Dear Dr. Ulleberg,

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 Jan 30 2026 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.

• A 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 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,

Susmita Lahiri (Ganguly)

Academic Editor

PLOS One

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When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

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I have read the journal's policy and the authors of this manuscript have the following competing interests: Coauthor MB are employed by Columbi Farms AS who has financial interests in the technology developed through the IPN Nutricycle project. All data interpretation and research findings were conducted independently and quality assured by SINTEF to ensure compliance with PLOS one guidelines. The other authors declare no competing interests.

We note that one or more of the authors are employed by commercial companies: Columbi Farms AS and Kytos

a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

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If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

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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

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #1: The manuscript is well written and highlights an important topic related to nutrient recovery and circular bioeconomy. The findings demonstrating that RO concentrate can be stored for six months without pasteurization are valuable. However, a few improvements are required:

Novelty clarification – The introduction should more clearly explain how this study advances existing research on RO/UF-based nutrient recovery.

Experimental limitations – Only one RAS source and batch were studied; please acknowledge this limitation and suggest future validation.

Statistical clarity – Figures should include statistical significance markers (p‐values, error bars clearly labelled).

Microbiological response – The significant viable cell increase after pasteurization needs deeper explanation.

Figures quality – Improve resolution and add clearer legends and units.

Reviewer #2: Your manuscript could be better if you compare your results with those main crops nutrient needs in Norway. Is interesting your foundings but it lacks of the support of the corps needs and more of what farmers need to improve their corps. This comparation will improve your work and give you more bases to have a more complete discussion obout your results. For now, your results are the preliminary part of what you really want to demonstrate with your research.

Reviewer #3: This is a valuable article in sustainable Aquacultur and Agriculture activities. Minor revisions are recommended.

I suggest to create a table for summerizing the chemical and microbial changes throughout the experimental period for both the Treated and the control Nutrient Concentrate.

**********

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

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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Attachments

Attachment

Submitted filename: PONE-D-25-37522.docx

Attachment

Submitted filename: PONE-D-25-37522 (reviewer comments).pdf

Dear Dr. Ulleberg,

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.

==============================  From Academic Editor :  download --> -->-->In addition, please add Data Availability Statement, placed before the Acknowledgment --> -->-->The data that support the findings of this study are openly available in OSF repository at https://osf.io/9bqpc/?view_only=b61606c3dd524ce3a9a3692b7dd7c81e -->--> -->-->While it has been stated as Reference 15, but the addition of the Data Availability Statement (before the Acknowledgment) will not just highlight the source of the data (as asked by Reviewer #3), but also to comply with https://journals.plos.org/plosone/s/data-availability -->--> -->-->Thank you.-->-->==============================?>

Please submit your revised manuscript by Apr 10 2026 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.

• A 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,

Dave Mangindaan

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The manuscript presents valuable findings with practical implications for aquaculture and sustainable agriculture.

Reviewer #2: The arguments in their results are very weak and require further review of the literature. Most of your assumptions lack of technical and literature support in each area of their results. I recommend conducting a more in-depth literary review to support your arguments and the evidence you've found in your results.

**********

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

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Reviewer #1: No

Reviewer #2: Yes:  Gerardo Rodríguez Quiroz

**********

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Attachments

Attachment

Submitted filename: minor revision.docx

Dear Dr. Ulleberg,

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.

From Academic Editor:

Please submit your revised manuscript by May 14 2026 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.

• A 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Assoc. Prof. Dave Mangindaan, PhD, MRSC, AMIChemE.

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: Major concerns:

• Experimental design is limited (single source, single batch; microbiology n=1), reducing reliability.

• Statistical analysis is inadequate; time-series data require more robust methods.

• Claims of “microbial stability” are somewhat overstated given observed fluctuations.

• Mechanisms behind nutrient loss after pasteurization are speculative and need validation.

• No agronomic validation (e.g., plant trials) to support fertilizer applicability.

Minor concerns:

• Improve clarity, reduce repetition, and refine language.

• Correct duplicated figure captions and improve figure presentation.

• Ensure consistency in units and terminology (avoid labeling as purely “organic fertilizer”).

Recommendation:

Major Revision – The study is promising but requires methodological strengthening and clearer interpretation before publication

Reviewer #2: (No Response)

**********

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

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Reviewer #1: No

Reviewer #2: Yes:  Gerardo Rodriguez Quiroz

**********

[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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Dear Dr. Ulleberg,

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 Jul 10 2026 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.

• A 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Dave Mangindaan

Academic Editor

PLOS One

Journal Requirements:

1. 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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #4: (No Response)

Reviewer #5: (No Response)

Reviewer #6: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #4: (No Response)

Reviewer #5: Partly

Reviewer #6: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: (No Response)

Reviewer #5: No

Reviewer #6: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: (No Response)

Reviewer #5: Yes

Reviewer #6: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: (No Response)

Reviewer #5: Yes

Reviewer #6: Yes

**********

Reviewer #1: all comments given are done appropriately. this is important paper in advanced aquaculture field where RAS technique is one of that.

