While Hg speciation measurements were beyond the scope of this study, we acknowledge their importance and propose that future research combining direct Hg speciation analyses with DOM compositional studies could further elucidate how Hg speciation interacts with DOM and microbial communities to influence Hg methylation.

To better understand, the corresponding sentences are added in the revised manuscript as below:

"Future research could integrate direct Hg speciation measurements with detailed DOM compositional analyses to better understand how specific DOM components and Hg species interact to influence microbial Hg methylation." (Mian text, Lines 334-336).

Comment 4: The discussion would improve by explaining how specific components of DOM influence the distribution of Hg-methylating gene communities?

Response: Thank you for this insightful comment. We agree that explaining how specific components of DOM influence the distribution of Hg-methylating gene communities would improve the discussion. To address this, we have revised the manuscript to elaborate on the mechanisms by which different DOM components, particularly low-molecular-weight organic acids, shape core Hg-methylating microbiomes.

Our study identified several key low-molecular-weight organic acids, such as oxalic acid, formic acid, and acetate, in paddy soils. These compounds play a pivotal role in stimulating core Hg-methylating microorganisms by providing essential carbon sources that fuel critical metabolic pathways. Specifically, Geobacter sulfurreducens and Desulfovibrio desulfuricans utilize acetate and fumarate in the TCA cycle (Hu et al., 2013; Liu et al., 2018), facilitating anaerobic respiration and electron transport processes that enhance Hg methylation rates. Similarly, methanogenic archaea like Methanoregula and Methanosarcina use formate and acetate through methanogenesis (Sakai et al., 2010; Schöne et al., 2022), further contributing to Hg methylation. These mechanisms suggest that low-molecular-weight organic acids selectively promote the growth and activity of Hg-methylating microorganisms, thereby influencing the distribution of Hg-methylating gene communities.

To better understand, the corresponding sentences are re-organized in the revised manuscript as follow:

"These findings demonstrate that DOM composition strongly influences microbial Hg methylation by stimulating key metabolic pathways. For instance, Geobacter sulfurreducens and Desulfovibrio desulfuricans use acetate and fumarate in the TCA cycle, supporting anaerobic respiration and electron transport that enhance Hg methylation (Hu et al., 2013; Liu et al., 2018b). Similarly, methanogenic archaea such as Methanoregula and Methanosarcina utilize formate and acetate through methanogenesis, further contributing to Hg methylation (Sakai et al., 2010; Schöne et al., 2022). Although metabolomic data were not included in this study, future research incorporating such analyses could provide valuable insights into how specific DOM components influence microbial metabolism and Hg methylation, revealing key metabolites and pathways such as acetate fermentation, methanogenesis, and electron transfer processes. This highlights how specific DOM components shape the core Hg-methylating microbiome and influence its role in MeHg production." (Mian text, Lines 321-329).

Comment 5: The conclusion needs to be strengthened. It currently restates results that are largely expected. Highlight the novel findings and their significance more clearly. Meanwhile, the environmental implication of this study can be discussed to demonstrate the impact of this study.

Response: Following the reviewer's suggestion, we have revised the conclusion to better emphasize the novel findings of this study and their broader significance. Specifically, we highlighted the role of DOM in influencing the core Hg-methylating microbiome and its direct impact on MeHg production. Additionally, we briefly discussed the environmental implications of our findings, focusing on the potential effects of human activities and climate change on DOM dynamics and Hg methylation processes.

To better understand, the corresponding sentences are re-organized in the revised manuscript as follow:

"This study provides novel evidence that DOM significantly influences MeHg production by altering the composition and stimulating the activity of the core Hg-methylating microbiome. While DOM regulates the composition of other members of the Hg-methylating microbiome, its impact on MeHg production is primarily mediated through the core Hg-methylating microbiome. Using metagenomic binning and pure incubation experiments, we demonstrated that low-molecular-weight DOM directly promotes MeHg production by enhancing the metabolic activity of core Hg-methylating microorganisms. These findings underscore the central role of the core Hg-methylating microbiome in Hg cycling and highlight DOM as a critical driver of microbial Hg methylation. As human activities and climate change continue to alter DOM composition and concentration, their influence on Hg methylation dynamics warrants further investigation to better predict and mitigate Hg-related environmental and health risks." (Mian text, Lines 351-359).

Reference

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