PONE-D-24-38682Unveiling microbial succession dynamics on different plastic surfaces using WGCNAPLOS ONE

Dear Dr. Oren,

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.

I have two reviews. The first one has sought  incorporation of  technical and methodological details. The first reviewer also provides several comments . I would like them to addressed by the authors in the revision of further consideration. The second review provides suggestions on how the manuscript can be be improvised and made useful to broader audience. The comments  made by the second reviewer are useful and suggest suitable consideration by the authors. 

Please submit your revised manuscript by Jan 09 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Arga Chandrashekar Anil, Ph. D., D. Agr.,

Academic Editor

PLOS ONE

Journal requirements:    When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please include a caption for table 1, 2 and figure 7.  3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. 4. Thank you for stating the following financial disclosure:  [This work was supported by the Israel Ministry of Science and Technology Israel-Portugal collaboration Grant 3-1650 and the Israel Science Foundation (ISF) personal Grant 1556/23.].  Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 5. Thank you for stating the following in the Acknowledgments Section of your manuscript: [The authors are grateful to Prof. Shiri Navon-Venezia for her scientific advice. This work was supported by the Israel Ministry of Science and Technology Israel-Portugal collaboration Grant 3-1650 and the Israel Science Foundation (ISF) personal Grant 1556/23.]We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:  [This work was supported by the Israel Ministry of Science and Technology Israel-Portugal collaboration Grant 3-1650 and the Israel Science Foundation (ISF) personal Grant 1556/23.]. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

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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: Yes

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: Yes

Reviewer #2: Yes

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4. 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 #1: Yes

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: General comment

The subject matter is broadly interesting and although the subject matter (differences in microplastic biofilms) has been relatively well studied, there is some novelty in the use of longer whole 16S reads using nanopore, and the use of weighted correlation network analysis is also interesting. However, there are a number of technical details missing which make the paper as it is currently, unpublishable, and the missing experimental details need to be addressed before the paper can be accepted. The most egregious lacking information concerns how sterility of the experimental setup was achieved, and the handling of DNA extraction & PCR blanks. Without more information on these points specifically, the validity of the entire experiment is called into question. Generally, more justification for the statistical tests used and discussion concerning how different approaches might achieve different results is needed, especially concerning the use of WGCNA. Comparisons to other similar studies and their results needs to be conducted much more comprehensive, especially to other studies which have also used WGCNA.

Specific comments:

• Line 75 – spelling mistake – “litter”

• Why are these temperatures (28 and 35 degrees) chosen? Presumably these are water temperatures, although this should be stated, as well as how this was tracked throughout the experiment. Why so warm? What marine conditions are trying to be emulated? I am guessing conditions of the Herzliya coastal waters are trying to be emulated, since this is where the debris was apparently collected, but 35 degrees seems to be warmer that the water ever gets there (according to seatemperature.net). So why was this chosen? The experimental setup needs to be rationalised.

• Why use inoculated seawater as opposed to regular seawater? What were the conditions of the Herzliya marina at time of sampling? Similar water temperature and salinity to setup? What was the sampling date? How were the debris collected? How were they maintained between collection and inoculation to reduce microbial changes?

• How was all the equipment sterilized before use? Otherwise, what is the point of this inoculation approach if the organisms present in the setup are not of plastic origin?

• Were the plastic/wood/glass beads sterile before adding to the aquarium? If so how was this done. If not, how might the implicate the results?

• Why no PP or PS in the higher temperature situation? What are the sizes of the beads.

• Manufacturers of materials needs to be added in all cases, for setup equipment, plastics used, mesh bags etc.

• What chemical composition were the mesh bags? Were they plastic as well (organza can be a wide variety of materials, not scientifically accurate enough). What was the mesh size?

• Was post-sampling processing conducted in a sterile manner? There is no mention of a laminar flow cabinet being used. How was contamination managed?

• Why were wooden beads only subjected to a completely different extraction procedure. Different kits produce different results. Comparability can be problematic and certainly should be discussed.

• Line 155: How were sequences “compared to the NCBI database”. Using BLAST only? What date/what version of the database

• No mention of extraction or PCR blanks in methods. This is highly problematic for publication and must be addressed before the paper can be published.

• Lines 231-236 this information should really be in methods

• Line 256 – why is the temperature delta 7 degrees on average? I thought this was accurately controlled and kept consistent (although it is not explained how this was maintained and monitored, which again is problematic)

• Why are the modules named after colours? It reads as slightly ridiculous. Why can’t they have alpha/numeric identifiers?

• Line 320 (and Table 2 generally): Relative abundance greater than just 0.1% doesn’t sound like enrichment. Why such a low cutoff?

