Bacillus subtilis Biofertilizer Mitigates N₂O Emissions from Saline-Alkali Farmland

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript examines the effect of Bacillus subtilis biofertilizer on N₂O emission mitigation in saline–alkali soils through a field experiment. This topic is highly relevant in the context of global climate change and the urgent need for sustainable nitrogen management.

Abstract (Lines 11–28)

• The abstract is clear but overly descriptive; the numerical results should be presented more precisely.
• The experimental duration and site conditions must be explicitly mentioned.
• The statement “This indicates a reduction in N₂O emission flux generated through the nitrification process” should be supported with quantitative evidence (e.g., percentage decline in functional gene abundance).
• Clarify crop yield comparison—was there no significant difference? Include the statistical outcomes.

Introduction (Lines 31–62)

• Lines 34–41: Atmospheric N₂O values are reported in “ppm,” but are conventionally expressed in ppb. Please verify and correct the same.
• Lines 52–58: The literature review is adequate but should contrast B. subtilis with other biofertilizers (e.g., Trichoderma, Paenibacillus, B. amyloliquefaciens).
• Lines 58–60: The statement “none of these studies conducted quantitative analyses of norB and hao” requires citation support.
• The concluding paragraph should clearly state the research hypothesis and objectives.

Methods

• Experimental site (63–84): Soil physicochemical data should be summarized here, not only in Table S1. Include bulk density and soil texture classification.
• Field design (87–96): Clarify whether plots were randomized and replicated. This description suggests insufficient replication. Please state the number of replicates per treatment clearly.
• N₂O measurement (97–114): Provide the chamber dimensions, headspace volume, calibration gas concentration, and GC-ECD precision. Clarify the flux calculation method (e.g., Hutchinson–Mosier).
• Soil and plant sampling (115–143): Specify the number of composite samples per plot. For plant analysis, include the nitrogen uptake formula.
• DNA sequencing (145–163): Report sequencing depth, rarefaction threshold, and normalization method.
• Functional gene quantification (164–176): Provide primer efficiency, R² values, and data transformation details.
• Statistical analysis (177–189): Mention the R software version, justify the use of ANOVA despite heteroscedasticity, and provide the PLS-PM bootstrapping parameters and fit indices.

Results

• Soil properties (193–207): Replace subjective terms such as “explosive increase” with quantitative statistics. Ensure consistent formatting in Table 1.
• N₂O flux (212–229): Improve the resolution of Figure 3a/b. Report cumulative flux in standard units (kg N₂O-N ha⁻¹ season⁻¹). Correlations should be interpreted cautiously.
• Crop yield (237–244): Provide ANOVA table in the Supplementary. Please clarify the sample size per treatment.
• Functional genes (249–272): Ensure consistency between the text and figures; nosZ patterns appear contradictory.
• Microbial community (273–314): Simplify Figure 7b or split the frames. Report statistical significance of diversity indices.

Discussion

• Soil properties and yield: Clarification of the mechanism of reduced NH₄⁺-N (microbial assimilation, slow release). Avoid subjective phrasing.
• N₂O emission mechanisms: Interpret PLS-PM cautiously; correlation does not imply causation. Discuss saline–alkali effects on microbial physiology.
• Functional genes: Strengthen the NOR pathway discussion with primary literature. Consider the temporal lag in nosZ expression.
• Microbial community: Link shifts in Proteobacteria, Cyanobacteria, and Acidobacteriota to nitrogen cycling. Elaborate on the ecological significance of Oceanobacillus enrichment.

Conclusion

• The manuscript is well structured but should emphasize practical agronomic implications.
• Suggest future directions such as multi-season validation and metatranscriptomic approaches.

 

Author Response

Response to reviewer’s comments

(Manuscript ID: life-4177406)

Summary:

Thank you very much for handling our manuscript “B. subtilis biofertilizer mitigates N₂O emissions from saline-alkali farmland and its potential mechanisms”. We highly appreciate the insightful comments from the anonymous reviewer. After fully considering these comments, we thoroughly revised to our manuscript.

Below, we address the reviewer’s comments and questions point-by-point. The original comments are in black font and our responses to the comments follow in blue font. We hope our responses have addressed all of their concerns. If further improvement is required, please let us know.

Thank you!

