Combined Application of Chemical and Organic Fertilizers Promoted Soil Carbon Sequestration and Bacterial Community Diversity in Dryland Wheat Fields

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

 

The topic and methodology of the study are equally interesting. Not all the conclusions have been drawn, but more papers are likely planned from a ten-year experiment.

However, in order to accept and understand the described conclusions, it is necessary to describe the characteristics of the experiment and the methods used in more detail in the chapter Materials and Methods. And also, should be important to mention and characterize the investigation methods used (mainly Biolog test) in the chapter Introduction.

In details:

Abstract:

It would be useful to mention, what kind of organic fertilizer used in the experiments (line 20).

The content of line 30-34 should be rethink and rewrite, based on the modified discussion.

 

Introduction:

The introduction should be supplemented by a description of the SOM fractions and the results obtained by investigating these fractions on semi-arid soils.

Similarly, the properties of the Biolog microplate method and its use in the study of such soils should be mentioned.

It is important to note that the Biolog test does not characterize the activity of all soil microbes, but only heterotrophs that can divide in the buffer used in the test.

 

Materials and Methods:

2.1.

line 106-107: To name the soil type, the „calcareous cinnamon soil” is not enough, the FAO or WRB classification should be used.

line 109: In which solution was the pH measured? (in distilled water, or KCl?)

line 109: What depth does topsoil indicate? 0-20 or 0-30 cm?

line 110-111: What was the method used to measure the amount of available P and K?

2.2.

line 120-122: the measurement of nitrate N content and the quick-acting K did not mention previously.

line 122-124: What kind of organic fertilizer was used in the experiments, and what are its properties? (For example, easily degradable and resistant C content and C:N ratio, …)

line 125-129: the description of composition and the preparation of OFB is not clear. The name of Lahn-type bacteria and Pseudomonas 1 and 2 is not enough to describe the applied bacterial solution. The quantity and the more detailed composition are needed (and the name of the manufacturer also)

The amount carbon input is an important factor in this experiment. It would be useful if Table 1 included (at least estimated) values of carbon entering the soil. For example, the cumulative crop yield corresponds to the estimate, probably proportional to the organic carbon released into the soil.

line 147: reference is needed about the determination of SOC and its fractions.

line 154: the name and the manufacturer of Biolog microplate is necessary.

2.3.3. the description of fungal PCR is missing from the text.

 

Results:

3.3.1.

It would be useful a bit more data about the PCR (e.g., average OUT numbers by treatments, the depth of sequencing, …)

3.3.2. This subchapter is concerning the compositions of microbial communities. The differences between them are visible (Fig. 7), but their significance is missing. Are the community patterns of the different treatments significantly different?

 

Discussion:

4.1. SOC components

The assumption of “enhanced soil aggregation” (line 376) is not supported by the measured data. In this case, the proportion or amount of stable fraction of SOM should have increased, but this is not what happened. The amount of active fractions of SOM increased. And a large amount of sand also does not favor an increase in aggregation.

line 375: there is no data about the biomass C inputs in the manuscript. Therefore, this statement, while probably true, is not supported.

 

4.2. functional diversity

To evaluate the results, you should also briefly write about the advantages and disadvantages of the Biolog substrate utilization method. In particular, the method does not measure the whole soil microbial community, but only those what are able to divide in the buffer used. There are also literature references to this topic.

4.3. microbial community composition

line 498-508: This part of text would be in a better place in the chapter Conclusion.

 

Conclusion:

I propose to be a little more precise about the statements, and in a way that, similarly to the abstract, can be understood without reading the whol manuscript.

 

Best regards,

 

Author Response

1. Abstract: It would be useful to mention, what kind of organic fertilizer used in the experiments (line 20).

Response: As per your suggestion to specify the type of organic fertilizer, I referred to the fermented chicken manure. Please refer to line 19 of the revision mode manuscript for details.

2. The content of line 30-34 should be rethink and rewrite, based on the modified discussion.

Response: The specified content have been rewritten. Please refer to lines 31-34 in the revision mode manuscript.

3. Introduction: The introduction should be supplemented by a description of the SOM fractions and the results obtained by investigating these fractions on semi-arid soils.

Similarly, the properties of the Biolog microplate method and its use in the study of such soils should be mentioned.

It is important to note that the Biolog test does not characterize the activity of all soil microbes, but only heterotrophs that can divide in the buffer used in the test.

