Review Reports - Biochar Exacerbates Nitrogen Loss in Drought-Affected Soil with Green Manure Application

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract

This section is fairly well structured, contains most of the necessary elements, and correctly identifies the research factors and the most important findings. I would suggest specifying which green manure crop the study concerns and on what type of soil the research was conducted.

Keywords

I suggest avoiding the repetition of keywords that are already included in the title of the manuscript.

Introduction

In my opinion, the introduction is well structured. The authors present the current state of knowledge, justify the importance of green manure, discuss the role of biowela and describe the role of moisture. However, I would suggest also paying attention to the specific taxonomic groups of soil microorganisms. Changes in soil conditions, such as the availability of organic matter and soil moisture, lead to changes in the populations of these groups, which in turn influence the decomposition of green manure. L89–90; L98–99 – repetition

Materials and Methods

This section is correctly structured. All the information necessary to replicate the experiment has been provided. My comments on this section are as follows: the incubation period was short, lasting only 60 days. The authors explain in a single sentence why this period was chosen, but in my opinion it is too short. Biochar is a material with a long-term effect, so many processes related to the stabilisation of organic matter, changes in the microbiome or nitrogen may not have had time to occur. Consequently, this makes it difficult to extrapolate the results to real field conditions. I suggest that the authors strengthen their argument regarding the 60-day period. Another issue is the fact that there were only three replicates, which is a very limited number for microbiological studies and sequencing. I also suggest addressing this point. Furthermore, it should be clarified why only two moisture variants were chosen. In the introduction, the authors mention a classification comprising more variants. I understand that certain threshold values were included, but this does not resolve the issue. Another important element that is missing is a more detailed description of the green manure. It was merely mentioned that it consisted of shredded alfalfa. What was its chemical composition – were only N and C specified? At what stage of growth was it harvested? The experimental mixture of soil and biochar was defined in terms of mass, whereas in the case of alfalfa, it was merely stated that it was air-dried. This is very imprecise and does not allow us to determine the actual amount of dry biomass that was introduced into the soil.

Results

The results are fairly sound and logical. The suggestions that arise after reading this chapter are:

- the significance levels for the obtained results are not specified

- in several lines, the authors include sentences more appropriate for the ‘Discussion’ section than for this section, e.g. lines 282–284; 362–364. In my opinion, these and other similar passages should be moved to the ‘Discussion’ section. I suggest reviewing the entire section in light of this issue

Discussion

The discussion is fairly well written, but in my opinion it lacks an explanation of the mechanisms of action; instead, it merely provides information about the effect, e.g. 396–398: why does biochar increase the activity of microorganisms during a drought? Furthermore, in my opinion, a key observation is the change in the ratio of fungi to bacteria over the course of the experiment. In the case of the microbiome, the interactions between taxonomic groups are more important to assess than their overall biomass or abundance. Changes in this ratio reflect changes in the soil. An increase in fungal content may indicate an accumulation of hard-to-decompose organic matter or the onset of green manure decomposition, as fungi are decomposition pioneers that prepare nutrients for subsequent groups of microorganisms. An increase in fungal content could also reflect other changes, such as pH. I suggest discussing this point in greater detail and drawing the appropriate conclusions. Furthermore, with regard to microorganisms, the authors assume in many instances that the presence of a particular bacterial species automatically implies high metabolic activity; however, this is not confirmed, as functional analyses (e.g. metagenomics or gene expression) would be required to establish this.

Conclusions

I suggest shortening the conclusions and focusing on the most important points, such as the fact that biochar does not always reduce nitrogen losses; the effect depends on soil moisture, and under drought conditions it may even increase nitrogen loss. In my opinion, this is a valuable observation, as many earlier manuscripts presented biochar primarily as a material that improves nutrient retention. I would also suggest highlighting limitations, such as the use of a single soil type and the need for testing under real-world (field) conditions, a longer incubation period, or a functional analysis of the microbiome.

Good luck

Author Response

Dear Editor and Reviewer,

Thank you very much for processing our manuscript (agronomy-4435956). We highly appreciate all comments and revision suggestions. All the comments from the three reviewers are valuable. We have made the revision following the reviewer’s comments. We have replaced the figures in the manuscript to make them clearer and more readable. We have improved the quality and clarity of our manuscript, and sincerely hope our revisions are satisfactory. Green represents explanation and response, while blue represents modification.

Reviewer 2:

Abstract

Response: We thank the reviewers for their comments. We have added the names of green manure crops and the types of soil.

“The impact of biochar on N conversion in drought-affected soil with green manure application has not been systematically investigated. Therefore, this study examined the effects of biochar produced at 350°C, 550°C, and 750°C (BC350, BC550, and BC750) on green manure (Medicago sativa L.) decomposition and N turnover in soil (Hapli-Udic Ferralosol) during a 60‑day laboratory incubation experiment under different moisture conditions (45% or 65% soil water-holding capacity (WHC)).”

Keywords

I suggest avoiding the repetition of keywords that are already included in the title of the manuscript.

Response: We thank the reviewers for their comments. We have revised the keywords.

“Keywords: Pyrolysis temperature; Drought; Green manure decomposition; Humification; Soil carbon sequestration”

Introduction

Response: We thank the reviewers for their comments. We have removed the repetitive parts (L98–99).

We have also added a discussion on how changes in soil conditions during green manure decomposition influence specific microbial taxonomic groups in the revised manuscript.

“Changes in soil environment also significantly affect the microbial community during green manure decomposition. Li et al. reported that environmental variables, including litter addition and soil depth, exerted strong selective pressure on the bacterial community, resulting in a pronounced enrichment of Actinobacteria and a concurrent suppression of Firmicutes; by comparison, the fungal community, composed primarily of Ascomycota and Basidiomycota, showed higher resilience, and such divergence in microbial community assembly ultimately contributed to differences in litter decomposition rates among treatments [10]. Wang et al. also found that the abundances of Acidobacteria and Bacteroidetes rose significantly during decomposition, while those of Actinobacteria and Chloroflexi decreased after decomposition [11].”

Materials and Methods

This section is correctly structured. All the information necessary to replicate the experiment has been provided. My comments on this section are as follows: the incubation period was short, lasting only 60 days. The authors explain in a single sentence why this period was chosen, but in my opinion, it is too short. Biochar is a material with a long-term effect, so many processes related to the stabilisation of organic matter, changes in the microbiome or nitrogen may not have had time to occur. Consequently, this makes it difficult to extrapolate the results to real field conditions. I suggest that the authors strengthen their argument regarding the 60-day period. Another issue is the fact that there were only three replicates, which is a very limited number for microbiological studies and sequencing. I also suggest addressing this point. Furthermore, it should be clarified why only two moisture variants were chosen. In the introduction, the authors mention a classification comprising more variants. I understand that certain threshold values were included, but this does not resolve the issue. Another important element that is missing is a more detailed description of the green manure. It was merely mentioned that it consisted of shredded alfalfa. What was its chemical composition – were only N and C specified? At what stage of growth was it harvested? The experimental mixture of soil and biochar was defined in terms of mass, whereas in the case of alfalfa, it was merely stated that it was air-dried. This is very imprecise and does not allow us to determine the actual amount of dry biomass that was introduced into the soil.