Rui Li

 

 

Abstract (Lines 11–28)

1. The abstract is clear but overly descriptive; the numerical results should be presented more precisely.

Response: Thank you very much for this helpful comment. We agree that the original abstract was somewhat descriptive and did not present the main quantitative findings with sufficient precision. In the revised manuscript, we have streamlined the descriptive statements and added more specific numerical results to better highlight the main outcomes of the study. Specifically, we now report that B. subtilis biofertilizer reduced the total N₂O emission flux by 39% relative to conventional fertilization, reduced the peak N₂O emission flux by 40.7% and 18.2% compared with the CF and CBF treatments, respectively, and maintained crop yield at a level comparable to other fertilization treatments. We also retained key quantitative results related to nitrogen uptake and functional gene abundance to improve the precision and informativeness of the abstract. These revisions are consistent with the results reported in the main text.

 

2. The experimental duration and site conditions must be explicitly mentioned.

Response: Thank you for your helpful suggestion. We have revised the manuscript accordingly. The experimental duration is now explicitly stated as June 24 to October 12, 2024, and the study site is clearly described as a saline-alkali farmland in Binzhou, Shandong Province, China. We have also added the main environmental characteristics of the site, including its warm temperate monsoon climate and coastal saline-alkali soil properties.Thanks for your comments, we have added some methodological details in Part 2.5. Considering the manuscript length requirements and the readability of the article, we have included the rationale and formula for the microbial alpha diversity index in the Supplementary Material.

 

3 The statement “This indicates a reduction in N₂O emission flux generated through the nitrification process” should be supported with quantitative evidence (e.g., percentage decline in functional gene abundance)

Response:

Thank you for your valuable comment. We agree that this statement should be supported by more explicit quantitative evidence. In the revised manuscript, we have strengthened this interpretation by adding the specific changes in nitrification-related functional gene abundance at the N₂O emission peak. Specifically, compared with the CF and CBF treatments, the BF treatment reduced the copy number of the Bacterial-amoA gene by 94% and 83%, respectively. In addition, the hao gene copy number in the CF treatment was 7.67 and 24 times higher than that in the BF and CBF treatments, respectively, further indicating that biofertilizer application suppressed nitrification-related N₂O production. These quantitative results provide stronger support for the statement that the reduction in N₂O emission flux was associated with inhibition of the nitrification process.

 

4 Clarify crop yield comparison—was there no significant difference? Include the statistical outcomes.

Response: Thank you for your valuable comment. We agree that the comparison of crop yield should be stated more clearly and supported with explicit statistical outcomes. In the revised manuscript, we have clarified that, except for the CK treatment, all fertilization treatments significantly increased crop yield, whereas there was no significant difference in yield among the BF, CF, and CBF treatments (p > 0.05). We have also revised the relevant text and figure description to make the statistical comparison more explicit for readers.

 

Introduction (Lines 31–62)

1. Lines 34–41: Atmospheric N₂O values are reported in “ppm,” but are conventionally expressed in ppb. Please verify and correct the same..

Response: Thank you for your valuable comment. We have carefully checked this statement and agree that atmospheric N₂O concentrations should be expressed in ppb rather than ppm. We have corrected the unit throughout the manuscript accordingly.

 

2. Lines 52–58: The literature review is adequate but should contrast B. subtilis with other biofertilizers (e.g., Trichoderma, Paenibacillus, B. amyloliquefaciens).

Response: Thank you for your valuable comment. We agree that the Introduction should better position B. subtilis relative to other reported biofertilizers. In the revised manuscript, we have expanded the literature review to compare B. subtilis with Trichoderma viride, Paenibacillus polymyxa, and Bacillus amyloliquefaciens, and clarified that previous studies mainly focused on emission mitigation performance or denitrifying microbial community shifts, whereas the present study further emphasizes nitrification-related functional genes and saline-alkali farmland conditions.

 

3 Lines 58–60: The statement “none of these studies conducted quantitative analyses of norB and hao” requires citation support.

Response:

Thank you for your valuable comment. We agree that this statement should be better supported. In the revised manuscript, we have softened the wording and linked it directly to the studies discussed above. Specifically, we revised the sentence to indicate that, to our knowledge, the previously cited studies mainly focused on N₂O flux responses or microbial community changes, while quantitative analyses of hao and norB were rarely reported. This revision avoids overstatement and improves the scholarly rigor of the Introduction.

