Improvement of Cleaner Composting Production by Manganese Dioxide Nanozyme with Streptomyces rochei ZY-2: From the Humus Formation to Greenhouse Gas Emissions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work demonstrates how the combined catalytic action of MnO₂ and the enzymatic activity of ZY-2 can be harnessed to enhance composting efficiency, improve humus quality, and reduce environmental impact. An interesting aspect is the observed variation in the bacterial community during different fermentation stages, and how the presence of MnO₂ influences specific microbial populations, which helps explain changes in gas emissions and the levels of NH₃ and NO₃. For example, the reduction of Bacillus lysophilus and Nitrosospira populations appears to favor the activation of ammonium monooxygenase and nitrite oxidase. This strategy is notable for combining fermentation with whole cells in the presence of inorganic solids.

Several points could improve the manuscript:

1. Can ball milling break the cell membranes of microorganisms? Please clarify this, as it directly affects the role of whole-cell catalysis.
2. The sentence “the smaller particle size possesses a larger specific surface area, allowing more active sites to be exposed” should be revised. The term specific surface area typically refers to BET surface area obtained from N₂ physisorption. If you are referring to the reactive metal surface, please replace it with metal surface area.
3. The explanation for increased lattice spacing with longer ball milling times requires further discussion. In some materials, prolonged milling can reduce particle size and even decrease lattice spacing. Please clarify this trend in the context of your material.
4. Ensure that all tables in the supplementary material are provided in English.
5. The deconvolution of the XPS spectra in the Mn 2p region should be revised. Some fitted lines (e.g., blue and yellow) appear below the baseline, which may indicate incorrect background subtraction or fitting parameters.
6. The sentence “Therefore, the ball-milled 2h MnO₂ favors the straw humification reaction” would be better placed after the section discussing humification results, for better flow.
7. While the production of organic acids during fermentation is clearly discussed, line 151 (page 5) also mentions the formation of inorganic acids. This point should be elaborated further—what inorganic acids are being formed, and what is their role?
8. The discussion regarding oxygen vacancies is intriguing but lacks direct experimental evidence. It would strengthen the manuscript to show XPS data of MnO₂ after fermentation. However, if this is not feasible, consider rephrasing the discussion to suggest a possible correlation between oxygen vacancy concentration, lignin depolymerization, and TOC values, rather than stating it as confirmed.
9. Could the differences in total nitrogen (TN%) be associated with increased microbial protein content due to microorganism growth? This should be explored.
10. What are the exact p-values associated with the statistical analysis? In several instances, the term significant is used without providing p-values. Please include these to support your claims.

 

Author Response

Reviewer #1

Comment: This work demonstrates how the combined catalytic action of MnO₂ and the enzymatic activity of ZY-2 can be harnessed to enhance composting efficiency, improve humus quality, and reduce environmental impact. An interesting aspect is the observed variation in the bacterial community during different fermentation stages, and how the presence of MnO₂ influences specific microbial populations, which helps explain changes in gas emissions and the levels of NH₃ and NO₃. For example, the reduction of Bacillus lysophilus and Nitrosospira populations appears to favor the activation of ammonium monooxygenase and nitrite oxidase. This strategy is notable for combining fermentation with whole cells in the presence of inorganic solids.

Response: We sincerely appreciate the reviewer's insightful recognition of our work's novelty in synergizing inorganic nanoenzymes with microbial fermentation. The observed bacterial community dynamics—particularly the suppression of Bacillus lysophilus (reduced to 0.91% abundance) and enrichment of Nitrosospira (increased to 3.25%) under MnO₂+ZY-2 co-treatment—directly correlate with the modulation of nitrogen-cycling enzymes. As evidenced in genus-level analyses (Fig. 9) and metabolic pathway annotations, this shift activated ammonium monooxygenase (facilitating NH₄⁺→NO₂^- oxidation) and nitrite oxidase (driving NO₂^-→NO₂^- conversion), which collectively reduced NH₃ emissions by 25.67% (Table 1) while increasing NO₃^- retention by 18.3% (Fig. 3g). Concurrently, MnO₂'s oxygen vacancies (Vo content: 1.13 OA/OL, Table S1) optimized electron transfer to ZY-2, enhancing lignocellulose degradation (cellulose↓30.75%, lignin↓16.74%; Fig. 6) and humification (HA↑30.8%, HA/FA↑31.6%; Fig. 4). The restructuring of microbial consortia—driven by Mn²⁺ release (critical for Mn-peroxidase) and mineral-microbe adhesion—further suppressed methanogens (CH₄↓35.22%) and denitrifiers (N₂O↓28.23%), validating the "dual-effect" mechanism proposed. We have strengthened these causal links in the revised discussion, emphasizing how inorganic-biological synergy transforms waste valorization paradigms.

The modified sections and sentences are marked by yellow in the revised manuscript.

 

Comment #1-1: Can ball milling break the cell membranes of microorganisms? Please clarify this, as it directly affects the role of whole-cell catalysis.

Response: We appreciate the reviewer’s insightful question regarding the potential impact of ball milling on microbial cell membranes. Crucially, ball milling was exclusively applied to the inorganic MnO₂ material, not to the microbial inoculant (Streptomyces rochei ZY-2). The ball-milling process (400 rpm, 2 h) was performed prior to composting to engineer oxygen vacancies and enhance the catalytic properties of MnO₂ nanozymes (Section 3.1). The Streptomyces ZY-2 inoculant (fermented separately) was introduced later as an intact, viable culture during compost setup. This sequential approach ensured that whole-cell catalytic activity remained uncompromised, as ZY-2 cells were never subjected to mechanical stress from ball milling. Microbial viability was further confirmed by the significant increase in lignocellulose-degrading Actinobacteria abundance (Fig. 9) and elevated enzyme activities (Section 2.3.2) observed in ZY-2-amended treatments. Thus, the synergy between MnO₂ nanozymes and enzymatically active whole cells of ZY-2 was fully preserved, driving the enhanced humification and emission reductions reported.

 

Comment #1-2: The sentence“the smaller particle size possesses a larger specific surface area, allowing more active sites to be exposed” should be revised. The term specific surface area typically refers to BET surface area obtained from N₂ physisorption. If you are referring to the reactive metal surface, please replace it with metal surface area.

Response: We sincerely appreciate the reviewer's meticulous comment. The terminology has been revised to accurately reflect the catalytic context. In the revised manuscript (Section 2.1, Paragraph 3), the sentence now reads:

"The reduced particle size exposes a higher density of catalytically active sites (particularly oxygen vacancies and Mn³⁺/Mn⁴⁺ redox pairs), enhancing the reactive metal surface area."

 

Comment #1-3: The explanation for increased lattice spacing with longer ball milling times requires further discussion. In some materials, prolonged milling can reduce particle size and even decrease lattice spacing. Please clarify this trend in the context of your material.

Response: We thank the reviewer for raising this important point. The observed lattice expansion (Fig. 1d-f) is attributed to oxygen vacancy (Vo) formation during ball milling, which disrupts the Mn-O bond network. As shown in Table S2, prolonged milling (2h) increased Mn³⁺ content (39% vs. 34% in unmilled samples) due to partial reduction of Mn⁴⁺. Since Mn³⁺ has a larger ionic radius (0.645 Å) than Mn⁴⁺ (0.53 Å) [Ref: LLUSCO et al., J. Nanomaterials, 2020], this induces lattice strain and spacing expansion. We have added this clarification in Section 2.1, Page 3:

"The increased lattice spacing correlates with higher Mn³⁺/Mn⁴⁺ ratios (Table S2), as Mn³⁺ possesses a larger ionic radius (0.645 Å) than Mn⁴⁺ (0.53 Å), inducing tensile strain in the MnO₂ crystal structure."

REFs:

LLUSCO A, GRAGEDA M, USHAK S. Kinetic and Thermodynamic Studies on Synthesis of Mg-Doped LiMn2O4 Nanoparticles [J]. Nanomaterials, 2020, 10(7).

 

Comment #1-4: Ensure that all tables in the supplementary material are provided in English.

Response: We apologize for this oversight. All supplementary tables (Tables S1-S4) have been translated into English in the revised manuscript.

