PONE-D-24-56633Novel method for combining microbial bioremediation with static magnetic fields to remediate mercury-contaminated SoilsPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Specific comments to the authors

The writing could benefit from clearer organization. Some sections, e.g. materials and methods, lack clarity, descriptions, and smooth transitions, making it difficult for readers to follow the work done. The study should provide more details on the experimental design, including controls and replicates. This information is crucial for assessing the reliability and validity of the results. The methods of statistical analysis used to evaluate the data should be clearly described. Without this, it is challenging to assess the robustness of the conclusions drawn from the results (specific tests, data transformation, etc.). There should be a stronger connection between the Materials and methods and the Results sections. For instance, specific details about how the treatments were applied should directly correlate with the results presented. Despite the promising findings presented, the work needs to be deeply revised, addressing these suggestions could enhance the clarity, rigor, and impact of the research.

Abstract:

1. I suggest revising the spelling and formatting, respecting the spacing throughout the text.

2. Do not use abbreviations, particularly in the case of bacterial isolate nomenclature.

3. When starting to refer to the results obtained, emphasize the observed effect (increase, decrease, etc.) on Hg removal instead of ‘The results demonstrated the impact of SMF on...’.

4. The statement that the combination of SMF and P. stutzeri ‘accelerated’ metal remediation in sterile soil, compared to SMF alone, suggests that the effect was observed in a shorter treatment time. If this was the case, I suggest clarification, otherwise ‘accelerated’ should be replaced by increased/promoted removal by x %, relative to SMF alone.

Introduction:

I suggest a clear delimitation of the impact of the problematic addressed, an expansion of previous studies (e.g. on the presence of resistance genes in the bacterial isolate used, use of SMF and their main results). I suggest reorganizing the section, including a bibliographical update and finally detailing clearly how the present article aims to contribute to solving the problem in question (mentioned vaguely in lines 75-76, 81-82 and 93-94). Additionally, data should be provided on the Hg levels (ppm, mg/kg) permitted/suggested by any regulatory or health control organism.

In particular:

1- Line 42, where it is argued that mercury is a bioaccumulative ‘toxin’, I suggest revision of the term. Hg can be classified as a ‘toxic’ metal, but not a toxin, which presupposes a biological origin of the compound in question. The above-mentioned citation of reference [1] does not claim that it is a toxin. I suggest adding its classification according to regulatory or health agencies/organizations, e.g. IARC.

2- I suggest broadening the discrimination between the geogenic origin of Hg and its use and consequent redistribution due to anthropogenic activity, perhaps by replacing citation 2 (line 44) or adding new citations. It would also enrich the introduction if the regional and global impact of the problem addressed were highlighted.

3- I suggest reorganizing what is included between lines 48 and 51, replacing citation 6 with a more current one, clarifying the impact of the different states that the metal can have, the predominant forms in the environments of interest and the relative toxicity of each form.

4- Line 59, replace ‘toxicant’ by ‘toxic metal’ or ‘pollutant’.

5- Line 61 mentions the ability of bacteria to convert Hg into ‘non-toxic’ forms, clarify which would be the starting (toxic) forms and those derived from microbiological transformation. Perhaps this should be placed on line 66, after quote 18, where these forms are mentioned without the clarification of their toxicity.

6- Line 63, reference is made to the mer operon as one of the ‘most intriguing’ aspects of resistance to this metal, however its components, mode of action and relationship of the bacterial isolate selected (P. stutzeri) with this operon (if there are gene components previously found/described in this isolate, etc.) are not clearly detailed.

7- Lines 72- 74, P. stutzeri is mentioned as one of the bacterial isolates previously used in Hg remediation studies, I suggest including the main results to be highlighted in these studies, allowing a priori to know the effectiveness of this isolate in the remediation of sites contaminated with this metal. In this point, quotes 25 and 26 should be included (adding concrete removal data), reorganize.

8- The objective of the work should be reformulated (unifying what is stated in lines 75-76, 81-82 and 93-94), detailing the context of Hg bioremediation and the novelty of the application of SMF. To state clearly how the present study intends to contribute to solve the problem of interest.

9- Lines 82-88 Rewrite reference to previous studies with SMF, highlighting the main results (numerical data, % removal, etc.). Line 85 mentions that SMF ‘can accelerate the treatment of water’, rewrite, mentioning removal/remediation and data. A study analyzing the role of SMF in wastewater is mentioned (citation 27), and previously no wastewater was detailed as a source of Hg contamination.

At this point in the introduction, it is not clear which Hg contaminated matrix is of interest to the present work.