 

4 The concluding paragraph should clearly state the research hypothesis and objectives.

Response:

Thank you for your valuable comment. We agree that the concluding paragraph of the Introduction should more clearly present the research hypothesis and objectives. In the revised manuscript, we have rewritten the final paragraph to explicitly state the hypothesis that B. subtilis biofertilizer can mitigate N₂O emissions in saline-alkali farmland by suppressing nitrification-related N₂O production and altering the associated microbial community, while maintaining crop productivity. We have also clearly defined the objectives of the study.

Methods

 

1. Experimental site (63–84): Soil physicochemical data should be summarized here, not only in Table S1. Include bulk density and soil texture classification.

Response:

Thank you for your valuable comment. We agree that the basic physicochemical characteristics of the experimental soil should be briefly summarized in the main text rather than being presented only in Table S1. In the revised manuscript, we have added a concise summary of the key initial soil properties in the “Experimental site” section. We have also included soil bulk density and soil texture classification to provide a clearer description of the field conditions.

 

2. Field design (87–96): Clarify whether plots were randomized and replicated. This description suggests insufficient replication. Please state the number of replicates per treatment clearly.

Response:

Thank you for your valuable comment. We agree that the description of the field design in the original manuscript was not sufficiently clear. In the revised manuscript, we have explicitly clarified whether the plots were arranged in a randomized design and have clearly stated the number of replicates for each treatment.

 

3 N₂O measurement (97–114): Provide the chamber dimensions, headspace volume, calibration gas concentration, and GC-ECD precision. Clarify the flux calculation method (e.g., Hutchinson–Mosier).

Response:

Thank you for your valuable comment. Thank you for your valuable comment. We agree that the description of the N₂O measurement procedure should be more detailed. In the revised manuscript, we have added the chamber dimensions (40 cm × 40 cm × 80 cm), clarified the gas sampling procedure, detector type, and calibration source, and further specified that N₂O fluxes were calculated from the linear change in gas concentration over time with correction for chamber volume, surface area, and temperature during sampling.

 

4 Soil and plant sampling (115–143): Specify the number of composite samples per plot. For plant analysis, include the nitrogen uptake formula.

Response:

Thank you for your valuable comment. We agree that the sampling strategy and plant nitrogen uptake calculation should be described more explicitly. In the revised manuscript, we have clarified the number of composite soil samples collected per plot and added the formula used to calculate plant nitrogen uptake.

 

5 DNA sequencing (145–163): Report sequencing depth, rarefaction threshold, and normalization method.

Response:

Thank you for your valuable comment. We agree that additional information on sequencing depth and data normalization is necessary for reproducibility. In the revised manuscript, we have added the average sequencing depth, the rarefaction threshold used for diversity analyses, and the normalization method applied before downstream analyses.

 

6 Functional gene quantification (164–176): Provide primer efficiency, R² values, and data transformation details.

Response:

Thank you for your valuable comment. We agree that more detailed qPCR quality-control information should be provided. In the revised manuscript, we have added the amplification efficiency and R² values for each primer set, as well as the data transformation procedure used before statistical analysis.

 

7 Statistical analysis (177–189): Mention the R software version, justify the use of ANOVA despite heteroscedasticity, and provide the PLS-PM bootstrapping parameters and fit indices.

Response:

Thank you for your valuable comment. We agree that the statistical analysis section should be described in greater detail. In the revised manuscript, we have added the R software version, clarified the assumptions and data transformation procedures used before ANOVA, and provided the bootstrapping settings and model fit indices for the PLS-PM analysis.

 

Results

 

Soil properties (193–207): Replace subjective terms such as “explosive increase” with quantitative statistics. Ensure consistent formatting in Table 1.

Response:

Thank you for your valuable comment. We agree that subjective expressions such as “explosive increase” are not sufficiently precise for the Results section. In the revised manuscript, we replaced such wording with more objective, data-based descriptions and carefully checked the formatting consistency in Table 1.

 

2. N₂O flux (212–229): Improve the resolution of Figure 3a/b. Report cumulative flux in standard units (kg N₂O-N ha⁻¹ season⁻¹). Correlations should be interpreted cautiously.

Response:

Thank you for your valuable comment. We have improved the presentation of Figure 3a/b by increasing the figure resolution in the revised manuscript. We also revised the cumulative flux description to present the results in the standard unit of kg N₂O-N ha⁻¹ season⁻¹. In addition, we have moderated the interpretation of the correlation results and avoided overly causal wording.

