Effect of In Situ Bioremediation of Soil Contaminated with DDT and DDE by Stenotrophomonas sp. Strain DXZ9 and Ryegrass on Soil Microorganism

Round 1

Reviewer 1 Report

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Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The topic and the approach of the study are interesting. Although the results presented could be considered complementary to a previous study of the authors, I believe that they could be sufficient to be presented as an autonomous new paper. However, the structure of the MS and the presentation/explanation of the results are insufficient ambiguous and need a major revision.

The Title and Abstract should be revised in order to be more to the point. Since the bioremediation results in terms of removal efficiency, is background information that was presented in a previous study of yours, and the focus of this study is complementary to those results, this must become evident and clearly stated. Otherwise, the study could be considered as repetition to a great extent. The Title could be e.g. “ Alterations on microbial communities, structure and enzymatic activity during combined microbial-plant bioremediation of contaminated with DDT and DDE”.

Likewise, the starting sentence of the abstract should be reversed to “ In the present study the changes in the microbial populations, the enzyme activity and bacterial community structure in contaminated soils was investigated during the bioremediation of using Stenotrophomonas sp. strain DXZ9 and ryegrass.”

The Introduction section must be revised accordingly, referring to the previous study and focusing on the aim of the study.

The Results section is rather poor and must be improved since results are presented resembling a technical report, with no correlation made or conclusion drawn.. According to the instruction provided in the template of the journal in the Results section “…a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn...” should be provided.

As such, par. 3.1 that provides background information that was presented in the previous study of the authors (ref. 18) should be presented differently noting that they are results presented previously. Table 1 should be, in my opinion, revised, providing only the initial concentrations of the substances, and for the next sampling periods, the relative decrease should be estimated and provided instead of the concentrations. Then those results can be briefly explained as it was made in the initial study, noting the differences among handlings and the assumption/conclusions of the previous study.

Then for all the next paragraphs, all results presented should be correlated with the degradation effect and explained in comparison.

The Discussion section must also be revised, focusing on discussing the findings of this study (and not the previous one) in comparison to other studies using microbial, plant, or combined bioremediation techniques. As such the focus should be on the changes of microbial structure, population size, and enzymatic activity and not on the actual effect of the bioremediation i.e. the degree or rate of the degradation of the pollutants.

All figures are of poor quality.

The use of language needs improvement. It is strongly advised the MS be checked by a native speaker.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript entitled "Effect of in situ bioremediation of soil contaminated with DDT and DDE by Stenotrophomonas sp. strain DXZ9 and ryegrass on soil microorganism" by Xie et al contains fundamental errors that cannot be rectified through revisions:

• The authors throughout the manuscript wrote wrong italics for scientific names
• The grammar/ language of the manuscript does not infer any meaning at many places (the comments as given earlier are not addressed yet).
• The expression of the units are not similar throughout the write-up
• Part of the present manuscript is already published earlier by the author (for example pot experiment) making the entire work more dilute and nothing more than a small extension of previous work.
• The most important section of the manuscript "Effect of bioremediation on soil microorganism quantity" have some UPGMA tree. Unfortunately, this is not a standard way to express microbial community. This is just a qualitative study on the community and does not infer any identification of the community before/ after bioremediation. The population change is majorly hypothetical.
• The conclusion of the work is "Bioremediation represented promising technology for decontaminating and restoring the ecosystem in sustainable manner, the characteristics of microbial community can directly display the ecological function of environmental system, it could be used to evaluate the effect of bioremediation on soil microorganism."; from this, it can be easily understood the focus of the manuscript is microbial community change! But is it an improvement? or another kind of interaction? Without identification, nothing makes it clear in the study.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have corrected the mistakes detected and they have followed most of the recommendations given. Therefore, as a consequence of the changes and corrections incorporated in this new version, the scientific quality of the manuscript has considerably improved. However, the figures are still of low quality.

Reviewer 2 Report

The revision of the MS is sufficient. Figures 4-7, are still of poor quality, though, please revise.