Response: We thank the reviewers for their comments. This study placed particular emphasis on the rapid release and turnover dynamics of carbon and nitrogen, processes that are largely concentrated within the 60 days following green manure incorporation—a period recognized as the rapid decomposition phase. As reported in the literature (Pan et al. 2011), the decomposition and mineralization of green manure typically proceed through three distinct phases: an early phase (0–15 d), a middle phase (15–60 d), and a late phase (beyond 60 d). In the early and middle phases, soil microbial activity remains high and nutrient turnover proceeds rapidly; beyond 60 days, the residual material is predominantly composed of recalcitrant substrates such as lignin, marking the transition to a slow decomposition phase. Accordingly, the 60‑day incubation period adopted in this study was designed to comprehensively cover the critical early and middle stages of green manure decomposition.

“This study focused on the rapid release and turnover dynamics of carbon and nitrogen, processes that were predominantly concentrated within the first 60 days after green manure incorporation-a period commonly referred to as the rapid decomposition phase [26]. The cultivation period of this study was relatively short (60 days), and the N in the biochar might not be released at all.”

We fully acknowledge that three replicates are relatively few for microbial sequencing given the complexity and variability of soil communities, and this is indeed a limitation of our work. Nevertheless, we believe that the consistent trends and statistically significant differences among treatments support the biological validity and reliability of our main conclusions regarding microbial community dynamics. In future experiments, we plan to adopt a minimum of 5-6 replicates per treatment to increase statistical power and strengthen the applicability of our results.

The slow decomposition of green manure in arid soils frequently leads to asynchrony between nutrient release and crop demand, a critical constraint that hampers the extensive use of green manure in water‑limited areas. To clarify the responses of green manure decomposition and soil C and N retention to moisture deficiency, we designed a comparative experiment encompassing both drought and well‑watered moisture regimes. In the opening paragraph of the Introduction, we have explicitly delineated the constraints imposed by drought on green manure application. In addition, we have also specified the moisture criteria for drought in the Introduction. We have also included the selection criteria for the soil moisture levels (i.e., 45% and 65% WHC) in the Materials and Methods section.

“Therefore, achieving rapid nutrient release from green manure while ensuring long-term nutrient retention in the soil represents a challenge that must be addressed for effective green manure application.”

“The percentage of soil moisture content in relation to the soil water-holding capacity (WHC) serves as an indicator of the drought degree. The following classification is used: no drought (>60%), mild drought (50%–60%), moderate drought (40%–50%), severe drought (30%–40%), and exceptional drought (<30%) [7].”

“Based on the drought classification criteria reported in previous literature [7], we selected two moisture conditions: a normal moisture group, which maintained soil moisture at 65% of the soil WHC, and a drought group, which maintained at 45% of the WHC.”

Alfalfa, cut at the peak of flowering, serves as a green manure that is high in protein and abundant in vitamins and minerals when returned to the field. The moisture content of air‑dried alfalfa was approximately 10%. Since the soil used in this study was also air‑dried, the application rates of all materials were calculated on an air‑dry weight basis, and thus the water content of the alfalfa was not deducted. We have made the corresponding additions in the Materials and Methods section.

“The green manure used was Medicago sativa L., which was harvested at the peak of flowering, air-dried and crushed to 2–3 cm pieces and is rich in protein, vitamins, and minerals. The moisture content of air‑dried alfalfa was approximately 10%.”

Results

The results are fairly sound and logical. The suggestions that arise after reading this chapter are:

- the significance levels for the obtained results are not specified

Response: We thank the reviewers for their comments. Significance levels have been explicitly indicated in the figure captions of the relevant bar charts. In addition, we have restructured the Results section by deleting the interpretive content (e.g., lines 282–284 and 362–364) and moving it to the Discussion section. Furthermore, we have carefully re‑examined the entire Results chapter to ensure that it strictly presents the experimental findings without any mechanistic interpretations.

Discussion

The discussion is fairly well written, but in my opinion, it lacks an explanation of the mechanisms of action; instead, it merely provides information about the effect, e.g. 396–398: why does biochar increase the activity of microorganisms during a drought? Furthermore, in my opinion, a key observation is the change in the ratio of fungi to bacteria over the course of the experiment. In the case of the microbiome, the interactions between taxonomic groups are more important to assess than their overall biomass or abundance. Changes in this ratio reflect changes in the soil. An increase in fungal content may indicate an accumulation of hard-to-decompose organic matter or the onset of green manure decomposition, as fungi are decomposition pioneers that prepare nutrients for subsequent groups of microorganisms. An increase in fungal content could also reflect other changes, such as pH. I suggest discussing this point in greater detail and drawing the appropriate conclusions. Furthermore, with regard to microorganisms, the authors assume in many instances that the presence of a particular bacterial species automatically implies high metabolic activity; however, this is not confirmed, as functional analyses (e.g. metagenomics or gene expression) would be required to establish this.

Response: We thank the reviewers for their comments. We have supplemented the Conclusions section with the requested additions based on the reviewer's comments. Please refer to the revised manuscript for details.

We suggest that biochar not only offers a suitable microhabitat for microorganisms but also promotes inter‑microbial electron exchange through its electron‑shuttling ability, while its hydrophilic nature helps maintain soil moisture, collectively leading to enhanced soil microbial activity.

“BC750 exhibited a pronounced promoting effect on NH₄⁺–N release from green manure under 65% soil WHC, whereas BC350 promoted NH₄⁺–N release under 45% soil WHC (Figure 2 and Figure 5). The promotion effect of BC350 and BC750 could be attributed to the water retaining capacity and EC of biochar, which facilitated the growth of microorganisms and the decomposition of green manure. BC750 exhibited the lowest H/C ratio of 0.24 and the highest EC (Table 2), suggesting it possessed high aromaticity and strong electron transfer capacity. Liu et al. demonstrated that biochar acted as an electron shuttle, thereby enhancing the mineralization rate of SOM [29]. It is therefore possible that the high EC of BC750 may have contributed to the accelerated decomposition of green manure observed in our study. BC350 exhibited the highest (O+N)/C ratio of 0.20, suggesting that it has the highest polarity and the largest number of hydrophilic groups (Figure 1a). This facilitated the soil water retention, which provided water for microbial activity and led to the decomposition and mineralization of green manure. Malinowski, et al also confirmed the result in this study [30]. Biochar co-application with green manure significantly increased CO2 emissions under WHC of 45% (Figure s2a) and significantly increased the MBC (Table 3), indicating that microbial activity was notably increased.”