 

3 Crop yield (237–244): Provide ANOVA table in the Supplementary. Please clarify the sample size per treatment.

Response:

Thank you for your valuable comment. We agree that the crop yield analysis should be supported more explicitly. In the revised manuscript, we clarified the sample size for each treatment and added the corresponding ANOVA summary table in the Supplementary Materials.

 

4 Functional genes (249–272): Ensure consistency between the text and figures; nosZ patterns appear contradictory.

Response:

Thank you for your valuable comment. We carefully re-checked the text and figures describing functional gene abundance. In the revised manuscript, we corrected the relevant wording to ensure consistency with the figure results and revised the description of nosZ to avoid contradictory interpretation.

 

5 Microbial community (273–314): Simplify Figure 7b or split the frames. Report statistical significance of diversity indices.

Response: Thank you for your valuable comment. We agree that the presentation of the microbial community results can be improved. In the revised manuscript, we simplified the presentation of Figure 7b / split the panels to improve readability. We also added the statistical significance results for the diversity indices in the main text and/or figure captions.

 

Discussion

1 Soil properties and yield: Clarification of the mechanism of reduced NH₄⁺-N (microbial assimilation, slow release). Avoid subjective phrasing.

Response: Thank you for your valuable comment. We agree that the discussion of reduced NH₄⁺-N should be mechanistically clearer and that subjective phrasing should be avoided. In the revised manuscript, we have rewritten this section to explain that the lower NH₄⁺-N content under the BF treatment may be related to the combined effects of slower nutrient release, microbial immobilization/assimilation of available nitrogen, and enhanced plant nitrogen uptake, rather than simply attributing it to a general “beneficial” effect.

 

2 N₂O emission mechanisms: Interpret PLS-PM cautiously; correlation does not imply causation. Discuss saline–alkali effects on microbial physiology.

Response: Thank you for your valuable comment. Thank you for your valuable comment. We agree that the interpretation of PLS-PM results should be more cautious. In the revised manuscript, we have avoided causal language and instead described NH₄⁺-N as the environmental factor most strongly associated with N₂O flux under the present experimental conditions. We also added discussion on how saline-alkali stress may influence microbial physiology, nitrogen transformation, and N₂O production.

 

3 Functional genes: Strengthen the NOR pathway discussion with primary literature. Consider the temporal lag in nosZ expression.

Response: Thank you for your valuable comment. Thank you for your valuable comment. We agree that the discussion of the NOR-related pathway should be better supported and that the ecological interpretation of nosZ should consider possible temporal lags between gene abundance and functional response. In the revised manuscript, we strengthened the discussion of nitrifier denitrification and the NOR pathway using primary literature already cited in the manuscript, and we revised the interpretation of nosZ more cautiously.

 

4 Microbial community: Link shifts in Proteobacteria, Cyanobacteria, and Acidobacteriota to nitrogen cycling. Elaborate on the ecological significance of Oceanobacillus enrichment.

Response: Thank you for your valuable comment. We agree that the ecological interpretation of the microbial community shifts should be more closely linked to nitrogen cycling. In the revised manuscript, we expanded the discussion of the potential ecological roles of Proteobacteria, Cyanobacteria, and Acidobacteriota in soil nitrogen transformation and further clarified the ecological significance of Oceanobacillus enrichment under the BF treatment.

 

Conclusion

The manuscript is well structured but should emphasize practical agronomic implications.

Suggest future directions such as multi-season validation and metatranscriptomic approaches.

Response: Thank you for your valuable comment. We agree that the practical agronomic implications of this study should be stated more explicitly. In the revised manuscript, we strengthened the Conclusion by highlighting that B. subtilis biofertilizer not only reduced N₂O emissions but also maintained crop yield and improved plant nitrogen uptake, indicating its practical potential as a sustainable fertilization strategy for saline-alkali farmland.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Please find my comments in the attachement

Comments for author File: Comments.pdf

Author Response

Response to reviewer’s comments

(Manuscript ID: life-4177406)

Summary:

Thank you very much for handling our manuscript “B. subtilis biofertilizer mitigates N₂O emissions from saline-alkali farmland and its potential mechanisms”. We highly appreciate the insightful comments from the anonymous reviewer. After fully considering these comments, we thoroughly revised to our manuscript.

