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In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

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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?

Reviewer #1: Yes

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: I Don't Know

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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.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: No

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This well-designed study provides valuable insights into bacterial dynamics in an A₂O wastewater treatment plant and its impact on the Tama River ecosystem. While the experimental approach and integration of microbial/chemical data are strengths, several areas require enhancement to clarify scientific foundations, methodological transparency, and environmental implications before publication.

See attached documents

Reviewer #2: Line 50, only NOB are mentioned, but to be correct also AOB should be mentioned too in the sentence, otherwise the statement is incomplete (or technically comammox nitrospira could be mentioned, but that is quite specific and not what the MS is about).

Line 52, I think it would be nice to mention the release of nitrous oxide here, but not strictly necessary if the authors prefer not to.

Line 68 to 70, the first half of the sentence simply states that these types of studies are important (without further elaboration), the second half then continues to state that it is also important for conservation and understanding of the bacterial communities along the river. Something seems to be missing or removed from the sentence to lead to this sentence structure. Please try to avoid stating that something is important without explaining why or for what.

Line 137, I think it would be better to report on the nitrogen removal efficiency (and potential S to) of the WWTP rather than just the figure showing the different concentrations. It gives the reader a clearer picture of the effectiveness of the WWTP.

Line 147, could the authors further elaborate on the interpretation of figure 3B? The temporal effects seem much stronger then the differences between the tanks, with for example 4s clustering close together, but for away from for example 13s which are on the other side of box.

Fig 6, How much natural variation is there when using ASVs to study the difference between bacterial communities in natural environments? Fig6 shows overlaps and unique ASVs for every sampling point, it would be interesting to see an additional sampling point that is not expected to be effected by the WWTP (say an S0) to see how much the overlap is with S1 as a consequence of natural variability rather then as a consequence of distance to the WWTP. This could be seen as a control sampling location. While I understand that it is now too late to redesign the study at this level, this question keeps me wondering and makes it more difficult to know how much of the observed differences are due to the distance from the WWTP and how much is due to natural variability. I think it would improve the manuscript if the authors could reflect on this.

Line 218, are they diluted or do other processes also play a role here? How much dilution do we expect on a few km of river, are there any tributary inflows? Is the effect caused by incomplete mixing of river and WWTP at S3 causing a higher fraction of the sample to be coming directly from the WWTP whereas S4 is better mixed? Could other processes explain the observation? Sedimentation/flocculation, environmental or competitive selection, predation?

Line 227, please define what you mean with a recovered bacterial community. The community is still clearly different from S1 (which can also be seen in Fig 6B) what conditions need to be met to consider the community recovered?

The data is available through the figures which is a nice way to present the data, however this also leaves the readers guessing for more exact values as it is difficult to know the exact intensity of a color gradient or the precise height of a bar in a bar chart. It would be better if this data is also available in the SI.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: Yes: Dr. Oussama BOUOUAROURDr. Oussama BOUOUAROURDr. Oussama BOUOUAROURDr. Oussama BOUOUAROUR

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewer comments .pdf

<p>The role of bacteria in wastewater treatment and the impact of treated wastewater on riverine bacterial ecosystems

PONE-D-25-23321R1

Dear Dr. Nishida,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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PLOS One

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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.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

**********

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

Reviewer #1: Yes

**********

Reviewer #1: This manuscript presents a well-designed study investigating bacterial dynamics within an A₂O wastewater treatment plant and their downstream effects on the River ecosystem. The authors have been highly responsive to the previous round of review comments, and the revised manuscript demonstrates substantial improvements in clarity, methodological transparency, and scientific rigor. I appreciate the thoroughness with which they have addressed each concern raised.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: Yes: Dr Oussama BOUOUAROURDr Oussama BOUOUAROURDr Oussama BOUOUAROURDr Oussama BOUOUAROUR

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