Characteristics of Enzyme Activities during Phytoremediation of Cd-Contaminated Soil
Round 1
Reviewer 1 Report
Review comments
The Authors for this study present an interesting case study about effects of phytoremediation on soil enzyme activities, the research is quite interesting. There are several parts throughout the manuscript need a major revision.
1. Introduction:
The introduction follows a funnel structure. It provides a wide background before discussing existing literature. Then, the gap in existing literature is discussed. Finally, the objective of the study is clearly stated and what the novelty is. The last paragraph usually provides the reader the aim and novelty of the study and which gap will be filled. However, the current introduction is inadequate on background, and it is unclear on what novelty and limitations.
2. 2.1 The plant types are recommended with abbreviations and specific Latin names.
3. 2.1 The background concentrations of cadmium in soils and the soil enzyme activity need to be given.
4. 2.1 and 2.2, some contents are repeated, such as the sampling location.
5. It is necessary to refine the experimental design and to give the quality control conditions of sample analysis and testing. QA/QC?
6. What is the relationship between different forms of soil cadmium and enzyme activities? How does this relate to the subject of this article? Further discussion by the author is needed.
7. What is the purpose of Table 1?
8. Line342-346, This part of the content is not significant, it is recommended to delete.
9. Some major references need to be updated in recent eight years.
10. Native English speaker to revise the manuscript for better flow.
Author Response
The Authors for this study present an interesting case study about effects of phytoremediation on soil enzyme activities, the research is quite interesting. There are several parts throughout the manuscript need a major revision.
Response: Thanks for the positive comment! We have revised the manuscript to further enhance its quality.
- Introduction:
The introduction follows a funnel structure. It provides a wide background before discussing existing literature. Then, the gap in existing literature is discussed. Finally, the objective of the study is clearly stated and what the novelty is. The last paragraph usually provides the reader the aim and novelty of the study and which gap will be filled. However, the current introduction is inadequate on background, and it is unclear on what novelty and limitations.
Response 1: According to the reviewer’s suggestion, we added background expression in the introduction part and remodified this part as you suggested.
- 2.1 The plant types are recommended with abbreviations and specific Latin names.
Response 2: The plant types have been replaced with Solanum nigrum L., Phytolacca acinose Roxb., and Bidens Pilosa L.
- 2.1 The background concentrations of cadmium in soils and the soil enzyme activity need to be given.
Response 3: Please see lines 79-81 in the revised manuscript. The background concentrations of research soil are Cd 0.15 mg/kg, urease 10.3 mg/g, protease 6.76 mg/g, catalase 5.28 mg/g, phosphatase 65.11 mg/g.
- 2.1 and 2.2, some contents are repeated, such as the sampling location.
Response 4: Repeated parts of the manuscript have been revised and integrated.
- It is necessary to refine the experimental design and to give the quality control conditions of sample analysis and testing. QA/QC?
Response 5: QA/QC was added as follows: "Analytical data quality was guaranteed through the implementation of laboratory QA/QC protocols, including the use of standard procedures, reagent blanks, percentage of recoveries, and analysis of replicates. All reagents used were of analytical grade (certified purity＞99.9 percent). The entire laboratory was cleaned using an HNO3(30 percent, v/v) bath overnight, followed by repeated rinsing with double distilled water."
- What is the relationship between different forms of soil cadmium and enzyme activities? How does this relate to the subject of this article? Further discussion by the author is needed.
Response 6: Different soil enzymes had different sensitivity to heavy metal pollution. Through the correlation analysis of heavy metal speciation and soil enzyme activity, the indicator relationship between them was obtained.
- What is the purpose of Table 1?
Response 7: By two-way ANOVA analysis of variance, it can better explain the relationship between soil enzyme activity、pollution concentration, and phytoremediation species.
- Line342-346, This part of the content is not significant, it is recommended to delete.
Response 8: It was checked and amended.
- Some major references need to be updated in the recent eight years.
Response 9: All references are checked and updated.
- Native English speaker to revise the manuscript for better flow.
Response 10: Someone with a higher level of English language was invited to amend the article.
Reviewer 2 Report
The manuscript by Lu et al. provides a novel study on characteristic changes of enzyme activities during phytoremediation of cadmium contaminated soil. The results are well presented and should be accepted for publication in Sustainability after a minor revision.