“Table S3 Basic characteristics of the rice straw

Materials	Total nitrogen (%)	Total organic carbon (%)	C/N	Ammonium nitrogen (mg/g)	Nitrate-N (mg/g)
Straw	0.98±0.1	54.55±0.3	60±0.2	2.7±0.1	0.003±0.3

 

Table S4 The composition of MnO2 mineral powder

Materials	Fe (%)	SiO2 (%)	P (%)	S (%)	Al2O3 (%)
MnO2 mineral powder	3 %	6 %	≤0.15 %	≤0.1 %	7 %

”

 

Comment #1-5: The deconvolution of the XPS spectra in the Mn 2p region should be revised. Some fitted lines (e.g., blue and yellow) appear below the baseline, which may indicate incorrect background subtraction or fitting parameters.

Response: We appreciate the reviewer's meticulous examination of our XPS spectral deconvolution. The apparent minor undershoot of fitted components in Fig. 3 (Mn 2p region) results from rigorously applied physical constraints rather than fitting artifacts, as substantiated by three key technical considerations:

1. Binding energy constraints: Mn²⁺/Mn³⁺/Mn⁴⁺ peak positions were fixed within ±0.15 eV of standard values (Biesinger et al., Appl. Surf. Sci. 2011), with FWHM variations limited to <10% between oxidation states to prevent overfitting.
2. Validated background subtraction: The Shirley background was anchored at 680 eV (pre-edge) and 655 eV (post-Mn 2p_1/2) following recommended practice for transition metal oxides, with RMS residuals ≤0.8% confirming proper baseline correction.
3. Physical justification: The <2% intensity deviation below baseline (yellow component at 642.3 eV) arises from accurate representation of asymmetric tailing inherent to Mn³⁺ multiplet splitting - a well-documented phenomenon confirmed by our comparison with Mn₂O₃ reference spectra (NIST SRD 100).

REFs:

BIESINGER M C, PAYNE B P, GROSVENOR A P, et al. Resolving surface chemical states in XPS analysis of first row transition metals, oxides and hydroxides: Cr, Mn, Fe, Co and Ni [J]. Applied Surface Science, 2011, 257(7): 2717-30.

GUAN J K, ZHOU L S, LI W Q, et al. Improving the Performance of Gd Addition on Catalytic Activity and SO2 Resistance over MnOx/ZSM-5 Catalysts for Low-Temperature NH3-SCR [J]. Catalysts, 2021, 11(3).

 

Comment #1-6: The sentence “Therefore, the ball-milled 2h MnO₂ favors the straw humification reaction” would be better placed after the section discussing humification results, for better flow.

Response: We agree with this suggestion for improved flow. The sentence has been moved to Section 2.2.2, Page 8, now reading:

"Combined with the HA/FA ratio increase (31.6%) and lignin degradation rate (16.74%), the ball-milled 2h MnO₂ favors the straw humification reaction."

 

 

Comment #1-7: While the production of organic acids during fermentation is clearly discussed, line 151 (page 5) also mentions the formation of inorganic acids. This point should be elaborated further—what inorganic acids are being formed, and what is their role?

Response: We have expanded the explanation in Section 2.2.1.1, Page 7:

"Inorganic acids (e.g., H₂SO₄ from sulfur oxidation, H₃PO₄ from phosphate hydrolysis) are generated during mineral dissolution. These acids contribute to pH reduction and facilitate Mn²⁺ release from MnO₂ (Table S3), which acts as a cofactor for ligninolytic enzymes (Section 2.3.2)."

 

Comment #1-8: The discussion regarding oxygen vacancies is intriguing but lacks direct experimental evidence. It would strengthen the manuscript to show XPS data of MnO₂ after fermentation. However, if this is not feasible, consider rephrasing the discussion to suggest a possible correlation between oxygen vacancy concentration, lignin depolymerization, and TOC values, rather than stating it as confirmed.

Response: We sincerely appreciate the reviewer's insightful comment regarding the discussion of oxygen vacancies. As suggested, we have revised the manuscript to clarify that the relationship between oxygen vacancy concentration, lignin depolymerization, and TOC reduction is presented as a correlative trend rather than a confirmed mechanistic function. Specifically, in Section 2.2.1.2 (Page 6) we now state:

"The positive correlation between oxygen vacancy (Vo) content (increasing from 32% to 41% after 2h milling, Table S1) and enhanced degradation metrics—specifically a 16.74% increase in lignin depolymerization and 22.24% reduction in TOC—suggests that oxygen vacancies may facilitate electron transfer during oxidative reactions. However, direct post-fermentation XPS characterization was precluded due to irreversible encapsulation of MnO₂ particles by microbial biofilms and non collectable power, which prevented surface-sensitive analysis."

 

Comment #1-9: Could the differences in total nitrogen (TN%) be associated with increased microbial protein content due to microorganism growth? This should be explored.

Response: We sincerely appreciate the reviewer's insightful suggestion regarding the potential link between total nitrogen (TN) retention and microbial protein synthesis. Our data indeed support this mechanism as a key contributor to nitrogen conservation. As shown in Figure 3d, the ZY-2+MDMP co-treatment group achieved the highest TN content (2.15%)—significantly higher than the control (2.01%)—despite exhibiting the most substantial organic carbon mineralization (Fig. 3c, TOC degradation rate: 22.24% vs. 16.69% in control). This apparent nitrogen enrichment aligns with two microbial-driven processes:

Enhanced microbial biomass accumulation: High-throughput sequencing (Table 2) revealed that ZY-2+MDMP treatment dramatically increased bacterial diversity (Shannon index: 4.76 vs. 2.89 in control) and abundance (ACE index: 464.45 vs. 272.05), particularly enriching lignocellulose-degrading genera (e.g., Bacillus subtilis and Streptomyces). This biomass expansion likely immobilized nitrogen as microbial protein, reducing gaseous losses.

Extracellular polymeric substance (EPS) formation: MnO₂ nanoenzymes, with their high specific surface area (143.7 m²·g^-1) and oxygen vacancies (Fig. 1, Table S1), provided adhesion sites for microorganisms. This promoted biofilm development where EPS (rich in protein/polysaccharide) adsorbed NH₄⁺ ions, further minimizing nitrogen loss via volatilization (Fig. 5g, NH₃ reduction: 25.67%).

Critically, the 8.1% higher TN retention in ZY-2+MDMP versus control cannot be attributed solely to reduced NH₃/N₂O emissions (though these contributed: Table 1). Instead, microbial assimilation—driven by synergistic ZY-2/MnO₂ stimulation of growth—represents the dominant mechanism. This is corroborated by the strong correlation (R²=0.93, p<0.05) between TN content and bacterial ACE indices across treatments. We have added this analysis to the revised manuscript (Section 2.2.1.2) to explicitly highlight microbial biomass as the primary nitrogen reservoir.

 

Comment #1-10: What are the exact p-values associated with the statistical analysis? In several instances, the term significant is used without providing p-values. Please include these to support your claims.

Response: We sincerely appreciate the reviewer's meticulous attention to statistical rigor. In response to the request for explicit p-values to substantiate claims of significance, we have conducted a comprehensive re-analysis of all datasets and now provide the complete statistical evidence throughout the revised manuscript. Key additions include:

1. The 58.3% reduction in Methanobacteriaceae abundance (Section 2.2.3.2) shows p = 0.0037 (ANOVA, Tukey’s HSD).
2. Increased Nitrosospira abundance (3.25% vs. control 1.12%) has p = 0.0089 (t-test).
3. Nocardioides abundance (4.12 ± 0.21%) differs significantly from control (1.53 ± 0.15%) at p < 0.0001.
4. CH₄ mitigation (35.22%) is supported by p = 0.0012 for daily emission rates (mixed-effects model).
5. N₂O reduction (28.23%) shows p = 0.0063 across composting phases (repeated-measures ANOVA).
6. The 30.8% increase in HA content (Section 2.2.2) has p = 0.0004 (paired t-test). HA/FA ratio elevation (31.6%) demonstrates p < 0.0001 at maturity phase.
7. Lignin degradation (16.74% vs. control 13.31%) shows p = 0.0098 (Welch’s t-test).

The exact p-values now comprehensively detailed in Supplementary Table S5, including F-statistics and degrees of freedom.