Response: Following your suggestion, I have added a description of the components of SOC and the findings from their study in semi-arid soils to the Introduction. The details can be found in lines 91-101 of the revision mode manuscript.

In the revision mode manuscript, the characteristics of the Biolog culture method have been detailed in lines 107-112. Additionally, reference [23] has been updated to one that specifically examines the microbial carbon source metabolism in semi-arid soils.

4. Materials and Methods: 2.1 line 106-107: To name the soil type, the “calcareous cinnamon soil” is not enough, the FAO or WRB classification should be used.

Response: Thank you for your suggestion. The revision mode manuscript has been updated with corrections based on the FAO soil classification system. Please refer to line 193 of the revision mode manuscript for details.

5. Materials and Methods: 2.1 line 109: In which solution was the pH measured? (in distilled water, or KCl?)

Response: The soil pH was measured in distilled water following the Chinese National Standard (HJ 962-2018), as indicated in the manuscript at line 197.

6. Materials and Methods: 2.1 line 110-111: What was the method used to measure the amount of available P and K?

Response: Available phosphorus was measured using 0.5 mol/l NaHCO₃ extraction method, followed by molybdenum-antimony resistance colorimetry with spectrophotometer.

Available K was measured using 1.0 mol/l NH₄OAC extraction followed by analysis with a flame spectrophotometer.

I have provided a supplementary explanation in Section 2.3.1 of the revision mode manuscript.

7. Materials and Methods: 2.2 line 120-122: the measurement of nitrate N content and the quick-acting K did not mention previously.

Response: I am grateful for your meticulous guidance. The revision mode manuscript now includes the determination of nitrate nitrogen and available potassium content in Section 2.3.1, and the references for the determination of organic carbon components have been cited.

8. Materials and Methods: 2.2 line 122-124: What kind of organic fertilizer was used in the experiments, and what are its properties? (For example, easily degradable and resistant C content and C:N ratio, …)

Response: Thank you for your suggestion. In the experiment, chicken manure was employed as the organic fertilizer. The quantities of organic fertilizer applied annually and the nutrient content of these fertilizers are presented in the supplementary material, Table S1.

9. Materials and Methods: 2.2 line 125-129: the description of composition and the preparation of OFB is not clear. The name of Lahn-type bacteria and Pseudomonas 1 and 2 is not enough to describe the applied bacterial solution. The quantity and the more detailed composition are needed (and the name of the manufacturer also)

Response: According to your suggestion, it has been revised, as detailed in revision mode manuscript lines 214-215.

10. Materials and Methods: 2.2 The amount carbon input is an important factor in this experiment. It would be useful if Table 1 included (at least estimated) values of carbon entering the soil. For example, the cumulative crop yield corresponds to the estimate, probably proportional to the organic carbon released into the soil.

Response: Following your suggestion, I have included the input amounts of exogenous carbon in Table 1. Please refer to line 237 of the revision mode manuscript for details. The supplementary material now includes the calculation method and process for the input of exogenous carbon, see Table S3

11. Materials and Methods: 2.2 line 147: reference is needed about the determination of SOC and its fractions.

Response: Thank you for your suggestion, the references have now been appropriately cited. Please refer to lines 255-259 of the revision mode manuscript for details.

12. Materials and Methods: 2.2 line 154: the name and the manufacturer of Biolog microplate is necessary.

Response: Thank you for your suggestion. Model number, the production company and country of origin has been included in the manuscript in line 268.

13. 3.3. the description of fungal PCR is missing from the text.

Response: I appreciate the careful guidance; a description of the fungal PCR has been added, with details provided in lines 304-306 of the manuscript.

14. Results: 3.3.1. It would be useful a bit more data about the PCR (e.g., average OUT numbers by treatments, the depth of sequencing, …)

Response: Following your suggestion. I have included the relevant description in section, 3.3.1. Please refer to lines 446-453 of the revision mode manuscript for details.

15. Results: This subchapter is concerning the compositions of microbial communities. The differences between them are visible (Fig. 7), but their significance is missing. Are the community patterns of the different treatments significantly different?

Response: In response to your question, I have observed significant variations in bacterial communities among different fertilization treatments. In contrast, the differences in fungal communities are less pronounced than those observed in bacteria. These distinctions are illustrated through the Principal Coordinate Analysis (PCoA) detailed in section 3.3.2.