10- Lines 89-91 The fact that there are previous studies combining bacterial isolation (P. stutzeri) with SMF for the treatment of contaminated sources (citation 25) detracts from the originality of the work, despite the differential nature - organic vs. inorganic - of the pollutant in question.

11- Line 92 I suggest changing the term ‘decontamination’ to ‘remediation’.

Materials and methods

1- Lines 97-101, give more details about the area sampled, why it was selected, land use history, coordinates, number of samples taken, extent of area sampled.

2- Lines 102-104, detail which properties (physical and chemical) were determined and provide data of the correspondent equipment used.

3- Line 106, detail which heavy metals and major elements are referred to, range of detection/quantification, units of expression of the results.

4- Line 111, clarify whether the analyzer used (DMA-80) measured total mercury or allows the speciation of this to be quantified, brand of equipment.

5- Lines 114-118, up to this point it is not clear why they decided to add Hg instead of working with the reference concentrations in the sampled area, or if they have not quantified the metal in these samples. It is not clear at what concentrations of the metal they want to work with (ppm, M, Mm), by adding 148 mg HgSO4 to 100 ml H2Od you have prepared a 5 mM solution of the metal (or of the salt, whose molecular weight is predominantly contributed by mercury), you should clarify what are the desired working concentrations, why they have been selected and how they were prepared. In line 117, it is not understood what is meant by ‘at 50-gram dose’, as it cannot be equivalent to the concentration of Hg present in 1 kg of soil when adding that solution (as understood in the text). I suggest revising and rewrite providing all the necessary data for the understanding of the selected methodology. I suggest modifying the title of this subsection, I do not consider the term ‘ground doping’ to be appropriate.

6- Lines 119-125, If the bacterial isolation was obtained by working group members, which I deduce from the reference provided, this should be clarified. Which sequences are available in GenBank, 16S?, complete genome sequence?, provide the corresponding accession/bioproject numbers. If there is a history of the presence of genes involved in Hg tolerance/resistance in this isolate add it beforehand in the Introduction section.

7- Lines 127-141, in the bioaugmentation assays the authors should explain why they do not use the liquid inoculum of the bacteria (or sterile medium in the corresponding treatments) and do the washes mentioned in lines 127 and 128. In this respect, the composition of the media used (g/L), incubation/growth conditions of the bacteria, CFU/ml of the culture that proceeded to centrifugation should be included. This centrifugation was in duplicate? what is the volume of growth media centrifuged?, what was the total bacterial count used?, 7x1010 CFU/ml, 1.75x109 CFU/ml?. In line 130 add the abbreviation ‘rpm’.

8- Line 132: 20 mg of soil in 40 ml of medium (with or without bacteria) corresponds mainly to a test in liquid medium (the volume of soil used is almost negligible), which considerably biases the conclusions that can be drawn for this matrix, on which the results are presented in the Abstract and throughout the work.

9- From what is written in lines 134 to 136 I interpret that there are 3 different treatments (in flask 1, flask 2, flasks 3+4), in this description the sterile soil without bioaugmentation is not included as a control, which I suggest adding. So far it is interpreted that the n of treatment replicates is 1 (for the first two treatments) and 2 for the non-sterile soil without bioaugmentation (flasks 3 and 4), which makes it very weak statistically. Whether what is stated in line 139 corresponds to replicates of these treatments should be clarified above, or whether it refers to temporal replicates of the trials. It is also not clarified that the soil sample used corresponds to the soil added with Hg, clarify, explain in detail all the procedures so that those who did not carry out the work can clearly understand the methodological steps followed.

10- Lines 138-139 ‘Bacterial counts and mercury analysis were conducted on a weekly basis’, detail here the methodology used for this purpose, did they quantify total Hg, speciation? If these parameters were measured weekly for a total period of 28 days there should be 4 sampling and data collection times (does not correspond with the times shown in Fig. 1-4), please clarify.

11- Lines 140-141, to which aseptic conditions do you refer? in which procedure were they used? through which materials or equipment were they achieved?

12- Lines 142-152, describe the equipment used to expose the samples to MFS, I suggest attaching a picture of it.

13- Lines 152-153 ‘prior to the commencement of the observation period’, refers to the soil samples prior to disposition in the previous bioaugmentation test, it is not easily detectable if it was the same test (as interpreted in the Abstract) or not, needs to be clarified, reformulated, at what time the soil samples were subjected to this field, pre or post dilution in the 40 ml of medium, I suggest to attach a picture of it.

14- Lines 156-162, why are treatments described again, can this section not be summarily unified with the one previously describing the different treatments in the Flasks, why are they now ‘Bottles’ 1-4, when I understand that they refer to the treatments already described.