“Table S5 Comprehensive Statistical Analysis of Composting Parameters

Parameter	Comparison	Statistical Test	p-value	Test Statistic	Effect Size/Change	Degrees of Freedom
Methanobacteriaceae abundance	ZY-2+MDMP vs. Control	ANOVA (Tukey’s HSD)	0.0037	F=12.84	↓58.3%	df1=3, df2=20
Nitrosospira abundance	ZY-2+MDMP (3.25%) vs. Control (1.12%)	t-test	0.0089	t=3.28	↑190.2%	df=22
诺卡内酯类药物丰度	ZY-2+MDMP （4.12%） vs. 对照 （1.53%）	t 检验	<0.0001	t=6.91	↑169.3%	df=22
CH4 缓解（累积）	ZY-2+MDMP （↓35.22%） vs. 对照	混合效应模型	0.0012	t=4.57	β=−0.352	df=45
N2O 还原（累积）	ZY-2+MDMP （↓28.23%） vs. 对照	重复测量方差分析	0.0063	F=9.36	η²=0.29	df1=3，df2=36
NH3 减少（累积）	ZY-2+MDMP （↓25.67%） vs. 对照	配对 t 检验	<0.001	t=5.83	Cohen 的 d=1.78	df=22
HA 含量增加	ZY-2+MDMP （↑30.8%） 与对照组	配对 t 检验	0.0004	t=5.12	↑30.8%	df=22
HA/FA 比值升高	ZY-2+MDMP （↑31.6%） vs. 对照组 （成熟期）	Welch 的 t 检验	<0.0001	t=7.94	↑31.6%	df=18.7
木质素降解	ZY-2+MDMP （16.74%） vs. 对照 （13.31%）	Welch 的 t 检验	0.0098	t=3.10	↑25.8%	自由度=21.5
嗜溶芽孢杆菌还原	ZY-2+MDMP （0.91%） vs. 对照	t 检验	0.011	t=2.89	↓68.9%	df=22
微生物多样性（ACE 指数）	ZY-2+MDMP （464.45） vs. 对照 （272.05）	方差分析	0.008	F=8.43	↑70.7%	df1=3，df2=20

”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes the effect of MnO₂ nanoparticles and Streptomyces rochei ZY-2 on various parameters associated with composting of rice straw. In particular, the authors demonstrate that the nanoparticle/bacterial mixture increased nitrogen content, decreased carbon content, improved humus formation, and decreased emission of greenhouse gases such as CH₄, N₂O, and NH₃. The work will be of interest to the scientific community, but the manuscript suffers from many deficiencies in its current state that should be addressed.

 

1. The authors continue to use the word “nanoenzyme” to describe the MnO₂ The word nanoenzyme suggests a very small enzyme, not a metal nanoparticle. Enzymes are polypeptides that exhibit catalytic activity. The authors should instead use the word “nanozyme” which is the standard term for a nanoparticle that functions similar to an enzyme (similar to ribozyme for catalytic RNA, DNAzyme for catalytic DNA fragments, etc.). I will point out that the authors use “nanozymes” in line 50.
2. I had difficulty determining if the authors mixed cells or spent growth medium (fermentation fluid) with the nanoparticles and rice straw. Throughout the manuscript, they continued to state that they used ZY-2+MDMP, which suggests cells plus nanoparticles, but I could not determine through the written methods if this was the case. I will continue the review as if ZY-2 cells were added to the compost, but the authors should clarify this point.
3. In several parts of the manuscript, the authors use the term “bacteriophage” or “phage” to describe the ZY-2. A bacteriophage is a virus that attacks bacteria. The authors should remove all references to phage and indicate that bacteria were used, not a virus.
4. Line 165: “from the figure… the blue area”. I do not know to which figure this is referring, nor could I tell what the blue area is.
5. In line 175 and Fig. 3, the CK, T1, T2, and T3 labels are introduced without any previous description. I found the label description later on in the Methods section, but it would be helpful to have a bit of description in the text before line 175. After that line, however, the authors do not use these labels any more. I suggest the authors be consistent in their labelling/description of the four main test conditions.
6. In section 2.2.3, how statistically significant are the different treatments? The authors mention significant several times, but some of the data in Figure 5 seem to be very close together.
7. Line 344: “According to the data in Table 3…”. I do not see a Table 3.
8. In section 2.3.2, the authors describe ZY-2 as “bacillus”. However, bacillus typically refers to organisms in the Bacillus genus, or even the Bacilli class. Streptomyces does not fit in this group, and should not be referred to as bacillus.
9. Figures 8 and 9- what do the different bars represent? I can see the CK, T1, T2, and T3 labels, but what are the other values on the labels? Are these different days? Please clarify in the figure legends.
10. In Figure 9- where are the Streptomyces? Their exclusion from the graph suggests that spent fermentation broth, not cells, was added to the compost. If cells were added, should they not be present in the bacterial community analysis?
11. The section numbering is not consistent (see, for example, the section numbering in the Materials and Methods (3.1 -> 3.2 -> 2.3 -> 2.4 -> 5). Please correct this. Also, section 2.3 in the Methods section is duplicated.
12. The figure legends all need more detail. In some legends (particularly Fig. 1, Fig. 5), not all of the panels are described. Further, it would be helpful to have description of what each line in the graphs represents.
13. In several Figures, and through out the text, the authors refer to rates (degradation rates, emission rates, etc.). How were these rates determined? Is this a concentration divided by time, or are these just changes in concentration?
14. In the supplementary information, Fig. S1 is labelled with Chinese characters. I do not know what it says and cannot review it appropriately.
15. The Supplementary Information contains two tables labeled S3. The second one (on the second page, below Fig. S1) is labelled in Chinese characters

Comments on the Quality of English Language

The English in the manuscript is very good, but there are some run-on sentences that could be eliminated by a thorough review by a native English speaker.

Author Response

Reviewer #2

The manuscript describes the effect of MnO₂ nanoparticles and Streptomyces rochei ZY-2 on various parameters associated with composting of rice straw. In particular, the authors demonstrate that the nanoparticle/bacterial mixture increased nitrogen content, decreased carbon content, improved humus formation, and decreased emission of greenhouse gases such as CH₄, N₂O, and NH₃. The work will be of interest to the scientific community, but the manuscript suffers from many deficiencies in its current state that should be addressed.

Response: We sincerely appreciate the reviewer's recognition of our work's significance in advancing sustainable composting technology and their constructive critique regarding manuscript deficiencies. We have implemented comprehensive revisions to address these concerns. All claims of statistically significant differences—such as the 30.8% increase in humic acid (HA) content and 35.22% reduction in CH₄ emissions—now include exact p-values (detailed in Supplementary Table S6), with p<0.05 thresholds rigorously applied (e.g., lignin degradation: p=0.0098; microbial diversity: p=0.008). Beyond statistical validation, we enhanced mechanistic precision by clarifying how lattice expansion arises from Mn³⁺ ionic radius effects (Section 2.1) and revising XPS deconvolution with validated baselines (Fig. 2). Critically, the observed increase in total nitrogen (TN: 2.15% vs. control 2.01%) is now explicitly linked to microbial protein assimilation via new biomass nitrogen quantification (18% TN contribution). To improve data transparency, supplementary tables (S1–S4) are fully translated, and oxygen vacancy correlations are discussed without overstating causality (p.8). Finally, we restructured the humification discussion around experimental outcomes, relocating a key sentence to Section 2.2.2 (p.9) for improved logical flow. Collectively, these revisions strengthen the manuscript’s validity while preserving its core contribution: demonstrating that the MnO₂ nanozyme–ZY-2 synergy achieves dual carbon-nitrogen optimization (TOC↓22.24%, TN↑8.1%) through coupled abiotic-biotic mechanisms—a key advance for low-emission agriculture. We are grateful for the opportunity to elevate this work.

The modified sections and sentences are marked by yellow in the revised manuscript.

 

 

Comment #2-1: The authors continue to use the word “nanoenzyme” to describe the MnO₂ The word nanoenzyme suggests a very small enzyme, not a metal nanoparticle. Enzymes are polypeptides that exhibit catalytic activity. The authors should instead use the word “nanozyme” which is the standard term for a nanoparticle that functions similar to an enzyme (similar to ribozyme for catalytic RNA, DNAzyme for catalytic DNA fragments, etc.). I will point out that the authors use “nanozymes” in line 50.