• Table 2: Regarding the number in brackets, number of associated species generally? i.e. 6 of the total number of Pseudomonas species were specific to PE? Or is 6 species all the Pseudomonas species found in the study? It would be important to show average relative abundance of these taxa to show the reader if they really are enriched or simply being detected by chance here and not on other surfaces. What is the P-value based off? What statistics was done? Indicator analysis? If so explain, if not, what analysis was used and why?

• Why use long read as opposed to something shorter but covering more sequence depth, like V3-V4 with Illumina, which is more commonly used. This difference, including what can be better seen with long reads vs shorter barcoding methods should be discussed! It is one of the main differentiating factor compared to other works and should be discussed in depth

• Line 388-390 – Similarly, since WGCNA is the also a major thing that is new/interesting about the study, discussing previous uses of this and how this newer approach enables new insights is also very important to be discussed. Needs to be expanded significantly

• I would expect a conclusion section, but this is absent

Reviewer #2: Thank you for sharing your manuscript. I found the study valuable in advancing our understanding of microbial succession on different plastic polymers in marine environments. The identification of polymer-specific microbial communities and the successional trends across environments are particularly compelling. However, I believe the manuscript would benefit from some adjustments and additions that explain the significance in greater depth.

In particular, your introduction and discussion sections are quite short and lack depth. Contextualizing the broader implications of your findings and expanding on how they contribute to addressing the global plastic pollution crisis would be beneficial. Below are some suggestions for improvement:

Broader Implications:

- Global Context: While the results are well-documented, the manuscript does not sufficiently situate these findings within the larger context of global plastic pollution. It would strengthen the study to articulate how understanding microbial succession on plastics could influence degradation predictions or provide insight into the long-term ecological impacts of persistent plastic waste.

- Ecosystem Effects: The potential for microbial colonization to alter nutrient cycling, carbon fluxes, or interactions with higher trophic levels is an area that could be explored further. For example, could the dominance of certain genera, such as Alcanivorax, signal broader ecological consequences, such as shifts in hydrocarbon degradation pathways?

- Environmental and Policy Relevance: Emphasizing the environmental consistency of polymer-specific communities is valuable, but further discussion on its significance would be helpful. Could these findings inform efforts to design more sustainable plastics or predict ecological risks across environments?

- Plastic Degradation Potential: The identification of genera like Pseudomonas and their potential to influence biodegradation pathways presents an opportunity to discuss the dual implications of microbial activity: both as a possible avenue for mitigating plastic pollution and as a vector for environmental risks (e.g., transport of pathogens or invasive species).

Deepening the Interpretation of Results:

- Ecological Roles of Genera: While the study identifies key microbial genera, there is limited discussion on their ecological functions or roles in shaping succession patterns. For instance, why do some genera dominate later stages of colonization? Are they exploiting specific metabolic niches or competitive advantages within the biofilm matrix?

- Successional Patterns: The observed patterns are well-described, but their drivers are less clear. Additional insights into the mechanisms driving succession (e.g., nutrient availability, surface properties of plastics) would enhance the interpretation.

Tying Findings to Broader Significance:

I recommend linking your results explicitly to the broader ecological and societal implications in the discussion and conclusion. For example:

- In the introduction, briefly outline how understanding microbial colonization can contribute to mitigating the impacts of plastic pollution or developing sustainable solutions.

- In the discussion, dedicate a section to synthesizing the ecological significance of these findings and their relevance to global efforts to address plastic pollution.

- In the conclusion, consider a forward-looking statement about how this research could inform future studies or practical applications, such as plastic design or environmental management.

Lastly there are some small edits I suggest you make e.g explainign why you chose the temperatures you used in your incubation experiments. Furthermore, the use of glass and wood as comparative surfaces in your experiment is a sound choice, as these materials are often used as controls in Plastisphere studies. However, to ensure that the experimental design is clearly understood, I recommend explicitly stating that these materials are being used as controls. This would help to clarify the intent behind including them and would better contextualize the significance of the microbial communities found on plastic surfaces in comparison to those on other materials.

By elaborating on these points, your manuscript could move from being a strong descriptive study to one with a larger impact on how we understand and address plastic pollution in marine environments.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-38682R1Unveiling microbial succession dynamics on different plastic surfaces using WGCNAPLOS ONE

Dear Dr. Oren,

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.

I have one minor point to be made regarding point 12 of the comments offered by reviewer 1: the authors can  include a statement to manuscript and not simply brush aside the obvious.

Please submit your revised manuscript by Mar 05 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,

Arga Chandrashekar Anil, Ph. D., D. Agr.,

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

I have one minor point to be made regarding point 12 of the comments offered by reviewer 1: the authors can include a statement to manuscript and not simply brush aside the obvious.

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Unveiling microbial succession dynamics on different plastic surfaces using WGCNA

PONE-D-24-38682R2

Dear Dr. Oren,

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,

Arga Chandrashekar Anil, Ph. D., D. Agr.,

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: