Long-Term Heavy Metal Pollution Induces Complex Differences in Farmland Topsoil and Rhizosphere Microbial Communities

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the Article Titled "Long-Term Heavy Metal Pollution Induces a Complex Difference on Topsoil and Rhizosphere Microbial Communities in Farmlands"

 

This study explores the bacterial and fungal communities in the topsoil and rhizosphere subjected to heavy metal (Cd, Cu, Pb, and Zn) pollution across four distinct types of tillage plots surrounding an abandoned 85 years old zinc smelter. The analysis delves into the intricate interplay between microorganisms, plants, and soil heavy metals, aiming to improve efforts of future soil remediation strategic approach. Abundance and diversity indices results indicate significant variations in bacteria and fungi diversity among four plots. The correlation analysis of microbial communities and heavy metals reveals that bacteria such as Pseudomonas and fungi including Chaetothyriales and Fusarium exhibit robust tolerance to heavy metal pollution. Conversely, bacteria like Vicinamibacteraceae, JG30_KF_CM45, RB41, Gaiella, MB-A2-108, 67-14, and Microvirga, along with fungi like Glomerellales, Hypocreales, Chaetomium, and Mortierella, display sensitivity to heavy metal toxicity. Utilizing a Structural Equation Model (SEM), the study identifies the inhibition of Zn and the promotion of Cd as influencing bacterial diversity, a modest inhibition of Cd on fungi diversity. The paper exhibits a well-structured and articulate presentation, with some minor suggestions to enhance its quality that are as follows:

 

-         Section “2.4. 16S and ITS rRNA analysis of topsoil and rhizosphere microbial communities” the title of this section can be rewritten in a simpler form example “2.4. microbial communities analysis of topsoil and rhizosphere”

-         Please give brief details for calculations of sobs and shannon indices and the way these indices used to reflect microbial community richness and diversity

-         In section 2.5. Statistical analysis, the authors used the kriging interpolation technique, I recommend them to mention what kind of kriging they used (ordinary kriging, simple, universal etc.)

-         There is no historical data about the base line of the studied areas regarding their heavy metals and microbial contents

-         In Figure 2 a1 and a2, please write the title of the vertical axes. Also try to keep the samples at the same order as appearing in Figure 2 b1 and b2 to make it easy for the reader to compare.

-         In page 6, the second paragraph, The authors wrote “In the CK group soil, the communitiy abundances of only bacterial genus Pseudomo-nas (3.37%)” please mention the figure you are discussing and check the correctness of the stated %. Please review the whole paragraph.

-         In Figure 3. Please show in the figure that (a) is bacteria, and (b) is fungi. The sample type is clear from the clustered tree.

 

 

 

 

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corrections highlighted changes in the re-submitted files.

Comments 1: Section “2.4. 16S and ITS rRNA analysis of topsoil and rhizosphere microbial communities” the title of this section can be rewritten in a simpler form example “2.4. microbial communities analysis of topsoil and rhizosphere” 

Response 1:We accepted that suggestion.

Comments 2:  Please give brief details for calculations of sobs and shannon indices and the way these indices used to reflect microbial community richness and diversity.

Response 2:" Sobs index were the observed richness (Operational Taxonomic Units, OTU). Shannon index, which could assess the richness and evenness of species composition in the sample, were calculated using the vegan package." These sentence were add as brief details of sobs and shannon indices.

Comments 3:  In section 2.5. Statistical analysis, the authors used the kriging interpolation technique, I recommend them to mention what kind of kriging they used (ordinary kriging, simple, universal etc.)

Response 3:We used the ordinary kriging interpolation and highlighted that in new revision.

Comments 4:  There is no historical data about the base line of the studied areas regarding their heavy metals and microbial contents.

Response 4: It's the first time research on microbial ecology in HMs polluted farmland around HZP, so no historical data can be used.

Comments 5: In Figure 2 a1 and a2, please write the title of the vertical axes. Also try to keep the samples at the same order as appearing in Figure 2 b1 and b2 to make it easy for the reader to compare. 

Response 5: The title of the vertical axes "OTU" was add in Figure 2 a1 and a2.

Comments 6: In page 6, the second paragraph, The authors wrote “In the CK group soil, the communitiy abundances of only bacterial genus Pseudomo-nas (3.37%)” please mention the figure you are discussing and check the correctness of the stated %. Please review the whole paragraph.

Response 6: "(Figure 2 (b))"was add at the end of last paragraph to mention.

Comments 7: In Figure 3. Please show in the figure that (a) is bacteria, and (b) is fungi. The sample type is clear from the clustered tree.

Response 7: We accepted that suggestion and revised that in revision.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Long-Term Heavy Metal Pollution Induces a Complex Difference on Topsoil and Rhizosphere Microbial Communities in Farmlands” is a very well written, compiled and interpreted article. It has discussed the impact of long-term heavy metal pollution on soil and rhizosphere microbial communities in farmlands using abundance and diversity index, correlation analysis of microbial communities and HMs, and Structure equation model (SEM)

 

·         There are a few grammatical errors and awkward phrasing.

·         In introduction, some sentences are quite long and complex. Consider breaking them down for better readability. For example, the sentence starting with "Meanwhile, they can be concentrated..." could be separated into two or more sentences for clarity.

·         Maintain uniformity in usage of acronyms, scientific names and also minor grammar issues.

Comments on the Quality of English Language

Minor editing of English language required 

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1:  In introduction, some sentences are quite long and complex. Consider breaking them down for better readability. For example, the sentence starting with "Meanwhile, they can be concentrated..." could be separated into two or more sentences for clarity.

Response 1: The long sentence has been revised to " Meanwhile, they can be concentrated and even converted into more toxic substances by soil organisms. That could change the soil physicochemical properties, pollute the soil water system through rainwater leaching and lead to soil degradation (Minnikova et al., 2017;Shao et al., 2020)."

Comments 2: Maintain uniformity in usage of acronyms, scientific names and also minor grammar issues.

Response 2: We checked this part, and modified them in revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The presented work examines the impact of soil contamination with heavy metals on the composition and number of soil bacteria and fungi. The article is interesting and well-written but requires a few corrections. Unfortunately, the text does not have line numbering, which makes it difficult to review.

1.      Introduction – „Heavy metals in soil are difficult to be degraded by microorganisms”  the word „degradated” should be replaced by „disposed of” – metal can not be degradated

2.      Introduction – „ …and even converted into more toxic substances…” Metal cannot be transformed into other substances, only into other forms

3.      The title of subchapter 2.2 should be changed to "Determination of carbon, phosphorus and nitrogen in soil samples" or "Determination of soil fertility parameters"

4.      Table 1 and Table 2 – There should be a legend under the tables explaining the sample designations

 

5.      Free spaces on pages 5, 7, 10 should be eliminated

Comments on the Quality of English Language

The article is linguistically correct, with a few typos and stylistic errors.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1:  Introduction – „Heavy metals in soil are difficult to be degraded by microorganisms”  the word „degradated” should be replaced by „disposed of” – metal can not be degradated

Response 1:  We use "utilized" replace "degraded".

Comments 2:  Introduction – „ …and even converted into more toxic substances…” Metal cannot be transformed into other substances, only into other forms

Response 2: We use "forms" replace "substances".

Comments 3: The title of subchapter 2.2 should be changed to "Determination of carbon, phosphorus and nitrogen in soil samples" or "Determination of soil fertility parameters"

Response 3: We changed that to "Determination of soil fertility parameters".

Comments 4:  Table 1 and Table 2 – There should be a legend under the tables explaining the sample designations

Response 4: We added "(Sample designations explanation: CK/SF/U/C corresponding to corn plot in reference area/ oil_sunflower plot/ uncultivated plot/ corn plot, R representing rhizosphere soil and S representing surface soil) under table 1.