Results

1- I suggest presenting the different results in accordance with what is described in the Materials and Methods section, without the divisions presented in the subtitles of lines 202-203, 224-225, 246.

2- Line 223 mentions a treatment that was not described in the Materials and Methods section, ‘non-bioaugmented sterile soil’.

Discussion

1- Line 264, I suggest replacing the term ‘decontamination’ with remediation.

2- Do you consider SMF as an ‘economically viable’ methodology? in which cases? for which matrices? would it be possible to use it on a large scale? discuss these, pros and cons of this methodology, potential impact on native microbiota. It remains to add to this section previous work carried out to remediate Hg, its main results, and contrasts with what is observed in the current work. A considerable part of the discussion refers to other contaminants, which are not the one of interest, nor is there any discussion on the mechanisms that allow P. stutzeri LBR to remediate the metal.

Reviewer #2: I have read the present manuscript authored by Naima Werfelli and colleagues with great interest and attention to detail. This manuscript explores a novel approach to enhancing microbial bioremediation by incorporating static magnetic fields to address mercury contamination in soils. Mercury, a persistent environmental pollutant, poses significant risks to ecosystems and human health.

Attached, I have provided some comments and suggestions that could help improve the article.

1. Consider reorganizing the content to progress from general mercury contamination issues to the specific focus of your study. For example:

• Mercury as an environmental pollutant (current impacts and sources).

• Current remediation challenges (limitations of traditional methods).

• Potential of bioremediation (your approach and objectives).

2. Some sentences are lengthy and contain multiple ideas. Consider breaking them down to improve clarity. For example: You mention that "a variety of bacteria have the capacity to resist toxic forms of mercury and subsequently convert them into non-toxic forms."

• It would be helpful to briefly explain the mechanisms involved in these conversions.

• You could also mention if P. stutzeri LBR has been compared with other strains for effectiveness.

3. The study objective is stated clearly at the end, but consider emphasizing what practical applications or next steps could follow from this research.

4. The material and method section is well-structured, but it could benefit from clearer subheadings and a more logical flow. Consider structuring it as follows for better readability:

• Soil Sampling and Preparation

• Soil Analysis (Physical, Chemical, and Heavy Metal Concentration)

• Bacterial Strain and Inoculation

• Bioaugmentation Tests

• Static Magnetic Field (SMF) Application

• Monitoring and Analysis

• Statistical Analysis

5. Specify if the mercury solution was mixed uniformly to ensure even distribution in the soil.

• Clarify why pH 7 was chosen and its significance in mercury solubility/stability.

• Indicate whether control groups were included for comparative purposes.

• The bacterial inoculum concentration (70 x 10⁹ cells) should mention if it was determined through OD measurements or CFU counting for better reproducibility.

• Mention if post-hoc tests were applied to detect significant differences between groups.

• Instead of "significance level of p < 0.05," consider rephrasing to "statistical significance was set at p < 0.05."

6. Ensure correct citation formatting, e.g.,

• "[4 , 5]" should be "[4,5]" (remove spaces before commas).

• Proper reference order for standards like "AFNORNF ISO 11265" should follow a uniform style.

• Some paragraphs contain excessive detail; simplifying them can make the section more reader-friendly.

• • Increase the clarity of tables and charts.

7. Summarize the key findings more clearly, highlighting the effectiveness of bioaugmentation and SMF's impact.

8. When presenting comparative results, such as the 49.36% and 72.49% remediation rates, consider briefly explaining why sterile soils showed a higher improvement compared to non-sterile soils. This helps provide context for the reader.

9. Clarify mechanisms where necessary; for instance, when discussing microbial competition in non-sterile soils, elaborate briefly on how this impacts mercury uptake.

10. Instead of saying "published studies," specify which studies to strengthen credibility.

11. A brief mention of potential future experiments to confirm these mechanisms could enhance the scientific rigor of the document.

12. Expand on future research opportunities, such as optimizing SMF parameters (intensity, duration), exploring its application in real-world contaminated sites, and assessing long-term environmental impacts.

13. Ensure uniform formatting in terms of punctuation, spaces, and abbreviations.

14. For example, journal names should follow a consistent format (e.g., Atmos. Environ. or Atmospheric Environment, but not a mix of both).

• Instead of writing the DOI as a URL, it's better to present it in the standard format: doi:10.xxxx/xxxx.

• Example:

https://doi.org/10.1016/S1352-2310(97)00293-8 → doi:10.1016/S1352-2310(97)00293-8

• Ensure a consistent style for author initials (e.g., "W.H. Schroeder" vs. "WH Schroeder").

• Remove unnecessary spaces between initials.

