Biochar Promotes Phosphorus Solubilization by Reconstructing Soil Organic Acid and Microorganism Networks

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors submitted an original research manuscript for publication, which scope fits to the journal. Biochar application for regulating P in soil, including studying organic acids and bacterial community, is not entirely new. However, the manuscript provided quite an interesting correlation among the organic acids, P fractions, and bacterial community. Considering this I quite some concerns that the authors better clarify in the manuscript:
1.  Almost all figures' text such as axis, inside legend, values are hard to read; they are extremely small fonts.

2. Pot experiment: This section is quite unclear to me it raised many questions:

-was it seed or seedling? if seedling, what was the control maintained so that the age of seedlings did not contribute the P/microbial profile later on the trial

-from exactly where the soil sample were collected? rhizosphere soil or soil that was not root touch? surface soil from pot or deep in the pot?

-what was the watering regime?

3. Microbial profile: it claimed "bacterial profile". were there not any archaea community from the 16S rRNA sequencing? if so, was that removed from OTU analysis? if not, it is always better to write "microbial community" containing bacteria and archaea 

4. Organic acids were identified and quantified without using an internal/external analytical standard? if so, the justification and quality control explanation are needed.

5. from biochar point of view I think two big concerns are:

-in discussion, it is argued "these" and "that" properties from biochar contribted to this and that such as production of organic acids. I understand this paper does not characterise biochar's functional groups. if authors have that data, it is better to put in Supplementary file. without this, it is very passive claim of the role of biochar for such a molecular level insights (bacterial community and organi acids)

-since this is for agronomy application, cost is a must component. provide cost of biochar production, pre-treatment cost before applying to soil and how that compare or make difference to the P budget in soil.

 

 

Author Response

Response to Reviewer 1 Comments

Comments and Suggestions for Authors: 
The authors submitted an original research manuscript for publication, which scope fits to the journal. Biochar application for regulating P in soil, including studying organic acids and bacterial community, is not entirely new. However, the manuscript provided quite an interesting correlation among the organic acids, P fractions, and bacterial community. Considering this I quite some concerns that the authors better clarify in the manuscript.

Reply: Thank you for your valuable insights and dedicated efforts to improve our manuscript. The followings presented below are a comprehensive, point-by-point response addressing each of your comments.

Comments 1: Almost all figures' text such as axis, inside legend, values are hard to read; they are extremely small fonts.

Response 1: Thank you for your valuable feedback. In response to your comment regarding the small fonts in the figures, we have increased the font size of the axis labels, legend text, and values to improve readability. The revised figures have been uploaded for your further review.

Comments 2: Pot experiment: This section is quite unclear to me it raised many questions:

-was it seed or seedling? if seedling, what was the control maintained so that the age of seedlings did not contribute the P/microbial profile later on the trial

-from exactly where the soil sample were collected? rhizosphere soil or soil that was not root touch? surface soil from pot or deep in the pot?

-what was the watering regime?

Response 2: Thank you very much for your valuable feedback. To clarify, in order to maintain consistency throughout the experiment, we used seeds rather than seedlings. We have added the following detail to the Materials and Methods section: "On the first day, ten seeds were sown in each pot." Thank you for your help in making the trial design clearer. (Material and methods section, line 127, page 3)

Thank you very much for your valuable feedback and for helping to improve the quality of our manuscript. In response to your question, the soil samples were collected from the rhizosphere of the plants within the pots. We have clarified this point in the revised manuscript to ensure better understanding. Also added references to rhizosphere soil sampling methods. We appreciate your thoughtful comments. (Material and methods section, line 131, page 3)

Thank you very much for your insightful feedback and for contributing to the improvement of our manuscript. In response to your question regarding the watering regime, we have added the following details to the Materials and Methods section: "All pots with soil were weighed at the beginning of the experiment and watered every morning to maintain the soil moisture content between 70% ~ 75%." (Material and methods section, line 129-131, page 3)

Comments 3: Microbial profile: it claimed "bacterial profile". were there not any archaea community from the 16S rRNA sequencing? if so, was that removed from OTU analysis? if not, it is always better to write "microbial community" containing bacteria and archaea 

Response 3: Thank you very much for your thoughtful feedbacks and for helping to improve the quality of our manuscript. In response to your comment, we have revised the manuscript by changing "bacterial community" to "microbial community". We appreciate your suggestion and believe this modification enhances the accuracy of the manuscript. Thank you again for your valuable input.