My comments:
-In the abstract: The term “enzyme activity recovery” should be briefly explained or replaced by another word to make it easy to follow and understand by readers.
-Keywords: “enzyme activity recovery” should be removed or replaced by another one.
-The specific situation of the study subjects needs to be explained to make the research objectives clearer.
-Sections 2.1 and 2.2 should be rephrased with more details how the experiments were done.
- Sections 2.1 and 2.2, more references of the experimental methods should be provided.
-Line 65, What is “recovery degree” ? Is that the percentage of the soil enzyme returned to its original value? Should be briefly explained.
-Line 79, is the concentration of cadmium solution mg/Kg? Should be specify.
-Section 3.6, 3.6.1 and 3.6.2 should be merged. The subject of 3.6 should be “correlations between cadmium and enzyme activities”.
-In the conclusions, the authors need to highlight the main finding of this study and make the conclusions as concise as possible.
- Tables and figures: The abbreviations should be defined in the table footnote/figure captions. In each figure, the different graphs should be marked with A, B, C.
Author Response
Responses to Reviewer
Comments:
The manuscript by Lu et al. provides a novel study on characteristic changes of enzyme activities during phytoremediation of cadmium contaminated soil. The results are well presented and should be accepted for publication in Sustainability after a minor revision.
Response: Thanks for the positive comment! We have revised the manuscript to further enhance its quality.
Minor comments:
- In the abstract: The term “enzyme activity recovery” should be briefly explained or replaced by another word to make it easy to follow and understand by readers.
Response1: It was checked and amended. It was modified as following " promoting enzyme activity ".
- Keywords: “enzyme activity recovery” should be removed or replaced by another one.
Response2: It was checked and amended. “Enzyme activity recovery” was replaced by "promoting enzyme activity".
- The specific situation of the study subjects needs to be explained to make the research objectives clearer.
Response3: It was modified in the revised manuscript.
- Sections 2.1 and 2.2 should be rephrased with more details how the experiments were done.
Response4: We made a supplement about the treatment described in the new added experiment design section. More details are in the revised manuscript.
- Sections 2.1 and 2.2, more references of the experimental methods should be provided.
Response5: We made a supplement about the treatment described in the newly added experiment design section. More details are in the revised manuscript.
- Line 65, What is “? Shou recovery degree” ? Is that the percentage of the soil enzyme returned to its original value be briefly explained.
Response6: “Enzyme activity recovery degree” is the percentage of the soil enzyme returned to its original value. More details are in the revised manuscript.
- Line 79, is the concentration of cadmium solution mg/Kg? Should be specify.
Response7: The concentration of cadmium solution mg/Kg means mg of heavy metals per kilogram of soil. Indicates the amount of heavy metals in the soil. The higher the content, the more serious the pollution. More details are in the revised manuscript.
- Section 3.6, 3.6.1 and 3.6.2 should be merged. The subject of 3.6 should be “correlations between cadmium and enzyme activities”.
Response8: It was checked and amended.
- In the conclusions, the authors need to highlight the main finding of this study and make the conclusions as concise as possible.
Response9: It was checked and amended.
- Tables and figures: The abbreviations should be defined in the table footnote/figure captions. In each figure, the different graphs should be marked with A, B, C.
Response10: We made supplements about the unspecified issues and abbreviations in the revised manuscript..
Reviewer 3 Report
The article (Characteristics of enzyme activities during phytoremediation of cadmium contaminated soil) is interesting and relevant to the journal’s scope. However, I feel a few flaws in different sections of the ms, as indicated below in the specific comments.
1. The authors should clarify the purpose of the article in the abstract In my view， the abstract has to be rephrased in this flow. What is the problem (research gap), how do you work to sort this problem (methodology), what are your findings (results), and what are your final take-home messages (conclusions)?
2. The introduction section contains various unnecessary statements. Remain focused on the topic. Begin with the broadest scope and get progressively narrower, leading steadily to the statement of objectives.
3. Material and Method section: Several points need to be clarified.
4. The title of the third part of the figure should be further refined and improved to better reflect the intention of the figure.