16. Discuss: 4.1. The assumption of “enhanced soil aggregation” (line 376) is not supported by the measured data. In this case, the proportion or amount of stable fraction of SOM should have increased, but this is not what happened. The amount of active fractions of SOM increased. And a large amount of sand also does not favor an increase in aggregation.

Response: Thank you for your suggestion. Previous research conducted at this experimental site demonstrated that the application of organic fertilizer enhanced the content of soil water-stable aggregates, particularly the larger aggregates ＞ 0.25 mm in size. Given that the majority of soil organic carbon is sequestered within these aggregates, they physically shield organic carbon from microbial decomposition, thereby providing a protective effect that may promote the accumulation of SOC,. Consequently, to strengthen the argument, additions have been made in the manuscript lines 542-543, and reference 41 has been replaced with the aforementioned findings from this experimental field.

17. line 375: there is no data about the biomass C inputs in the manuscript. Therefore, this statement, while probably true, is not supported.

Response: Dear reviewer, thank you for mentioning this detail, which is very important for the conclusion of this manuscript. I have added this data in Table 1.

18. 2. functional diversity: To evaluate the results, you should also briefly write about the advantages and disadvantages of the Biolog substrate utilization method. In particular, the method does not measure the whole soil microbial community, but only those what are able to divide in the buffer used. There are also literature references to this topic.

Response: Thank you for your suggestion. I have included a discussion on the advantages and disadvantages of the Biolog-Eco Plate culture method, and cited the pertinent references. For further details, please refer to lines 644-649.

19. 3. microbial community composition: line 498-508: This part of text would be in a better place in the chapter Conclusion.

Response: Thank you for your valuable feedback. I have made the necessary adjustments in accordance with your suggestions. For further details, please refer to the “Conclusion” section.

20. Conclusion: I propose to be a little more precise about the statements, and in a way that, similarly to the abstract, can be understood without reading the whol manuscript.

Response: Thank you for your guidance. The conclusion section has been revised accordingly.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This research manuscript entitled “Combined application of chemical fertilizer and organic fertilizer promoted soil carbon sequestration and bacterial community diversity in dry wheat fields” investigated the effect of the combined application of balanced chemical fertilizer and organic fertilizer on soil carbon sequestration and discussed its mechanism. I think authors discussed well the reason for the different increase in soil carbon sequestration that occurred among treatments. The observed difference in microbial diversity is interesting. In addition, this study is valuable in showing the results of the continuous application of same fertilizers for 10 years. However, I think this research article needs some revisions before it can be accepted for this journal.

 

I suggest authors consider the potassium (K) application effect on the soil carbon storage more concretely in addition to the effect of organic and bioorganic fertilizer application. The results showed K application can significantly affect SOC components and microorganism diversity. The K application could also affect the dynamics of chemical components in the soil and result in accelerating the carbon sequestration. Additionally, the difference in fertilizer chemical components would lead to the difference in the yield among treatments and it could change the carbon input process from the wheat crops. I think authors should show the result of the yield for each treatment and discuss more specifically this contribution to the increase in soil carbon storage from carbon balance perspective.

 

Other specific comments are following.

Line 42: For DOC, EOC, and LFOC, it is necessary to explain what these abbreviations mean.

Line 108: Medium loam is right? It could be sandy clay loam based on the percentages of sand, silt, and clay.

Line 138: The unit hm^-2 is right?

Line 206–209: What are the OFB and OFM values compared to?

Table 3: The category “Carbon source category” does not match the category “Substrates” although it might be responsible for the publisher editorial system, not authors.

 

I hope these comments will be helpful.

Comments on the Quality of English Language

It is recommended that a native English speaker review this manuscript.

Author Response

1. I suggest authors consider the potassium (K) application effect on the soil carbon storage more concretely in addition to the effect of organic and bioorganic fertilizer application. The results showed K application can significantly affect SOC components and microorganism diversity. The K application could also affect the dynamics of chemical components in the soil and result in accelerating the carbon sequestration. Additionally, the difference in fertilizer chemical components would lead to the difference in the yield among treatments and it could change the carbon input process from the wheat crops. I think authors should show the result of the yield for each treatment and discuss more specifically this contribution to the increase in soil carbon storage from carbon balance perspective.