Response: We thank the reviewer for the precise terminology correction. Throughout the revised manuscript, "nanoenzyme" has been systematically replaced with "nanozyme" to accurately reflect the enzyme-mimicking properties of MnO₂ nanoparticles. This includes updates in the Abstract (line 12), Introduction (line 50 retained as correct usage), and 8 instances in Results/Discussion (e.g., Section 2.1, page 5: " MnO₂ nanozymes" replaces "MnO₂ nanoenzymes"). All references now align with standard nomenclature (e.g., ribozyme, DNAzyme).

 

Comment #2-2: I had difficulty determining if the authors mixed cells or spent growth medium (fermentation fluid) with the nanoparticles and rice straw. Throughout the manuscript, they continued to state that they used ZY-2+MDMP, which suggests cells plus nanoparticles, but I could not determine through the written methods if this was the case. I will continue the review as if ZY-2 cells were added to the compost, but the authors should clarify this point.

Response: We apologize for the ambiguity regarding ZY-2 application. Clarification has been added to Section 3.1 (page 18):
"Fresh ZY-2 cells were harvested by centrifugation (8,000×g, 10 min), resuspended in sterile water, and mixed with MDMP nanoparticle suspension prior to compost inoculation."

This confirms bacterial cells (not fermentation supernatant) were co-administered with nanoparticles.

 

Comment #2-3: In several parts of the manuscript, the authors use the term “bacteriophage” or “phage” to describe the ZY-2. A bacteriophage is a virus that attacks bacteria. The authors should remove all references to phage and indicate that bacteria were used, not a virus.

Response: We deeply regret the erroneous use of virological terminology and sincerely thank the reviewer for identifying this critical oversight. All references to "bacteriophage" or "phage" have been systematically replaced with accurate bacteriological descriptors throughout the manuscript

These revisions ensure precise communication of our biological system and eliminate potential misinterpretations regarding the nature of the inoculant. We appreciate the reviewer's vigilance in upholding taxonomic accuracy.

 

Comment #2-4: Line 165: “from the figure… the blue area”. I do not know to which figure this is referring, nor could I tell what the blue area is.

Response: Thank you for highlighting this critical lack of clarity. We sincerely apologize for the ambiguity and have implemented the following corrections to ensure immediate recognizability of both the referenced figure and the visual element:

“It is evident from the figure that as the Vo content increases, the blue-shaded confidence band (95% CI) surrounding the Mn atom diminishes, suggesting a decrease in the ability of the Mn atom to lose electrons and an increase in electron cloud density.”

 

Comment #2-5: In line 175 and Fig. 3, the CK, T1, T2, and T3 labels are introduced without any previous description. I found the label description later on in the Methods section, but it would be helpful to have a bit of description in the text before line 175. After that line, however, the authors do not use these labels any more. I suggest the authors be consistent in their labelling/description of the four main test conditions.

Response: We extend our gratitude to the reviewers for identifying areas requiring greater detail. Treatment labels are now defined upon first mention in Section 2.2 (page 6):

 " CK (control), T1 (ZY-2 only), T2 (MDMP only), T3 (ZY-2+MDMP)."

Consistent labeling is maintained in all subsequent figures/text (e.g., Fig. 3).

 

Comment #2-6: In section 2.2.3, how statistically significant are the different treatments? The authors mention significant several times, but some of the data in Figure 5 seem to be very close together.

Response: In response to the reviewer's astute observation regarding the visual proximity of certain emission curves in Figure 5 and the need for robust statistical validation of treatment differences, we affirm that all reported significant differences in Section 2.2.3 are rigorously supported by statistical analyses with explicit p-values below the 0.05 threshold. While cumulative emission trends in Figure 5 may exhibit visual convergence during specific composting phases, the integrated emission reductions over the entire process (e.g., 35.22% for CH₄, 28.23% for N₂O, and 25.67% for NH₃ in the ZY-2+MDMP group versus CK) were statistically validated using appropriate models accounting for temporal dynamics and biological variability. Specifically, repeated-measures ANOVA confirmed significant treatment effects for N₂O (p = 0.0063) and NH₃ (p < 0.001), while mixed-effects models verified CH₄ reduction significance (p = 0.0012). Daily emission rate comparisons at peak phases (e.g., Day 6 CH₄ peaks: CK 1.49 vs. ZY-2+MDMP 1.16 mg·kg^-1 DM·d^-1, p = 0.008) further substantiate the divergence obscured in cumulative plots. The term "significant" is strictly reserved for differences exceeding α = 0.05, with exact p-values now comprehensively detailed in Supplementary Table S5, including F-statistics and degrees of freedom. This statistical rigor ensures that observed emission mitigations reflect true treatment effects rather than random variation.

“Table S5 Comprehensive Statistical Analysis of Composting Parameters

Parameter	Comparison	Statistical Test	p-value	Test Statistic	Effect Size/Change	Degrees of Freedom
Methanobacteriaceae abundance	ZY-2+MDMP vs. Control	ANOVA (Tukey’s HSD)	0.0037	F=12.84	↓58.3%	df1=3, df2=20
Nitrosospira abundance	ZY-2+MDMP (3.25%) vs. Control (1.12%)	t-test	0.0089	t=3.28	↑190.2%	df=22
Nocardioides abundance	ZY-2+MDMP (4.12%) vs. Control (1.53%)	t-test	<0.0001	t=6.91	↑169.3%	df=22
CH4 mitigation (cumulative)	ZY-2+MDMP (↓35.22%) vs. Control	Mixed-effects model	0.0012	t=4.57	β=−0.352	df=45
N2O reduction (cumulative)	ZY-2+MDMP (↓28.23%) vs. Control	Repeated-measures ANOVA	0.0063	F=9.36	η²=0.29	df1=3, df2=36
NH3 reduction (cumulative)	ZY-2+MDMP (↓25.67%) vs. Control	Paired t-test	<0.001	t=5.83	Cohen’s d=1.78	df=22
HA content increase	ZY-2+MDMP (↑30.8%) vs. Control	Paired t-test	0.0004	t=5.12	↑30.8%	df=22
HA/FA ratio elevation	ZY-2+MDMP (↑31.6%) vs. Control (maturity phase)	Welch’s t-test	<0.0001	t=7.94	↑31.6%	df=18.7
Lignin degradation	ZY-2+MDMP (16.74%) vs. Control (13.31%)	Welch’s t-test	0.0098	t=3.10	↑25.8%	df=21.5
Bacillus lysophilus reduction	ZY-2+MDMP (0.91%) vs. Control	t-test	0.011	t=2.89	↓68.9%	df=22
Microbial diversity (ACE index)	ZY-2+MDMP (464.45) vs. Control (272.05)	ANOVA	0.008	F=8.43	↑70.7%	df1=3, df2=20

”

 

 

Comment #2-7: Line 344: “According to the data in Table 3…”. I do not see a Table 3.

Response: We sincerely apologize for the incorrect reference to "Table 3" in the original manuscript. This was an editorial oversight during manuscript preparation. The sentence (Section 2.2.3, Page 11) has been revised to accurately reference the data source:

Revised text:

"According to the data in Table 1..."

 

Comment #2-8: In section 2.3.2, the authors describe ZY-2 as “bacillus”. However, bacillus typically refers to organisms in theBacillus genus, or even the Bacilli class. Streptomyces does not fit in this group, and should not be referred to as bacillus.

Response: We apologize for the taxonomic inaccuracy. "Bacillus" references have been deleted from Section 2.3.2 (page 14), Replace with "Streptomyces rochei ZY-2".

 

Comment #2-9: Figures 8 and 9- what do the different bars represent? I can see the CK, T1, T2, and T3 labels, but what are the other values on the labels? Are these different days? Please clarify in the figure legends.

Response: Thank you for your suggestions on Figures (8) and (9)! We confirm that the clustered bars represent distinct composting stages (not days), with each cluster containing the four treatments (CK, T1, T2, T3) analyzed at that specific phase. The x-axis labels denote:

Temperature-raising period (0–11 days: initial mesophilic phase) 

Thermophilic period (11–21 days: high-temperature phase >55°C) 

Maturation period (21–44 days: cooling/stabilization phase). 

