Responses of N₂O, CO₂, and NH₃ Emissions to Biochar and Nitrification Inhibitors Under a Delayed Nitrogen Application Regime

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript studies the effect of different fertilization rate, as well as amendment with biochar and nitrification inhibitor DMPP on greenhouse gas (GHG) and NH3 emissions, as well as on soil properties and crop yield.

The subject is very relevant. The design of the study is appropiate and the collected data is sufficient.

The analysis of the data is extensive in the number of analysis performed (ANOVA, correlation analysis, random forest, PCA), however it is not clear the purpose of so many different analysis, as it seems to overlap. In some cases the analysis does not seem suitable for the intended purpose (PCA to study "the effects of biochar and DMPP treatments").

Essential information on the analyses performed is missing (what were the models used for the ANOVA and random forest, what post-hoc test was applied after ANOVA, what correlation analysis method was used.

There also seem to be incoherences between the results presented in the tables and those described in the text.

The inclusion of the results for N0 and N2 is distracting. I would rather present the comparison between N0, N1 and N2 shortly in the beginning and INDEPENDENTLY from the study of the effects of DMPP and biochar (as biochar and DMPP were only applied to N1).

> See detailed comments in the PDF document.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer Comments

Dear reviewer:

We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript (Agriculture-3246386). The reviewer comments are laid out below in italicized font and specific concerns have been numbered. Our response is given in normal font and changes/additions to the manuscript are given in the blue text.

I have made direct corrections to the minor grammatical issues you raised in the paper.

Comments 1: This abbreviation MUST be changed, as it can be easily confused with dinitrogen. Why not N180 and N270?

Response 1: We have changed the abbreviation to N₁₈₀ and N₂₇₀.

Comments 2: Be careful, DMPP keeps NH4 from being oxidized, but doesn't increase its concentration! Was there "an increase" or a higher value of NH4 with DMPP?

Response 2: We have corrected that part (Line 88-93).

Comments 3: As the study of the effect of biochar and DMPP is made in comparison with N1, I would rather leave NO and N2 out. Or, if to present their results, I would make it in short way in separate tables or figures (only NO, N1, N2), and then focus on the effects of DMPP and biochar in comparison with N1 as control. This would make the whole Results and Discussion part easier to understand.

Response 3: As you mentioned, the results of biochar or DMPP treatment presented in the article are compared with those under N₁₈₀ treatment. We have taken your advice into account and focus on the effects of DMPP and biochar in comparison with N1 at section 3.8(426-438). The foundation of our research is that the application of reduced N application (N₁₈₀) may lead to a reduction in N gas emissions compared to conventional N (N₂₇₀); however, it may also result in decreased crop yields. Therefore, we advocate for the integration of N₁₈₀ with synergistic agents. The objective of our study was to assess whether ‘N₁₈₀ + synergist model’ provides advantages in yield maintenance relative to conventional nitrogen applications (N₂₇₀). Consequently, the data from N₂₇₀ treatment remains included.

Comments 4: How often were these samples collected? In the beginning and the end, or also in between?

Response 4: Soil samples were collected subsequent to the acquisition of N₂O gas and at the time of wheat harvest (Line 217).

Comments 5: Better not to mix abbreviations (WFPS) with complete names (volumetric water content, soil bulk density) and default values (2.65). Why not just?

WFPS = VWC / (1 - BD/PD)

Response 5: We have corrected (Line232-234).

Comments 6: Write the name of the kit and in parenthesis the producer and location:

Name of the kit (JingKang Biological Engineering Technology Co., Ltd., location)

Response 6: We have revised (237-243).

Comments 7: PCA is a tecnique for dimensionality reduction, which gives hints of the relationships between variables, but doesn't allow to study "effects". Change this with a more appropiate word like "relationships", for example.

Response 7: We have corrected (Line 251).

Comments 8: You can specify here if you performed the correlation analysis according to Pearon, Spearman or another method. If not, you should specify if when the results are presented.

Response 8: The correlation analysis was conducted according to Pearson.

Comments 9: This is confusing. Which model was used in each case? As there are two factors (nitrification inhibitor and biochar) you would expect the use only of the two-way ANOVA.

Response 9: We have delineated the random forest model, principal component analysis, and two-way ANOVA objects (Line247-255).

Comments 10: What post-hoc analysis was performed after ANOVA to get the groups (letters) for the different treatments?

Response 10: ANOVA was conducted following the test of variance homogeneity, mean comparisons were conducted using the least significant difference test, with a significance threshold at P<0.05 (Line249-251).

Comments 11:  random forest analysis, What was/were the model/s used? Which was the dependent variable?

Response 11: Random forest analysis utilizing the ‘rfPermute’ package (Line 255). In alignment with your recommendation, we conducted a random forest analysis for each of the three gases and assessed the relative significance of soil properties and enzyme activity indicators in relation to the emissions of these gases (Figure. 9)(Line 418).

Comments 12: In fact, as you have a treatment without fertilization (N0), you should use a three-way ANOVA with fertilization as another factor, if you want to include in the analysis N0.

Response 12: Thanks for your suggestion, to investigate the potential interaction between biochar and DMPP, we conducted a two-factor analysis of variance. Our primary focus was on the interactions between N₁₈₀-based biochar and DMPP; therefore, we conducted a two-factor ANOVA, using N₁₈₀ as the reference variable, as we have stated in Response 3.

Comments 13: Define abbreviations (SOC, TN). This has to be done in all figures.

Response 13: We have Define abbreviations (267-268).

Comments 14: This plot is very difficult to "read" (it is impossible to differentiate the trends between the treatments).First, you could add lines between the measurement points of the same treatment (as you did for Figure 2). This would show, for example, that N0 remains low for the three parameters. Second, you could separate treatments in different columns (see facet plots), for example, for treatments with and without biochar). Third, the symbols have to be consistent between figures for the same treaments (eg. N2 cannot have a star in Figure 1 and a circle in Figure 2).

