Xanthan- and Gelatine-Based Composites Used as Nursery Groundcovers: Assessment of Soil Microbiology and Seedling Performance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer’s comments

 

In the article “Xanthan- and gelatine-based composites used as nursery groundcovers: assessment of soil microbiology and seedling performance”, Valentini et al. use novel groundcovers for tree seedlings. These novel groundcovers can improve soil moisture retention, prevent competition, while being economically and practically viable. In a nursery experiment, plant growth, weed establishment, soil characteristics, and microbial activity and community composition are assessed. The authors deploy various appropriate methods to investigate these questions and have produced a novel and interesting research paper and a valuable contribution to sustainable afforestation.

 

The Introduction gives a succinct summary of the increasing challenges with reforestation and afforestation and gives a good summary of the field. However, the aims and hypotheses should be stated more clearly at the end of the introduction. The method section is clearly written and does not require any additional work. The experimental design is thorough and well replicated.

 

The results would benefit from some editing and refining (see specific comments below). What I find especially lacking from the results is a deeper analysis of the taxonomic composition of the fungal and bacterial communities in the soil samples, and I think the paper would benefit from 1) doing a differential abundance analysis, and 2) linking significantly differentially abundant taxa to the functional results reported from the CLPP assay. Also a discussion of the larger implications of changes in microbial community composition from the use of different groundcovers is lacking.

 

Finally, make sure that the claims you state in your conclusions are backed by your results.

 

After these revisions I believe the paper fit to be published in “Sustainability”.

 

Specific comments

 

Introduction

 

Overall fine, but in the last paragraph of the introduction the aims for the study should be presented more clearly, so in brief what you did exactly and what questions/hypotheses you hoped to answer. Take the first sentence of the conclusion as a guidance.

 

Methods

 

L219: what are these potential unwanted variables?

 

Results

 

General comment: I was going to comment on that there was too much discussion in the results, but then realized that this is a Results and Discussion section, please change the heading to Results and Discussion to make this clear.

 

L300: For someone not familiar with the dataset, having the sample names “T1, T2” etc is not so informative, and I find myself having to scroll back to table 2 to check which name belongs to which sample again and again. As a suggestion, using abbreviations like XW (for Xanthan gum + wood fibres) and GW (gelatine + wood fibres) would already make it easier to follow, and T5 can just be written as “control” in the text and figures without adding too many characters to the manuscript.

 

Figure 3: same thing here, having another indicator for treatment than just Tx would make the figure nicer and more informative to look at. The same goes for all the other figures and tables.

 

L306: …than the former?

 

L326-330: The previous result sections were written in past tense, here it should be past tense as well.

 

L328: What does “some influence” mean? Is this a statistically significant association or not?

 

Table 5: I feel like these values could be better represented as a figure. Would make it easier and more illustrative to compare among boxplots than having to read through the table, at least in my opinion. The other tables are fine.

 

L354: I cannot follow this sentence, the ORP value changed in the control when you added groundcovers for the other treatments? Please reword.

 

L357: change to “increased” (past tense).

 

L359: same

 

L361: Remove the word “additionally”, as the next sentence is explaining/discussing your data and not listing more results.

 

L363/364: The sentence starting with “Higher microbial activity…” doesn’t really add much information, I would just add citation [59] to the previous sentence, as the fact that increased CO2 emissions indicate microbial activity should be quite obvious.

 

L364/365: Here it is unclear whether you are discussing your own findings or the literature, please reword.

 

L364-376: In this whole paragraph the same statements and citations are being repeated over and over again. Please rewrite and shorten.

 

L377: Please add which data this analysis is done on (the soil’s physicochemical properties)

 

Figure S2: It almost seems like T4 also is separate from T1,3,5 ? Why choose a clustering threshold where only T2 is separated?

 

L393: I am not previously familiar with this method, but isn’t Fig 5A showing a soil extract, taken at ONE timepoint, on a 96-well plate with incubation time on the x axis? How can this graph possibly indicate a change in “soil conditions” over time if you only have samples from one timepoint at the end of the growing season? This makes no sense to me, shouldn’t the analysis of this figure focus on the slopes of the curves between the different treatments? From the error bars it looks like there is no significant difference between the groundcover treatments. Please rewrite this or explain how this can show “improved soil conditions” over time.