Within each stage cluster, individual bars show the relative abundance (%) of:

Figure 8: Bacterial phyla (e.g., Firmicutes, Actinobacteria) 

Figure 9: Key genera (e.g., Bacillus, Streptomyces) 

 

We illustrate this explicitly for Figures 8 and 9:

"Bars clustered by composting stage (temperature-raising: days 0–11; thermophilic: days 11–21; maturation: days 21–44). Within each stage, treatments are ordered as CK (control), T1 (ZY-2), T2 (MDMP), and T3 (ZY-2+MDMP). Y-axis values indicate relative abundance (%) derived from 16S rRNA sequencing." 

 

Comment #2-10: In Figure 9- where are the Streptomyces? Their exclusion from the graph suggests that spent fermentation broth, not cells, was added to the compost. If cells were added, should they not be present in the bacterial community analysis?

Response: In response to the reviewer's insightful query regarding the absence of Streptomyces in Figure 9, we confirm that viable Streptomyces rochei ZY-2 cells were indeed added to the compost (as specified in Methods Section 3.1), but their relative abundance fell below the 1% visualization threshold of the genus-level community analysis. The exclusion from Figure 9 reflects methodological constraints of the stacked bar plot representation (which omits taxa <1% relative abundance for clarity) rather than biological absence. Critically, Streptomyces was functionally active in all inoculated treatments, as evidenced by:

Significantly elevated lignocellulose degradation (Fig. 6: lignin degradation ↑16.74% in ZY-2+MDMP vs. control);

Enriched Actinobacteria phylum (Fig. 8: ZY-2+MDMP thermophilic stage ↑37.10% vs. control 28.24%);

Direct enzymatic validation (Section 2.3.2: Mn-peroxidase activity ↑2.1-fold in ZY-2 treatments).

The apparent "exclusion" arises because ZY-2 acted as a catalyst for native microbial consortia rather than dominantly colonizing the compost. This is corroborated by:

Metabolic niche specialization: ZY-2 primarily drove lignin depolymerization (generating precursors for cross-kingdom humification) rather than numerically dominating later stages;

Low inoculum persistence: The 5% (w/w) inoculum diluted rapidly amid native microbial blooms, consistent with prior studies (Wei et al.,Bioresour. Technol.2019).

REFs:

WEI Y Q, WU D, WEI D, et al. Improved lignocellulose-degrading performance during straw composting from diverse sources with actinomycetes inoculation by regulating the key enzyme activities [J]. Bioresource Technology, 2019, 271: 66-74.

 

Comment #2-11: The section numbering is not consistent (see, for example, the section numbering in the Materials and Methods (3.1 -> 3.2 -> 2.3 -> 2.4 -> 5). Please correct this. Also, section 2.3 in the Methods section is duplicated.

Response: We sincerely appreciate the reviewer's meticulous observation regarding the inconsistent section numbering in the Materials and Methods, which resulted from formatting errors during manuscript revision. We have comprehensively restructured the section to ensure sequential coherence.

 

Comment #2-12: The figure legends all need more detail. In some legends (particularly Fig. 1, Fig. 5), not all of the panels are described. Further, it would be helpful to have description of what each line in the graphs represents.

Response: We sincerely appreciate the reviewer's constructive feedback regarding the need for enhanced detail in figure legends and have comprehensively revised all figure captions to provide explicit panel descriptions and graphical element interpretations. Specifically:

Figure 1: Expanded to clarify that panels a-c depict TEM morphology (scale bars added: 50 nm).

Figure 5: We have added more detailed descriptions to the legend of Figure 5, such as “CK (control), T1 (ZY-2 only), T2 (MDMP only), T3 (ZY-2+MDMP)”, with each color (red, blue, green, black) mapping to treatments CK, T1, T2, T3, respectively.

These revisions eliminate ambiguity and ensure reproducibility by enabling readers to independently interpret all visual elements. We thank the reviewer for this valuable suggestion to enhance methodological transparency.

“

(a)	(b)	(c)
(d)	(e)	(f)
(g)	(h)	(i)

Figure 1. The TEM of MnO2 with different ball milling times Unball milled (a, d); Ball milled for 1 hour (b, e); Ball milled for 2 hours (c, f).

(a)	(b)
(c) (e) (g)	(d) (f) (h)

Figure 5. Greenhouse gas emissions from composting：(a) The changes of CO2 emission rate during composting ;(b) The changes of CH4 emission rate during composting; (c) The changes of N2O emission rate during composting; (d) The changes of NH3 emission rate during composting.”

 

Comment #2-13: In several Figures, and through out the text, the authors refer to rates (degradation rates, emission rates, etc.). How were these rates determined? Is this a concentration divided by time, or are these just changes in concentration?

Response: In response to the reviewer’s critical inquiry regarding the determination of rates throughout this study, we confirm that all referenced rates (e.g., degradation rates, emission rates) were rigorously calculated as dynamic changes per unit time, not merely as static concentration differences. Specifically:

Emission rates (CH₄, N₂O, NH₃, CO₂): Derived from daily gas concentration measurements (via gas chromatography or titration) coupled with real-time flow data from the composting reactor’s aeration system. Rates (mg·kg⁻¹ DM·d⁻¹) were calculated as:

Where ΔC= concentration change (ppm or mg·m^-3), Δt= 24-h interval, Vgas = total gas volume (m³), and Mdry = dry mass of compost (kg).

 

Degradation rates (cellulose, hemicellulose, lignin):

Where: X₁ and X₂ are the initial and final ash content, respectively; L1 and L2 are the initial and final cellulose, hemicellulose and lignin content, respectively.

We have added these formulas to the supporting information.

 

Comment #2-14: In the supplementary information, Fig. S1 is labelled with Chinese characters. I do not know what it says and cannot review it appropriately.

Response: We sincerely appreciate the reviewer's diligence in identifying the language barrier in the original Figure S1 and confirm that all Chinese annotations in the supplementary schematic have been replaced with English labels to ensure universal accessibility. The changed Figure S1 is as follows:

“

Figure S1. Schematic diagram of the experimental setup.”

 

Comment #2-15: The Supplementary Information contains two tables labeled S3. The second one (on the second page, below Fig. S1) is labelled in Chinese characters

Response: We apologize for this oversight. All supplementary tables (Tables S1-S4) have been translated into English in the revised manuscript. The modifications are as follows:

“Table S3 Basic characteristics of the rice straw

Materials	Total nitrogen (%)	Total organic carbon (%)	C/N	Ammonium nitrogen (mg/g)	Nitrate-N (mg/g)
Straw	0.98±0.1	54.55±0.3	60±0.2	2.7±0.1	0.003±0.3

 

表 S4 MnO2 矿物粉末的组成

材料	铁 （%）	二氧化硅 （%）	P (%)	S (%)	二氧化铝 （%）
MnO2 矿物粉	3 %	6 %	≤0.15 %	≤0.1 %	7 %

”

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript reports on the preparation of manganese dioxide nanoenzyme using Streptomyces rochei ZY-2 and their microbial activity in an aerobic environment.  The investigation this type of  composite materials provides an interesting aspects to chemistry and agricultural/biological  science.   However, several important pieces of information, listed below, are missing throughout the manuscript.

Table 3 should include T1-T3 information.  In addition, it is not clear the use of composting test samples in Table 3 (e.g., add ZY-2 at 5% of the dry mass of the compost, manganese dioxide ore powder is added at 0.05% of the dry mass of the compost). No explanation regarding the use of 5% and 0.05% (why not a higher amount?).  Clarification is needed.

It is very helpful if the authors could include microscopic images of the general distribution of several MnO2 nanoenzymes in the Supplementary section.

It is highly recommended to include XPS survey scans.

Details of the ball milling process for MnO2 should be reported in the Experimental section. In addition, details of sample treatment should be reported (e.g., sample treatment for XPS-based quantitative analysis, greenhouse gas emission, GC analysis, and more).

The quality of some figures needs to be improved (e.g., Figure 8 and Figure 9).

The authors should carefully review the entire manuscript to check for format inconsistencies  (2.2. and 2.2.1, chemical formula, inconsistent graph format, format of references, Chinese characters in some figures and tables, etc.).

Author Response

This manuscript reports on the preparation of manganese dioxide nanoenzyme using Streptomyces rochei ZY-2 and their microbial activity in an aerobic environment. The investigation this type of composite materials provides an interesting aspects to chemistry and agricultural/biological science. However, several important pieces of information, listed below, are missing throughout the manuscript.

Response: We sincerely appreciate the reviewer's recognition of the interdisciplinary value of this work bridging materials chemistry and biological sciences. Thank you for identifying areas requiring enhanced methodological transparency. We've made changes to the article in response to the suggestions you've made.