In addition, following the reviewer's suggestion, we have supplemented the discussion on the changes in the fungal-to-bacterial ratio during the experiment. The increase in fungal abundance may indicate the onset of green manure decomposition, as fungi act as early decomposers that provide available substrates for subsequent microbial populations, thereby enhancing overall microbial activity.

“Furthermore, under 45% WHC, the increase in the MBC/MBN ratio during the end-stage of incubation indicated a rise in the relative abundance of fungi compared with bacteria. This expansion of the fungal community may signal an acceleration of green manure decomposition [32]. As early decomposers, fungi are capable of breaking down recalcitrant organic compounds and providing substrates for subsequent microbial populations, thereby potentially enhancing overall microbial activity [33].”

Response: We thank the reviewers for their comments. We acknowledge that our experimental design did not include direct analyses of functional genes or microbial activities, which represents a limitation of this study. In future work, we will carefully follow the reviewer's recommendations and incorporate functional analyses into our experimental protocols.

Conclusions

Response: We thank the reviewers for their comments. We have rewritten the conclusion.

“This study showed that the electron-transfer capacity of BC750 and the water retention capacity of BC350 appeared to promote green manure decomposition and increased soil NH₄⁺–N content (>5%) under the experimental conditions. The addition of biochar under 65% soil WHC was associated with increased MBC and humus substances. This was accompanied by a greater than 20% increase in soil total humus acid-N content following the combined application of biochar and green manure, which may have facilitated the turnover and conversion of soil N to stabilized forms. However, under 45% soil WHC, the addition of biochar was associated with an apparent increase in TN loss (>11%) under laboratory drought conditions. This could be tentatively attributed to a relatively low degree of humification and limited formation of macroaggregates, which may have resulted in a reduced capacity for soil N sequestration.

When applied as a water-retention agent under drought affected laboratory conditions, biochar was observed to accelerate the decomposition of green manure. However, insufficient water supply limited the effective operation of soil N immobilization pathways. Therefore, rational water management is essential for minimizing total N loss. Meanwhile, biochar modification is also needed to improve N stability in soil, either by facilitating abiotic humification or by enhancing the adsorption and binding of soil N to mineral surfaces. Efficient N management plays a key role in mitigating agricultural non‑point source pollution and curbing greenhouse gas emissions. Several limitations should be acknowledged: the laboratory incubation design, short duration (60 days), no field validation, and the absence of functional analysis of microbial communities. These factors limit direct extrapolation to field conditions. Long‑term field trials, functional microbial profiling, and quantitative partitioning of N loss pathways are required to bridge these knowledge gaps in future investigations.”

References

Pan F, Lu J, Liu W,Geng M (2011) Study on characteristics of decomposing and nutrients releasing of three kinds of green manure crops. Journal of Plant Nutrition and Fertilizers 17: 216-223 (in Chinese). https://doi.org/10.11674/zwyf.2011.0130.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Corrections and suggestions are in the attached file.

What is the main question addressed by the research?

This study investigated the impact of biochar on nitrogen loss in drought-affected soils amended with green manure. Although biochar can promote green manure decomposition and N release, it may exacerbate total N loss under drought conditions. The stable conversion of N into associated humus occurs only under normal moisture conditions.

Do you consider the topic original or relevant to the field? Does it address a specific gap in the field? Please also explain why this is/ is not the case.

Yes, the study showed that the co-application of biochar and green manure resulted in a total soil N loss exceeding 11% under drought conditions—a notable finding, given that biochar is generally considered beneficial for N retention. The BC350 and BC750 biochars promoted the decomposition and mineralization of the green manure due to their hydrophilicity (enhancing water retention) and electron-transfer capacity. Biochar addition increased soil NH4+-N content by 5.58% (with BC350) and 38.37% (with BC750) compared to green manure alone. The rise in soil pH induced by biochar application, combined with the drought-driven inhibition of stable soil organic matter and macroaggregate formation, led to high soil N loss. The conversion of released N into humus-associated N occurs only under normal moisture conditions.

What does it add to the subject area compared with other published material?

This study highlights that the impact of biochar on N transformation in drought-affected soil amended with green manure had not been systematically investigated previously. It elucidates the key pathways governing C and N sequestration in drought-affected soils subjected to the co-application of green manure and biochar. The study demonstrates that, while biochar addition can promote green manure decomposition and the release of available N, it can also exacerbate total N loss from the soil. Stable conversion into humus-associated N requires normal moisture conditions for the N released from the co-applied green manure and biochar.

What specific improvements should the authors consider regarding the methodology? What further controls should be considered?

The authors should consider including a biochar-only control group to evaluate its direct impact on soil properties. It would be both interesting and necessary to examine in greater detail the exact pathways of nitrogen loss from the soil, including total emissions of NH3, N2, and N2O.

Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed? Please also explain why this is/is not the case.

Yes, the conclusions are consistent with the evidence and arguments presented. Biochar with high electron-transfer capacity (BC750) and biochar with high water-holding capacity (BC350) promoted green manure decomposition and increased soil NH4+-N content. Biochar addition at 65% water-holding capacity (WHC) increased soil microbial biomass (MBC) and humic substances, facilitating the conversion of soil N into stabilized forms. At 45% WHC, biochar addition led to greater total N loss (>11%), attributed to a low degree of humification and limited macroaggregate formation, which reduced the soil's N-sequestering capacity. Limited water availability under drought conditions restricted microbial N-sequestering pathways, despite the biochar-induced acceleration of green manure decomposition. Nitrogen fertilizer management strategies in arid environments should focus on enhancing biochar's capacity for inorganic N fixation or facilitating the formation of more stable organic N compounds.

Are the references appropriate?

Yes, they agree with the investigation's proposals

Any additional comments on the tables and figures.

No.

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewer,

Reviewer 3:

Improve the text by inserting the values obtained for the mentioned variables.

Response: We thank the reviewers for their comments. We have added the observation data and revised the wording.

“The results indicated that the enhanced soil N loss could be attributed to both the biochar‑induced rise in soil pH (>8%) and the drought‑driven suppression of stable organic matter (e.g., >20% reduction in humus acid) and macroaggregate formation.”

Replace; the words are shown in the title.

Response: We thank the reviewers for their comments. We have revised the keywords.

“Keywords: Pyrolysis temperature; Drought; Green manure decomposition; Humification; Soil carbon sequestration”

Long paragraph; shorten and split into two paragraphs.
Long paragraph; shorten and split into two paragraphs.
Long paragraph; shorten and split into two paragraphs.

Response: We thank the reviewers for their comments. We have revised the Introduction and Experimental Design sections by condensing and splitting the paragraphs, and have reorganized the logical flow, as per your suggestion. Please refer to the revised manuscript for details.

Include information on the chemical and physical attributes of the soil, particularly the organic matter (OM) content.

Response: We thank the reviewers for their comments. We have supplemented the relevant soil property data in the Materials and Methods section, as per your suggestion.