 

L403-406: Please be more specific, these two sentences do not discuss these results sufficiently.

 

L408: This paragraph can be expanded to describe the clustering (T2 alone, T4+T5, T1+T3) in its entirety + the implications.

 

L421-430: Is this paragraph and the diversity analysis based on the sequencing data or the SAWCD values? The text for the supplementary figure implies the latter (“on the results reported in Figures 5a and 5b”), while further down you write about “microbial taxa”. Which is it? The CLPP assay does not yield any information on taxa.

 

L434+436: *Planctomycetes

 

L448: bacterial genome? You only have 16S amplicon data.

 

L448-L453: Very repetitive, please shorten and combine these sentences.

 

L457: What does “maximized for T2” mean? That Agaricomycetes had the highest relative abundance in the T2 treatment?

 

L457: The sentence lacks a period and citation, and it could benefit from additional clarity or detail.

 

L461: Here it should be mentioned that both Fraxinus and Alnus are AM plants, so differences in abundance of Glomeromycota between treatments would be interesting to look into further.

 

Figure 6: Please update the figure so that all the texts and text labels are readable. Also, from the clustering it looks like you have some interesting results here that I would like to see explored further. The clusterings for bacteria and fungi look different, with T2 vs T4 separating from the other samples in the nMDS analysis. I would suggest running a differential abundance analysis to find out which OTUs are statistically significantly different between the treatments.

 

L466-467: Do you see any changes in the abundance of fungi with lignocellulolytic capabilities or not? You have taxonomic data, why are you speculating?

 

L477: *lower richness

 

L480: Which findings “of the earlier section”?

 

L483: Why is this section called “alteration in soil N pool” when what you have looked at is nifH gene expression?

 

L488: Higher than what? You did not find any significant differences. Unfortunately, this whole section feels disconnected from the rest of the paper and currently doesn’t really add much to the overall story. Either move to supplementary materials or rewrite so that it matches the overall context better.

 

Conclusions

 

L499-503: Make clearer what refers to ash and what to alder.

 

L507: Please specify what soil properties were changed.

 

L511: here you claim “the groundcover made of xanthan gum (T1) appeared to boost the diversity of soil microorganisms and their capacity to fix nitrogen”, I cannot find any support for these claims in your results.

Comments on the Quality of English Language

Overall good, but make sure to fix inconsistencies in tense (past/present), and that all sentences and paragraphs are clear and not too repetitive.

Author Response

In the article “Xanthan- and gelatine-based composites used as nursery groundcovers: assessment of soil microbiology and seedling performance”, Valentini et al. use novel groundcovers for tree seedlings. These novel groundcovers can improve soil moisture retention, prevent competition, while being economically and practically viable. In a nursery experiment, plant growth, weed establishment, soil characteristics, and microbial activity and community composition are assessed. The authors deploy various appropriate methods to investigate these questions and have produced a novel and interesting research paper and a valuable contribution to sustainable afforestation.

 

The Introduction gives a succinct summary of the increasing challenges with reforestation and afforestation and gives a good summary of the field. However, the aims and hypotheses should be stated more clearly at the end of the introduction. The method section is clearly written and does not require any additional work. The experimental design is thorough and well replicated.

 

The results would benefit from some editing and refining (see specific comments below). What I find especially lacking from the results is a deeper analysis of the taxonomic composition of the fungal and bacterial communities in the soil samples, and I think the paper would benefit from 1) doing a differential abundance analysis, and 2) linking significantly differentially abundant taxa to the functional results reported from the CLPP assay. Also a discussion of the larger implications of changes in microbial community composition from the use of different groundcovers is lacking.

 

Finally, make sure that the claims you state in your conclusions are backed by your results.

 

After these revisions I believe the paper fit to be published in “Sustainability”.

 

Specific comments

 

Introduction

 

Overall fine, but in the last paragraph of the introduction the aims for the study should be presented more clearly, so in brief what you did exactly and what questions/hypotheses you hoped to answer. Take the first sentence of the conclusion as a guidance.