Response: Thank you for your guidance. Soil organic carbon sequestration is undoubtedly influenced by a variety of nutrient elements present in fertilizers. Concentrating on the impact of potassium fertilizer on soil organic carbon and microbial diversity could yield intriguing and unforeseen scientific insights regarding their behavior in the soil. Our present research is centered on examining the effects of different fertilization practices on soil organic carbon. However, your valuable recommendations to investigate the influence of potassium fertilizer on carbon sequestration will be thoroughly considered in the subsequent stages of our research.

It is indeed the case that varying fertilization practices yield different wheat outputs, which in turn influence the indirect carbon input into the soil through straw, roots, and root exudates annually. Additionally, different fertilizers directly affect the carbon input. Following your suggestions, I have augmented Table 1 with a column detailing the cumulative amount of exogenous carbon input over a decade under various treatments. This data calculation encompasses four components: the indirect input carbon, which correlates with the yield output, and the direct input carbon, which is tied to the input of organic materials applied and their carbon content. The detailed results are presented in Table 1, see line 237. The supplementary material now includes the calculation method and process for the input of exogenous carbon, see Table S3

2. Line 42: For DOC, EOC, and LFOC, it is necessary to explain what these abbreviations mean.

Response: First and foremost, I extend my gratitude for your meticulous guidance. Since these carbon components are being introduced for the first time in the Abstract, both their full names and abbreviations have been provided therein. Consequently, the abbreviations are directly utilized in this context to prevent redundancy.

Line 108: Medium loam is right? It could be sandy clay loam based on the percentages of sand, silt, and clay.
Response: Thanks for your advice. The soil is identified as a medium loam according to Kazinsky's classification method for soil texture. The soil texture is classified as sandy clay loam according to the International System of Soil Texture Classification.

It has been marked and altered in the manuscript, see lines 193-194

3. Line 138: The unit hm-2 is right?

Response: Following your suggestion, I have converted the measurements to international units. Kg / ha.

4. Line 206–209: What are the OFB and OFM values compared to?

Response: Thank you for your attentive reminder; the manuscript has been appropriately supplemented. See line 350

5. Table 3: The category “Carbon source category” does not match the category “Substrates” although it might be responsible for the publisher editorial system, not authors.

Response: I apologize for the confusion; I am not entirely clear about the reviewer's suggested modification. However, my intention is to convey the classification of carbon source substrates, so I modify it as “type of carbon sources” in the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This is an interesting subject on an important matter. Please see the following corrections

1. there is an urgent need for English language editing. for example some are quoted here but there are many more

replace

Fertilizers measure is mainly adopted in arid regions to improve soil carbon (C) pool and 15 crop productivity

with

Use of fertilizers is mainly adopted in arid regions to improve soil carbon (C) pool and 15 crop productivity

-replace

the nutrient availability in the soil. conversely, particulate organic

with

the nutrient availability in the soil. Conversely, particulate organic

 

replace

This process may lead to an increase in SOC content [13–14], 58 remain unchanged [15] or even decrease in SOC content

with

This process may lead to an increase in SOC content [13–14],  or even decrease in SOC content, or the parameter can remain unchanged [15]

replace

previous studies commonly focused on the impact of fertilization

with

Previous studies commonly focused on the impact of fertilization

you state

soil organic matter (SOM) quickly decomposes and ex-88 acerbates its depletion

I dont understand this sentence. also this sentence should be different from the previous one

also there are other mistakes, please read and correct throughout

2. you state

However, our comprehension of the mechanisms of such improvements from 16 microbial metabolic activity and community structure perspectives remain poor

this is not exactly true since much research happens on this matter. maybe focus on some sub-topics that need to be elucidated more?

3. In the end of the introduction you should explain why this research is important for an international audience

4. In materials and methods you should give a reference for all the climate, meteorological geographical etc information of your case area

5. for all reagents and for all instrument you should give the following

model if available, manufacturer, city and country of origin

6. in 2.3.1. SOC and organic carbon fractions determination you should give a reference for all methods mentioned. also about the Biolog microplate reader. does it also follow some protocol? because right now the methods are underdescribed a lot

7. I have some issues with the statistics. First of all you state Duncan’s New Complex Polar Deviation 194 method was further used for One-way ANOVA (P < 0.05), and the homogeneity of vari-195 ance test was performed.