Response 14: In accordance with your recommendation, we have added lines between the measurement points of the same treatment and revised Figure 1 to distinctly illustrate the various processes for enhanced clarity (Fig.1, Line 277).

Comments 15: The truncated y-axis is misleading: First, the lines connecting the points SHOULD NOT be solid if the axis is truncated (they should be truncated too).I think it is not a good idea to have double truncation in the same plot (Figure 2.b). There are alternatives to the truncated y-axis: for example, two plots side-by-side, one where you show everything into scale, and a second, where you "zoom" only in a part of the plot (for example, the interval 0-200 in Figure 2.a)

Response 15: We have revised Figure 2 to distinctly illustrate the various processes for enhanced clarity (Fig.2, Fig.3, Line 305, 309).

Comments 16: I would certainly show the cumulative emissions as a barplot, where the differences between the treatments would be easily noticeable.

Response 16: We have revised the table of cumulative emissions to a barplot (Fig. 4, Line 321).

Comments 17: Add space between the plot and new paragraph.

Response 17: We have revised.

Comments 18: No need to add "Note" in the caption of a figure.

Response 18: We have revised (Line 345).

Comments 19: Again, the symbols and colors should be consistent for the treatments among figures. There could be an effect of Simpson's paradox, but you don't have enough points in each treatment to conclude this.

Response 19: We have revised the illustration (Fig. 5, Line344).

Comments 20: All abbreviations should be explained in the caption of each figure (e.g. WFPS)

Response 20: We have revised (Table. 2, Line331)

Comments 21: What exactly are these? The yield of wheat?

Response 21: Wheat harvest index (yield, above ground N uptake and NUE) (Line 431).

Comments 22: When you add letters (a, b, c, d) you have to explain to what they refer in the caption. In this case, what each plot represents.

I think the PCA and the RF should be presented separately, as they are completely different tecniques. Plot "a" can be removed, as it is completely uninformative.

Response 22: In accordance with your suggestion, we removed Plot 'a' and subsequently generated the PCA and random forest analysis charts, respectively (Line 416-418).

Comments 23: I would represent the factor loadings differently. Although a table is more informative, usually they are presented as arrows.

Response 23: We appreciate your suggestion and have retained this load diagram to more effectively illustrate the contributions of various indicators to the principal components.

Comments 24: You are performing the PCA with both dependent (gas CE, yield) and independent variables in the analysis, which is not ideal.

Personally, I would perform the PCA (or an alternative) for the gas fluxes alone, and then add the other data (enzyme activity, soil properties) a posteriori as environmental data (similarly as the function "envfit" does in R language).

Response 24: In accordance with your suggestion, we excluded the yield index and conducted a principal component analysis on gaseous emissions, enzyme activity, and soil properties. Subsequently, a random forest analysis was performed for each of the three gases, respectively (Line 416, 418).

Comments 25: Which was the dependent variable?

N2O? CO2? NH3? The analysis should be performed separately for each gas.

Yield is without a doubt one dependent variable (a "Result" of the experiment, it should not be included in the model.

Response 25: We excluded the yield analysis and conducted a separate examination of the three gases (Line 418).

Comments 26: Which ones? I assume you refer to post-hoc tests after the two-way ANOVA, which one was used?

Response 26: Two-factor ANOVA on both biochar and DMPP (Line 427-429).

Comments 27: This doesn't make sense. If the effect of DMPP is not significant, why addition of DMPP is a "valid measure for reduction of N2O emissions"?

Response 27: We misdescribed it, and we've corrected it (Line 514).

Comments 28: When you are discussin your results, it is helpful to add references to them (in which figure or table they are presented?).

Response 28: We have added references.

Comments 29: I assume you refer to Table S1. From what I see, for both N1 and the treatments with DMPP, the HAO values have the group letter "a", meaning they are not different, so I don't understand how their activity was supressed by DMPP.

Response 29: We have corrected it (Line518-519).

Comments 30: From what I interpret in Table 1 for N2O, both N1D and the treatments of N1D with biochar have the group letter "c", so again, they are not different. Therefore, biochar didn't "enhance the repression of N2O by DMPP".

Response 30: We have corrected it (Line 520-521).

Comments 31: From Table S1, HAO has "a" for both N1D and both treatments with biochar - they are not different. For NOO, only N1DB15 is different from N1D.

Response 31: We recheck the enzyme activity data and reconstructed the Bar chart, and we have revised this part (Line 377, Line 524-525).

Comments 32: Not clear: from Table S1, NXR activity as significantly lower for both treatments ("b") than in N1 and N1D ("a").

Response 32: We have corrected it (Line 526-527).

Comments 33: Does correlations analysis support this?

Response 33: Random forest analysis support this conclusion. In accordance with your suggestion, and given that the results of the random forest analysis and correlation analysis exhibit partial overlap, we have decided to retain the random forest analysis in order to enhance the conciseness of the paper, while omitting the section on correlation analysis (Line 535-536).

Comments 34: I can't find soil temperature included in the correlation analysis. From where comes this statement?

Response 34: We did not conduct a correlation analysis between soil temperature and CO₂ emissions, however, the results presented in the two charts indicate a gradual increase in CO₂ emissions corresponding to rising temperatures (Fig. 3, Fig S1b).

Comments 35: I cannot find Figure 6.

Response 35: We have corrected it (Line 572).

Comments 36: For NH3, compared with application of DMPP and biochar separately; N1 has group "ab", so it is not significantly higher than DMPP+biochar.

Also for pH, the synergistic increased effect of DMPP+biochar is not true for all combinations (Table 1).