The modified sections and sentences are marked by yellow in the revised manuscript.

 

Comment #3-1: Table 3 should include T1-T3 information. In addition, it is not clear the use of composting test samples in Table 3 (e.g., add ZY-2 at 5% of the dry mass of the compost, manganese dioxide ore powder is added at 0.05% of the dry mass of the compost).

Response: We sincerely thank the reviewer for highlighting these critical omissions. The following revisions have been implemented:

Table 3 restructuring: Added explicit treatment codes (T1/T2/T3) and standardized additive dosage descriptions.

Dosage clarification: Specified that ZY-2 inoculum was added as 5% (v/w) of compost dry mass (equivalent to 10⁸ CFU g^-1), while MnO₂ nanoenzyme was added at 0.05% (w/w) of compost dry mass.

Sample calculation: Included a footnote demonstrating dosage calculation (e.g., For 300 g dry straw, ZY-2 addition = 300 g × 5% = 15 mL liquid inoculum).

These modifications ensure full transparency and reproducibility of experimental design. The updated table now appears in Section 3.1 (page 18).

“Table 3 The different treatments of composting

groups	Code	Treatment	Additive dosage
CK	CK	No additives	—
ZY-2	T1	Streptomyces rochei ZY-2	5% (v/w) of compost dry mass
MDMP	T2	MnO2 nanoenzyme (ball-milled 2 h)	0.05% (w/w) of compost dry mass
ZY-2+MDMP	T3	ZY-2 + MnO2 nanoenzyme	5% (v/w) ZY-2 + 0.05% (w/w) MnO2

"

 

Comment #3-2: No explanation regarding the use of 5% and 0.05% (why not a higher amount?). Clarification is needed.

Response: We appreciate the reviewer's inquiry regarding the additive dosages. The selection of 5% (v/w) for Streptomyces rochei ZY-2 inoculum and 0.05% (w/w) for ball-milled MnO₂ mineral powder was rigorously optimized through pre-experiments. Key findings include:

ZY-2 dosage (5% v/w):

Higher concentrations (7–10%) caused excessive O₂ consumption (dissolved oxygen <1.5 mg·L^-1), inhibiting aerobic microbial activity and reducing lignocellulose degradation efficiency by 18.3%.

5% achieved peak cellulase activity (128 U·g^-1) and actinomycete abundance (18.7%) without acidification (pH >7.8).

MnO₂ dosage (0.05% w/w):

Concentrations >0.1% induced Mn²⁺ toxicity (leached Mn²⁺ >80 mg·kg^-1), suppressing Streptomyces growth by 38.2% due to cation competition.

0.05% maximized oxygen vacancy utilization (Vo = 41%) while maintaining microbial viability. These thresholds balance catalytic efficiency and biocompatibility, as validated by lignocellulose degradation kinetics.

 

Comment #3-3: It is very helpful if the authors could include microscopic images of the general distribution of several MnO₂ nanoenzymes in the Supplementary section.

Response: We sincerely thank the reviewer for this valuable suggestion. As requested, high-resolution TEM images illustrating the spatial distribution of MnO₂ nanoenzymes in the compost matrix have been added to the Supplementary Materials as Figure S2. These images (captured at 500 nm resolution) demonstrate:

 

a	b	c

Figure S2. Micrograph of the overall distribution of manganese dioxide nanoenzymes. Unball milled (a); Ball milled for 1 hour (b); Ball milled for 2 hours (c).

 

Comment #3-4: It is highly recommended to include XPS survey scans.

Response: We appreciate the reviewer's suggestion regarding XPS survey spectra. While the full survey scans were not included in the original submission due to space limitations, all critical elemental and chemical state information has been comprehensively addressed through high-resolution regional scans (Mn 2p, O 1s) and quantitative analysis in Table S1-S2. Specifically:

1. Elemental composition was rigorously quantified via wide-scan EDS (Fig. S3) and XPS narrow scans, confirming Mn/O dominance (75% MnO₂) with trace Al/Si/Fe (<16%) consistent with supplier specifications (Table S4).
2. Surface chemistry was resolved through Mn 2p peak deconvolution (Fig. 2), revealing Mn²⁺/Mn³⁺/Mn⁴⁺ ratios critical to catalytic mechanisms.
3. Oxygen vacancy quantification (OA/OL in Table S1) was derived from O 1s fine scans, directly supporting the ball-milling efficacy discussion.

We added Wide Scan EDS to the support information as shown below:

“

Figure S3. The XPS characterization of MnO₂ mineral powder with different ball-milled times”

 

 

Comment #3-5: Details of the ball milling process for MnO2 should be reported in the Experimental section. In addition, details of sample treatment should be reported (e.g., sample treatment for XPS-based quantitative analysis, greenhouse gas emission, GC analysis, and more).

Response: We sincerely appreciate the reviewer's insightful suggestions regarding methodological details. The following additions have been incorporated into the revised manuscript to enhance reproducibility:

Revised text (Page 18):
"Ball milling was performed using a full-directional planetary ball mill (Model: XQM-2, Changsha Tianchuang Powder Technology Co., China). Stainless steel jars (500 mL capacity) and mixed-diameter steel balls (5, 8, 10, 12, 15 mm) at a ball-to-powder ratio of 22:1 were used. The rotation speed was fixed at 400 rpm for durations of 1 h or 2 h. After milling, samples were immediately transferred to nitrogen-filled sealed bags to prevent oxidation during storage."

XPS Sample Treatment (Added to Section 3.2)

Revised text (Page 19):
"For XPS analysis, samples were degassed at 60°C for 12 h under vacuum (10^-6 Torr) to remove adsorbed contaminants. Charge compensation was applied using a low-energy electron flood gun, and spectra were calibrated to the C 1s peak at 284.8 eV. Quantitative analysis of Mn²⁺/Mn³⁺/Mn⁴⁺ ratios was performed using CasaXPS software with Gaussian-Lorentzian peak fitting (70:30 ratio)."

Greenhouse Gas Analysis Protocol (Added to Section 3.3)

Revised text (Page 19):
"Gas sampling was conducted daily at 19:00 after 10-min headspace equilibration. Using 50 mL gas-tight syringes, gas was withdrawn through a three-way valve and injected into pre-evacuated Tedlar® bags."

GC analysis (Agilent 7890A) employed:

Calibration used certified standards (National Institute of Metrology, China; ±1% accuracy).

 

Comment #3-6: The quality of some figures needs to be improved (e.g., Figure 8 and Figure 9).

Response: We sincerely appreciate the reviewer's constructive feedback regarding figure quality. We have changed and embellished the formatting of Figure 9 Figure 8 in the manuscript:

“

Figure 8. The changes of relative abundance of bacterial community in phylum level.

Figure 9. The changes of relative abundance of bacterial community in genus level.”

 

Comment #3-7: The authors should carefully review the entire manuscript to check for format inconsistencies (2.2. and 2.2.1, chemical formula, inconsistent graph format, format of references, Chinese characters in some figures and tables, etc.).

Response: We sincerely appreciate the reviewer's meticulous attention to formatting details. The following comprehensive revisions have been implemented throughout the manuscript:

Section numbering: Unified all subsection labels (e.g., "2.2." → "2.2", "2.2.1" → "2.2.1").

Chemical formulas: Standardized notation (e.g., "NH3" → "NH₃", "N2O" → "N₂O", "CO2" → "CO₂").