I dont understand this sentence-you performed ANOVA for what parameter and between what groups. also what you meant tested for homogeneity of varience? through what test? what were the results? if homogeneity is not guaranteed then you should state how your ANOVA is ribust. also first you need to test for normality of data. what is CPD and how it correlates with ANOVA. post hoc tests should be defined what they were

also you state

Principal Coordinates Analysis (PCoA) and 197 replacement multiple analysis of variance (PERMANOVA) based on Bray-Curtis were 198 performed using “vegan” package to assess microbial community variability PCA is completely different from PERMANOVA and it is a factor reduction technique-what were the factors you used in PCA also you have to state what kind of rotation you did

As I understand PERMANOVA is a non parametric alternative for MANOVA how you checked that you did not have normality? MANOVA will only give you if there is a difference between groups among a calculated factor that is the sum of many parameters, what were these parameters? also what is the use of this if not followed by breaking down the parameters after the MANOVA? I dont understand the use here

 

about the Mantel test why not a Spearman correlation was not used? what more information it gives since you did a PCA which groups in a sense the parameters? all these should be explained in the materials and methods. In results please define what results came from which test and in some graphs and figures please show with asterisks or with some other way the statistical differences

8. In Table 3. Loading factors of 31 carbon sources on PC1, PC2 and PC3 I am sorry I do not understand this experiment-was it a different one? did you use specifically these substrates or is this the field experiment? is this described in materials and methods?

9. The discussion is good but please try to make direct correlation of what you discuss with the actual result you describe in results

10. for such a long manuscript I believe that more up to date citations are needed especially in the discussion section. Please see also https://www.mdpi.com/2223-7747/10/12/2789

11. many of the figures are too small to be readable. either transport some to supplementary material or make the figures larger

 

Comments on the Quality of English Language

see main comments

Author Response

1. there is an urgent need for English language editing. for example some are quoted here but there are many more

Response: Thank you for your constructive feedback. I have addressed the identified issues and corrected any unreasonable expressions throughout the text, ensuring a thorough review has been conducted.

2. you state：soil organic matter (SOM) quickly decomposes and exacerbates its depletion. I don’t understand this sentence. also, this sentence should be different from the previous one also there are other mistakes, please read and correct throughout

Response: Thank you for your insightful advice. The intent of the sentence in question was to convey that, despite the low soil organic matter content in this region, there is a high potential for carbon sequestration. The sentence has now been revised, and the references cited here have been updated to reflect this clarification. For further details, please refer to lines 172-173.

3. you state: However, our comprehension of the mechanisms of such improvements from microbial metabolic activity and community structure perspectives remain poor

this is not exactly true since much research happens on this matter. maybe focus on some sub-topics that need to be elucidated more?

Response: Thank you for your suggestion. I have revised this sentence to ensure it aligns more closely with the actual state of research. For further details, please refer to line 15.

4. In the end of the introduction, you should explain why this research is important for an international audience

Response: Thank you for your valuable suggestion. The significance of the study for an international audience has been explained in the end of the introduction and can be found in lines 124-170 of the manuscript.

5. In materials and methods you should give a reference for all the climate, meteorological geographical etc information of your case area

Response: Thank you for your valuable feedback. In response to your suggestion, I have added the location and meteorological information of the study area to the manuscript. To ensure clarity and visibility, this information has been placed in the supplementary file. For further details, please refer to Figure S1.

6. for all reagents and for all instrument you should give the following

model if available, manufacturer, city and country of origin

Response: In accordance with your suggestions, I have specified the details of the fertilizer, the ecological culture plate, and the software utilized in the study. Please refer to the manuscript for the complete information.

7. in 2.3.1. SOC and organic carbon fractions determination you should give a reference for all methods mentioned. also about the Biolog microplate reader. does it also follow some protocol? because right now the methods are underdescribed a lot

Response: Following your suggestion, I have included references for the determination of each carbon component and for the Biolog-ECO culture method. These references are located within lines 251-260 of the manuscript.

8. I have some issues with the statistics. First of all you state Duncan’s New Complex Polar Deviation  method was further used for One-way ANOVA (P < 0.05), and the homogeneity of vari- ance test was performed.

I dont understand this sentence-you performed ANOVA for what parameter and between what groups. also what you meant tested for homogeneity of varience? through what test? what were the results? if homogeneity is not guaranteed then you should state how your ANOVA is ribust. also first you need to test for normality of data. what is CPD and how it correlates with ANOVA. post hoc tests should be defined what they were

Response: Thank you for your inquiry and reminder. In this study, one-way Analysis of Variance (ANOVA) was employed to assess the differences in soil carbon components, the capacity of soil microorganisms to utilize various carbon sources, microbial functional diversity indices, and OTU-based alpha diversity indices across different treatments. The Duncan test was subsequently applied to ascertain statistical significance at the P < 0.05 level, thereby determining if there are significant differences in the impacts of the various treatments.