Comments 4: Organic acids were identified and quantified without using an internal/external analytical standard? if so, the justification and quality control explanation are needed. 

Response 4: Thank you for your valuable comment. In response to your question, organic acids in this study were identified and quantified using standards with internal standard quantification. All target organic acids were individually prepared at gradient concentrations, with succinic acid-D4, cholic acid-D4, and salicylic acid-D4 serving as internal standards. Standard curves were generated based on the linear relationship between peak area and concentration. The linear regression was plotted with the ratio of the chromatographic peak area of the target standard solution to the internal standard peak area on the vertical axis and concentration on the horizontal axis. The linearity (R²) for all analytes was greater than 0.99, indicating good linearity. To calculate the sample concentration, the ratio of the sample analyte's mass spectrometry peak area to the internal standard peak area was substituted into the linear equation to determine the concentration results. We hope this explanation addresses your concerns regarding the quantification method and quality control. Thank you again for your constructive feedback.

Comments 5: from biochar point of view I think two big concerns are:

-in discussion, it is argued "these" and "that" properties from biochar contribted to this and that such as production of organic acids. I understand this paper does not characterise biochar's functional groups. if authors have that data, it is better to put in Supplementary file. without this, it is very passive claim of the role of biochar for such a molecular level insights (bacterial community and organi acids)

-since this is for agronomy application, cost is a must component. provide cost of biochar production, pre-treatment cost before applying to soil and how that compare or make difference to the P budget in soil.

Response 5: Thank you for your valuable feedback. In response to your comment, we have now included the infrared spectroscopy data describing the functional groups of biochar in the supplementary file, as per your suggestion. We hope this addresses your concern and strengthens the claims made in the discussion section. Thank you again for your helpful suggestions. (Material and methods section, line 114-115, page 3)

Thank you very much for your valuable feedback. In response to your question regarding the cost and pre-treatment of biochar, the cost of biochar production is approximately 1800 RMB per ton.  In practical production, biochar typically does not require pretreatment.  This is based on previous research from our team, which found that untreated biochar can enhance the phosphorus content in soil.  However, due to the phosphorus content inherent in untreated biochar, it remains unclear whether biochar directly supplies phosphorus to the soil or indirectly enhances soil phosphorus availability.  The aim of our current study is to demonstrate that, in addition to directly providing phosphorus to the soil, biochar can also indirectly improve soil phosphorus availability through its inherent material properties. In response to your comment, we have added the following statements in the discussion section: "Conventional phosphorus fertilizers, when applied to the soil, are largely fixed, resulting in low efficiency of phosphorus uptake by plants. Biochar, through its inherent material properties, can enhance soil phosphorus availability, playing an irreplaceable role in agricultural production." (Discussion section, line 476-479, page 14)

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear,

The paper Biochar Promotes Phosphorus Solubilization by Reconstruction of Soil Organic Acid and Bacteria Networks deals with the impact of biochar on soil organic acids and bacterial community structure, as well as P fractions, in the presence of different cultivated plants.

The paper is well written, results are clearly presented.

However, there are some issues regarding establishment of experiment and methodology.

The results are based on the short-term experiment in pots. In addition, the soil per pot is only 500g, and the plants were soybean, maize and mung bean, which are quite large. The plants were followed for 25 days.

Is this experimental design adequate? Is it applied earlier in the papers? Please provide references. In addition, please provide references for 2.3.3. Measurement of Organic Acids and soil sampling for the analyses performed.

Is this short period adequate for measurement of biochar effect on soil structure?

The initial soil pH is quite high, 8.3. Whether the application of biochar (pH 9.13) affected the change in soil pH value after plants cultivation?

I recommend to resolve the previous mentioned tasks.