Comments 5: Free spaces on pages 5, 7, 10 should be eliminated.

Response 5: We checked that and modified them in revision.

Comments 6: The article is linguistically correct, with a few typos and stylistic errors.

Response 6: We checked this part and made some modification.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript “Long-Term Heavy Metal Pollution Induces a Complex Difference on Topsoil and Rhizosphere Microbial Communities in Farmlands” requires some revision.

 

General suggestions.

Please, make sure that the text is edited in accordance with the requirements of the journal:   Correspondence: author: Dr. Qili Yang. E-mail: [email protected] – looks like e-mail is written via different font size.

 References must be numbered in order of appearance in the text. In the text, reference numbers should be placed in square brackets [ ].

Please, revise some sentences to make them shorter.

 

Abstract

The problem statement in the abstract is too broad (“The function recovery of farmland after long-term heavy metal pollution remains unclear”). It does not imply the relevance of the work presented. The conclusion of the work in the abstract is not clear to understand the output of the research.   

Please, avoid unidentified abbreviations in abstract (HMs).

 

Keywords

No need capital letters. Some keywords are too general. It is advised to give more precise ones.

 

Introduction

The sentence is formulated not very correctly: “Heavy metals in soil are difficult to be degraded by microorganisms”, since metals are non-biodegradable.  The authors also report this in the previous sentence. It is better to revise.

The information in the sentence is too general : “Li et al. (2020) demonstrated that microbial community com-position varied among depth layers in a typical Pb/Zn smelting area, and Proteobacteria was the most dominant phylum in different depth layers”, since Proteobacteria include a lot of microbial species. Thus, there is no opportunity to evaluate the impact of metals on microorganisms. Please, revise.

There was no any information in the introduction section on metal-plant interaction, however, they appear in the summary of this section: “Therefore, improving the interaction between resistant and beneficial rhizosphere soil microorganisms and heavy metal tolerant plants can en-hance plant biomass and tolerance against heavy metals for green remediation of HMs contaminated farmland”. It is confusing, what relationship plants have to the research.

The research deals with “a typical agricultural region impacted severely by a sizeable zinc smelter”.

It would be better to add some information in the introduction to highlight the state-of-art is some similar sites. The information presented now is quite general and does not give an idea about such studies and the need for such work.

Except for individual objectives, it would be better to formulate the goal of the work.

 

Materials and Methods

Once mentioned (Introduction), there is no need to duplicate abbreviation in brackets:  Huludao Zinc Plant (HZP), toxic heavy metals (HMs).

Please, revise to make it more simple: “Besides, the fourth plot was selected as reference place marked as CK group, which was a cornfield ten km away from research area westward, and CK_S was surface soil and CK_R was corn rhizosphere soil in there”.

 

Results and discussion

Figure 2.  Captions for figures b1 and b2 are too small.

It is also better to regroup the text to avoid free space between figures and text.

The authors give the information: “As can be seen from the heatmap (figure 4b), bacteria Pseudomonas and fungi Chaeto-thyriales and Fusarium showed red in the heavy metal block, indicating a positive correlation between microbial community abundance and soil heavy metal content. Chaetothy-riales showed a significantly positive correlation with Cd, Pb and Zn …”. It would be helpful if authors could give some explanation of such correlation.

 

Conclusion

It is known that microbial resistance to metals depends not only on their presence in, for example, soil. It also depends on the form of metals in soil (soluble, insoluble) as well as on the presence of sources of carbon and energy for microorganisms.

The authors write: “the abundance of microbial species in soil of crop-tillage plots was significantly higher than that of untillage plots, and the corn plot soil in HMs contaminated areas had more abundant microbial species than un-contaminated area soil”. So, the question appears if untilled plots with no metal contamination had low microbial diversity comparing to corn plot soil, maybe it is the question not only of presence of metals, but also presence of additional nutrients in agricultural soil that could be fertilized and provide microbial growth?