Response 36: "DMPP+biochar" significantly reduced NH₃, compared with application of DMPP and biochar separately. The present findings indicate a significant negative correlation between soil pH and NH3 loss, as the N180D treatment also contributes to an increase in soil pH. Therefore, we propose that the reduction in NH3 emissions cannot be solely attributed to the elevated soil pH; rather, it is likely due to the enhanced NH₄⁺/NH₃ uptake capacity of biochar (Line 572-575).

Comments 37: From Table 1, TN has "a" for all the treatments.

Response 37: We have corrected it (Line 586).

Comments 38: As biochar is added to N1, it should be compared with N1!

This confirms that N0 and N2 should be removed from all comparisons of biochar and DMPP effect.

Response 38: We have revised this description (Line 589-590).

Comments 39: What was the criteria to select this?

Response 39: Based on the distribution of N2O gas emission flux, we established a threshold of 110 μg/(m²·h) to differentiate between high and low N2O emission fluxes, which has been incorporated into the figure (Line 644).

Comments 40: This is confusing. These are soil chemical properties and bulk density. And by "wheat", you probably mean grain and straw yield. Is "Above-ground N uptake" included here too? Be specific.

Response 40: In accordance with your suggestion, and given that the results of the random forest analysis and correlation analysis exhibit partial overlap, we have decided to retain the random forest analysis in order to enhance the conciseness of the paper, while omitting the section on correlation analysis.

 Comments 41: Heatmap of what? What is represented in the map? What are the units (-3 to +3)?

Response 41: In accordance with your suggestion, and given that the results of the random forest analysis and correlation analysis exhibit partial overlap, we have decided to retain the random forest analysis in order to enhance the conciseness of the paper, while omitting the section on correlation analysis.

Comments 42: Very nice pattern (PC1 is differentiating amount of biochar, and PC2 addition of DMPP). But it is difficult to interpret because we don't know what data was used to make the PCA. What data is presented here?

Response 42: We have included a description of the data (Line 649-651).

Comments 43: The captions should be more descriptive. E.g. "Enzymatic activity in (...) for the different treatments". Also, the abbreviations have to be defined.

Response 43: We have made revisions in Figure 7 (Line 376).

Comments 44: This table seems very important to be in the Supplementary materials. I would move it to the main text.

Response 44: We have implemented your suggestions by converting the table into a bar chart, which has been presented in the main body of the paper (Fig. 7; Line 376).

Comments 45: I would separate this into groups: I would present soil chemical properties and microbial activity separately.

As the matrix is simmetric, it can be simplified., I would keep only the cumulative fluxes in the rows (x axis).

Response 45: Given that the results of the random forest analysis and correlation analysis exhibit partial overlap, we have decided to retain the random forest analysis in order to enhance the conciseness of the paper, while omitting the section on correlation analysis.

Comments 47: Where Table S2 and S3 obtained by post-hoc analysis of the SAME ANOVA model? This is relevant as the interaction between these two factors is significant.

Is the data presented in this table adding something to the data presented in Table 2? If not, it could be removed.

Response 47: Table S2 and S3 obtained by post-hoc analysis of the SAME ANOVA model, we have removed it.

We tried our best to improve the manuscript and made some changes marked in blue in revised paper which will not influence the content and framework of the paper. We appreciate for Editors/Reviewers’ warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Yours sincerely

Haizhong Wu

21-Oct, 2024

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript by Liu and coworkers presents a comprehensive study of the role of different fertilizer additives on the emission of CO₂, N₂O and NH₃ in a specific agricultural soil. The applications were performed twice during the cropping period - at the beginning of sowing and a second time through top-dressing at a time of high nutrient demand by wheat crops.

Aim of this field study was to evaluate the impact of biochar and DMPP, both in isolation and combination of the GHG emissions and how these additives impact on the 'bug' communities or enzymes, respectively, responsible for N transformations in this particular soil as well as on the soil's physical and chemical properties.

In a nutshell, the study finds that biochar in combination with DMPP (N1DN15) increases the NUE and grain yield. However, the performance strongly depends on the application rates of N fertilizer, DMPP and biochar with some treatments being detrimental to NUE and GHG emissions.

I have a series of comments/questions/suggestions.

1) In general, how many decimal places are really required and useful with regards to the error of the measurements? My suggestion is to round all %age numbers up to the closest integer. Whether the CO₂ emissions increase by 2.9% or 3% does not make a difference for the 'message' to be conveyed.

2) line 74: is biochar really a nitrification inhibitor? By which mechanism would it deactivate AMO?

3) line 144: "conventional" and 'optimal" N comes a bit out of the blue. What does this mean? Please provide rationale/explanation.

4) line 212: where does the value of 2.65 g cm^-3 for the mean soil density come from? Please provide rationale/explanation.

5) Please provide soil and biochar characteristics as both can vary substantially, and the experimental results could be very specific to THAT soil and biochar.

6) Figure 1 and S1 - is there a correlation between WFPS and soil NH₄⁺-N, NO₃^--N  and DOC for the various treatments?

7) Table 3: units for NH₄⁺-N, NO₃^--N and inorganic N (what is this???) need to be provided. The WFPS data appear to be strange, haven't the parameter been measured simultaneously? In other words, why is the value for WFPS different in the different control experiments?

8) Figure 4: What is the reason for the different responses to the various treatments after the first application (11-13 Feb), compared to the essential similar responses after the second application?

9) Figure 5: all panels are way too small, cannot decipher them.

10) line 486: the lower activity of HOA is likely due to the deactivation of AMO, since they are connected through an electron-shuttle process.

11) line 491: I don't understand the rationale of the 'synergistic' effect between biochar and DMPP. This needs some explanation. Also line 592: it's not 'synergistic' but 'combined' applications, the effects might be synergistic (but again, how?)

12) line 544: should be NH₃ capture and not emission.

13) Figure S1 uses different symbols for the treatments.