 

The authors thank the reviewer for the very precise revision and for the useful comments. The last paragraph of the introduction was rewritten and the hypothesis of the study were clarified.

 

Methods

 

L219: what are these potential unwanted variables?

 

 

The authors thank the reviewer for the comment: the sentence was rewritten.

 

 

Results

 

General comment: I was going to comment on that there was too much discussion in the results, but then realized that this is a Results and Discussion section, please change the heading to Results and Discussion to make this clear.

           

The authors agree with the reviewer: the title was changed in order to make clear that it was a “Results and Discussion” section.

 

L300: For someone not familiar with the dataset, having the sample names “T1, T2” etc is not so informative, and I find myself having to scroll back to table 2 to check which name belongs to which sample again and again. As a suggestion, using abbreviations like XW (for Xanthan gum + wood fibres) and GW (gelatine + wood fibres) would already make it easier to follow, and T5 can just be written as “control” in the text and figures without adding too many characters to the manuscript.

 

Figure 3: same thing here, having another indicator for treatment than just Tx would make the figure nicer and more informative to look at. The same goes for all the other figures and tables.

 

The authors agree with the reviewer and decided to revise the nomenclature of the samples as suggested: Figures, Tables and coding along the text were revised accordingly.

 

L306: …than the former?

 

Revised.

 

L326-330: The previous result sections were written in past tense, here it should be past tense as well.

 

The authors thank the reviewer for the remark, the section was revised. 

 

L328: What does “some influence” mean? Is this a statistically significant association or not?

 

The authors thank the reviewer for the remark, the section was revised in order to avoid confusion. 

 

Table 5: I feel like these values could be better represented as a figure. Would make it easier and more illustrative to compare among boxplots than having to read through the table, at least in my opinion. The other tables are fine.

           

The authors thank the reviewer for the suggestion: data of Table 5 were represented in a Figure to improve the clarity due to the large amount of data.

 

 

L354: I cannot follow this sentence, the ORP value changed in the control when you added groundcovers for the other treatments? Please reword.

 

The authors agree with the reviewer: the sentence was rewritten.

 

L357: change to “increased” (past tense).

 

Revised.

 

L359: same

 

Revised.

 

L361: Remove the word “additionally”, as the next sentence is explaining/discussing your data and not listing more results.

 

Revised.

 

L363/364: The sentence starting with “Higher microbial activity…” doesn’t really add much information, I would just add citation [59] to the previous sentence, as the fact that increased CO2 emissions indicate microbial activity should be quite obvious.

 

The authors thank the reviewer for the suggestion: the sentence was rewritten.

 

L364/365: Here it is unclear whether you are discussing your own findings or the literature, please reword.

 

The authors thank the reviewer for the suggestion: the paragraph was revised and shortened.

 

 

L364-376: In this whole paragraph the same statements and citations are being repeated over and over again. Please rewrite and shorten.

 

The authors thank the reviewer for the suggestion: the paragraph was revised and shortened.

 

L377: Please add which data this analysis is done on (the soil’s physicochemical properties)

 

Revised.

 

Figure S2: It almost seems like T4 also is separate from T1,3,5 ? Why choose a clustering threshold where only T2 is separated?

 

The clustering analysis was performed following the selection of the optimum number of clusters based on the elbow method, further supported by the Silhouette method. Both of these along with 1000 bootstrapping were able to imply, that the datasets had minimized within-cluster variance and that samples are closely grouped in two clusters. Based on this, 2 clusters were selected and the ellipse implies the 95% confidence interval. The lacking information was added to the caption of the Figure S2.

 

L393: I am not previously familiar with this method, but isn’t Fig 5A showing a soil extract, taken at ONE timepoint, on a 96-well plate with incubation time on the x axis? How can this graph possibly indicate a change in “soil conditions” over time if you only have samples from one timepoint at the end of the growing season? This makes no sense to me, shouldn’t the analysis of this figure focus on the slopes of the curves between the different treatments? From the error bars it looks like there is no significant difference between the groundcover treatments. Please rewrite this or explain how this can show “improved soil conditions” over time.