5. There should be clear instructions under the icon and the graph should be self-naming.
6. In results, better to add the numeric description of results (% variations) instead of just adding the data values for easy understanding of the readers. Discussion should be merely based on the observed findings. Not just a review of the literature. Answer the question posed in the introduction and correlate your finding with the existing knowledge.
7. The authors need to strengthen the conclusions. What are your real findings and what are your judgements of these findings Please add future vision to the conclusion and improvement ideas to the readers.
8. Check whether the format of all references is according to the journal format.
9. Language needs substantial improvement. There are several grammatical and typo mistakes throughout the manuscript.
Author Response
The article (Characteristics of enzyme activities during phytoremediation of cadmium contaminated soil) is interesting and relevant to the journal’s scope. However, I feel a few flaws in different sections of the ms, as indicated below in the specific comments.
Response: Thanks for the positive comment! We have revised the manuscript to further enhance its quality.
- The authors should clarify the purpose of the article in the abstract In my view，the abstract has to be rephrased in this flow. What is the problem (research gap), how do you work to sort this problem (methodology), what are your findings (results), and what are your final take-home messages (conclusions)?
Response 1: The abstract of this manuscript followed a flow as research background, objective, methodology, results, and conclusions. We made some changes to the abstract to make it more concise, and more details are in the revised manuscript.
- The introduction section contains various unnecessary statements. Remain focused on the topic. Begin with the broadest scope and get progressively narrower, leading steadily to the statement of objectives.
Response 2: It was checked and amended accordingly.
- Material and Method section: Several points need to be clarified.
Response 3: We made a supplement about the treatment described in the added experiment design section. More details are in the revised manuscript.
- The title of the third part of the figure should be further refined and improved to better reflect the intention of the figure.
Response 4: It was checked and amended accordingly.
- Here should be clear instructions under the icon and the graph should be self-naming.
Response5: We made supplements about the unspecified issues and abbreviations.
- In results, better to add the numeric description of results (% variations) instead of just adding the data values for easy understanding of the readers. Discussion should be merely based on the observed findings. Not just a review of the literature. Answer the question posed in the introduction and correlate your finding with the existing knowledge.
Response 6: We added a numeric description to the results and amended the discussion in the revised manuscript.
- The authors need to strengthen the conclusions. What are your real findings and what are your judgements of these findings Please add future vision to the conclusion and improvement ideas to the readers.
Response 7: It was done in the revised manuscript.
- Check whether the format of all references is according to the journal format.
Response 8: It was checked and amended accordingly.
- Language needs substantial improvement. There are several grammatical and typo mistakes throughout the manuscript.
Response 9: Someone with a higher level of English language was invited to amend the article.
Reviewer 4 Report
Comments for author File: 
Comments.pdf
Author Response
Abstract There are several sentences that has to many “different” (e.g., Lines 11-12). I suggest to change them not to have too many same words, and to have more detailed information.
Response: It was checked and amended accordingly.
Lines 21-22: “… and the recovery degree of different … different under different..” -> Please revise the sentence so that it has actual information. What plant species had the highest recovery degree? How much more when compared to other species?
Response: “… and the recovery degree of different … different under different..” was replaced with “ different grass species recovered to varying degrees under various Cd solution stresses”. Four soil enzyme activities were employed as evaluation indices and principal component analysis was applied, to pick the grass species that will recover the highest. The promoting enzyme activity of Solanum nigrum L. was found to be the highest (Comprehensive score=0.185) under high concentration cadmium stress, while the promoting enzyme activity of Phytolacca acinosa Roxb. was found to be the highest (Comprehensive score=0.312) under low concentration cadmium stress.
Introduction
Line 35: You are using cadmium as full name throughout the entire manuscript, but I suggest to change it to Cd at some point to make the sentence more concise and understandable.
Response: It was checked and amended accordingly. Cadmium was replaced with Cd.
Lines 39-42: Providing more information on what forms of cadmium (weak acid, reduced, oxidized, residual) play important role in this environmental and health risks seems to be helpful.
Response: More details are in the revised manuscript.
Lines 43-66: Soil extracellular enzyme is indeed an indicator of important ecological processes, but I think that is a lack of contextualization. Why is it important to measure extracellular enzymes in soil where phytoremediation is being applied?
For example, •
Can exoenzymes be an indicator of how much plants are adapting to the toxic (contaminated) environment? If so, citation?