• Ensure all references have complete citation details, such as volume, issue, page numbers, and publication year.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Comments to the Editor.docx

Attachment

Submitted filename: Reviewer comments.docx

PONE-D-24-56633R1Novel method for combining microbial bioremediation with static magnetic fields to remediate mercury-contaminated SoilsPLOS ONE

Dear Dr. Abbes,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 10 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Naga Raju Maddela, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: Review Comments to the Author

Overall Assessment

This study presents a novel and promising approach to mercury bioremediation using Pseudomonas stutzeri LBR combined with static magnetic fields (SMF). The methodology is largely rigorous, and the findings could significantly advance sustainable soil decontamination strategies. However, revisions are required to address inconsistencies, clarify methods, and ensure compliance with journal policies.

Major Technical Concerns

Inconsistent Results Reporting (Page 2, Lines 33–39; Page 10–12)

The Abstract states SMF accelerated remediation by 49.36% (non-sterile) and 72.49% (sterile). In contrast, Results sections report 81.77% (non-sterile) and 87.17% (sterile) reductions in Hg concentration.

Action: Reconcile these discrepancies. Define how "acceleration" is calculated (e.g., percentage increase in remediation rate vs. control). Provide a unified metric throughout.

Unclear Experimental Controls (Page 7, Lines 133–141)

Flasks 3–4 are described as "non-sterile soil + M9" without specifying if P. stutzeri LBR was included. This ambiguity complicates interpretation of bioaugmentation effects.

Action: Explicitly label control groups (e.g., *"Flask 3: Non-sterile soil + M9 (no bacteria)"*).

Unsupported Mechanistic Claims (Page 15, Lines 331–334)

The proposal that SMF enhances mercuric reductase (MerA) activity lacks experimental validation (e.g., enzyme assays).

Action: Include MerA activity data under SMF exposure OR reframe this as a testable hypothesis (e.g., "SMF may stimulate MerA, warranting further study").

Methodology & Reporting Improvements

Ambiguous Hg Dosing (Page 6, Line 118)

The protocol (148 mg HgSO₄ in 100 mL solution added to 1 kg soil) does not specify the final soil Hg concentration.

Action: State Hg concentration post-doping (e.g., "resulting in X mg Hg/kg soil").

Background Metal Levels (Page 9, Table 1)

Elevated Fe (6504 mg/kg) and Cr (341.03 mg/kg) suggest potential soil contamination. Their impact on Hg remediation is unaddressed.

Action: Briefly discuss whether background metals influence Hg bioremediation dynamics.

Data Availability (Throughout)

Raw data (bacterial counts, Hg measurements) are not deposited in a public repository.

Action: Add a Data Availability Statement (e.g., "Data are available in [repository name] at [DOI/link]").

GenBank Accession (Page 6, Line 125)

The P. stutzeri LBR sequence is cited as deposited in GenBank, but the accession number is missing.

Action: Provide the GenBank accession number in the Methods.

Language & Presentation

Terminology & Notation:

Italicization: Inconsistent formatting of bacterial names (e.g., Pseudomonas stutzeri vs. Pseudomonas stutzeri).

Action: Italicize all genus/species names (e.g., P. stutzeri LBR).

Exponents: Bacterial counts use "10º" (e.g., 27 × 10º CFU/mL) instead of "10⁹" (Page 14, Lines 298–302).

Action: Correct to "10⁹" throughout.

Redundancy: Phrases like "non-bioaugmented non-sterile soil" (Page 10, Lines 210–214) are repetitive.

Action: Simplify to "non-bioaugmented soil."

Typos:

"borne on plasmids" → "born on plasmids" (Page 3, Line 64).

Ethics & Policies

Funding Declaration (Page 16, Line 345):

Funding sources are not acknowledged.

Action: Declare funding (e.g., "This work was supported by [Agency]") or state "No funding was received."

Recommendations

Strengths: Innovative SMF application, robust bioremediation outcomes (>80% Hg reduction), and clear potential for environmental impact.

Revisions Needed: Address major technical inconsistencies (Point 1), clarify methods (Points 2, 4, 7), and comply with data-sharing policies (Point 6).

Suggested Minor Edits: Improve terminology consistency (Point 8) and correct typos (Point 9).

Decision: Major Revision required prior to reconsideration.

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Reviewer #3: Yes:  Dr. Mutiu A. Alabi

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Attachments

Attachment

Submitted filename: Peer Review Report for PONE.pdf

Novel method for combining microbial bioremediation with static magnetic fields to remediate mercury-contaminated soils

PONE-D-24-56633R2

Dear Dr. Abbes,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Naga Raju Maddela, Ph.D

Academic Editor

PLOS ONE

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