Responses of Soil Microbial Communities to Biogas Slurry Irrigation in Paddy Fields: Interactions with Environmental Factors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review Remarks on: Responses of soil microbial communities in a paddy soil irrigated with biogas slurry (Water-3506090). The manuscript presents significant contributions to biogas slurry in paddy soils to enhance soil fertility and microbial functionality. However, revisions are necessary to improve methodological section, figure quality and the relevance of the findings.

 

Remarks:

• The title of the manuscript is not representing whole indices considered in the experiment, the interaction of biogas slurry with heavy metals and environmental factors a paddy soil are also performed.
• Quantitative results must be added in abstract in comparative; what changes Redundancy analysis indicated, must be added in numerical values.
• Keywords are repeated with titles, must be reduced with unique keywords.
• The introduction needs careful revision. Superfluous information is added, many facts are not supported by key findings. The introduction part lists a lot of existing studies but does not make clear overview connected with objective present work. Second, the contribution of the whole research is not clear, and it is scattered in the last paragraph. In the last paragraph, although the author mentions the goal of this study and slightly mentions the shortcomings of the existing research, some arguments in it are not convincing. This section should further stress on strengthening the relationship between BS application and microbial community. Incorporating citations to recent studies is highly recommended.
• Could the author clarify storing samples at -80 °C for microbial community analysis? Isn’t it affect microbial functionality and density?
• I wonder author analyzed total concentrations of various heavy metals and metalloids, however, did not well discussed its relevance in results and discussion.
• In discussion, author similar with introduction have given just superfluous sentences the citations; however, did not compared the current results key findings of those papers with cited.
• Methodology needs more clarity in term of the selection of 0-3 years of BS irrigation. Are these durations based on regional management practices or previous studies? It is not clear from the current explanation, how the “chessboard layout method" for soil sampling has been utilized? Elaborate on the parameters used in redundancy analysis (RDA). It is also not clear how the environmental factors are selected. Explain the U-shaped recovery of fungal diversity after 3 years. Is this due to microbial adaptation, changes in soil redox conditions, or shifts in nutrient availability?
• Define technical terms like redundancy analysis and alpha diversity indices briefly upon first mention. Improve figure resolution and label readability (axis labels in Figures 1–4 is too small, colors in Figure 3 heatmap could be more distinct).
• If the authors used only dose of BS, i.e., 450 tons per hectare? What is the base using this dose and is this applied each year? The clarity is required details on the level of moisture throughout the year after paddy harvesting, how the field moisture was maintained for consequently 3 years.

The manuscript presents timely and impactful research on microbial responses to biogas slurry in paddy soils. With revisions to enhance methodological transparency, figure quality, and practical relevance, this study will make a valuable contribution to Water. Minor revisions are recommended.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript (water-3506090) presents valuable research on the impacts of biogas slurry irrigation on soil microbial communities in paddy fields. The experimental design and analytical methods employed are comprehensive, and the findings regarding microbial community dynamics provide important insights. However, several areas require improvement before the manuscript can be accepted for publication. Specifically, the theoretical framework needs strengthening, statistical analyses must be clearly presented, detailed microbial diversity data should be provided, correlations with environmental factors need clarification, and the presentation of results should be enhanced. Addressing these points will significantly improve the manuscript's clarity and impact. Therefore, I recommend a major revision.

Specific Comments
1.  In the Abstract, please clarify the specific bacterial and fungal diversity indices used in your study when stating BS irrigation significantly reduced bacterial diversity.
2.  In Section 2.1, please provide more details about the control fields (ZG0) - were they irrigated with regular water or received other treatments?
3.  The heavy metal analysis methodology in Section 2.2 is well described, but please specify the detection limits for the ICP-MS analysis, particularly for Cd measurements.
4.  In the Results section (not visible in the provided excerpt), ensure that all tables' statistical significance indicators (lowercase letters) are consistent with the actual statistical analyses performed.
5.  In Table S1, all Cd concentration values (0.26±0.02, 0.13±0.01, 0.22±0.01, and 0.13±0.01 mg·kg⁻¹) are marked with the same lowercase letter "a", indicating no significant differences. However, these values appear visually distinct with minimal overlap. Please verify your statistical analysis and correct the significance indicators if necessary, as these concentrations likely represent statistically different groups at p < 0.05. 
To improve the theoretical framework of the manuscript, consider the following suggestions: (1) In the abstract, describe the research background, problem statement, and research objectives in detail to make it clearer. (2) explain how BS irrigation affects the research topic in the introduction, using relevant literature to support your points. Clearly state the research hypotheses and objectives. (3) In the discussion section, examine the effects of BS irrigation on microbial communities. Analyze the results in context, discuss study limitations, and suggest ideas for future research.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper is well written and clear to the reader.

However, there are minor corrections in the Abstract, Introduction, Materials and Methods and in Results and Discussion.