Below, we address the reviewer’s comments and questions point-by-point. The original comments are in black font and our responses to the comments follow in blue font. We hope our responses have addressed all of their concerns. If further improvement is required, please let us know.

Thank you!

Rui Li

 

 

1. Title: the full name of bacteria Bacillus subtilis should be used in the title.

Response: Thank you for your valuable comment. We agree that the full bacterial name should be used in the title for clarity and scientific accuracy. In the revised manuscript, we have replaced the abbreviated form with the full name Bacillus subtilis in the title.

 

2. Title: sounds bad grammatically; my propositions are (1) shortened “Bacillus subtilis biofertilizer mitigates N₂O emissions from saline-alkali farmland”, (2) “Potential mechanisms of N₂O emissions mitigation from saline-alkali farmland by Bacillus subtilis biofertilizer”, (3) “Effect of Bacillus subtilis biofertilizer on N₂O emissions from saline-alkali farmland, biological properties of the soil and crop yield”, (4) “N₂O emission mitigation from saline-alkali farmland by Bacillus subtilis biofertilizer and its potential mechanisms”, but I am aware there might be another valuable proposition for the title.

The experimental duration and site conditions must be explicitly mentioned.

Response: Thank you for your valuable comment and for providing several helpful suggestions for improving the title. We agree that the original title was not sufficiently concise and grammatically polished. In the revised manuscript, we adopted your first suggestion and revised the title to: “Bacillus subtilis biofertilizer mitigates N₂O emissions from saline-alkali farmland.” We believe this title is clearer, more concise, and better reflects the main finding of the study.

 

3 Key words: the primary idea of key words was to broaden the range of topic words beyond those used in the title; please replace the words which were used in the title with another ones that will bring us closer to the essence of the paper, e.g. “soil biome”, “nitrogen cycling”.

Response:

Thank you for your valuable comment. We agree that the keywords should extend beyond the wording used in the title and better reflect the core scientific content of the manuscript. In the revised manuscript, we replaced the title-overlapping keywords with more informative terms related to the main mechanisms and research focus of the study, such as nitrogen cycling; soil microbial community; functional gene; Nitrification, saline-alkali soil, N₂O mitigation. These revised keywords better capture the essence of the manuscript and improve its discoverability.

 

4 Introduction: The Introduction chapter should present not only a significance and research idea about the experiment but should also explain clearly the background of the experiment. Please broaden the Introduction adding some paragraphs discussing:

1. a) the role of bacteria in the soil and particularly in the soil N cycling,
2. b) the characteristics of studied organisms (why just B. subtilis was chosen? Is this B. subtilis fertilizer commercially available or was developed by the authors?),
3. c) what is the role of functional genes amoA, hao, norB and nosZ and how the analysis of their presence can increase our knowledge on bacterial activity in the environment.

Response:

Thank you for your valuable comment and for these thoughtful suggestions. We agree that these aspects are important for understanding the broader background of the study. However, considering the need to maintain a concise and focused Introduction, we did not substantially expand this section with additional background paragraphs. Instead, we kept the Introduction centered on the research gap, hypothesis, and objectives of the present study, while the functional relevance of the investigated genes and the ecological interpretation of microbial responses are further addressed in the Results and Discussion sections. In addition, the rationale for selecting Bacillus subtilis is that it is a widely used plant growth-promoting and environmentally adaptable biofertilizer microorganism with potential relevance to nitrogen transformation processes in saline-alkali soils. We hope that the current structure provides a balanced presentation of background information and study focus.

 

5 Introduction: The Introduction chapter should present not only a significance and research idea about the experiment but should also explain clearly the background of the experiment. Please broaden the Introduction adding some paragraphs discussing:

1. a) the role of bacteria in the soil and particularly in the soil N cycling,
2. b) the characteristics of studied organisms (why just B. subtilis was chosen? Is this B. subtilis fertilizer commercially available or was developed by the authors?),
3. c) what is the role of functional genes amoA, hao, norB and nosZ and how the analysis of their presence can increase our knowledge on bacterial activity in the environment.

Response: Thank you for your valuable comment and for this constructive suggestion. We agree that clearly stated research questions can improve the logical structure of the Introduction. However, in the present manuscript, we chose not to further restructure the Introduction into an explicit “research question” format. This is because we believe the current Introduction already presents the research background, identifies the main knowledge gap, and leads naturally to the objectives of the study. In addition, the subchapters in Section 4 were written to interpret the major findings rather than to directly mirror a predefined list of research questions. Therefore, to maintain the current organization and readability of the manuscript, we did not make further changes in this regard.