 

The BIOLOG assay is an analysis of the microbial metabolic potential based on the carbon source utilization pattern on the EcoPlates. Soil samples, collected at one time point, were used in this study to compare the effect of different treatments on microbial metabolic potential at the end of the growing season. A higher AWCD represents higher microbial metabolic potential. Figure 6A shows the general trend of metabolic potential among the treatments rather than a detailed comparison of individual treatments. The term "improved soil conditions" in this context refers to differences in microbial functionality resulting from the soil amendments themselves at the end of the growing season, not changes throughout an entire growing season. The required information has been added to the revised version between line no. 383-389.

 

 

L403-406: Please be more specific, these two sentences do not discuss these results sufficiently.

           

This section has been revised between the line no. 400-404.

 

L408: This paragraph can be expanded to describe the clustering (T2 alone, T4+T5, T1+T3) in its entirety + the implications.

 

The section has been revised between the line no. 405-411.

 

L421-430: Is this paragraph and the diversity analysis based on the sequencing data or the SAWCD values? The text for the supplementary figure implies the latter (“on the results reported in Figures 5a and 5b”), while further down you write about “microbial taxa”. Which is it? The CLPP assay does not yield any information on taxa.

 

            This section has been revised between line no. 422-436.

 

L434+436: *Planctomycetes

           

            Revised.

 

L448: bacterial genome? You only have 16S amplicon data.

 

            This was written by mistake, and revised in the modified manuscript in line no 451-452.

 

L448-L453: Very repetitive, please shorten and combine these sentences.

           

            This section has been revised between line no. 447-455.

 

 

L457: What does “maximized for T2” mean? That Agaricomycetes had the highest relative abundance in the T2 treatment?

 

            This implies the highest abundance of Agaricomycetes in T2, revised in line no. 459.

 

L457: The sentence lacks a period and citation, and it could benefit from additional clarity or detail.

 

            This section has been revised between line no. 459-460.

 

L461: Here it should be mentioned that both Fraxinus and Alnus are AM plants, so differences in abundance of Glomeromycota between treatments would be interesting to look into further.

 

No significant changes in the Glomeromycota were found through an ANOVA analysis. This section has been added in the line no 465-466.

 

Figure 6: Please update the figure so that all the texts and text labels are readable. Also, from the clustering it looks like you have some interesting results here that I would like to see explored further. The clusterings for bacteria and fungi look different, with T2 vs T4 separating from the other samples in the nMDS analysis. I would suggest running a differential abundance analysis to find out which OTUs are statistically significantly different between the treatments.

 

The shared bacterial and fungal classes (Figure S5) and differential abundance of bacterial and fungal classes have been added to the revised version (Figure S6 and S7) has been added to the revised version. (Line no. 471-472; 481-491).

 

L466-467: Do you see any changes in the abundance of fungi with lignocellulolytic capabilities or not? You have taxonomic data, why are you speculating?

 

The abundance of lignocellulolytic fungal communities has been added and addressed in line no. 471-472 (Figure S4).

 

L477: *lower richness

           

            Revised

 

L480: Which findings “of the earlier section”?

 

            The authors thank the reviewer for the remark: the sentence was revised.

 

L483: Why is this section called “alteration in soil N pool” when what you have looked at is nifH gene expression?

 

            The heading of the section has been revised in line no 500.

 

L488: Higher than what? You did not find any significant differences. Unfortunately, this whole section feels disconnected from the rest of the paper and currently doesn’t really add much to the overall story. Either move to supplementary materials or rewrite so that it matches the overall context better.

 

This section has been revised to meet the better integration with other sections (Line no. 501-507).

 

Conclusions

 

L499-503: Make clearer what refers to ash and what to alder.

 

L507: Please specify what soil properties were changed.

 

L511: here you claim “the groundcover made of xanthan gum (T1) appeared to boost the diversity of soil microorganisms and their capacity to fix nitrogen”, I cannot find any support for these claims in your results.

 

The conclusions were revised and rewritten according to the comments.

 

Comments on the Quality of English Language

Overall good, but make sure to fix inconsistencies in tense (past/present), and that all sentences and paragraphs are clear and not too repetitive.