Best regards,

Author Response

Response to Reviewer 2 Comments

Comments and Suggestions for Authors: 
The paper Biochar Promotes Phosphorus Solubilization by Reconstruction of Soil Organic Acid and Bacteria Networks deals with the impact of biochar on soil organic acids and bacterial community structure, as well as P fractions, in the presence of different cultivated plants. The paper is well written, results are clearly presented. However, there are some issues regarding establishment of experiment and methodology. 

Reply: Thank you for your valuable insights and dedicated efforts to improve our manuscript. The followings presented below are a comprehensive, point-by-point response addressing each of your comments.

Comments 1: The results are based on the short-term experiment in pots. In addition, the soil per pot is only 500g, and the plants were soybean, maize and mung bean, which are quite large. The plants were followed for 25 days. Is this experimental design adequate? Is it applied earlier in the papers? Please provide references. In addition, please provide references for 2.3.3. Measurement of Organic Acids and soil sampling for the analyses performed.

Response 1: Thank you for your valuable feedback. In response to your concerns, previous studies have shown that biochar can influence the early growth of field crops. Similar experimental designs, including pot experiments, have been used to evaluate biochar's effects on seedling growth. As per your suggestion, we have now cited relevant studies with comparable designs as references. We hope this addresses your concern regarding the experimental setup. (Material and methods section, line 129, page 3)

Thank you for your valuable comment. In response to your request, we have now added the relevant references for soil sampling in section 2.3.3 "Measurement of Organic Acids and Soil Sampling for the Analyses Performed." These references provide supporting documentation for the methods used in our study. We hope this addresses your concern. Thank you again for your helpful suggestions. (Material and methods section, line 132, page 3; line 198, page 5)

Comments 2: Is this short period adequate for measurement of biochar effect on soil structure?

Response 2: Thank you very much for your insightful comment. In response to your question regarding the adequacy of the short experimental period for measuring biochar's effect on soil structure, we appreciate your observation. Based on previous researchs, biochar is a highly porous, carbon-rich material that, when applied to soil, can directly enhance soil porosity and aeration, thereby improving soil structure. Following your suggestion, we have now included an explanation of this characteristic of biochar in the introduction to provide better context for the experiment. We hope this addition addresses your concern. Thank you again for your valuable feedback. (Introduction section, line 73-74, page 2)

Comments 3: The initial soil pH is quite high, 8.3. Whether the application of biochar (pH 9.13) affected the change in soil pH value after plants cultivation?

Response 3: Thank you for your valuable feedback. In response to your question about whether the application of biochar (pH 9.13) affected the soil pH, we appreciate your observation. Based on our previous research, biochar, being an alkaline material, tends to increase soil pH. High pH conditions, however, are generally unfavorable for phosphorus dissolution in soil. Therefore, we hypothesize that biochar may promote the dissolution of phosphorus under high pH conditions by regulating microbial activity and organic acid secretion. We have added this perspective to the discussion section to further clarify this point. We hope this addition addresses your concern. Thank you again for your helpful comments. (Discussion section, line 449-450, page 14)

 

 

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors, you have done god work on role of biochar in P solubilization.

There are some issues in the manuscript, which need to be addressed.

1. You have mentioned 'millet', what type of millet you have cultivated, eg., finger millet or something else.
2. You have treated your biochar with NaCl and NaHCO₃  for removal of P, is this practice is viable for a farmer. How would you recommend your findings to a common farmer to adopt biochar for enhancing P uptake.
3. You have mentioned the pH of your treated biochar 9.13, how this alkaline pH will help in solubilization of P. May be this high pH neutralize the organic acids production by soil bacteria. 
4. You have used calcium superphosphate as a source of P in your experiment, don't you think that this form of P fertilizer is less soluble in water, instead single superphosphate would have been a better choice.
5. You may include following references; Bai, K., Wang, W., Zhang, J. et al. Effects of phosphorus-solubilizing bacteria and biochar application on phosphorus availability and tomato growth under phosphorus stress. BMC Biol 22, 211 (2024). https://doi.org/10.1186/s12915-024-02011-y
6. Lu, L., Qin, W., Wu, M. et al. Biochar promotes FePO₄ solubilization through modulating organic acids excreted by Talaromyces pinophilus. Carbon Res. 4, 27 (2025). https://doi.org/10.1007/s44246-025-00193-w
7. https://doi.org/10.1016/j.biortech.2024.130329
8. Overall the manuscript is interesting, at some places grammatical errors have been observed. 