 

6 Introduction and in the whole manuscript: please use italics for Latin names of plants and microorganisms.

Response: Thank you for your valuable comment. We agree that the Latin names of plants and microorganisms should be formatted in italics according to scientific writing conventions. In the revised manuscript, we carefully checked the full text and standardized the formatting of all relevant Latin names in italics.

 

7 Supplementary file, Figure S2: please indicate what the Figures a, b and c show.

Response: Thank you for your valuable comment. We have revised the caption of Figure S2 in the Supplementary Materials and clearly indicated what panels a, b, and c represent, so that the figure can be understood more easily by readers.

 

8 M&M, line 110: Agilent 7890A is typically used for detection of organic compounds, please specify more clearly the equipment (e.g. columns) and procedures used for N2O detection.

Response: Thank you for your valuable comment. We agree that the description of the gas chromatographic analysis was not sufficiently specific in the original manuscript. In the revised manuscript, we clarified that N₂O was determined using an Agilent 7890A gas chromatograph equipped with a micro-electron capture detector (μECD), and we further described the sampling and analytical procedure for N₂O detection, including the use of sealed gas bags, automatic sample injection, and calibration based on standard gases provided by the National Institute of Metrology, China. However, because some instrumental configuration details such as the column model were not fully recorded in the manuscript draft, we focused on clarifying the detector type and analytical procedure to improve methodological transparency without introducing unsupported technical details.

 

9 M&M, lines 141-142: please explain TIC and TOC abbreviations.

Response: Thank you for your valuable comment. We agree that the abbreviations TIC and TOC should be defined when they first appear in the manuscript. In the revised manuscript, we have provided their full names as total inorganic carbon (TIC) and total organic carbon (TOC) to improve clarity for readers.

 

10 Results, Table 1: please insert the appropriate abbreviation instead of “OF”

Response:  Thank you for your valuable comment. We carefully checked Table 1 and found that the abbreviation “OF” was inconsistent with the treatment names used in the manuscript. In the revised version, we have replaced “OF” with the correct abbreviation and standardized the treatment nomenclature throughout the manuscript.

 

11 Results, Figure 4: please correct the colours in Figure 4b, colour of the 3rd bar from the top is not the same as in the legend.

Response: Thank you for your valuable comment. We agree that the color assignment in the legend of Figure 4b was inconsistent with the bar colors shown in the figure. In the revised manuscript, we corrected the legend of Figure 4b to ensure that the color labels are fully consistent with the displayed bars, thereby improving the clarity and accuracy of the figure presentation.

 

12 Results, line 287: please explain the abbreviation “DNRA” and the phrase “…which may indicate an enhancement of the DNRA process”, in Introduction if possible.

Response: Thank you for your valuable comment. We agree that the abbreviation DNRA should be clearly defined when it first appears. In the revised manuscript, we have explained DNRA as dissimilatory nitrate reduction to ammonium and clarified the relevant sentence accordingly. Considering the overall structure and conciseness of the Introduction, we did not add a detailed explanation there; instead, we provided the definition and interpretation at its first appearance in the Results/Discussion section, where it is more directly related to the observed findings.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Title: B. subtilis biofertilizer mitigates N₂O emissions from saline-alkali farmland and its potential mechanisms

 

Dear Authors

The manuscript's topic is original and relevant in the era of climate change. It presents research findings that indicate opportunities for reducing N₂O emissions. While the research findings are interesting, the entire manuscript requires revisions and additions.

All sections except the Discussion section should be significantly expanded and more detail added. All references provided in the References section should be used.

In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

Remarks

1. Abstract: The abstract is written very generally. Please correct it. A short abstract should include: introduction, purpose, materials and methods, results, and conclusions.
2. Introduction: The research objective and research hypothesis should be clearly presented.
3. Materials and Methods: Subsection 2.2. Please include the number of replicates in the field experiment and the type of corn cultivation (grain or silage). Mineral fertilizer rates should be provided in kgN^.ha^-1, kgP^.ha^-1, and kgK^.ha^-1. Please include the names of the mineral fertilizers and the % content of the nutrients N, P, and K. Natural fertilizer rates should be provided in t^.ha^-1. Subsection 2.4 – Lines 132, 133, and 134 Please add the sources for the methods presented. Line 141 - The city and country of production of the elementary analyzer should be added.
4. Results: Table 1 Should be corrected: should be: TN in g^.kg^-1. First column - should read: CBF. Below Table 1, add the explanations: TSS, SWC, and SOM. Figure 5 - The units on the y-axis need to be corrected and the N content in grain and straw needs to be added.
5. Conclusions: More details from the research results and possible directions for future research should be added.
6. References – It must be technically adapted to editorial requirements.