Reviewer #2: (No Response)

Reviewer #4: This work recovered plant nutrients from salmon RAS effluent using a RO system and studied the storage stability of the resulting RO concentrate over six months. It is concluded that RAS NC is stable when stored at room temperature without light for six months, regardless of pasteurization. Some more comments are:

1. Flocculation, UF and RO are used to treat the RAS effluent. More discussion and data on the system should be provided. For example, what are the size, rejection, recovery of the UF and RO units? What are the requirements for plant nutrients in terms of composition and concentration? How is RO concentrate compared to the requirements?

2. Why is pasteurization (heat treatment) carried out at 80 oC for 10 minutes? Is this an optimum condition?

3. Visual assessment of physical characteristics revealed no change in precipitation. Why is precipitation highlighted as the reason for reduced Fe concentration?

4. It is hypothesized that the observed increase in the solution’s pH has resulted in

precipitation of iron and other dissolved ions. This claim needs to be supported with calculation. The pH is increased from 6.97 to 7.37. How much precipitation can happen with this change of pH?

5. With heat treatment, why is the pH increased from 6.97 to 7.37?

6. It is said that EC indicates temporal fluctuations in the degree of mineral precipitation during the storage period. Why are there temporal fluctuations?

7. The viable cell load increased slightly in the unpasteurized control towards the end of the storage period, which may indicate the onset of spoilage. Do this mean the pasteurized sample have longer storage stability?

8. UF is effective in removing microorganisms. Can you use UF to pasteurize the sample, in steady of 80 oC for 10 minutes?

Reviewer #5: The author needs to address these to improve their study:

1. There is substantial research on using RO after effluent treatment, with a strong focus on the resulting concentrate. Pasteurization has also been used to improve the stability of the effluent. So, what is actually the novelty of this work compared to those reported works? What is the main scientific contribution of recovering liquid fertilizer from RAS effluent using reverse osmosis?

2. I also did not find why RO is selected over other nutrient concentration technologies, such as nanofiltration, forward osmosis, membrane distillation, evaporation, or adsorption. There is no systematic approach to selecting RO in their work.

3. What about RAS composition before RO treatment? I found only RAS NC composition and characteristics. They need to compare the composition before and after RO treatment.

4. Figure 1 shows physical characteristics. I was thinking it was something like density and other physical properties. But it is basically only the image of the RAS. This is not physical characteristics. It is just physical appearance. So better to use another caption to avoid misleading about something else.

5. Does the recovered RO concentrate meet basic requirements for use as a liquid fertilizer? I mean, let’s say by comparing the recovered fertilizer with commercial liquid fertilizers?

6. Under the tested dark-storage conditions, pasteurization did not significantly affect the measured microbial load, intact bacterial cell concentration, or bacterial diversity of the RO-concentrated RAS liquid fertilizer. But it is not sufficient on its own to support a broad practical or commercial claim that pasteurization is unnecessary or obsolete as they concluded. They should test specifically for safety-relevant microorganisms, not only total viable cells to provide strong claim on

7. Could the process support circular economy principles in aquaculture-agriculture integration?

Reviewer #6: 1. Please add a flowchart to show more clearly the experimental process.

2. The conclusion that the concentrate is “microbiologically stable” may be somewhat overstated considering the observed increase in viable cell load and reduction in bacterial diversity toward the end of the storage period in the unpasteurized samples. These trends may indicate the early onset of spoilage, and the discussion should acknowledge this limitation more cautiously.

3. Although the study demonstrates storage stability, the practical fertilizer applicability of the recovered concentrate is not experimentally validated. No plant growth trials, nutrient uptake studies, or agronomic performance assessments were conducted to confirm the effectiveness of the product as an alternative fertilizer source. Including preliminary cultivation experiments would substantially enhance the practical significance of the manuscript.

**********

what does this mean?). 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 Privacy Policy

Reviewer #1: No

Reviewer #2: Yes:  Gerardo Rodriguez Quiroz

Reviewer #4: No

Reviewer #5: No

Reviewer #6: No

**********

[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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Liquid fertilizer recovered from the effluent water of recirculating aquaculture system by reverse osmosis exhibits storage stability independent of pasteurization

PONE-D-25-37522R4

Dear Dr. Ulleberg,

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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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sovik Das

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #6: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #6: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #6: Yes

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #6: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #6: Yes

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Reviewer #1: author have given all answered. Accept the paper. its focused on advanced aquaculture system and this system is sustainable.

Reviewer #2: All the recommendations had been addressed. I hope your paper could contribuite to the aquaculture in the further future

Reviewer #6: Authors have added necessary explanations and figures, and they have responded appropriately to all my comments.

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

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Reviewer #1: No

Reviewer #2: No

Reviewer #6: No

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