 

Comments on the Quality of English Language

1. The authors should check the manuscript for grammatical errors.

Author Response

Response to Reviewer 3 Comments

Comments and Suggestions for Authors: 
You have done god work on role of biochar in P solubilization.There are some issues in the manuscript, which need to be addressed. 

Reply: Thank you for your valuable insights and dedicated efforts to improve our manuscript. The followings presented below is a comprehensive, point-by-point response addressing each of your comments.

Comments 1: You have mentioned 'millet', what type of millet you have cultivated, eg., finger millet or something else.

Response 1: Thank you for your valuable comment. In response to your question, we cultivated foxtail millet in this study. We have now replaced the term "millet" with "foxtail millet" throughout the manuscript for clarity. We hope this resolves your concern. Thank you again for your helpful feedback.

Comments 2: You have treated your biochar with NaCl and NaHCO3  for removal of P, is this practice is viable for a farmer. How would you recommend your findings to a common farmer to adopt biochar for enhancing P uptake.

Response 2: Thank you for your valuable feedback. In response to your question, in practical agricultural production, biochar typically does not require pre-treatment. Based on our previous research, untreated biochar has been found to improve the availability of phosphorus in soil. However, untreated biochar itself also contains phosphorus. The aim of our current study is to demonstrate that biochar not only directly provides phosphorus to the soil but also indirectly increases the availability of phosphorus in the soil. We hope this explanation helps clarify the potential benefits of biochar in farming practices. Thank you again for your comments.

Comments 3: You have mentioned the pH of your treated biochar 9.13, how this alkaline pH will help in solubilization of P. May be this high pH neutralize the organic acids production by soil bacteria.

Response 3: Thank you for your insightful comment. Based on our previous research, biochar, as an alkaline material, increases soil pH. While high pH conditions are generally unfavorable for phosphorus dissolution, we hypothesize that biochar may promote phosphorus solubilization under high pH conditions by regulating microbial activity and organic acid secretion. Following your suggestion, we have added this perspective to the discussion section to further clarify the potential mechanisms at play. Thank you again for your valuable input. (Discussion section, line 449-450, page 14)

Comments 4: You have used calcium superphosphate as a source of P in your experiment, don't you think that this form of P fertilizer is less soluble in water, instead single superphosphate would have been a better choice.

Response 4: Thank you for your comment. Upon reviewing your suggestion, we realized that the use of "calcium superphosphate" in the manuscript was due to a translation error. The fertilizer used in the experiment was actually single superphosphate. We have corrected this in the revised manuscript. We appreciate your careful attention to this detail. Thank you again for your helpful feedback.

Comments 5: You may include following references; Bai, K., Wang, W., Zhang, J. et al. Effects of phosphorus-solubilizing bacteria and biochar application on phosphorus availability and tomato growth under phosphorus stress. BMC Biol 22, 211 (2024).

Response 5: Thank you very much for your helpful suggestion. I appreciate your recommendation to include the reference "Effects of phosphorus-solubilizing bacteria and biochar application on phosphorus availability and tomato growth under phosphorus stress." I have now cited this paper in the manuscript. (Discussion section, line 434, page 13)

Comments 6: Lu, L., Qin, W., Wu, M. et al. Biochar promotes FePO4 solubilization through modulating organic acids excreted by Talaromyces pinophilus. Carbon Res. 4, 27 (2025). https://doi.org/10.1007/s44246-025-00193-w

Response 6: Thank you very much for your valuable suggestion and for helping to improve the quality of our manuscript. In response to your recommendation, I have now cited the following reference: Biochar promotes FePO4 solubilization through modulating organic acids excreted by Talaromyces pinophilus. (Discussion section, line 439, page 13)

Comments 7: https://doi.org/10.1016/j.biortech.2024.130329

Response 7: I appreciate your recommendation to include the reference "Biochar application can enhance phosphorus solubilization by strengthening redox properties of humic reducing microorganisms during composting." I have now cited this paper in the manuscript. Thank you again for your valuable input in improving the quality of the manuscript. (Discussion section, line 393, page 12; line 461, page 14)

Comments 8: Overall the manuscript is interesting, at some places grammatical errors have been observed.