Why is the “recovery rate” of the extracellular enzyme activity important? What does it indicate?
Papers you cited might be discussing this part, and I think it is important to incorporate those in your introduction and rationalize the reason for this study. If you had specific hypothesis, presenting hypothesis might be a great way to explain it too.
Response: We added Tang H. et al. and Pan F. H. et al 's studies to elaborate on the importance of Soil extracellular enzyme indicators. More details are in the revised manuscript.
Materials and Methods
Line 79: What cadmium solution? What chemical was used (e.g., CdCl2) and how much volume of solution was added to how much amount of the soil? What is 0, 5, and 10 mg/kg Cd solution? Is it mg/L? When you use this in the results, it seems that the treatments are 0, 5, and 10 mg Cd /kg soil.
Response: Cd contaminated solution with CdCL2. The concentrations of Cd used in the experimental design were 5mg/kg and 10mg/kg, respectively. After conversion, the mass of CdCl2 required per 1000g of soil was 8.17mg; and 116.34mg.
Line 97:
1) In M&M, how you calculated degree of recovery should be provided.
Response: Degree of recovery= (enzyme activity in contaminated soil on certain day / enzyme activity in uncontaminated soil at beginning)*100
2) Also, how did you sample the soil at 30 and 60 days of growth? I think the rhizosphere soil (soil near the plant roots) will have higher enzyme activity thus the sampling method should have been consistent.
Response: The soil used in the experiment was non-rhizosphere soil. Cut a small hole in one side of a pot, and when sampling time comes, remove just enough soil from the pot for the experiment.
Line 103: Statistical analysis
1) Please provide more information on how you dealt with the statistical assumption for ANOVA. (I personally think you should use REPEATED MEASURES from GLM instead of simple ANOVA.)
Response: Your suggestion is very good, and We think the analysis of ANOVA can support my following discussions. We added some information dealing with the statistical assumption for ANOVA in the revised manuscript.
2) More description is needed for membership function approach and principal component analysis. For example, what type of factors were used for PCA?
Response: We added some descriptions to the membership function approach and principal component analysis, more details are in the revised manuscript.
Results
Lines 134-145: The sentence is too complicated; please break it into several sentences. Does this mean that you did not have controls which did not have any herbaceous plants but kept for 60 days for repeated enzyme activity measurement?
Response: The complicated sentence was modified using shorter ones. The aim of this part is to sort out which specie plant with best remediation effect, so our data were supportable for it without soil control after 60 days.
Line 146: Which part of your result show that the Cd inhibition effect is only temporary? I think that expression can only be used when the enzyme activity level is bounced back to the original levels.
Response: Your suggestion is very good. It was checked and amended accordingly.
Line 152-153: “… the difference of urease activity … was different” -> Please rephrase so that readers can clearly understand your intention.
Response: It was checked and amended accordingly.
Line 157: The best recovery effect on soil urease activity -> What is the recovery effect? Should be defined and be described in the methods as well.
Response: It was checked and amended accordingly.
Figure 2: Please provide the standard errors of the degree of recovery as well.
Response: The average value is used to calculate the degree of recovery, so no standard errors are marked.
Lines 167-170: The explanation for the upper case and the lower case is the same. Please double check and correct them.
Response: It was checked and amended accordingly.
Line 204: “Cadmium pollution concentration” -> change it to “Cd pollution level” or “Cd concentration” (please check the other parts of the manuscript too – for example, Line 222)
Response: It was checked and amended accordingly.
Line 208: “Soil catalase activity increased under phytoremediation” -> Can this be demonstrated from your dataset? You did not have controls (contaminated soil without plants) with 60 days of time period.
Response: It was checked and amended accordingly.
Discussion
Overall discussion can be largely improved by discussing the possible reasons for different recovery rate of various enzymes studied here, and its environmental significance.
Lines 302-304: I think this is an important sentence that should go to the introduction. Also, have you measured the decrease of Cd concentration from the soil in 30 and 60 days?
Response: It was checked and amended accordingly.
Lines 305-306: “Liao J. et al. concluded that urase had the most significant effect on heavy metal pollution” -> Sentence is not clear and cannot find the reason of citing this study. Does Urease have impact on metal pollution? Isn’t urease something that is affected by metal pollution? And most significant effect on heavy metal pollution among what?