“The soil is classified as Hapli-Udic Ferralosol (Chinese Soil Taxonomy, CST), which is characterized by its high acidity and low organic matter content. The soil had total C (TC) and TN contents of 7.49 g/kg and 0.76 g/kg, respectively, and a bulk density of approximately 1.22 g/cm³.”

Insert Figure 6 into the RESULTS section.
Insert Figure 7 into the RESULTS section.

Response: We thank the reviewers for their comments. We have moved Figure 6 to a suitable location within the Results section, and have made corresponding revisions to the relevant text in the Discussion section. We consider that Figure 7, which displays the PLS‑PM results derived from the data, provides mechanistic interpretations rather than direct observations. As such, we have retained it in the Discussion section and do not see a need to relocate it. Please refer to the revised manuscript for details.

Improve the text by inserting the values obtained for the mentioned variables

Response: We thank the reviewers for their comments. We have added the observation data and revised the wording. Please refer to the revised manuscript for details.

As a suggestion, it would be interesting for the authors to outline future studies that could support or further elaborate on the results obtained in the investigation

Response: We thank the reviewers for their comments. We fully agree that clearly outlining future research directions in the Discussion section helps to better highlight the broader implications of our study. Accordingly, we have added a brief section on future perspectives at the end of the Discussion in the revised manuscript, summarizing potential follow‑up studies to further validate and elaborate on our findings.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript could become suitable for publication after substantial revision, with particular emphasis on strengthening the experimental justification, moderating unsupported claims, improving statistical treatment, and clearly distinguishing evidence-based conclusions from hypotheses. This would considerably enhance the scientific rigor and reliability of the study.

Good Luck.

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewer,

Reviewer 1:

Abstract

The abstract states that biochar increased nitrogen loss by more than 11%, but the study never directly measured the major nitrogen loss pathways. Only soil nitrogen disappearance was calculated. No measurements were performed for NH₃ volatilization, N₂ emission, NO emission and nitrate leaching.

Response: We thank the reviewers for their comments. In this study, we calculated total nitrogen loss by comparing soil total nitrogen contents before and after the 60‑day incubation period. It should be noted that this was a pot experiment, and no water leakage was observed from the bottom of the pots throughout the incubation period; therefore, nitrate leaching can be excluded as a possible loss pathway. In addition, we measured N₂O emissions and found that they were negligible under drought conditions. Combined with the microbial community analysis, the increased abundance of complete denitrifying bacteria may have promoted complete denitrification. Thus, the most likely pathways of soil nitrogen loss in this study are NH₃ volatilization and N₂ emissions. Therefore, in the Abstract, we only reported that soil total nitrogen loss exceeded 11% under drought conditions, but did not specify the various pathways of nitrogen loss.

Mechanisms (hydrophilicity, electron-transfer) that promote decomposition are speculative without context on how they differ by pyrolysis temperature (BC350 vs. BC750). As well as the study did not directly measure water retention by each biochar, electron transfer and microbial respiration associated with electron shuttling.

Response: We thank the reviewers for their comments. We have supplemented the Abstract with a description of how pyrolysis temperature affects the hydrophilicity and electron‑transfer capacity of biochar.

“Due to low‑temperature biochar (BC350) possesses hydrophilicity (thereby enhancing water retention) due to its surface oxygen‑containing functional groups, while high‑temperature biochar (BC750) relies on the aromatic conjugated π‑electron system for electron transfer, both BC350 and BC750 promoted green manure decomposition and mineralization.”

In the present study, we directly determined the hydrophilicity and electrical conductivity of each biochar (Table 2). It has been well documented that as the pyrolysis temperature increases, the content of oxygen‑containing functional groups and the hydrophilicity of biochar tend to decrease, while pH, specific surface area, carbon content, and electrical conductivity (EC) increase accordingly (Fan et al. 2022; Uchimiya et al. 2011; Zhang et al. 2017). Furthermore, the elevated CO₂ emissions measured under drought conditions (Figure S4) with the addition of biochar also directly indicated increased microbial activity. Nevertheless, microbial respiration associated with electron‑shuttling processes was not directly measured in this study, which may represent a limitation; however, this does not undermine the robustness of our primary finding that biochar co‑application facilitated green manure decomposition.

The abstract should clearly mention the incubation study's 60-day duration and laboratory conditions, because readers may otherwise assume this was a field experiment.

Response: We thank the reviewers for their comments. We have added a statement regarding the 60‑day laboratory incubation condition to the Abstract.

“Therefore, this study examined the effects of biochar produced at 350°C, 550°C, and 750°C (BC350, BC550, and BC750) on green manure decomposition and N turnover in soil during a 60‑day laboratory incubation experiment under different moisture conditions (45% or 65% soil water-holding capacity (WHC)).”

Introduction

Although the literature review is generally adequate, the Introduction lacks a clearly defined knowledge gap; rather than merely summarizing previous studies on biochar, drought, and green manure, the authors should explicitly identify inconsistencies in previous findings, contradictory reports, and remaining research gaps to better justify the study objectives and hypotheses.

Response: We thank the reviewers for their comments. We have clarified the limitations of green manure application in the first paragraph of the Introduction.

In the third paragraph of the Introduction, we have supplemented the statement that the effects of biochar on nitrogen transformation are closely related to its properties, and that biochars with different properties exert considerably different effects on soil nitrogen transformation. Furthermore, while many studies have examined the role of biochar properties in regulating soil N transformation, the majority of these studies have concentrated only on the dynamics of available N, which constitutes a minor portion of soil N. In contrast, the loss of total N and its comprehensive discussion under organic N (e.g., green manure) addition have received far less attention.

“The effects of biochar on N transformation vary with its properties, which are closely related to the pyrolysis temperature. While many studies have examined the role of biochar properties in regulating soil N transformation, the majority of these studies have concentrated only on the dynamics of available N, which constitutes a minor portion of soil N. In contrast, the loss of total N and its comprehensive discussion under organic N (e.g., green manure) addition have received far less attention.”

In the fourth paragraph of the Introduction, we have clarified that low‑temperature biochar benefits green manure decomposition and N transformation under sufficient moisture, but its water‑retention efficacy and regulatory mechanisms under drought conditions are still largely unknown and warrant further investigation.

“Although existing research has shown that biochar promotes green manure decomposition and N turnover under favorable moisture conditions, whether such positive effects are sustained under drought and the associated mechanisms remain poorly understood, with systematic investigations still lacking. We speculated that the hydrophilicity of biochar may accelerate the decomposition of green manure and promote N release under drought condition, but may not promote its transformation into a more stable N form, potentially resulting in N loss from drought affected soil.”

In the fifth paragraph of the Introduction, we have supplemented the discussion regarding the effects of high‑temperature biochar on soil nitrogen transformation; however, its contribution to nitrogen immobilization warrants further in‑depth study.