English was revised and the manuscript checked.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

First of all, congratulations on such an interesting research topic and the results. Your work is very interesting, but I have a few comments.

1. 'References' section to be revised. Please get familiar with the citation rules and make the necessary changes. I am reckon that this is a very important aspect - authors should adapt to the requirements of a given journal and ensure as few editorial corrections as possible.  I did not find a single correctly cited item in the entire list.

 

2. Please correct the way you write species of microorganisms... sometimes you use italics, sometimes you don't. Pay attention to the fonts in whole text, formatting of subpoints (bold, italics, size) and and also the way of describing tables (lines 153 vs 160).

3. I think that the purpose of the research in the entire "Introduction" section is a little bit lost - please make it more visible, so that the reader can easily focus on what exactly you were doing.

4. The abberviation of xanthan gum appears in "2. Materials" and you used it only 2 times in the paper - please decide whether it makes sense to keep two abbreviations or replace the full names with abbreviations. You did not use the abbreviation for gelatine. Why? The nomenclature at work should be uniform. Please provide arguments for this point.

5. The "Results" section is visually prepared to a very high standard, as it is clear and easy to read - good job!  However, this section should only contain your own results and in my opinion, any speculation and deliberation can be moved to a separate "Discussion" section, which does not exisit in your paper.  Frankly speaking, the article definitely lacks an extended discussion, as well as a comparison of the results with previous studies and progress made so far.

   

Author Response

First of all, congratulations on such an interesting research topic and the results. Your work is very interesting, but I have a few comments.

1. 'References' section to be revised. Please get familiar with the citation rules and make the necessary changes. I am reckon that this is a very important aspect - authors should adapt to the requirements of a given journal and ensure as few editorial corrections as possible.  I did not find a single correctly cited item in the entire list.

The authors thank the reviewer for the encouraging words and for the comments. All the references were revised formatting using the citation style provided by MDPI.

2. Please correct the way you write species of microorganisms... sometimes you use italics, sometimes you don't. Pay attention to the fonts in whole text,formatting of subpoints (bold, italics, size) and and also the way of describing tables (lines 153 vs 160).

The authors thank the reviewer for the comment: formatting and fonts were revised. Moreover also the description of Tables was corrected.

3. I think that the purpose of the research in the entire "Introduction" section is a little bit lost - please make it more visible, so that the reader can easily focus on what exactly you were doing.

            The authors thank the reviewer for comment. Also according to the comment of Reviewer 1, the introduction was revised and the purpose and of the work was clarified.

4. The abberviation of xanthan gum appears in "2. Materials" and you used it only 2 times in the paper - please decide whether it makes sense to keep two abbreviations or replace the full names with abbreviations. You did not use the abbreviation for gelatine. Why? The nomenclature at work should be uniform. Please provide arguments for this point.

The authors perfectly agree with the reviewer that the use abbreviations should be consistent and motivated. The abbreviation for xanthan gum was therefore removed from the manuscript.

5. The "Results" section is visually prepared to a very high standard, as it is clear and easy to read - good job!  However, this section should only contain your own resultsand in my opinion, any speculation and deliberation can be moved to a separate "Discussion" section, which does not exisit in your paper.  Frankly speaking, the article definitely lacks an extended discussion, as well as a comparison of the results with previous studies and progress made so far.

The authors thank the reviewer for the comment but they believe that the discussion of results, due to the complexity of the work, is clearer if performed in the same section. Therefore a single “Results and Discussion” section is present in this work. Anyway the authors tried to clarify, simplify (in some cases, according to Reviewer 1) and extend the discussion of the results.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

in my view the manuscript has improved significantly since the first time I read it. I appreciate that my comments, questions and thoughts were put into consideration. However, there are still a few comments from my side before the manuscript gets published:

Introduction

 

-              Good that the specific aims are now listed at the end of the introduction, for aim 3) I would replace “structure” with “community composition”

 

Results and Discussion

-              L 551 and onwards: Here I am missing some deeper exploration of the differential abundance results. My recommendation would be to focus on the comparisons that were separated in the clustering, so for bacteria GW vs all the other treatments and for fungi WC vs all the other treatments (see also my comment below for Figure S6). Then try to connect these differentially abundant taxa with the taxa you were discussing in the previous section (3.3.2). If you are just going to say “we did find differentially abundant bacteria and fungi” you may as well skip that part completely imo.