Response: This expression was moved to Introduction.
Line 306: The findings -> Our findings (It seems like you are still talking about Liao Jie et al.)
Response: It was checked and amended in the revised manuscript.
Line 307-308: “although it increased following phytoremediation” -> Again, how did you measure phytoremediation? Did you measure the decrease of Cd concentration from soil during the studied period (30 and 60 days)?
Response: The conversions of Cd form in the soil during the study period (30 days and 60 days) were determined. Personally, I don't think the amount of heavy metal pollution will change in a short time. The purpose of this study was to discuss the morphological changes in contaminated soil after a short period of phytoremediation.
Line 313: “soil protease activity was less than 0 day (P < 0.05)” -> How can the enzyme activity be negative?
Response: The soil protease activity was less than 0 day (P < 0.05) doesn’t mean negative. After heavy metal contamination, the protease activity of soil decreased, and the detected value was lower than the protease content in contaminated soil.
Line 320: “certain microorganisms” -> What microorganisms?
Response: Fungus, and actinomycetes had the greatest advantages and could improve the soil environment enzyme activities.
Line 321-322: How do you know it is from plant self-healing? What about the microorganism healing? If you did not have the same treatments of Cd levels and time periods without any plants, how can you separate the effect of microorganisms and plants on the enzyme activity?
Response: After 60 days of growth, the plant was almost dead. Therefore, ignoring the influence of rhizosphere microbial community on soil enzyme activity.
Lines 342-346: The two sentences collide with each other. PK fertilizer, oxalic acid, and biochar on Cd content was not covered in this study.
Response: It was checked and amended in the revised manuscript.
Grammar and Miscellaneous
Line 8-9: Suggest to change “soil enzyme activities of three herbs,” to “enzyme activity of the soil planted with three herb species,”.
Response: It was checked and amended in the revised manuscript.
Line 9: Suggest to change “pot experiment was conducted to …” -> “We conducted pot experiments to study…”
Response: It was checked and amended in the revised manuscript.
Lines 89-97: Please provide the references. e.g., Lines 93-95: Guan, S. Y., Zhang, D. S., & Zhang, Z. M. (1986). Soil enzyme and its research methods; Agriculture (pp. 274– 297).
Response: It was checked and amended in the revised manuscript.
Lines 99-100: Why did you not use General Linear Model’s repeated measures? Was this two-way ANOVA conducted separately for each day? If so, please clarify. Also, Tukey’s LSD was only used when the factor was significant (< 0.05) is it correct? If so, please clarify this too.
Response: We believe that our two-way ANOVA statistics is valid and more details were added in the revised manuscript.
Line 106: heavy metal cadmium: I think you can delete heavy metal. Please do in the other part of the manuscript too.
Response: It was checked and amended in the revised manuscript.
Lines 106-107: under various growth cycles-> under different Cd concentration and growing periods
Response: It was checked and amended in the revised manuscript.
Line 110: … Bidens pilosa were low -> Please provide the actual numbers.
Response: The actual numbers were added in the manuscript of the paper.
Line 111: Higher than what? Please clarify.
Response: The content of reduced and residual state Cd are higher than the other two state (the weak acid state and oxidation state) in the contaminated soil, after 30 days and 60 days of phytoremediation
Line 112-118: Why don’t you provide these numbers in Fig. 1 with the bars? I think it will be much easier for readers to understand your results. Also, the format of comma is different from what it is supposed to be (please check this throughout the manuscript).
Response: It was checked and amended in the revised manuscript.
Line 122: … in different cycles -> You are calling day of plant growth (at 30 and 60 days) as cycles, but I suggest to change the term into day of plant growth or alike. “Cycle” can give misunderstandings (Line 173 too)
Response: It was checked and amended in the revised manuscript.
Line 317: “which is compatible with the changing law of soil protease” -> which has similar temporal trend with that of protease,
Response: It was checked and amended in the revised manuscript.
Line 325: delete “, according to Zhao Zhiyu et al.” I think other sentences that has unnecessarily mentioning author names can be removed and simplified too
Response: It was checked and amended.
Round 2
Reviewer 1 Report
The authors have revised the manuscript as Comments, and the manuscript has been sufficiently improved to warrant publication in Sustainability in my submission.