“A clear understanding of the mechanisms underlying biochar‑mediated soil N immobilization under drought conditions is currently lacking. Water limitation may adversely affect long‑term immobilization by inhibiting microbial activity and mass diffusion. We propose that biochar with higher aromaticity may stimulate microbial activity via enhanced electron transfer through enhanced electron transfer, thereby accelerating green manure decomposition. Importantly, the enhanced microbial activity promoted the production and accumulation of microbial necromass. These microbial residues can be physically protected by occlusion within soil aggregates, or transformed via microbial processes into stable organic N pools, such as humus acid‑N, thereby facilitating long-term N stabilization in soils.; meanwhile, biochar with elevated pH may further increase soil pH and stimulate NH₃ volatilization, representing a competing loss pathway.”

Hypothesis 3 could better link pH elevation explicitly to NH₃ volatilization and denitrification.

Response: We thank the reviewers for their comments. We have made the necessary changes.

“(3) under drought condition, biochar application further increases soil pH, which may enhance NH₃ volatilization and promote denitrification, collectively exacerbating soil total N loss.”

The rationale for choosing 350/550/750°C is weak—justify based on property gradients (polarity vs. aromaticity/EC).

Response: We thank the reviewers for their comments. We have made necessary additions to the Introduction section of the article.

“Low-temperature biochar (e.g., pyrolyzed at 350°C) exhibits strong hydrophilicity due to its surface richness in polar oxygen-containing groups, which engage in firm hydrogen bonding with water molecules [16]. The large specific surface area and the abundance of the functional groups of biochar can enhance the WHC of soil [17] and the greater hydrophilicity of lower-temperature biochar improved soil water retention [18], which in turn enhanced microbial activity [19]. Li et al. demonstrated that biochar addition during the composting process retained moisture, consequently promoting the decomposition of organic matter and accelerating NH₄⁺–N and NO₃^--N production [20], while also increasing WHC and facilitating humification [21]. Although existing research has shown that biochar promotes green manure decomposition and N turnover under favorable moisture conditions, whether such positive effects are sustained under drought and the associated mechanisms remain poorly understood, with systematic investigations still lacking. We speculated that the hydrophilicity of biochar may accelerate the decomposition of green manure and promote N release under drought condition, but may not promote its transformation into a more stable N form, potentially resulting in N loss from drought affected soil.

As the pyrolysis temperature increases, the content of oxygen‑containing functional groups and the hydrophilicity of biochar tend to decrease, while pH, specific surface area, C content, and electrical conductivity (EC) increase accordingly [22]. At elevated temperatures (e.g., 750°C), biochar develops extensive graphite‑like sheet structures composed of aromatic rings. The delocalized conjugated π‑electrons residing on these rings can migrate freely throughout the C skeleton, thereby establishing an efficient pathway for electron transfer [23]. Consequently, biochar can function as an electron shuttle, reducing the electron transfer distance and improving the efficiency of electron transfer across microbial interfaces [24], thereby promoting N conversion. However, Liu et al. found that the liming effect of biochar in the topsoil increased soil pH, thereby exacerbating NH₃ volatilization losses in soils with neutral pH [25]. A clear understanding of the mechanisms underlying biochar‑mediated soil N immobilization under drought conditions is currently lacking. Water limitation may adversely affect long‑term immobilization by inhibiting microbial activity and mass diffusion. We propose that biochar with higher aromaticity may stimulate microbial activity via enhanced electron transfer through enhanced electron transfer, thereby accelerating green manure decomposition. Importantly, the enhanced microbial activity promoted the production and accumulation of microbial necromass. These microbial residues can be physically protected by occlusion within soil aggregates, or transformed via microbial processes into stable organic N pools, such as humus acid‑N, thereby facilitating long-term N stabilization in soils.; meanwhile, biochar with elevated pH may further increase soil pH and stimulate NH₃ volatilization, representing a competing loss pathway.”

The claim that the aromatic structure promotes nitrogen stabilization is not sufficiently supported by the existing literature; the authors should provide a clearer mechanistic explanation of how biochar aromaticity influences microbial immobilization, humification, and soil aggregate formation, rather than presenting these processes as unsupported hypotheses.

Response: We thank the reviewers for their comments. We have revised the speculative statements in the Introduction accordingly. The original version primarily emphasized that the high aromaticity of biochar could facilitate electron transfer in soil, potentially promoting soil nitrogen transformation and enhancing nitrogen immobilization under normal moisture conditions. Following the reviewer’s suggestion, we have rephrased the relevant statements at the end of the Introduction as follows: the effects of high‑temperature biochar on soil nitrogen transformation are variable, and further studies are needed to elucidate its specific role in soil nitrogen transformation.

Methodology

No biochar-only treatment is a noted limitation, but it weakens attribution (e.g., biochar N contribution or pH effect alone).

Response: We thank the reviewers for their comments. We have already explained this in the Materials and Methods section and the Conclusion. The incubation period of this study was relatively short (60 days), and the nitrogen in the biochar might not have been fully released, as indicated by the absence of a significant decrease in biochar nitrogen content after incubation (Table 1). Furthermore, since this study primarily focused on the effects of combined biochar and green manure application on soil nitrogen turnover, we did not include a biochar‑only control group. Admittedly, the lack of a biochar‑only control may have overlooked the direct effects of biochar on soil properties; however, this limitation does not compromise the reliability of our conclusions.

The use of only three replicates provides relatively low statistical power given the inherent variability associated with microbial sequencing, greenhouse gas measurements, and soil heterogeneity; therefore, the authors should acknowledge and discuss this limitation and its potential implications for the reliability and generalizability of the results.

Response: We thank the reviewers for their comments. We consider that the significant differences observed among treatments, which were consistent across multiple parameters, still provide meaningful and reliable insights into the trends of nitrogen dynamics. In addition, three replicates are also commonly adopted in most published soil incubation studies, for example, Li et al. (2016) and Pan et al. (2011).

The application ratio of 2:1.6:100 (biochar: green manure: soil) is difficult to interpret and compare with previous studies; therefore, the authors should express the application rates using standard units, such as Mg ha⁻¹, % (w/w), or g kg⁻¹ soil, to improve clarity and facilitate comparison with existing literature.

Response: We thank the reviewers for their comments. We have made the necessary revisions as requested.

“Biochar and green manure were incorporated into soil at rates equivalent to 2% (w/w) and 1.6% (w/w) of the soil dry weight, respectively. The mixed materials (totaling 4 kg) were then filled into the pots.”

The manuscript does not provide sufficient justification for selecting 45% and 65% water-holding capacity (WHC) as the experimental moisture levels; the authors should explain whether these thresholds were based on field measurements, previous literature, or established drought classifications to strengthen the rationale for the experimental design.

Response: We thank the reviewers for their comments. In the Introduction, we have clearly defined the drought classification standards, and further supplemented the rationale for selecting the two moisture levels in the Materials and Methods section.

“Based on the drought classification criteria reported in previous literature[7], we selected two moisture conditions: a normal moisture group, which maintained soil moisture at 65% of the soil WHC, and a drought group, which maintained at 45% of the WHC.”