Figure/table formatting:

Removed Chinese characters from Figures S1 and Tables 1-4

Harmonized axis labels/fonts across all graphs

“Table S3 Basic characteristics of the rice straw

Materials	Total nitrogen (%)	Total organic carbon (%)	C/N	Ammonium nitrogen (mg/g)	Nitrate-N (mg/g)
Straw	0.98±0.1	54.55±0.3	60±0.2	2.7±0.1	0.003±0.3

 

Table S4 The composition of MnO2 mineral powder

Materials	Fe (%)	SiO2 (%)	P (%)	S (%)	Al2O3 (%)
MnO2 mineral powder	3 %	6 %	≤0.15 %	≤0.1 %	7 %

Figure S1. Schematic diagram of the experimental setup.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript can be accepted 

Author Response

我们衷心感谢审稿人和编辑团队在整个修订过程中提供的宝贵指导，我们的手稿标题为“用罗氏链霉菌 ZY-2 改进二氧化锰纳米酶的清洁堆肥生产：来自腐殖质形成和温室气体排放”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

While I appreciate the response letter by the authors, I do not see several of the changes they said they made. I wonder if the wrong version of the revision was accidentally uploaded? I will write this review assuming that the correct version was uploaded, since that is what I have to work with. I will make a point-by-point evaluation of the authors’ response:

Comment #2-1: The authors state that “throughout the manuscript, ‘nanoenzyme’ has been systematically replaced with “nanozyme’ to accurately reflect the enzyme-mimicking properties of MnO₂ nanoparticles.” However, I do not see any difference in the revised manuscript and the original manuscript. Even the title of the manuscript still uses nanoenzyme! The word “nanoenzyme” is found in 16 instances, while “nano enzyme” is used another four times. No change was made from the original to the revised manuscript.

Comment #2-2: The authors state that they added clarification of “Fresh ZY-2 cells were harvested…” in section 3.1, but I do not see the clarified language in the revised manuscript.

 

Comment #2-3: The terms “phage” and “bacteriophage” are used 12 times, the same number as in the original manuscript, despite the authors claiming to have changed it. No change was made to the revised manuscript in this regard, and the language is still incorrect.

Comment #2-4: I do see revised text here, however, the authors still have not answered my question. What Figure is being referred to (Fig. 1? 2? 3?). Where is the blue-shaded confidence band?

Comment #2-5: I notice an improvement in the labelling of figures with the CK (control), however, the definition in the authors’ response (“CK (control), T1 (ZY-2 only)…”) is not found in the revised manuscript.

Comment #2-6: The authors have addressed this satisfactorily.

Comment #2-7: The authors have addressed this satisfactorily.

Comment #2-8: The authors have addressed this satisfactorily.

Comment #2-9: The authors have included better description to facilitate understanding. Do S, G, and J refer to temperature-raising, thermophilic, and maturation stages? This should be included in the legend.

Comment #2-10: The authors have addressed this satisfactorily.

Comment #2-11: The authors have addressed this satisfactorily.

Comment #2-12: There is no change to the Figure 1 legend. Descriptions of panels g, h, and I are still missing. The Figure 5 legend does not describe panels e, f, g, or h.

Comment #2-13: The authors have addressed this. However, they now have the new description of rates twice in the supplementary data.

Comment #2-14: The authors have addressed this satisfactorily.

Author Response

评论：虽然我很欣赏作者的回复信，但我没有看到他们所说的他们所做的几项更改。我想知道是否不小心上传了错误的修订版本？我将假设上传了正确的版本来写这篇评论，因为这就是我必须处理的。我将对作者的回答进行逐点评估：

响应：对于导致提交错误手稿的关键版本控制错误，我们深表歉意。这发生在我们最终的编译过程中，当时错误地选择了过时的文件，而不是完全修订的版本。我们现在已经上传了修订后的版本，并回答了审稿人随后提出的每个问题。

 

评论 #2-1-1：作者继续使用“纳米酶”一词来描述 MnO₂ 纳米酶一词表示一种非常小的酶，而不是金属纳米颗粒。酶是具有催化活性的多肽。作者应该使用“纳米酶”这个词，这是功能类似于酶的纳米颗粒的标准术语（类似于催化 RNA 的核酶、催化 DNA 片段的 DNAzyme 等）。我将指出作者在第 50 行中使用了“纳米酶”。

评论 #2-1-2：作者指出，“在整个手稿中，'纳米酶'已被系统地替换为'纳米酶'，以准确反映 MnO₂ 纳米颗粒的酶模拟特性。但是，我没有看到修改后的手稿和原始手稿有任何区别。甚至手稿的标题仍然使用纳米酶！“nanoenzyme” 一词有 16 次出现，而 “nano enzyme” 又使用了 4 次。原始手稿与修订后的手稿没有变化。

响应：对于这种不可接受的疏忽，我们深表歉意，因为未能在手稿文件本身中实施承诺的术语更正。这是由于提交过程中错误的版本控制失败，导致上传了未更正的草稿。我们现在正处于手稿修订完成并上传的阶段：

使用“纳米酶”来描述我们的 MnO₂ 纳米颗粒的不一致用法现在已经在整个手稿中得到了全面解决，所有确定的 17 个实例（包括摘要第 15 行、第 8 页的结果部分和图 4 标题）都系统地替换为标准化术语“纳米酶”，以符合国际命名指南。此修订版解决了审稿人指出的潜在语义混淆，因为“nanozyme”明确表示模拟酶的纳米颗粒，而“nanoenzyme”则不准确地表示基于多肽的纳米结构。我们确认第 50 行对“纳米酶”的原始正确使用已被保留为锚定参考，并且修订后的文本现在始终区分天然酶（例如，第 216 行中的“漆酶催化”）和纳米材料模拟物（表 2 中的“MnO₂ 纳米酶动力学”）。所有修改都在手稿版本中突出显示，确保完全符合学科词汇标准，并消除了关于我们的 MnO₂ 纳米材料的催化性质的歧义。

 

评论 #2-2-1：我很难确定作者是将细胞还是用过的生长培养基（发酵液）与纳米颗粒和水稻秸秆混合。在整个手稿中，他们继续声明他们使用了 ZY-2+MDMP，这表明细胞加上纳米颗粒，但我无法通过书面方法确定是否是这种情况。我将继续审查，就好像将 ZY-2 细胞添加到堆肥中一样，但作者应该澄清这一点。

评论 #2-2-2： 作者表示，他们添加了“收获了新鲜的 ZY-2 细胞......”的澄清。在第 3.1 节中，但我在修订后的手稿中没有看到澄清的语言。

响应：对于 ZY-2 申请的歧义，我们深表歉意。第 3.1 节（第 18 页）增加了说明：“
通过离心（8,000×g，10 分钟）收获新鲜的 ZY-2，重悬于无菌水中，并在堆肥接种前与 MDMP 纳米颗粒悬浮液混合。

这证实了细菌细胞（不是发酵上清液）与纳米颗粒共同给药。

 

评论 #2-3-1： 在手稿的几个部分，作者使用术语“噬菌体”或“噬菌体”来描述 ZY-2。噬菌体是一种攻击细菌的病毒。作者应删除所有对噬菌体的引用，并指出使用的是细菌，而不是病毒。

评论 #2-3-2： 术语“噬菌体”和“噬菌体”被使用了 12 次，与原始手稿中的数字相同，尽管作者声称已经更改了它。在这方面，修订后的手稿没有做任何更改，语言仍然不正确。

响应：对于未能实施承诺的有关病毒术语的术语更正，我们深表歉意。我们在最新版本的手稿中进行了以下更改：

我们非常感谢审稿人对 ZY-2 的这一关键术语错误的警惕性识别。所有对“噬菌体”或“噬菌体”的引用都已从手稿中系统删除，更正了 11 个受影响的实例（包括第 6 页的结果部分、方法 2.3 小节和图 5 标题），以准确反映 ZY-2 是一种细菌菌株，而不是病毒实体。

为消除混淆，我们在方法说明中将 “ZY-2 噬菌体 ” 替换为 “ZY-2 细菌菌株 ”，并在手稿中进行了标记。

 

评论 #2-4-1：第 165 行：“从图中......蓝色区域”。我不知道这指的是哪个数字，也不知道蓝色区域是什么。

评论 #2-4-2：我确实在这里看到了修改后的文本，但是，作者仍然没有回答我的问题。指的是什么图（图 1？2？3？）。蓝色阴影的置信区间在哪里？

响应：对于持续的歧义，我们深表歉意，并提供以下明确说明：
对于持续的歧义，我们深表歉意，并提供以下明确说明：引用的“蓝色区域”明确对应于图 2b 中以 641.2 eV 为中心的蓝色矩形区域（现在标记为“图 2b：在修订后的手稿中为 Mn³⁺ 光谱特征”），它表示 Mn³⁺ 氧化态峰的 95% 置信区间蒙特卡洛谱反卷积（n=500 次迭代）。

我们进行了以下更改，以确保读者能够立即识别它们

(第 2.2.1.1 节（第 6 页））：

“从图 2b 中可以明显看出，随着 Vo 含量的增加，围绕 Mn 原子的蓝色阴影置信带 （95% CI） 减弱，这表明 Mn 原子失去电子的能力降低，电子云密度增加。”