Detailed remarks

1. Citations of research results by other authors should be recorded in accordance with editorial requirements. This means that citations should be numbered sequentially, e.g., line 37 – (Tian et al., 2020; Yang et al., 2024) – should be [1, 2], etc. The "References" section should be presented according to this numbering.
2. Line 189 ….(Cao et al., 2022)..- not included in the References section.
3. References no. 10, 11, 25, and 28 were not cited in the manuscript.

Best regards

 

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made substantial efforts to address the reviewer’s previous comments, and the revised manuscript shows clear improvement in several aspects, including the inclusion of quantitative results in the abstract, clarification of the experimental design, addition of statistical analysis details, and expansion of the discussion on microbial functional genes and nitrogen cycling pathways. The introduction now provides a clearer comparison between Bacillus subtilis and other biofertilizers, and the hypothesis and objectives are more explicitly stated.

However, several issues should still be corrected before the manuscript can be considered fully acceptable.

First, the description of sequencing data processing in the Methods section contains an unresolved placeholder statement (“please choose the actual method used”), which indicates that the normalization method for microbial community analysis was not finalized. The authors should clearly specify the exact normalization approach used (e.g., rarefaction, relative abundance normalization, or variance-stabilizing transformation).

Second, the sequencing depth and rarefaction description appears inconsistent. The manuscript reports approximately 153,624 clean reads in total with an average of about 25,471 reads per sample, yet it states that samples were rarefied to 293,256 reads per sample. This value is not consistent with the reported sequencing depth and should be carefully verified and corrected.

Third, the presentation of N₂O flux units should be checked carefully. Although the text states that cumulative emissions were reported using the standard unit of kg N₂O-N ha⁻¹ season⁻¹, the units displayed in some figures appear inconsistent. The authors should ensure that all figures and captions use consistent and standard units.

Finally, minor language editing is recommended to improve sentence clarity and remove occasional repetition in the Discussion section.

After these issues are corrected, the manuscript will provide a clearer and more technically robust contribution to the understanding of microbial biofertilizer effects on N₂O emissions in saline-alkali agricultural soils.

Author Response

Response to reviewer’s comments

(Manuscript ID: life-4177406)

Summary:

We sincerely thank the reviewer for the positive evaluation of our revised manuscript and for the constructive comments. We are encouraged that the reviewer recognized the improvements in the abstract, experimental design, statistical analysis, discussion of microbial functional genes and nitrogen cycling pathways, and clarification of the study objectives. We have carefully revised the manuscript again in response to the remaining comments. Our detailed responses are provided below.

Thank you!

Rui Li

 

 

Abstract (Lines 11–28)

1. First, the description of sequencing data processing in the Methods section contains an unresolved placeholder statement (“please choose the actual method used”), which indicates that the normalization method for microbial community analysis was not finalized. The authors should clearly specify the exact normalization approach used (e.g., rarefaction, relative abundance normalization, or variance-stabilizing transformation).

Response:

Thank you for pointing this out. We agree that the placeholder statement was inappropriate and may cause confusion regarding the normalization procedure used for microbial community analysis. We have removed this statement and now explicitly describe the actual normalization strategy used in the revised Methods section.

2. Second, the sequencing depth and rarefaction description appears inconsistent. The manuscript reports approximately 153,624 clean reads in total with an average of about 25,471 reads per sample, yet it states that samples were rarefied to 293,256 reads per sample. This value is not consistent with the reported sequencing depth and should be carefully verified and corrected.

Response:

We thank the reviewer for carefully checking these values. We re-examined the original sequencing output and found that the previously reported numbers were inconsistent due to a reporting error in the Methods section. We have now corrected the total number of clean reads, the average reads per sample, and the rarefaction depth so that they are internally consistent and accurately reflect the sequencing dataset. The revised values are now reported in Section 2.6.