The description of the greenhouse gas measurements is insufficient for reproducibility. Although a closed-bottle incubation method was used to measure CO₂ and N₂O emissions, essential methodological details—including headspace volume, container sealing procedure, gas sampling protocol (static or dynamic), sampling intervals, and analytical procedures—are missing. Furthermore, the authors should clearly describe how gas fluxes and cumulative emissions were calculated and provide the corresponding equations used in the analysis.

Response: We thank the reviewers for their comments. The detailed measurement and calculation methods are provided in the agronomy-4435956-supplementary.

“Text S1: The moisture setting and material composition are as previously operation. The incubator is maintained at relative air humidity around 60%, a temperature of 25℃, with a photoperiod regime of 12 h of light exposure and 12 h of darkness. During the routine incubation, the caps of all screw‑cap bottles were kept loose to allow gas exchange. The volume of the head cavity of the caps is approximately 200 mL. For gas sampling, the caps were tightened. Before gas collection, zero‑grade air was flushed into the bottles for 10 min, and then all bottles were sealed for 2 h (from 9:00 to 11:00 a.m.). Subsequently, gas samples were withdrawn into gas bags via the gas exchange method. Exchange the gas in the threaded bottle with N₂. Exchange 100 mL of gas each time. Gases were collected on 1th, 2th, 3th, 5th, 7th, 10th, 13th, 16th, 20th, 25th, 30th, 35th, 40th, 45th, 50th, 55th, and 60th day. CO₂ and N₂O concentration were measured using a gas chromatograph (8890 GC System, Agilent, America). Daily CO₂/N₂O emission was calculated as: Daily emission = average flux during the 2‑h measurement × 24 h. Cumulative emissions were then computed by integrating the daily emissions over the 60‑day incubation period using the trapezoidal rule [Σ (daily emission × interval between adjacent sampling days)].”

The nitrogen balance calculation is incomplete because "Loss-N" was estimated by difference (Initial TN − Final TN), implicitly assuming that all unaccounted nitrogen was lost from the system. However, a portion of the nitrogen may have remained in dissolved organic nitrogen, microbial residues, dissolved ammonium, or other unidentified organic pools. Therefore, the term "Loss-N" is potentially misleading and should be replaced with a more appropriate term (e.g., "unaccounted N") or its limitations should be clearly acknowledged and discussed.

Response: We thank the reviewers for their comments. In this study, "loss‑N" was estimated by the difference method (initial total nitrogen minus final total nitrogen). This calculation accounts for all nitrogen‑containing fractions in the soil, including dissolved organic N, microbial necromass, ammonium‑N, and other unidentified organic N forms; therefore, no N pool is omitted from the estimation. Nevertheless, we agree that the term "loss‑N" may be imprecise, as it does not specify the actual pathways of nitrogen loss. To avoid ambiguity, we have revised the term to " Apparent Loss-N " in the revised manuscript.

Results

The Results and Discussion are not clearly separated, as the Results section frequently includes mechanistic explanations and interpretations rather than focusing solely on the experimental observations. The authors should present the findings objectively in the Results section and move the interpretation and discussion of underlying mechanisms to the Discussion section to improve the manuscript's organization and readability.

Response: We thank the reviewers for their comments. We have restructured the Results and Discussion sections: the Results section now presents only the objective findings, whereas all explanations regarding underlying mechanisms and extended interpretations have been consolidated into the Discussion section, which has improved the logical flow and overall readability of the manuscript.

Although nitrogen dynamics are discussed extensively, the statistical comparisons among sampling dates are unclear. Because the measurements were collected repeatedly over time, a repeated measures ANOVA or an equivalent mixed-effects model would be more appropriate than conducting multiple one-way ANOVAs, as it accounts for temporal dependence and reduces the risk of inflated Type I error.

Response: We thank the reviewers for their comments. We fully agree that conducting multiple independent one‑way ANOVAs on repeated measurement data is not appropriate. Accordingly, we have revised our statistical analysis by employing a repeated‑measures ANOVA to properly account for temporal dependence and reduce the risk of inflated Type I error. The relevant sections in the Methods and Results have been updated accordingly. The new analysis yielded results consistent with our original conclusions, but with more robust statistical support. We have already provided the additional information in the Materials and Methods section.

“The statistical analysis was performed using SPSS version 25.0 (IBM Corporation, Armonk, NY, America). To account for repeated measurements taken over the 60‑day incubation period, a repeated‑measures analysis of variance (ANOVA) was conducted. When a significant main effect or interaction was detected, post‑hoc comparisons were carried out using Tukey’s honestly significant difference (HSD) test to control the family‑wise error rate. For variables measured only at a single time point, one‑way ANOVA was used, followed by Tukey’s HSD test for multiple comparisons. All data are presented as mean ± standard error (SE), and statistical significance was set at p < 0.05. To quantify the direct and indirect effects of multiple drivers on soil humus, we applied partial least squares path modeling (PLS-PM) using the “plspm” package in R. The structural model was constructed based on hypothesized causal relationships among latent constructs. All manifest variables were modeled in reflective mode (mode A). Model performance was evaluated using R² values and the goodness-of-fit (GOF) index.”

The microbial community analysis reports only taxonomic relative abundance, which does not provide direct evidence of microbial functional activity. Therefore, conclusions regarding functional processes, such as enhanced denitrification, are not sufficiently supported and should either be substantiated with functional analyses (e.g., functional gene abundance or activity measurements) or presented more cautiously as potential interpretations rather than definitive conclusions.

Response: We thank the reviewers for their comments. We acknowledge that 16S rRNA gene amplicon sequencing solely reveals taxonomic relative abundance, which reflects community composition rather than direct in-situ metabolic activity. Accordingly, we have rigorously revised our manuscript throughout to decouple taxonomic shifts from definitive functional claims. Replaced definitive statements (e.g., “enhanced denitrification occurred”) with cautious correlative interpretations (e.g., “suggesting a potential for enhanced denitrification,” or “indicating a taxonomic shift favoring putative denitrifying lineages”). Added a prominent paragraph in the Discussion section explicitly outlining this methodological limitation, emphasizing that while the community structure shifted towards known denitrifies, direct functional assays are required to confirm actual activity, and we have marked this as a priority for future research. In this study, the changes in relative abundance from microbial community analysis were used to indirectly support the accelerated decomposition of green manure and enhanced denitrification observed in our experiment, rather than serving as direct evidence. The changes in soil nitrogen fraction contents were the primary direct evidence for assessing soil nitrogen retention and loss in this study. The absence of direct analyses of functional genes or microbial activities in our experimental design constitutes a limitation of this work. The corresponding revisions have been made in the Discussion section; please refer to the revised manuscript for details.