Reviewer 3 Report

The authors now have made substantial changes and the manuscript looks much better than the earlier version.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript entitled "Effect of in situ bioremediation of soil contaminated with DDT 2 and DDE by Stenotrophomonas sp. strain DXZ9 and ryegrass on 3 soil microorganism" by Xie et al address a very important and timely topic DDT-DDE bioremediation, which may have a big general interest among the audiences. However, the manuscript is written in a way that makes it hard to read. The sentences are not complete, often they are repetitive, and the methodology is not sound enough for the claim. Here are some of the points among many:

1. Line no. 34 "where agricultural usage was banned in 1983" and "historical residues", line no. 43-44, Line no. 46 "are the focus of debate in the world", Line no. 52-54, Line no. 107-110 do not imply any meaning. There are many more other than these where the big, long sentences are not generating any meaning.
2. In many parts of the manuscript, for a single explanation, an unnecessarily huge number of references are added; some of which are Line no. 37, Line no. 58, and more. 
3. The referencing is not fine in many places; for example, Line no. 65 "Megharaj" may be replaced as Megharaj et al.
4. Line no. 146-147 mentions a table but the table is not in place.
5. Many parts of the manuscript have jumbled up words that are hard to understand again; for example Line no. 40 in soil in China, Line no. 50 Inoculation highly effective degrading bacteria, Line no. 72 remediation contaminated soil, Line no. 134 Soil bacterial community structure diversity, and more.
6. The subheadings need to be improved; for example 2.1 "Supplies and Chemicals" may be changed to "Microorganism and Chemicals". Stenotrophomonas sp. strain DXZ9 is referred to be described in reference no. 36 but the accession number needs to be mentioned. Microorganisms need to be mentioned first, then the soil parameters and other chemicals. 
7. "2.4 Microbial enumeration and enzymatic activity of tested soil" is the base of the study but, only microbial load is counted, sequencing or community structure is not mentioned. Only microbial enumeration is not sufficient to detect a community structure change.
8. The enzyme activity in soil that has been studied in this manuscript is polyphenol oxidase, urease, catalase, and dehydrogenase". Why particularly these 4 were chosen are neither mentioned.
9. The UPGMA phylogenetic tree does not infer any community.
10. The pot experiment design of ryegrass is neither mentioned. Reference no. 36 is a work done by the authors. The present manuscript is just a part/ extension of the same work. However, a detailed soil microbial community analysis is missing.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The present study examined the changes in the structure of the bacterial community contaminated with DDT and DDE soil and on the overall enzymatic activity of microorganisms, during the bioremediation process via Stenotrophomonas sp and ryegrass, either solely applied or in combination. The experimental part in terms of DDT and DTU removal efficiency during different handlings as well as the mathematical modeling of the removal process was presented in a previous study (ref. 36). As such Table 1, presents the results of figure 2 of ref. 36 and for that reason the authors include those results in the Materials and method and section.

In my opinion, part of this background information (e.g. the % of reduction and reduction rate) should indeed be included in the MS with reference to the already published study and the authors should elaborate on the new information generated which provides complementary knowledge to the previous study. The results of the present study, on the other hand, while being interesting and presented rather well are not efficiently discussed and explained. I believe the overall structure of the MS should be reconsidered.

Abstract:

 The whole abstract should be rewritten. The sentences are too long and complicated, with many grammatical and syntax errors making it hard for the reader to follow. E.g. Lines 9-10: change to “ In the present study the bioremediation of contaminated soils using Stenotrophomonas sp. strain DXZ9 and  ryegrass was investigated, focusing on the changes in the microbial populations, the enzyme activity and bacterial  community structure in the soil”

Line 31: DDT, Please explain abbreviations when appearing for the first time in the text or in an appendix

Line 35: What is meant by “historical residues”?

Line 36-37: Please rephrase avoiding repetition of information

Line 37: DDE, Please explain abbreviations when appearing for the first time in the text or in an appendix

Lines 39-40: “...concentrations of DDT and DDE that have been detected in soils in China are 1.92 mg·kg-1, and 0.84 mg·kg-1 [10,16], respectively.” instead of “The maximum residual concentration of DDT is 1.92 mg·kg-1, and the maximum residual concentration of DDE is 0.84 mg·kg-1 [10,16] in soil in China.”