Response 8: Thank you for your positive feedback and constructive comments. In response to your observation regarding grammatical errors, we have thoroughly reviewed the manuscript and corrected the identified issues. We believe these revisions have improved the clarity and readability of the paper. Thank you again for your helpful suggestions. (Material and methods section, line 147-148, page 4; Results section, line 274, page 7; Discussion section, line 448, page 14)

 

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors improved the manuscript following much of the suggestions. However, I assume it is fair to say:

-the fonts in most of the figures are still far from being readable.

-Cost and practical implications of biochar is not for my considering, it is better to add in the manuscript. It will help to understand the practical aspect of this biochar for the intended application.

-better to clarify microbes containing bacteria and archaea. it is usually bacteria in your case I think and if you know it what percentage of bacteria, you can make more compelling writing.

-biochar spectra as SI file is good, but what is the relation of this properties for the organic acids/P related changes, and that should be in the discussion?

 

Author Response

Response to Reviewer 1 Comments

Comments and Suggestions for Authors: 

Authors improved the manuscript following much of the suggestions. However, I assume it is fair to say.

Reply: Thank you for your valuable insights and dedicated efforts to improve our manuscript. The followings presented below are a comprehensive, point-by-point response addressing each of your comments.

Comments 1: the fonts in most of the figures are still far from being readable.

Response 1: Thank you for your helpful comment. According to your suggestion, we have revised the figures by adjusting the small and unclear fonts to improve readability. The figure texts have been enlarged and optimized to ensure clarity in the revised manuscript.

Comments 2: Cost and practical implications of biochar is not for my considering, it is better to add in the manuscript. It will help to understand the practical aspect of this biochar for the intended application. 

Response 2: Thank you for your valuable suggestion. In response, we have revised the manuscript to include a discussion on the cost and practical implications of biochar. Specifically, we added the following content: “The cost of biochar production is approximately 1800 RMB per ton, and in practical agricultural production, biochar typically does not require pretreatment. Previous studies have shown that untreated biochar can enhance phosphorus availability in soil [15]. However, due to the phosphorus inherently present in untreated biochar, it remains unclear whether biochar directly supplies phosphorus to the soil or indirectly enhances soil phosphorus availability. The objective of this study is to demonstrate that, in addition to directly providing phosphorus to the soil, biochar can also indirectly improve soil phosphorus availability through its inherent material properties.” We appreciate the reviewer’ s valuable comment, which we believe has improved the applicability of the manuscript. (Discussion section, line 480-487, page 16)

Comments 3: better to clarify microbes containing bacteria and archaea. it is usually bacteria in your case I think and if you know it what percentage of bacteria, you can make more compelling writing. 

Response 3: Thank you for your valuable comment. According to your suggestion, we have clarified the composition of microbial domains. Based on the phylum-level taxonomic classification, we added that the majority of the OTUs were assigned to the domain Bacteria (approximately 99.97%), while a smaller proportion (approximately 0.03%) belonged to Archaea. (Material and methods section, line 172-176, page 4)

Comments 4: biochar spectra as SI file is good, but what is the relation of this properties for the organic acids/P related changes, and that should be in the discussion? 

Response 4: Thank you for your valuable feedback. In response to your comment, we have now included a discussion on the relationship between the spectral properties of biochar and the changes in organic acids and phosphorus. This addition has been made in the Discussion section, as per your suggestion. We hope this addresses your concern and enhances the clarity of the manuscript. Thank you again for your helpful comments. (Discussion section, line 389-392, page 14; line 450-452, page 15)

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript is substantially improved. The authors explained all questions in satisfactory way.

Author Response

We sincerely thank the reviewer for the positive feedback and recognition of our revisions. We greatly appreciate your constructive comments, which helped us improve the quality and clarity of our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors

You have revised the manuscript as suggested by reviewrs

Author Response

We sincerely thank the reviewer for the positive feedback and recognition of our revisions. We greatly appreciate your constructive comments, which helped us improve the quality and clarity of our manuscript.