“Humicola is known to promote lignocellulose decomposition and soil humic acid formation. Wang et al. also found that biochar addition increased the abundance of Humicola, which in turn promoted organic matter decomposition, possibly because Humicola can produce thermostable cellulase, hemicellulase, ligninase, and amylase, and thus contributes significantly to the decomposition process [39]. Under 65% WHC, the abundance of Humicola in GB350, GB550, and GB750 was higher than that in GM (Figure 6), which is consistent with the possibility that biochar addition may have facilitated green manure decomposition and soil humus formation.”

“The decomposition of green manure can substantially deplete oxygen within soil pores, leading to the development of localized hypoxic conditions, which in turn favor denitrification as the prevailing pathway [51]. Biochar co-application with green manure increased the abundance of Arthrobacter, Streptomyces, and Bacillus [52], which likely promoted denitrification and exacerbated soil N loss (Figure 6), thereby further confirming our speculation. This is consistent with our observation that N₂O emissions were negligible under drought (Figure S2b), suggesting that the N removed from the soil may have been predominantly converted to N₂ via complete denitrification rather than emitted as N₂O.”

TC/TN declines under drought are key but require error bars and clear % loss attribution to GM vs. native SOM.

Response: We thank the reviewers for their comments. Error bars have been clearly indicated in our figures. We fully agree with the reviewer regarding the importance of attributing TC/TN losses to the added green manure rather than to native soil organic matter. However, the soil used in this study had relatively low organic matter content, and given the short incubation period, the changes in native soil carbon and nitrogen were likely limited. In addition, gas flux measurements showed that CO₂ and N₂O emissions from the unamended soil were extremely low (Figure S2), further supporting that the observed carbon and nitrogen losses were predominantly derived from the added green manure. Furthermore, the carbon and nitrogen contents of biochar showed little change before and after incubation (Table 1), indicating that the decrease in soil TC and TN could be mainly attributed to the decomposition and loss of green manure.

The presentation of the dominant microbial taxa in Figure 5 is informative; however, the discussion should more explicitly link changes in microbial community composition to functional processes, such as relating the abundance of putative denitrifies to the observed N₂O and N₂ emissions. In addition, the ecological significance of the shifts in dominant fungal phyla, particularly Ascomycota and Mucoromycota, should be interpreted more comprehensively and supported by relevant literature.

Discussion

Several conclusions are speculative, as the authors attribute nitrogen loss to processes such as NH₃ volatilization, complete denitrification, and microbial electron transfer without directly measuring these mechanisms. These interpretations should be clearly presented as hypotheses or potential explanations rather than confirmed mechanisms.

Response: We thank the reviewers for their comments. We sincerely thank you for this critical and constructive comment. We fully agree that attributing nitrogen loss to mechanisms without direct measurements is inappropriate, and we have thoroughly revised the Discussion section accordingly in the revised manuscript. Specifically, we have made the following modifications:

1.Replaced deterministic statements with cautious, speculative language. 2.Explicitly framed mechanistic interpretations as “potential explanations”. 3.Added a clear limitation statement: In the appropriate place within the Discussion, we have explicitly stated that “this study did not directly measure NH₃ volatilization, N₂ emissions, or microbial electron transfer processes; therefore, the relevant inferences should be interpreted with caution.”

The manuscript frequently interprets correlation as causation. For example, the positive correlation between Humicola abundance and nitrogen retention does not demonstrate that Humicola was responsible for nitrogen sequestration. The authors should avoid causal statements unless supported by direct experimental evidence.

Response: We thank the reviewers for their comments. We thank the reviewer for the careful suggestions regarding causal inference. We fully agree that correlation alone is insufficient to establish causation. Accordingly, we have systematically revised the entire manuscript.

“Humicola abundance was positively correlated with soil TN contents (r = 0.68, p < 0.05), and soil total humus acid-N content was also positively correlated with soil TN contents (r = 0.89, p < 0.01) (Figure S5). Taken together, these correlative patterns suggest that under 65% soil WHC, biochar addition may have enhanced humification and promoted the integration of N-containing organic compounds into the complex molecular skeleton of humus acids, which could potentially contribute to mitigating soil N loss.”

“Furthermore, there was a significant positive correlation between the MBC/MBN ratio and the total humus acid and humic acid content in soil (r = 0.64, p < 0.05, r = 0.65, p < 0.05) (Figure S5). These correlative patterns suggest that the succession of microbial communities toward a fungal-dominant state may be associated with the retention of soil C and N.”

The Discussion would benefit from a broader comparison with international studies rather than relying predominantly on literature that supports the present findings. In particular, contradictory reports on the effects of biochar on nitrogen retention and loss should be critically discussed to provide a more balanced interpretation.

Response: We thank the reviewers for their comments. We fully agree that citing only literature that supports our conclusions would result in a less comprehensive and less objective interpretation. Accordingly, we have revised the Discussion section of the manuscript. Please refer to the revised manuscript for details.

“FTIR spectrum also revealed that the molecular structure of soil humic acid aliphatic, young, and simple (Figure S3b), and also promoted the continuous renewal of humus [37]. Qian et al. demonstrated that biochar application in subtropical orchard soils not only enhanced N retention, forming a temporary organic N reservoir and reducing inorganic N leaching potential, but also improved soil N holding capacity through its porous structure and abundant functional groups [38]. Humicola is known to promote lignocellulose decomposition and soil humic acid formation. Wang et al. also found that biochar addition increased the abundance of Humicola, which in turn promoted organic matter decomposition, possibly because Humicola can produce thermostable cellulase, hemicellulase, ligninase, and amylase, and thus contributes significantly to the decomposition process [39]. Under 65% WHC, the abundance of Humicola in GB350, GB550, and GB750 was higher than that in GM (Figure 6), which is consistent with the possibility that biochar addition may have facilitated green manure decomposition and soil humus formation.”

“Microbial secretions released during green manure decomposition, together with biochar itself, can promote soil aggregate formation. By enhancing the "microbial C pump" effect [45] and enriching humification‑related microbial taxa [46], biochar facilitates the conversion of C and nitrogen into stable microbial residues and humus.”

“The higher content of microaggregates (Figure S4b) inhibited the diffusion of soil oxygen, thereby creating a suitable habitat for complete denitrification by denitrifying bacteria. The decomposition of green manure can substantially deplete oxygen within soil pores, leading to the development of localized hypoxic conditions, which in turn favor denitrification as the prevailing pathway [51].”

“In addition, Sun et al. also reported that under water‑saving drip irrigation, the water‑holding capacity of biochar sustains a soil moisture regime conducive to denitrification, potentially increasing N₂O emissions [53]; concurrently, reduced irrigation diminishes the dilution of NH₄⁺–N, and the elevation of soil pH caused by biochar may further enhance NH₃ volatilization [54].”

The role of biochar aromaticity is overstated throughout the Discussion. Although aromaticity is known to influence biochar stability, the present study does not directly demonstrate that it enhanced nitrogen immobilization. Therefore, such conclusions should be presented more cautiously and supported by appropriate evidence.