Lines 42-44: Unclear meaning. Please rephrase

Lines 45-46: Please rephrase

Line 46: “ The plant-microbes combined remediation of soil” instead of “ The plant-microbial technique”

Lines 49-50: Is this really new? It seems that is quite some literature on the topic

Lines 56-72: This part is very generic. You should probably focus on the remediation via bacterial species such as Stenotrophomonas and plants, either solely or in combination.

Line 73: “… the combined remediation of contaminated soil was studied using the hyper-accumulating perineal plant ryegrass and the DDT degrading bacterial strain Stenotrophomonas sp. DXZ9…”  instead of “…the combined remediation contaminated soil 72 has been done with hyperaccumulator ryegrass and DDT degradating strain DXZ9…”

Lines 72-79: Is this one sentence? Please rewrite, focusing on the novelty of your study.

Materials and methods:

The section must be partly rewritten since important information is not actually provided (e.g. information about the origin and handling of the microorganism, information about the plant, the soil samples etc.) whereas results are presented that should be omitted.

Lines 85-87: Please omit

Lines 87 – 88: Please move information to a separate paragraph entitled e.g. “Microorganisms” where you should provide also all background information on the bacterial strain used (isolation, maintenance, pre-culturing, etc.)

Lines 88-89: Please move information to a separate paragraph entitled e.g. “Experimental site ” or “Soil samples” or sth similar providing information on the type, origin, etc. of the soil samples.

Please add a paragraph providing information about the plant

Lines 91-92: Please omit repetition of information. “…of six treatments with 5 replicates each, namely…” instead of “…of six treatments with 5 replicates each, The 30 pots were divided into the six treatment groups, and the six treatments were…”

Lines 92-93: Please provide at least some information on what the different letters stand for!

Lines 101-115: Please move to the results section

Line 131: Please analyse the abbreviation (DGGE) either here or in the appendix  

Lines 132-133: Please provide some info with respect to primers used, bioinformatics analysis etc.

Line 136: How about the initial structure of the soil population? It’s quite crucial to provide this info since it is the starting point for all handlings!

Line 139: Please omit “In this paper,”

Lines 146-195: Please concentrate this information in a few lines only, providing the outlook of the experimental results of reference 36 concerning the reduction of DDT/DDE, and focusing of the comparison (and advanced efficiency) of combined B/G handling with the other handlings.

Lines 202-203: Figure 1 does not only illustrate microbial population chances of the combined handling but of all handling and controls, too.

Line 203: bacterium instead of strain

Line 203:” …has greatly…” instead of “…was greatly…”

Lines 216-218: Please rephrase

Lines 339-342: Please move info in the respective M&M section

Figure 3: The quality of the photo of the electrophoresis gel is rather poor. I am afraid that the info depicted in Figure 3b is not clearly detected. Figure 3c: shouldn’t handling 1 and 4 (containing S) be more closely related?

Legends of Figs.4, 5 and 6: “.. in the bacterial…” do you mean in the “bacterial population structure?”

Lines 428-484: The discussion is rather generic. More emphasis should be given on the explanation and correlation of the specific outcomes of the study with previous studies. For example, how does bacterial colonization of contaminated soils together with plants affect fungal, bacterial, etc. populations?

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

A report for: ijerph-1489271 Effect of in situ bioremediation of soil contaminated with DDT and DDE by Stenotrophomonas sp. strain DXZ9 and ryegrass on soil microorganism

I have reviewed the manuscript that summarized the bioremediation of contaminated soils using Stenotrophomonas sp. strain DXZ9 and ryegrass. This type of studies about bioremediation are always interesting, spacially if author stated it represented promising tools for decontaminating and restoring the ecosystem in sustainable ways without causing much harm to the biodiversity,. The subject fall within the general scope of the journal and the title clearly reflects the contents of the paper. So, I am afraid it is difficult to recommend this manuscript for publication in the present form; some paragraphs could be improved as suggested below.

Keywords: Phytoremediation; Ryegrass; Bioremediation; Soil microorganism; Organochlorine pes- 27 ticide. Change the keywords. Not use the words of the title as keywords

-Line 37. s [2,27,39,43,45,]. I'm not sure this is the way the journal demands

-Line 72-80. It is neccesary to rewrite, since what appears is a kind of summary.

-Line 83 ..were purchased from Shenyang Research Institute of Chemical Industry, China. It is impoirtant?