-              L 560/561: this is worded in a confusing way, with the first sentence (no significant difference) seemingly contradicting the other sentence, so first I thought the second sentence was about the ITS data (also because both Table S2 and S3 are mentioned in the first sentence). Please rewrite to clarify.

 

Supplementary Material

-              Table S1: Sample identifiers not changed

-              Figure S4,S5: same

-              Figure S6: the text labels in the volcano plots are far too small to be readable. As a suggestion, maybe it would be better to focus on the comparisons that were identified as relevant from the nMDS plots in Fig 7? So e.g. for bacteria it would be GW vs all the other treatments while for fungi that would be WC vs all the other treatments. The other comparisons are not significantly different based on your ordination analyses, so looking at differential abundance would be less interesting imo.

Comments on the Quality of English Language

Mostly fine, but the manuscript would benefit from an additional language check before resubmission.

Author Response

Trento (Italy), January 21^st 2025

 

Dr Nick Yang

Assistant Editor

Polymers

 

Subject: second revised version of the manuscript No. sustainability-3255080 entitled “Xanthan- and gelatine-based composites used as nursery groundcovers: assessment of soil microbiology and seedling performance” by Francesco Valentini, Alessandro Sorze, Jaime Coello, Laura Ros, Atif Aziz Chowdhury, Federica Piergiacomo, Giulia Casapiccola, Lorenzo Brusetti, Janine Bösing, Sebastian Hirschmüller and Andrea Dorigato.

 

Dear Professor Yang,

Thank you very much for sending us the second report of the referee 1 on our manuscript. We have prepared a revised version of it, in which we have addressed all the comments/questions raised by the referee. We gratefully acknowledge the reviewer for his accurate revision work, useful to prepare a better version of the manuscript. Main modifications were edited in red in the text of the manuscript and summarized below for your convenience. We hope that this revised version of the manuscript could be finally accepted for publication.

 

Yours sincerely,

 

Francesco Valentini

University of Trento

Department of Industrial Engineering

Via Sommarive, 9 - 38123 Trento, tel. +390461282539

e-mail: francesco.valentini@unitn.it

 

 

 

Reviewer 1

Dear Authors, in my view the manuscript has improved significantly since the first time I read it. I appreciate that my comments, questions and thoughts were put into consideration. However, there are still a few comments from my side before the manuscript gets published:

Introduction

-              Good that the specific aims are now listed at the end of the introduction, for aim 3) I would replace “structure” with “community composition”

The authors agree with the reviewer, the sentence was modified as suggested.

Results and Discussion

              L 551 and onwards: Here I am missing some deeper exploration of the differential abundance results. My recommendation would be to focus on the comparisons that were separated in the clustering, so for bacteria GW vs all the other treatments and for fungi WC vs all the other treatments (see also my comment below for Figure S6). Then try to connect these differentially abundant taxa with the taxa you were discussing in the previous section (3.3.2). If you are just going to say “we did find differentially abundant bacteria and fungi” you may as well skip that part completely imo.

This section has been modified to meet the requirements in the revised version (line no 503-512).

-              L 560/561: this is worded in a confusing way, with the first sentence (no significant difference) seemingly contradicting the other sentence, so first I thought the second sentence was about the ITS data (also because both Table S2 and S3 are mentioned in the first sentence). Please rewrite to clarify.

This section has been modified between the line no 513-519 in the revised version.

 

Supplementary Material

-              Table S1: Sample identifiers not changed

Revised

-              Figure S4,S5: same

Revised

-              Figure S6: the text labels in the volcano plots are far too small to be readable. As a suggestion, maybe it would be better to focus on the comparisons that were identified as relevant from the nMDS plots in Fig 7? So e.g. for bacteria it would be GW vs all the other treatments while for fungi that would be WC vs all the other treatments. The other comparisons are not significantly different based on your ordination analyses, so looking at differential abundance would be less interesting imo.

The Figure S6 and S7 were revised removing non-relevant comparisons.

Author Response File: Author Response.docx