 

Author Contributions

Please, revise to meet the template example.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please, revise some sentences to make them shorter.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1: Please, make sure that the text is edited in accordance with the requirements of the journal: Correspondence: author: Dr. Qili Yang. E-mail: [email protected] – looks like e-mail is written via different font size.

Response 1: I have checked that and they are all in same font size in our uploaded manuscript.

Comments 2:  References must be numbered in order of appearance in the text. In the text, reference numbers should be placed in square brackets [ ].

Response 2: We rearranged all references and cited them according the requirements of the journal in revised manuscript.

Comments 3: Please, revise some sentences to make them shorter.

Response 3: We checked that and modified long sentences to short ones in revised manuscript.

Comments 4: The problem statement in the abstract is too broad (“The function recovery of farmland after long-term heavy metal pollution remains unclear”). It does not imply the relevance of the work presented. The conclusion of the work in the abstract is not clear to understand the output of the research. 

Response 4: We changed the statement with "The microbial effect of farmland after long-term heavy metal pollution remains unclear."

Comments 5: No need capital letters. Some keywords are too general. It is advised to give more precise ones.

Response 5: We changed those letters and made some modification in this part.

Comments 6: The sentence is formulated not very correctly: “Heavy metals in soil are difficult to be degraded by microorganisms”, since metals are non-biodegradable.  The authors also report this in the previous sentence. It is better to revise. 

Response 6: We use "utilized" replace "degraded".

Comments 7: Once mentioned (Introduction), there is no need to duplicate abbreviation in brackets:  Huludao Zinc Plant (HZP), toxic heavy metals (HMs). 

Response 7: We revised that in revision.

Comments 8: Please, revise to make it more simple: “Besides, the fourth plot was selected as reference place marked as CK group, which was a cornfield ten km away from research area westward, and CK_S was surface soil and CK_R was corn rhizosphere soil in there”.

Response 8:The sentence was changed to "Besides, the fourth plot was selected as reference place (CK group), which was a cornfield ten km away from research area westward. Its surface soil was marked as CK_S and CK_R represent rhizosphere soil."

Comments 9: 

Captions for figures b1 and b2 are too small. It is also better to regroup the text to avoid free space between figures and text.

Response 9: We enlarged the Captions for figures b1 and b2.

Comments 10: The authors give the information: “As can be seen from the heatmap (figure 4b), bacteria Pseudomonas and fungi Chaeto-thyriales and Fusarium showed red in the heavy metal block, indicating a positive correlation between microbial community abundance and soil heavy metal content. Chaetothy-riales showed a significantly positive correlation with Cd, Pb and Zn …”. It would be helpful if authors could give some explanation of such correlation.

Response 10: We discussed the correlation in the following paragraph and this positive correlation was a relative result comparing with the negative effect.

Comments 11: The authors write: “the abundance of microbial species in soil of crop-tillage plots was significantly higher than that of untillage plots, and the corn plot soil in HMs contaminated areas had more abundant microbial species than un-contaminated area soil”. So, the question appears if untilled plots with no metal contamination had low microbial diversity comparing to corn plot soil, maybe it is the question not only of presence of metals, but also presence of additional nutrients in agricultural soil that could be fertilized and provide microbial growth?

Response 11: I think your question is logically right, but our research mainly aimed to discuss the microbial ecosystem's response in long-term HMs polluted farmland. That part could be discussed in next stage research.

Comments 12:   Author Contributions  Please, revise to meet the template example.

Response 12: We made some modification to meet the template example.

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript was improved. There is one suggestion: it is more useful to use references with DOI which is not included in the manuscript. It is suggested to add DOI to the list of references where it is possible.

Author Response

Thank you very much for taking the time to review this manuscript, again. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1: There is one suggestion: it is more useful to use references with DOI which is not included in the manuscript. It is suggested to add DOI to the list of references where it is possible.

Response 1: I have added all DOI to our references in the new uploaded manuscript, except one published in Chinese.