-Figures 1 to 6 Essential, it is necessary to improve it

-Line 449-484 Sentence too long, fragment it

-Please, I suggest to to rewrite all the conclusions. -Line 486-488 The removal rates were to 81% for DDT and 55% for DDE (69% for both) in Treatment 486 S+G+D+B, the populations of bacteria in the soil increased sharply, and microbial activity 487 was remarkably improved in the bioremediation with ryegrass-microbe. Rewrite since it is perhaps the most important thing in the article 

I wish those changes will contribute to improve your paper.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors, though the revised version of the manuscript has addressed several points suggested earlier, but still, there are many points that need to be taken care of for further consideration, which is as follows:

1. The reference numbers inside the manuscript do not follow any style (for example, 1, 2, 3..... and so on; it is arranged totally haphazard)
2. Several lines throughout the manuscript do not have any reference, where proper references need to be added (line no. 33, 41, 46, 49, 66, 69, 417)
3. In several parts of the manuscript, one line has been referred to with an unnecessarily high number of references, please choose the proper and correct one, or else explain them (Line no. 48, 55)
4. The subheadings at many places do not create any meaning; for example "Microorganism Strain DXZ9" may be replaced as simply "Study Microorganism", "Microbial enumeration and community structure, enzymatic activity of tested soil" as "Microbial community structure, and enzymatic activity of tested soil", "Data analysis and statistical methods" as "Statistical Analysis". 
5. The methodologies are not well explained. Specifically, sections 2.2, 2.3, and 2.4 are just cited with the references. The authors may explain it more so that study can be replicable. 
6. The authors are suggested to give the pot experiments images of the 2.3 experimental design set.
7. Table 1 is unnecessarily given two times; once as Table 1 and another Table 1. Cont. Please make them as one. The a, b, c in the values of the table are not explained. Please add that too.
8. As mentioned earlier and not addressed till now, The UPGMA phylogenetic trees are given in different study sets; but in none of the cases, the phylogenetic information of the bacterial community is written. Please write down the community composition from the 16S sequencing results.
9. The "Discussion" section needs thorough improvement; it is very hypothetical and theoretical now. Please interrelate the discussion with the result data. 
10. The "Conclusion" section needs to be to the point, informative concluding the results obtained along with some future prospects. 

Reviewer 2 Report

I do not think that the revision is sufficient and it seems to have been made rather hasty. The results of this study are complementary to a previous study and therefore in my opinion these results should be used explanatory and complementary to the previous ones and emphasis should be placed on describing a possible mechanism rather than just descriptively citing results and comparing them with other studies (not that relevant in some cases. You talk about microbial populations and plants. How about their metabolisms, interactions (among microbial species and between microorganisms and ryegrass)? You study enzymatic activity but you hardly discuss the results. The discussion is in general; somewhat talkative, providing at several points information that is not directly related to the purpose of the study. I think it should be rewritten. Moreover, there are also many problems with using the language. I suggest the text be revised in its entirety by a local speaker Some comments below

Line 14-15: It’s rather removal efficiency than rate

Lines 15-21: Too long sentence and hard to follow the meaning. Pleas rephrase

Line 22: Who/what represented?

Lines 22-23: “… it is very significant to propose new approaches and technological bottlenecks to promote DDT biodegradation” No clear meaning, please rephrase

Lines 41-42: I do not think that this was the target of the study, since those results are presented in your previous studies. The elucidation of microbial activity and dynamics is the actual target

Line 44: please omit “in the world”

Line 47: “ was proven to be quite promising” instead of  “..was promising.”

Lines 73-75: Please rephrase

Line 76-77: I do not think that this argument is correct

Lines 124-125: Please omit since analyses is provided below

Lines 416-422: This information should be included in the introduction section, not the discussion

Lines 422-424: Please rephrase

Line 427-429: This argument is generic. In order to be consistent with the above mentioned information you should compare the remediation results among ryegrass handling and sole bacterial handlings.

Line 438: Do you mean “it was shown”?

Line 441: was nearer? What does that mean?

Lines 445-446: have you also taken into account the effect of root secretion in your study?

Line447:  Is the chemical structure of chlorpyrifos similar to DDT?