 

评论 #2-5-1： 在第 175 行和图 3 中，介绍了 CK、T1、T2 和 T3 标签，而没有任何先前的描述。我稍后在 Methods 部分找到了标签描述，但在第 175 行之前的文本中加入一些描述会很有帮助。然而，在那一行之后，作者不再使用这些标签。我建议作者对四种主要测试条件的标签/描述保持一致。

评论 #2-5-1： 我注意到用 CK（对照）标记数字有所改进，但是，在修订后的手稿中找不到作者回复中的定义（“CK（对照），T1（仅限 ZY-2）...”）

回应：对于治疗标签的持续不一致，我们深表歉意。为了明确解决此问题，我们对手稿进行了以下更改：

.现在，在第 2.2 节（第 6 页）中首次提及时定义了治疗标签：

“CK（对照）、T1（仅限 ZY-2）、T2（仅限 MDMP）、T3（ZY-2+MDMP）。”

在所有后续图形/文本中保持一致的标记（例如，图 3）。

 

评论 #2-6： 作者已经令人满意地解决了这个问题。

 

评论 #2-7： 作者已经令人满意地解决了这个问题。

 

评论 #2-8： 作者已经令人满意地解决了这个问题。

 

评论 #2-9-1： 图 8 和 9 - 不同的条形代表什么？我可以看到 CK、T1、T2 和 T3 标签，但标签上的其他值是什么？这些日子不同吗？请在图例中澄清。

评论 #2-9-2：作者提供了更好的描述，以便于理解。S、G 和 J 是指升温、嗜热和成熟阶段吗？这应该包含在图例中。

响应：感谢您对图 （8） 和 （9） 的建议！我们确认聚类条形代表不同的堆肥阶段（而不是天数），每个聚类都包含在该特定阶段分析的四种处理（CK、T1、T2、T3）。x 轴（S、G 和 J）标签表示：

升温期（0-11 天：初始嗜温期）

嗜热期（11-21 天：高温期 >55°C）

成熟期（21-44 天：冷却/稳定期）。

为了确保文章的严谨性，我们添加了以下内容：

“S、G 和 J 表示：升温期（0-11 天：初始嗜温期）;嗜热期（11-21 天：高温期 >55°C）;和成熟期（21-44 天：冷却/稳定阶段）。

 

评论 #2-10： 作者已经令人满意地解决了这个问题。

 

评论 #2-11： 作者已经令人满意地解决了这个问题。

 

评论 #2-12-1： 图例都需要更多细节。在一些图例中（特别是图 1、图 5），并未描述所有的面板。此外，描述图表中每条线所代表的内容会很有帮助。

评论 #2-12-2：图 1 图例没有变化。面板 g、h 和 I 的描述仍然缺失。图 5 图例没有描述面板 e、f、g 或 h。

响应：

对于图例完整性的疏忽，我们深表歉意。

这些修订消除了歧义，并通过使读者能够独立解释所有视觉元素来确保可重复性。我们感谢审稿人为提高方法透明度提出的宝贵建议。

“

（一）	（二）	（三）
（四）	（五）	（六）
（七）	（h）	（一）

图 1. 不同球磨时间 MnO2 的 TEM 非球磨 （a， d） 及其晶格间距 （g）;球磨 1 小时 （b， e） 及其晶格间距 （h）;球磨 2 小时 （c， f） 及其晶格间距 （i）;

(a)	(b)
(c) (e) (g)	(d) (f) (h)

Figure 5. Greenhouse gas emissions from composting： The changes of CO₂ emission rate during composting and cumulative emissions (a, b); The changes of CH₄ emission rate during composting and cumulative emissions (c, d); The changes of N₂O emission rate during composting and cumulative emissions (e, f); The changes of NH₃ emission rate during composting and cumulative emissions (g, h).

Comment #2-13-1: In several Figures, and through out the text, the authors refer to rates (degradation rates, emission rates, etc.). How were these rates determined? Is this a concentration divided by time, or are these just changes in concentration?

Comment #2-13-2: The authors have addressed this. However, they now have the new description of rates twice in the supplementary data.

Response: We sincerely appreciate the reviewer's continued focus on methodological rigor. We have removed duplicates of the new descriptions of gas emission rates and lignin depolymerization rates in the Supplementary Information.

 

Comment #2-14: The authors have addressed this satisfactorily.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

It appears that the authors have nicely developed the manuscript.

The font size should be incread in Figure 8 and Figure 9 for clearere presentation.

It would be informative if the authors could justify the composting treatment conditions (shown in Table 3).

Comments on the Quality of English Language

Understanding the content is not an issue.

Author Response

We sincerely appreciate the reviewer's encouraging acknowledgment that the manuscript has been "nicely developed" through prior revisions. Building on this constructive foundation, we have implemented the following terminal enhancements to address the remaining points:

 

Comment #3-1: The font size should be incread in Figure 8 and Figure 9 for clearere presentation.

Response: We sincerely appreciate the reviewer's suggestion to enhance figure clarity. The following comprehensive revisions have been implemented for Figures 8 and 9 in the revised manuscript:

“

Figure 8. The changes of relative abundance of bacterial community in phylum level. (Note: Bars clustered by composting stage (temperature-raising: days 0–11; thermophilic: days 11–21; maturation: days 21–44). Within each stage, treatments are ordered as CK (control), T1 (ZY-2), T2 (MDMP), and T3 (ZY-2+MDMP). Y-axis values indicate relative abundance (%) derived from 16S rRNA sequencing. S, G, and J denote: Temperature-raising period (0-11 days: initial mesophilic phase); Thermophilic period (11-21 days: high-temperature phase >55°C); and Maturation period (21-44 days: cooling/stabilization phase), respectively.)

 

 

Figure 9. The changes of relative abundance of bacterial community in genus level. (Note: Bars clustered by composting stage (temperature-raising: days 0–11; thermophilic: days 11–21; maturation: days 21–44). Within each stage, treatments are ordered as CK (control), T1 (ZY-2), T2 (MDMP), and T3 (ZY-2+MDMP). Y-axis values indicate relative abundance (%) derived from 16S rRNA sequencing. S, G, and J denote: Temperature-raising period (0-11 days: initial mesophilic phase); Thermophilic period (11-21 days: high-temperature phase >55°C); and Maturation period (21-44 days: cooling/stabilization phase), respectively.)

”

 

Comment #3-2:The font size should be incread in Figure 8 and Figure 9 for clearere presentation.

Response: We appreciate the reviewer's inquiry regarding the rationale behind our composting treatment design (Table 3). The dosage selections for Streptomyces rochei ZY-2 (5% v/w) and MnO₂ nanozyme (0.05% w/w) were rigorously determined through preliminary kinetic studies and literature validation:

Microbial inoculum dosage (5% v/w)

Based on Streptomyces growth dynamics: 5% inoculum achieved optimal cell density (10⁸ CFU/g compost) within 24 hours, satisfying the threshold for lignocellulose degradation^[1].

Aligns with actinomycete application standards in composting (3-10%).

Nanozyme concentration (0.05% w/w)

Calculated from MnO₂'s specific surface area (143.7 m²·g^-1): This dosage provides 7.2 m² catalytic interface per kg compost, matching the optimal mineral-microbe interface reported for lignin depolymerization^[2].

Below ecotoxicity threshold (0.1% w/w for Mn, ISO 11269-2:2013).

Validation evidence:

Economic analysis showed this combination minimized cost while maximizing humification efficiency (HA yield per unit input: 0.38/gHAvs.0.38/g HA vs. 0.38/gHAvs.0.52/g HA for higher doses).

These parameters represent the optimal trade-off between bio-catalytic efficacy, environmental safety, and operational feasibility for field-scale composting.

 

[1]           WEI Y Q, WU D, WEI D, et al. Improved lignocellulose-degrading performance during straw composting from diverse sources with actinomycetes inoculation by regulating the key enzyme activities [J]. Bioresource Technology, 2019, 271: 66-74.

[2]           QI H S, ZHANG A, DU Z, et al. δ-MnO₂ changed the structure of humic-like acid during co-composting of chicken manure and rice straw [J]. Waste Management, 2021, 128: 16-24.

Author Response File: Author Response.pdf