Response: We thank the reviewers for their comments. We agree with the reviewer’s point that, in the original Introduction, we speculated that biochar aromaticity might promote the conversion of soil nitrogen to humus acid‑N. After careful consideration of the entire manuscript and subsequent revisions, we have revised this speculation and now clearly state that biochar aromaticity may enhance microbial activity by facilitating electron transfer among microorganisms, thereby accelerating green manure decomposition. This perspective is also reflected in Discussion section 4.1.

“BC750 exhibited the lowest H/C ratio of 0.24 and the highest EC (Table 2), suggesting it possessed high aromaticity and strong electron transfer capacity. Liu et al. demonstrated that biochar acted as an electron shuttle, thereby enhancing the mineralization rate of SOM [29]. It is therefore possible that the high EC of BC750 may have contributed to the accelerated decomposition of green manure observed in our study.”

Regarding biochar‑enhanced soil nitrogen immobilization, we consider that the primary mechanisms are likely associated with the formation of soil macroaggregates and the provision of suitable habitats for relevant microorganisms (such as Humicola and Mucoromycota), which together create a favorable microenvironment for microbial fixation of soil carbon and nitrogen. Relevant discussions have been supplemented in Discussion section 4.2.

“Qian et al. demonstrated that biochar application in subtropical orchard soils not only enhanced N retention, forming a temporary organic N reservoir and reducing inorganic N leaching potential, but also improved soil N holding capacity through its porous structure and abundant functional groups [38]. Humicola is known to promote lignocellulose decomposition and soil humic acid formation. Wang et al. also found that biochar addition increased the abundance of Humicola, which in turn promoted organic matter decomposition, possibly because Humicola can produce thermostable cellulase, hemicellulase, ligninase, and amylase, and thus contributes significantly to the decomposition process [39]. Under 65% WHC, the abundance of Humicola in GB350, GB550, and GB750 was higher than that in GM (Figure 6), which is consistent with the possibility that biochar addition may have facilitated green manure decomposition and soil humus formation.”

“Correspondingly, the abundance of Mucoromycota was higher under 65% soil WHC, which may further promote the fixation of SOM, and the addition of biochar further enhanced this condition. Mucoromycota has the capacity to produce extensive mycelium and contribute more stable organic C pools [42]. Their enrichment under 65% WHC, particularly with biochar addition, suggests that favorable moisture conditions may enhance their role in promoting soil structure and organic matter retention. This is consistent with the observed increase in stable organic C under 65% WHC in our study. In this study, the observed increase in stable organic C may have favored soil N sequestration. The MBC/MBN ratio serves as an indicator of soil microbial community [43]. At the end of cultivation, the addition of biochar resulted in a notable enhancement in MBC/MBN ratio (Table 3), indicating a high level of activity within the fungal community. Fungal hyphae and abundant extracellular secretions can serve as cementing substances that promote the formation of agglomerates and form physical barriers (Figure S4a), which are more conducive to the fixation of soil organic C and N [44]. Microbial secretions released during green manure decomposition, together with biochar itself, can promote soil aggregate formation. By enhancing the "microbial C pump" effect [45] and enriching humification‑related microbial taxa [46], biochar facilitates the conversion of C and nitrogen into stable microbial residues and humus.”

The Discussion does not adequately consider alternative mechanisms that may explain the observed nitrogen dynamics, including adsorption–desorption processes, microbial immobilization, dissolved organic nitrogen, and microbial necromass. These potential pathways should be discussed alongside the proposed mechanisms to provide a more comprehensive interpretation of the results.

Response: We thank the reviewers for their comments. We greatly appreciate the reviewer's constructive suggestions regarding alternative mechanisms that may explain the observed nitrogen dynamics. In our Discussion, we did mention pathways such as adsorption–desorption, microbial immobilization, dissolved organic nitrogen, and microbial necromass.

“The MBC/MBN ratio serves as an indicator of soil microbial community [43]. At the end of cultivation, the addition of biochar resulted in a notable enhancement in MBC/MBN ratio (Table 3), indicating a high level of activity within the fungal community. Fungal hyphae and abundant extracellular secretions can serve as cementing substances that promote the formation of agglomerates and form physical barriers (Figure S4a), which are more conducive to the fixation of soil organic C and N [44].”

However, our discussion primarily focused on three key aspects: (1) the promoting effect of biochar co‑application on green manure decomposition under drought conditions; (2) the enhancement of soil humification by biochar under normal moisture conditions, which facilitated soil carbon and nitrogen immobilization; and (3) the combined effects of biochar‑induced high pH and increased abundance of denitrifying microorganisms under drought conditions, which contributed to soil nitrogen loss. Consequently, the nitrogen transformation pathways raised by the reviewer were discussed to a lesser extent in this study.

Conclusion

The conclusions extend beyond experimental evidence. Statements such as "biochar exacerbates nitrogen loss" should be revised to more accurately reflect the study conditions, for example, "biochar increased apparent nitrogen loss under laboratory drought conditions," thereby avoiding overgeneralization beyond the scope of the experiment.
The Conclusion should explicitly acknowledge the main limitations of the study, including the laboratory incubation design, short experimental duration, absence of plants, lack of leaching processes, and the absence of field validation. These limitations should be clearly stated, as they restrict the direct extrapolation of the findings to agricultural field conditions.

Response: We thank the reviewers for their comments. We have rewritten the conclusion.

Statistics

Because measurements were collected repeatedly over a 60-day incubation period, the statistical analysis should account for temporal dependence. A repeated-measures ANOVA or, preferably, a linear mixed-effects model would be more appropriate than independent one-way ANOVAs, as these approaches better accommodate repeated observations and provide more robust inference.
The use of Fisher's least significant difference (LSD) test for multiple comparisons may increase the risk of Type I error. A more conservative post hoc procedure, such as Tukey's honestly significant difference (HSD) test or another appropriate multiple-comparison correction, is recommended to improve the reliability of the statistical analysis.

References

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Li M, Liu M, Li Z, Jiang C,Wu M (2016) Soil N transformation and microbial community structure as affected by adding biochar to a paddy soil of subtropical China. Journal of Integrative Agriculture 15: 209-219. https://doi.org/10.1016/S2095-3119(15)61136-4.

Uchimiya M, Wartelle L H, Klasson K T, Fortier C A,Lima I M (2011) Influence of pyrolysis temperature on biochar property and function as a heavy metal sorbent in soil. J. Agric. Food. Chem. 59: 2501-2510. https://doi.org/10.1021/jf104206c.

Zhang H, Chen C, Gray E M,Boyd S E (2017) Effect of feedstock and pyrolysis temperature on properties of biochar governing end use efficacy. Biomass Bioenergy 105: 136-146. https://doi.org/10.1016/j.biombioe.2017.06.024.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,
Thank you very much for the effort you put into revising the manuscript based on the suggestions from the first round of reviews. In my opinion, the manuscript has been significantly improved. I have no further comments.