Overall, this field study gives some insight how different treatments could affect NUE, specifically GHG emissions. However, it is not clear how general the results are or whether the findings are specific for this particular soil and crop (given also that some of the data obtained here are contradict with literature data). I would like the authors to tone down the message, because it needs more studies in different soils and under different conditions to establish whether N1DB15 is the best treatment. 

Author Response

Response to Reviewer Comments

Dear reviewer:

We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript (Agriculture-3246386). The reviewer comments are laid out below in italicized font and specific concerns have been numbered. Our response is given in normal font and changes/additions to the manuscript are given in the blue text.

Comments 1: In general, how many decimal places are really required and useful with regards to the error of the measurements? My suggestion is to round all %age numbers up to the closest integer. Whether the CO₂ emissions increase by 2.9% or 3% does not make a difference for the 'message' to be conveyed.

Response 1: Thanks for your suggestion, we have revised % numbers up to the closest integer. 

Comments 2: line 74: is biochar really a nitrification inhibitor? By which mechanism would it deactivate AMO?

Response 2: We express regret for our oversight, what we intend to convey is that “N-based fertilizers are applied in conjunction with a synergistic agent, such as biochar, nitrification inhibitors (NIs), or a combination of dual inhibitors, to mitigate N loss by regulating the process of N conversion”. Furthermore, our findings indicate that biochar enhances the activity of AMO (Line 76).

Comments 3: line 144: "conventional" and 'optimal" N comes a bit out of the blue. What does this mean? Please provide rationale/explanation.

Response 3: “conventional N” refers to the quantity of conventional nitrogen fertilizer (270 kg N ha⁻¹) applied by local farmers, while “optimal N” denotes the ideal annual N input for winter wheat production in this region, as determined by our team's experimental findings conducted in the study area over the past two years.

Comments 4:  line 212: where does the value of 2.65 g cm-3 for the mean soil density come from? Please provide rationale/explanation.

Response 4: The value of 2.65 (g cm⁻³) denotes the density of soil particles, we have supplemented the literature [38] to provide a detailed explanation (Line 234).

Comments 5: Please provide soil and biochar characteristics as both can vary substantially, and the experimental results could be very specific to THAT soil and biochar.

Response 5: We have provided the indices of soil texture, cation exchange capacity (CEC), and available phosphorus, while also augmenting the parameters for biochar contact angle, specific surface area and pore volume (Lines 144-147, Lines 173-174).

Comments 6: Figure 1 and S1 - is there a correlation between WFPS and soil NH4+-N, NO3--N and DOC for the various treatments?

Response 6: No significant correlation was observed between WFPS and soil NH₄⁺-N, NO₃⁻-N, and DOC across the different treatments.

Comments 7: Table 3: units for NH4+-N, NO3⁻-N and inorganic N (what is this???) need to be provided. The WFPS data appear to be strange, haven't the parameter been measured simultaneously? In other words, why is the value for WFPS different in the different control experiments?

Response 7: We have provided units for NH₄⁺-N, NO₃⁻-N and inorganic N. Inorganic N was defined as the sum of NH₄⁺-N and NO₃⁻-N (Line 281).

WFPS =volumetric water content/ (1 - soil bulk density/2.65), The dynamic changes in soil water content during different monitoring periods resulted in corresponding variations in WFPS within the control experiment treatment.

Comments 8: Figure 4: What is the reason for the different responses to the various treatments after the first application (11-13 Feb), compared to the essential similar responses after the second application?

Response 8: From 11 Feb to 13 Feb, as illustrated in Fig S1a (Line 639), continuous rainfall totaling 6.5 mm was recorded in the study area. This precipitation markedly suppressed NH₃ volatilization, thereby contributing to the observed variability in responses across different treatments following the initial N application.

Comments 9: Figure 5: all panels are way too small, cannot decipher them.

Response 9: We have revised Figure 5 and increased the size of all panels(Line 416-417).

Comments 10: line 486: the lower activity of HOA is likely due to the deactivation of AMO, since they are connected through an electron-shuttle process.

Response 10: Thank you for your valuable suggestions, we have elucidated the influence of AMO on the activity of the HAO enzyme.

Comments 11: line 491: I don't understand the rationale of the 'synergistic' effect between biochar and DMPP. This needs some explanation. Also line 592: it's not 'synergistic' but 'combined' applications, the effects might be synergistic (but again, how?)

Response 11: We appreciate your insightful review comments. We acknowledge that our previous expression lacked precision. Specifically, the combination of biochar and DMPP exhibits a synergistic effect on gas emissions, rather than the synergistic application of these two components. We have implemented the required revisions to enhance clarity on this matter (Line 630).

Comments 12: line 544: should be NH3 capture and not emission.

Response 12: We have corrected “emission” into “capture” (Line 578).

Comments 13: Figure S1 uses different symbols for the treatments.

Response 13: Thanks for your careful checks, we have used uniform symbols for the treatments (Fig. S1) (Line 642).

We tried our best to improve the manuscript and made some changes marked in blue in revised paper which will not influence the content and framework of the paper. We appreciate for Editors/Reviewers’ warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Yours sincerely

Haizhong Wu

19-Oct, 2024

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This study, "Responses of N2O, CO2, and NH3 emissions to biochar and nitrification inhibitors under a delayed nitrogen application regime", investigates an important topic in agricultural greenhouse gas emissions and nitrogen management. The way the experiments were set up to compare different mixes of biochar, DMPP nitrification inhibitor, and nitrogen application rates taught us a lot. However, there are some issues with data presentation, statistical analysis, and interpretation that should be addressed:

- The rationale for the specific biochar application rates (7.5 and 15 t/ha) is not clearly explained. A justification based on previous literature should be provided.

- Only one growing season was studied. Multi-year data would strengthen the conclusions about longer-term effects (based on data availability).

- More details are needed on the gas sampling and analysis methods, including sampling frequency, time of day, and quality control measures, etc.