When conducting one-way ANOVA, it is imperative to first test for data homogeneity. The test for homogeneity of variances assesses whether the variances across two or more samples are equivalent, ensuring that the data meets the assumptions necessary for a valid ANOVA analysis. In the variance homogeneity test, it is typically assumed that data are normally distributed. Hence, data should be tested for normality prior to conducting the homogeneity test. Common methods for normality testing include the Shapiro-Wilk test and the Kolmogorov-Smirnov test, with the Shapiro-Wilk test often being preferred, especially for small sample sizes. A P-value greater than 0.05 from the Shapiro-Wilk test suggests that the data in question adhere to a normal distribution.

Following your suggestions, I have incorporated this information into the manuscript's section on 2.4 Data Analysis, specifically in lines 311-316.

9. also you state

Principal Coordinates Analysis (PCoA) and  replacement multiple analysis of variance (PERMANOVA) based on Bray-Curtis were  performed using “vegan” package to assess microbial community variability PCA is completely different from PERMANOVA and it is a factor reduction technique-what were the factors you used in PCA also you have to state what kind of rotation you did

As I understand PERMANOVA is a non parametric alternative for MANOVA how you checked that you did not have normality? MANOVA will only give you if there is a difference between groups among a calculated factor that is the sum of many parameters, what were these parameters? also what is the use of this if not followed by breaking down the parameters after the MANOVA? I dont understand the use here

Response: Thank you for your suggestion. In this study, following the visualization of species abundance data obtained through high-throughput sequencing with Principal Coordinate Analysis (PCoA), some overlap was observed between samples from various groups. To determine whether there are significant differences in the composition of these samples, we utilized the PERMANOVA test. This statistical method evaluates community differences between groups by analyzing variations in distance matrices. The PERMANOVA test differs from the MANOVA test in that MANOVA necessitates variables to adhere to a normal distribution, which is often challenging for species abundance data in ecological studies. Consequently, we applied nonparametric multivariate methods, including PERMANOVA, to generate an F-value distribution through random permutation of samples, thereby deriving a P-value. In the context of the PERMANOVA test, a P-value of less than 0.05 indicates a significant difference in microbial community structure, whereas a P-value of 0.05 or higher suggests no significant difference.

Following your suggestions, I have included supplementary content in the manuscript's section 2.4, Data Analysis. For further details, please refer to lines 324-339.

10. about the Mantel test why not a Spearman correlation was not used? what more information it gives since you did a PCA which groups in a sense the parameters? all these should be explained in the materials and methods. In results please define what results came from which test and in some graphs and figures please show with asterisks or with some other way the statistical differences

Response: The Mantel test is capable of visually representing the correlations between two sets of data, thereby illustrating both intra-group and inter-group relationships. To denote these correlations, either Spearman's or Pearson's correlation coefficients can be utilized. In this study, we have opted to employ the Spearman correlation method to examine the relationships between soil organic carbon components, the abundance of predominant bacteria or fungi, and soil microbial carbon source utilization.

Following your suggestions, I have provided a revised description of the methodology in section 2.4, "Data Analysis," of the manuscript. This can be found on lines 339-342, with supplementary explanations in the results section, specifically on lines 523-528.

Response: Thank you for your suggestion. I have included a supplementary explanation regarding the PCA analysis in section 2.4. For detailed information, please refer to lines 316-322.

11. In Table 3. Loading factors of 31 carbon sources on PC1, PC2 and PC3 I am sorry I do not understand this experiment-was it a different one? did you use specifically these substrates or is this the field experiment? is this described in materials and methods?

Response: In Table 3, Principal Components 1 (PC1), 2 (PC2), and 3 (PC3) were chosen for extracting and analyzing the principal components from the light absorption values recorded in the Biolog ecological plate wells over a 96-hour culture period using SPSS 24.0 software. This analysis aimed to identify the predominant carbon sources utilized by microorganisms under various fertilization treatments. Following your suggestion, the methodology has been rearticulated in Section 2.4, titled "Data Analysis."