The specific concerns are given in the attached reviewer's report

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing the review comments and revising the manuscript. While I appreciate the improvements made, particularly in clarifying the diversity indices (shannon index) in the abstract and providing detailed control field treatments, there remain some critical issues that require attention.

My primary concern relates to the statistical analysis of the Cd concentration data presented in Table S1. The current presentation shows values of 0.26±0.02a, 0.13±0.01a, 0.22±0.01a, and 0.13±0.01a (n=3), all marked with the same significance indicator 'a'. This statistical representation appears problematic for several reasons. First, there is a two-fold difference between the highest (0.26) and lowest (0.13) values. Second, the standard deviations are relatively small (±0.01-0.02), indicating minimal overlap between groups. For instance, the range for 0.26±0.02 (0.24-0.28) shows no overlap with 0.13±0.01 (0.12-0.14). These distinct separations suggest that these groups likely represent statistically different populations at p < 0.05.

Author Response

1. Thank you for addressing the review comments and revising the manuscript. While I appreciate the improvements made, particularly in clarifying the diversity indices (shannon index) in the abstract and providing detailed control field treatments, there remain some critical issues that require attention.

My primary concern relates to the statistical analysis of the Cd concentration data presented in Table S1. The current presentation shows values of 0.26±0.02a, 0.13±0.01a, 0.22±0.01a, and 0.13±0.01a (n=3), all marked with the same significance indicator 'a'. This statistical representation appears problematic for several reasons. First, there is a two-fold difference between the highest (0.26) and lowest (0.13) values. Second, the standard deviations are relatively small (±0.01-0.02), indicating minimal overlap between groups. For instance, the range for 0.26±0.02 (0.24-0.28) shows no overlap with 0.13±0.01 (0.12-0.14). These distinct separations suggest that these groups likely represent statistically different populations at p < 0.05.

RE: Thank you for your thorough evaluation and valuable feedback regarding the statistical presentation of Cd concentrations in Table S1. We sincerely apologize for the initial oversight and have rigorously re-analyzed the data to address your concerns. Below is a detailed response to your comments:

（1）You highlighted that:“Cd concentrations (0.26±02a, 0.13±0.01a, 0.22±0.01a, 0.13±0.01a) were labeled with the same letter ‘a’, despite apparent numerical differences and non-overlapping standard deviations, suggesting potential statistical significance at p < 0.05.”We fully agree with your observation. The raw data ranges (mean ± SD) for Cd concentrations are as follows: ZG0: 0.24 - 0.28, ZG1/ZG3: 0.12 - 0.14, ZG2: 0.21 - 0.23. These ranges exhibit no overlap, which strongly supports the presence of statistically significant differences among the groups.

（2）Our initial analysis employed single-level significance labeling (p < 0.05). To more accurately reflect the robustness of the observed differences, we have now implemented a two-tiered significance labeling system: Lowercase letters (a, b, c): Indicate differences at p < 0.05, Uppercase letters (A, B, C): Indicate differences at p < 0.01.The revised Table S1 was as follows:

Table S1. Soil heavy metals in paddy fields irrigated with BS over different years (mg·kg^-1)

sample	Pb	Cd	Cr	Cu	Zn	Ni	Hg	As
ZG0	54.63 ±0.08b/B	0.26 ±0.02a/A	80.97 ±0.07b/B	38.06 ±0.13c/C	129.62 ±0.45b/AB	21.41 ±0.16b/B	0.16 ±0.01b/B	13.87 ±0.11a/A
ZG1	41.40 ±0.11c/C	0.13 ±0.01c/B	70.19 ±0.23d/D	43.89 ±0.29b/B	128.60 ±0.46c/B	23.65 ±0.22a/A	0.16 ±0.00b/B	11.16 ±0.04c/C
ZG2	64.99 ±0.12a/A	0.22 ±0.01b/A	76.00 ±0.12c/C	46.03 ±0.07a/A	130.71 ±0.56a/A	21.16 ±0.15b/B	0.22 ±0.01a/A	13.15 ±0.03b/B
ZG3	34.48 ±0.26d/D	0.13 ±0.01c/B	82.93 ±0.24a/A	24.04 ±0.18d/D	78.42 ±0.23d/C	21.33 ±0.10b/B	0.10 ±0.00c/C	9.74 ±0.01d/D

Note:Values are presented as the mean ± standard deviation (n = 3). Different lowercase letters  indicate a significant difference at p < 0.05, uppercase letters indicate differences at p < 0.01 in the same column.

Once again, thank you for your meticulous review and for contributing to the improved quality of this work.

 

Attachments:

（1）Supplementary statistical output (Tukey HSD results for heavy metal concentrations).

（2）Manuccript -v2 (Updated Table S1, with dual-letter labels).

Author Response File: Author Response.pdf