- The method for calculating cumulative emissions is not sufficiently described (needs to be added).

- ANOVA results should be presented for all major comparisons (Results and discussion section).

- The discussion of enzyme activities (section 3.5) is difficult to follow. A table summarizing the key enzyme changes would improve clarity.

- The mechanisms proposed for changes in emissions are sometimes not very convincing. More direct evidence (e.g. microbial community analysis) would strengthen these arguments.

- Figure 2 is hard to read due to overlapping lines. Consider splitting into separate panels. Similar comment for Figures 4, S1, and S2. Figure 5 needs to be more clearly presented, as we can't read the text or the numbers.

- The conclusion that "N1DB15 treatment is the optimal strategy" seems overly strong given the single-season nature of the study. This should be qualified.

Minor comments:

- Line 45-47: The statement about N2O's contribution to global warming potential needs updating with more recent IPCC values (from the Sixth Assessment Report (AR6), which was finalized in March 2023).

- Line 136-137: More details on soil characteristics (e.g. CEC, texture) would be helpful for comparison to other studies.

- Line 413-417: The comparison to previous biochar studies needs more nuance. Factors like biochar feedstock and production temperature likely explain some of the differences observed.

While this study provides useful data on a relevant topic, revisions to improve clarity, statistical rigor, and mechanistic explanations would strengthen the manuscript considerably. Thus, I would recommend revising the manuscript with major changes.

Comments on the Quality of English Language

Overall, the paper demonstrates a good command of scientific English, however, there are minor areas where improvements could enhance clarity and readability.

Author Response

Response to Reviewer Comments

Dear reviewer:

We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript (Agriculture-3246386). The reviewer comments are laid out below in italicized font and specific concerns have been numbered. Our response is given in normal font and changes/additions to the manuscript are given in the blue text.

Comments 1: The rationale for the specific biochar application rates (7.5 and 15 t/ha) is not clearly explained. A justification based on previous literature should be provided.

Response 1: We incorporated insights from the literature to establish a foundation for the varying ratios of biochar addition (Line 105-110). “He et al. proposed that the application of biochar at a rate of 7.5 t ha⁻¹ combined with dual inhibitors constitutes the most effective strategy for reducing N₂O, NH₃, and N leaching. In addition, Huang et al. identified through an integration of field experiments and model predictions that the application of 15 t ha⁻¹ biochar in conjunction with N fertilizer represents an optimal approach for enhancing soil fertility and optimizing wheat yields in northern China”.

Comments 2: Only one growing season was studied. Multi-year data would strengthen the conclusions about longer-term effects (based on data availability).

Response 2: We appreciate your valuable suggestion. While long-term data from field experiments are undoubtedly more persuasive, this study was conducted under a delayed N application regime. The seasonal data obtained elucidate the emission patterns across various treatments and their impacts on soil properties within the context of this regime.

Comments 3: More details are needed on the gas sampling and analysis methods, including sampling frequency, time of day, and quality control measures, etc.

Response 3: We have incorporated a comprehensive description of the gas sampling and analysis methods, including specific details on sampling frequency, timing, and quality control measures (Line 186-211).

Comments 4: The method for calculating cumulative emissions is not sufficiently described (needs to be added).

Response 4: We have added the method for calculating cumulative emissions (Line 213-215).

Comments 5: ANOVA results should be presented for all major comparisons (Results and discussion section).

Response 5: ANOVA results have presented for all major comparisons in Table S2 (Line 645-670).

Comments 6: The discussion of enzyme activities (section 3.5) is difficult to follow. A table summarizing the key enzyme changes would improve clarity.

Response 6: We have refined the discussion regarding enzyme activities (Section 3.5) (Line368-392) and substituted the original table with Figure 7 (376), which offers a more intuitive representation.

Comments 7: The mechanisms proposed for changes in emissions are sometimes not very convincing. More direct evidence (e.g. microbial community analysis) would strengthen these arguments.

Response 7: We sincerely appreciate your valuable advice, the investigation of the laws and mechanisms governing gas emissions should be conducted from a microbial perspective. This represents a crucial avenue for our future research on NH₃ and greenhouse gas emissions, as well as their underlying microbial mechanisms.

Comments 8: Figure 2 is hard to read due to overlapping lines. Consider splitting into separate panels. Similar comment for Figures 4, S1, and S2. Figure 5 needs to be more clearly presented, as we can't read the text or the numbers.

Response 8: In response to your insightful review suggestions aimed at enhancing the readability of the figures, we have undertaken a revision of Figures 2, 4, and S1. In accordance with your recommendations, the data processing has been organized into three distinct categories and plotted separately. Additionally, we have refined Figure 5 to convey its contents with greater clarity. The purpose of Figure S2 is to elucidate the relationship between N₂O flux and soil WFPS through a scatter plot represented by a fitted exponential model.

Comments 9: The conclusion that "N1DB15 treatment is the optimal strategy" seems overly strong given the single-season nature of the study. This should be qualified.

Response 9: We appreciate your suggestion and would like to emphasize that the results of this experiment have been confirmed exclusively during a single growing season of wheat crops, as stated in the conclusion. Additionally, further long-term field experiments are necessary to incorporate various soil types and environmental factors in order to assess the effects of biochar and nitrification inhibitors (Lines 630-632).

Comments 10: Line 45-47: The statement about N2O's contribution to global warming potential needs updating with more recent IPCC values (from the Sixth Assessment Report (AR6), which was finalized in March 2023).

Response 10: We present supplementary insights derived from the IPCC (2023) concerning greenhouse gas emissions, including N₂O (Line 42-45).

Comments 11:  Line 136-137: More details on soil characteristics (e.g. CEC, texture) would be helpful for comparison to other studies.

Response 11: We have refined the description of soil characteristics pertaining to soil texture, cation exchange capacity (CEC), and available phosphorus (Lines 144-147).

Comments 12: Line 413-417: The comparison to previous biochar studies needs more nuance. Factors like biochar feedstock and production temperature likely explain some of the differences observed.

Response 12: In accordance with your suggestion, we conducted a literature search and found that the pyrolysis temperature of biochar markedly affects the nitrification rate in soil, with temperatures between 400 and 500 degrees Celsius demonstrating a significant capacity to enhance soil nitrification. This observation further elucidates why biochar promotes N₂O emissions in the soil within this study (Line 452-454).

We sincerely appreciate the time and effort invested by the reviewers in evaluating our manuscript. We look forward to any additional feedback or suggestions.

Yours sincerely

Haizhong Wu

19-Oct, 2024

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The comments from the first revision have been addressed.

From the revised version (it was not identified in the original submitted manuscript), it has been clarified the post-hoc test used was Fisher's LSD. This test is not suitable when more than three treatments are considered and should be replaced with an appropriate test.

Please, see this and other comments in the comments of the PDF file.

Comments for author File: Comments.pdf

Author Response

Dear reviewer:

We would like to express our sincere gratitude to the reviewers for their invaluable feedback (Agriculture-3246386), particularly regarding data processing, which has significantly enhanced our ability to refine the data analysis in this paper. The comments from the reviewers are presented below in italicized font, with specific concerns numbered accordingly. Our responses are provided in standard font, while modifications and additions to the manuscript are highlighted in blue text.

Comments 1: - The grammar of the sentence is confusing. The name of the kit is still not included.

Response 1: We have revised this sentence (Line 250-261), and incorporated the materials provided with the kit in Table S1 (Line 664-665).

Comments 2: Significance level should be identified with "α".

Response 2: We have revised it to "α" (Line 268).

Comments 3: This test is NOT appropriate if you have more than 3 groups.  You can find many sources discussing this issue. For example, see first answer:

https://www.researchgate.net/post/When_can_we_use_Fishers_Least_Significant_Difference_LSD_test.

You can consider applying Tukey HSD test.

Response 3: Thank you for your insightful revisions regarding the statistical methodology. In accordance with your suggestions, we have revised the Tukey HSD test on the data presented in this paper (Line 268-269).

Comments 4: I assume you are performing two different ANOVA tests:

1. one-way ANOVA with Treatment as factor
2. two-way ANOVA with DMPP and biochar as factor.

You have to clarify this.

I think it would be more correct to perform a single test: a three-way ANOVA with fertilization rate, DMPP and biochar as factors.

Response 4: We have clarified one-way ANOVA with Treatment as factor, while two-way ANOVA with DMPP and biochar as factor (Line 264-266).

In alignment with your suggestion, our analysis of the effects of biochar and DMPP will be exclusively centered on comparisons with N₁₈₀, while excluding N₀ and N₂₇₀ to highlight the significance of biochar and DMPP. In all diagrams pertaining to ANOVA, it is categorized into two classes: one category comprised six treatments with an identical application of N(N₁₈₀), and the other consisting of three treatments (N₀, N₁₈₀, N₂₇₀) with varying N levels.

Comments 5: In your Response 12, you say "we conducted a two-factor ANOVA, using N180 as the reference variable, as we have stated in Response 3.". I am not sure what a "reference variable" is in ANOVA... I assume you are performing a one-way ANOVA with Treatment as factor. This is a very poor model. You perform a two-way ANOVA afterwards to test the effect of biochar and dmpp. There is only different treatments which are compared equally.

In all tables and figures, I see that all the treatments are compared. How can you compare treatments with different fertilization rate if you don't include this in the ANOVA? If you include N0 and N270, you NEED to include fertilization rate in the model (three-way ANOVA with fertilization, DMPP and biochar).

Response 5: We appreciate your insights regarding our statistical analysis. In accordance with your suggestion, our examination of the effects of biochar and DMPP will focus exclusively on comparisons with N₁₈₀, while omitting N₀ and N₂₇₀ to underscore the significance of biochar and DMPP. In all diagrams related to ANOVA, it is classified into two categories: one category comprises six treatments with an identical application of nitrogen (N₁₈₀), while the other includes three treatments (N₀, N₁₈₀, N₂₇₀) with varying N levels.

Comments 6: according to a Fisher LSD post-hoc test.

Response 6: We have revised it (Line 287).

Comments 7: What means this?

Response 7: The same below represented in the chart below all convey this meaning, such as α<0.05. We have removed this sentence.

Comments 8: Figure.2, Please, correct the font size in the upper figure. Also, increase separation of the truncation (to make more visible that the lines are truncated).

Response 8:  We have corrected the upper figure, and also increased separation of the truncation (Fig.2; Line 326).

Comments 9: Figure.3, Again, correct font size of upper figure.

Response 9: We have made revisions based on your valuable feedback (Fig.3; Line 328).

Comments 10: Add the treatments below the x-axis. Then you can remove the color legend. The caption is incomplete. See comments in previous figure.

Response 10: We have added the treatments below the x-axis and removed the color legend (Line 340). We have revised the caption (Line 341-342).

Comments 11: You have to add the units in the figure's captions too.

Response 11: We  have added the units in the figure's captions (Line 397).

Comments 12: If the genes are not ordered in any speical way, you could group them by approximate mean enzymatic activity to make the plots more comptact: around 50 U/L, around 150-200 U/L, with only NOD on a separate scale. You can use a double axis plot for NOD, instead of the trncated axis solution.

You can also use trellis plots.

Response 12: In accordance with your suggestion, we have reorganized the enzyme activity map and implemented double coordinates in place of the truncated axis solution (Fig.7 Line 396).

Comments 13: Principal component analysis, random forest algorithm.

Response 13: We have revised it (Line 424).

Comments 14: This should go to Material and Methods or to the caption of Figure 8.