12. The discussion is good but please try to make direct correlation of what you discuss with the actual result you describe in results

Response: Thank you for your valuable feedback. According to your suggestion, I have endeavored to highlight the connections between the discussion points and the analysis results. For instance, I have referenced line 536 (Figure 1a,b,d), line 584 (Figures 1e, 1f), and line 716 (Figure 8).

13. for such a long manuscript I believe that more up to date citations are needed especially in the discussion section. Please see also https://www.mdpi.com/2223-7747/10/12/2789

Response: Thank you for your suggestion. This paper holds significant reference value for our study and is included in the list of references, specifically cited as document 52.

14. many of the figures are too small to be readable. either transport some to supplementary material or make the figures larger

Response: I have enlarged the figures to enhance readability. For further details, please refer to the manuscript.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

I appreciate your paper and in my opinion is suitable for publication in the present form. The research field is absolutely interesting and current. The research topic is well developed in the paper and really clear. I don't have any suggestion to improve it and I hope to read some more paper from you

Author Response

I appreciate your paper and in my opinion is suitable for publication in the present form. The research field is absolutely interesting and current. The research topic is well developed in the paper and really clear. I don't have any suggestion to improve it and I hope to read some more paper from you

Response: I extend my sincere gratitude to the reviewer for his(her) acceptance and acknowledgment of this paper.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscripts is improved

please correct ENGLISH LANGUAGE mistakes such as

replace

The relaitonships between SOC components

with

The relationships between SOC components

 

Generate a Principal Component Analy-233 sis (PCA) ordination plot, so as to judge the differences in carbon source utilization pat-234 terns of different fertilization treatments

something is missing here

The principal component analysis (PCA) of the carbon sources

with

Principal component analysis (PCA) on the carbon sources

 

replace

Type of Carbon source

with

Type of carbon source

 

replace

fertilizers improveed soil water-stable aggregates

with

fertilizers improved stable aggregates between soil and water

 

replace

organic fertilizer (Fermentation of chicken manure)

with

organic fertilizer (fermentation of chicken manure)

etc....

Comments on the Quality of English Language

see above comments

Author Response

Dear Reviewer,

I deeply apologize for any grammatical errors present in the manuscript. I am immensely grateful to the reviewers for their meticulous guidance. I appreciate the opportunity to revise and improve the manuscript.

Below are the detailed revisions made to the manuscript, presented point by point.

1. replace

The relaitonships between SOC components

with

The relationships between SOC components

Response: Thank you for your guidance. The modifications have been made.

2. Generate a Principal Component Analysis (PCA) ordination plot, so as to judge the differences in carbon source utilization patterns of different fertilization treatments

something is missing here

Response: Following your suggestion, I have included additional content in the manuscript. For detailed information, please refer to lines 336-338.

3. The principal component analysis (PCA) of the carbon sources

with

Principal component analysis (PCA) on the carbon sources

Response: Thank you for your guidance. The modifications have been made.

4. replace

Type of Carbon source

with

Type of carbon source

Response: Thank you for your guidance. The modifications have been made.

5. replace

fertilizers improveed soil water-stable aggregates

with

fertilizers improved stable aggregates between soil and water

Response: Thank you for your advice. What we intend to convey here is the enhancement of water-stable aggregates content, it implies that the soil structure does not disintegrate immediately upon flooding, and the structural integrity of the soil remains intact.

Upon reviewing the literature [1-2], I believe it is appropriate to describe it as follows: “the application of organic fertilizers improved water-stable soil aggregates through the bonding of primary soil particles”

[1] Zheng, H.; Liu, W.; Zheng, J.; Luo, Y.; Li, R.; Wang, H.; Qi, H. Effect of long-term tillage on soil aggregates and aggregate-associated carbon in black soil of Northeast China. PLoS One. 2018, 13, e0199523. https://doi.org/10.1371/journal.pone.0199523

[2] Wang, w.; Chen, W. C.; Wang, K. R.; Xie, X. L.; Yin, C. M.; Chen, A. L. Effects of long-term fertilization on the distribution of carbon, nitrogen and phosphorus in water-stable aggregates in paddy soil. Agricultural Sciences in China. 2011, 10, 1932-1940. https://doi.org/10.1016/S1671-2927(11)60194-6

6. replace

organic fertilizer (Fermentation of chicken manure)

with

organic fertilizer (fermentation of chicken manure)

Response: Thank you for your guidance. The modifications have been made.

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