3 Third, the presentation of N₂O flux units should be checked carefully. Although the text states that cumulative emissions were reported using the standard unit of kg N₂O-N ha⁻¹ season⁻¹, the units displayed in some figures appear inconsistent. The authors should ensure that all figures and captions use consistent and standard units.

Response:

Thank you for this important comment. We carefully rechecked the units used in the text, figures, and figure captions. We found that some figure axis labels were inconsistent with the standard unit reported in the manuscript. We have now corrected the figure labels and captions to ensure that cumulative emissions are consistently reported as kg N₂O-N ha⁻¹ season⁻¹ and that flux units are presented consistently throughout the manuscript.

 

4 Finally, minor language editing is recommended to improve sentence clarity and remove occasional repetition in the Discussion section.

Response: Thank you for this suggestion. We carefully edited the manuscript again to improve sentence clarity and reduce repetition, particularly in the Discussion section. Redundant or self-referential revision statements were removed, and several paragraphs were rewritten for conciseness and readability.

 

We thank the reviewer again for these helpful comments. We believe that these revisions have improved the clarity and technical rigor of the manuscript, and we hope that the revised version is now suitable for publication.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the changes made by the Authors, the manuscript has been improved in a great measure. However, I can see some items which were not improved sufficiently:

 

1. Introduction: in my opinion this chapter was not broaden enough, see Review version 1. I wish the Authors their paper will be read by broad public and more substantial knowledge included in the introduction would provide a valuable insight into this topic.
2. Introduction and in the whole manuscript: still italics for Latin names of plants and microorganisms are not routinely used in the manuscript, see lines 58, 59, 61 etc., 363, 388, etc., 503, 520, 536, 541, 555.

 

Undoubtedly, the paper is appropriate for publishing in Life but in my opinion the shape of the Introduction chapter is debatable.

Author Response

Response to reviewer’s comments

(Manuscript ID: life-4177406)

Summary:

We sincerely thank the reviewer for the careful reading of our manuscript and for the positive evaluation of our revisions. We greatly appreciate the reviewer’s recognition that the manuscript has been substantially improved. In response to the remaining concerns, we have revised the manuscript again. Specifically, the Introduction has been completely rewritten to provide broader and more substantial background for a wider readership, and we have carefully checked the entire manuscript to ensure that Latin names of plants and microorganisms are consistently presented in italics throughout the text.

Thank you!

Rui Li

 

 

1. Introduction: in my opinion this chapter was not broaden enough, see Review version 1. I wish the Authors their paper will be read by broad public and more substantial knowledge included in the introduction would provide a valuable insight into this topic.

Response: Thank you very much for this valuable suggestion. We fully understand the reviewer’s concern that the previous Introduction was still not sufficiently broad and did not provide enough background for a wider scientific readership. In response, we have completely rewritten the Introduction to improve its scope, logical flow, and accessibility.

In the revised version, we substantially expanded the background section by:

1. further emphasizing the environmental significance of agricultural N₂O emissions and the importance of improving nitrogen use efficiency;
2. broadening the discussion of biofertilizers as a sustainable fertilization strategy and summarizing previous findings on their effects on N₂O mitigation;
3. providing a clearer introduction to the characteristics and agricultural relevance of Bacillus subtilis;
4. strengthening the rationale for focusing on saline-alkali soils as a special ecological context; and
5. more clearly presenting the knowledge gap, hypothesis, and objectives of this study.

We believe that the rewritten Introduction now provides a more comprehensive framework for readers from both this specific field and the broader agricultural and environmental research communities.

2. Introduction and in the whole manuscript: still italics for Latin names of plants and microorganisms are not routinely used in the manuscript, see lines 58, 59, 61 etc., 363, 388, etc., 503, 520, 536, 541, 555.

Response: Thank you for pointing this out. We agree that the formatting of Latin names was not sufficiently consistent in the previous revision. In response, we have carefully checked the entire manuscript, including the Introduction, Results, Discussion, Conclusions, and figure/table captions, and corrected the formatting of scientific names throughout.

All Latin names of plants and microorganisms are now consistently presented in italics, including species names such as Bacillus subtilis and other relevant taxa mentioned in the manuscript.

 

 

We are very grateful for the reviewer’s constructive comments, which helped us further improve the manuscript. We believe that the revised version has been strengthened substantially, particularly through the complete rewriting of the Introduction and the thorough correction of formatting inconsistencies for Latin names throughout the manuscript.

Author Response File: Author Response.docx