Response 14: We have revised it.

Comments 15: Why in Figure 8.a) they are 79.2 and 11.1 %?

Response 15: The object of principal component analysis varies.  the original Fig 8 conducted principal component analysis on soil properties and enzyme activities. The original Figure S4 presents the principal component analysis encompassing soil properties, gas emissions, enzyme activity, and wheat harvest index.

Comments 16: Figure.8, you are still including dependent (gas fluxes) and independent variables in the PCA.

If you look at Figure 8.a) you can see that the big % explained by PC1 is the result of including N0 in the analysis. But considering that N0 didn't receive fertilization, it is nor surprising that it differs in almost all parameters.

The differences in the other treatments from the PCA are relatively small (11.1% for PC2). Also if to look at Figure 8.b) we can see that the PC1, explaining 80% of variability, is affected by most of the variables.

From what I would conclude that the PCA analysis is not really giving any important information.

Response 16: In response to your review comments, we have removed the dependent variable (gas fluxes) from the principal component analysis and restructured both the principal component analysis diagram and the loading diagram. The revised principal component analysis excluded N₀ and N₂₇₀ treatments, incorporating only six treatments with an equivalent amount of nitrogen applied (Fig.8; Line 441). Furthermore, certain elements of the original Fig. S4 are redundant with the new Figure 8; therefore, the original Fig. S4 has been removed.

Comments 17: Figure 9, It would be interesting to see these parameters in the same order for the three gases, then we would clearly see the differences between gases.

Response 17: Thank you for your affirmation; we have revised it based on your previous review comments.

We sincerely appreciate the time and effort invested by the reviewers in evaluating our manuscript. We look forward to any additional feedback or suggestions.

Best regards

Haizhong Wu

3-Nov, 2024

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript investigates the effects of biochar and nitrification inhibitors on greenhouse gas emissions under delayed nitrogen application in wheat cultivation. While the authors have made improvements following the previous review round, there are still some concerns that need to be addressed.

- The authors have added two-way ANOVA analysis as requested (Table 5), which is an improvement, but there are still issues:

• The presentation of statistical results in Table 5 needs better formatting and clarity
• Some p-values are reported inconsistently (sometimes with "<" and sometimes with exact values)
• The degrees of freedom (DF) columns seem incorrect for a two-way ANOVA with these treatments

- Reviewer 1's concern about unsupported conclusions has been partially addressed:

• The authors modified statements about CO2 and NH3 emissions
• However, some conclusions in the abstract still overstate the statistical significance of their findings
• The discussion about enzyme activity has been improved but still includes some unsupported generalizations

- The enzyme activity measurement section (Lines 235-244) still lacks sufficient detail about:

• Specific assay conditions
• Quality control measures
• Standard curves or reference materials used

- Several figures need improvement:

• Figure 1: Axis labels are inconsistent in formatting (The authors could rotate the x-axis labels by 45 degrees to improve readability and prevent overlapping)
• Figure 2 & 3: Hard to distinguish between curves and error ranges. Can be made more clear.
• Figure 8: PCA plot needs better labeling and explanation of loadings

- Soil sampling procedure needs more detail about:

• Sample handling
• Storage conditions
• Time between sampling and analysis

- Specific Comments:

1. Lines 16-32: The abstract contains several strong claims that aren't fully supported by the statistical analysis presented.
2. Lines 140-148: The soil characterization section should include information about soil sampling depth replication.
3. Lines 235-244: The enzyme analysis section needs substantial expansion, particularly regarding quality control measures.
4. Lines 400-440: The discussion of biochar effects needs better integration with the statistical results presented.

The manuscript has improved since the first submission but requires further revision before it meets publication standards. The authors should pay particular attention to improving the statistical analysis and ensuring that the data presented fully supports all conclusions.

Decision Recommendation: Major Revision

Comments on the Quality of English Language

The manuscript requires moderate English language editing to improve clarity and readability. Several complex sentences need simplification, and some paragraphs contain awkward phrasing that impedes understanding. Key issues include inconsistent use of technical terms, redundant expressions, incorrect article usage (a/an/the), subject-verb agreement errors, and inconsistent formatting of units and chemical formulas. 

Author Response

Comments 1: - The authors have added two-way ANOVA analysis as requested (Table 5), which is an improvement, but there are still issues:

The presentation of statistical results in Table 5 needs better formatting and clarity

Some p-values are reported inconsistently (sometimes with "<" and sometimes with exact values)

The degrees of freedom (DF) columns seem incorrect for a two-way ANOVA with these treatments

- Reviewer 1's concern about unsupported conclusions has been partially addressed:

Response 1: We standardize the format of p-values (Line 454-455).

We re-evaluated the two-factor variance results, the degrees of freedom (DF) columns correct for a two-way ANOVA with these treatments.

Comments 2: The authors modified statements about CO2 and NH3 emissions

However, some conclusions in the abstract still overstate the statistical significance of their findings

The discussion about enzyme activity has been improved but still includes some unsupported generalizations

- The enzyme activity measurement section (Lines 235-244) still lacks sufficient detail about:

Specific assay conditions

Quality control measures

Standard curves or reference materials used

Response 2: We have rewritten the summary and corrected some inappropriate descriptions (Line 23-32).

We have enhanced the preservation conditions of soil for the assessment of enzyme activity (Line 226-29). Additionally, we enhanced the composition of the enzyme kit (Table S1, Line 672-674) and outlined enzyme activity assay procedure (Table S2, Line 674-677).

We have provided quality control measures for the assessment of enzyme activity (Line 254-360), and supplied standard curve for the enzymatic activity (Fig.S1, Line 653-658).

Comments 3: - Several figures need improvement:

Figure 1: Axis labels are inconsistent in formatting (The authors could rotate the x-axis labels by 45 degrees to improve readability and prevent overlapping)

Figure 2 & 3: Hard to distinguish between curves and error ranges. Can be made more clear.

Figure 8: PCA plot needs better labeling and explanation of loadings

Response 3: We have rotated the x-axis labels by 45 degrees to improve readability and prevent overlapping (Fig.1, Line 293).

We extended the length of the ordinates in Figures 2 and 3, and adjusted their proportions to enhance the readability of the graphs (Fig.2, Line 321; Fig.3, Line 324).

We have revised figure 8 (Line430), and refined its description (Line 420,425).

Comments 4: - Soil sampling procedure needs more detail about:

Sample handling

Storage conditions

Time between sampling and analysis

Response 4: We outlined the procedures for the collection and processing of soil samples, as well as the time interval between sampling and analysis (Lines 219-229)..

Comments 5: - Specific Comments: Lines 16-32: The abstract contains several strong claims that aren't fully supported by the statistical analysis presented.

Response 5: We have refined the descriptions in the abstract, fully supported by the statistical analysis presented (Line 23-32).

Comments 6: Lines 140-148: The soil characterization section should include information about soil sampling depth replication.

Response 6: We have provided detailed information about soil sampling depth replication (Line 143-145).

Comments 7: Lines 235-244: The enzyme analysis section needs substantial expansion, particularly regarding quality control measures.

Response 7: We have outlined the quality control measures for enzyme analysis, and included the components of the enzyme kit, the procedure for conducting enzyme activity tests, and the standard curve for assessing enzyme activity (Line 254-260; Fig.S1, Line653; Table.S1, Line 673; Table.S1, Line 675).

Comments 8: Lines 400-440: The discussion of biochar effects needs better integration with the statistical results presented.

Response 8: We have revised the discussion on the effects of biochar (Line 420-425; Line 442-444).

Comments 9: Comments on the Quality of English Language

The manuscript requires moderate English language editing to improve clarity and readability. Several complex sentences need simplification, and some paragraphs contain awkward phrasing that impedes understanding. Key issues include inconsistent use of technical terms, redundant expressions, incorrect article usage (a/an/the), subject-verb agreement errors, and inconsistent formatting of units and chemical formulas.

Response 9: We have invited a native English speaker to assist in enhancing the language quality of this paper, and hope the correction will meet with approval.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

The submitted manuscript entitled “Responses of N2O, CO2, and NH3 emissions to biochar and nitrification inhibitors under a delayed nitrogen application regime” assessed the application impact of the treatment on those gas emissions. Although, the topics in the study is suitable for the Journal of Agriculture, the manuscript cannot be accepted for publication because of the discussion not based on the data and insufficient description of methods.

First, there are many explanations did not based precisely on collected data. For example, there are no significant difference in CO2 and NH4 emission between N1 and N1DB15 treatment in Table 3, while authors concluded that N1DB15 treatment effectively mitigates emissions of these gas in Abstract (Line 31-32). Also, in the discussion of enzyme activity, biochar application alone did not always stimulates soil nitrification because of no difference in AMO and NXR between N1 and N1B7.5 (Table S1). Discussion in scientific paper should be constructed depending of the collected data. In addition, there are many missing information in terms of materials and method. Management practice of wheat cultivation was not explained, so, we can not consider the influence of N uptake by plant on N2O emissions. There are also no information how to measure grain yield and biomass of the straw in the manuscript. Also, there are many careless miss of spelling, style of the abbreviations, low resolution of figures, etc. I recommend to check the manuscript more carefully before the submission.

Followings are some of the minor comments

P4Line172: Need to provide each detectors for N2O and CO2 analysis.

P4Line188: Potassium collide is “KCl”, but not KCL

P5Line 201: What the meaning of the value, 2.65?

P5Line211-216: No information of the statistical analysis of random forest and correlation analysis

P6Figure6a and c: “-1” should be superscripted and “+” should be removed from the label of y-axis

P8Table3: there are two tables with same table number and title (table 3).

P8Table3: there are two treatment with same name (N1DB7.5) in the first table 3.

P10Line325-326: Table S1 did not show the correlation.

P10Line341: There are no “HXR” in the table.

P12Line369-380: No data was provided about the correlation analysis.

P17Figure S2: What the meaning of the title?

Comments on the Quality of English Language

Authors should check all text not only English grammar, but all description of the text style.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting study that investigated the impacts of delayed nitrogen application, biochar, and nitrification inhibitors on soil N2O, CO2 and NH3 emissions. They set three levels of N application, three biochar concentrations, and two DMPP levels. They found that reduced N application increased CO2, but reduced N2O and NH3. Biochar application increased N2O and NH3. Adding DMPP reduced N2O and CO2 emissions, but increase NH3.

The overall experimental design was good. Results were presented relatively clear. The manuscript reads well. But I have one major issue related to data analysis. The experiment used an incomplete experimental design with three treatment factors (N, biochar, and DMPP). However, in data analysis, the authors only test the simple treatment effects. I would suggest the authors to conduct contrast tests to detect the impacts of biochar, DMPP when a significant overall ANOVA test is significant. For example, N1 versus N1B7.5, N1B15, N1DB7.5, and N1DB15 test whether adding biochar influenced the response variable. N1B7.5 and N1DB7.5 versus N1B15 and N1DB15 test whether different amounts of biochar application. Similarly, N1B7.5 and N1B15 versus N1DB7.5 and N1DB15 test the DMPP impacts with biochar addition. The current analysis only tested the eight simple treatment effect. I recommend Major revision.

Specific comments:

L24: add respectively after emissions.

L202: Please add a little bit more details on enzyme activity measurements.

L212-216: The experiment used a non-balanced experimental design (full factor design would have 3 x 3 x 2=18 treatments). Please provide more details on data analysis and how the impacts of biochar, DMPP were detected.

Fig. 2: How were dynamics of soil N2O and CO2 considered in the ANOVA?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf