PONE-D-23-17073Screening and diversity of culturable HNAD bacteria in the MBR sewage treatment systemPLOS ONE

Dear Dr. Li

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PLOS ONE

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Editor Comments:

Nitrogen removal is a crucial process in wastewater treatment and is often challenged by several factors. Heterotrophic nitrifying-aerobic denitrifying (HNAD) bacteria have unique biological characteristics that could help to achieve more efficient treatment processes. The paper reports an interesting work. but authors should emphasize its importance for the wastewater field. A more integrated presentation of the results, perhaps joining table 2 and 3 would be preferable. Also, the discussion should be improved, better exploring for example the importance of these kind of bacteria and their potential use for devising strategies for improving systems nitrogen removal. In addition, during the revision process, authors are advice to perform a revision of the English to turn the manuscript message more clear.

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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: This manuscript reported the diversity and denitrification capacity of heterotrophic nitrifying and aerobic denitrifying (HNAD) bacteria isolated from activated sludge. The pure HNAD bacteria were isolated by gradient dilution and underlining. The identification and diversity analysis of HNAD bacteria were based on the 16S rDNA sequencing and phylogenetic analysis. The nitrogen removal capacity of HNAD bacteria was evaluated by heterotrophic nitrification and denitrification media. Some specific comments were proposed as below:

1. L44. The traditional nitrification process utilized autotrophic nitrifiers rather than heterotrophic nitrifiers.

2. L111-112. How was the bacterial suspension with suitable dilution gradients cultivated?

3. L145-147. Alkaline potassium persulfate digestion is used to measure total nitrogen. How are nitrate nitrogen and ammonia nitrogen measured in this manuscript?

4. L163. There are 53 HNAD strains reported in the manuscript, but only 26 in Table 1.

5. What are the differences and novelties between the isolated HNAD bacteria and those reported?

Reviewer #2: The manuscript PONE-D-23-17073 described the diversity study on cultureable HNAD bacteria the MBR sewage treatment system. Some interesting data and resluts were obtained, and these showed that 53 HNAD strains were isolated from the activated sludge, and they could be classified as 26 species in 7 genera with Acinetobacter as the dominant one, among 2 phyla, suggesting a relative high diversity of HAND bacteria in the system. The results also indicated that some of the HAND strains were quite efficient in removing nitrogen, refering that they might be potential in denitrification treatment of wastewater. Therefore, this ms might be acceptable after revising with following comments:

Abstract: Heterophic nitrifying ability of isolateds HAND strains shuold be also tested and presented.

Table 1 could be deleted

Tables 2 & 3: Growth (OD600) of the strains under these conditions needs to be also presented.

L 28: (bacterial strain)

L 44: Heterotrophic nitrifiers?

L 85: LB broth

L 87: Enrichment medium

This manuscript needs further language editing.

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Reviewer #1: No

Reviewer #2: No

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Screening and diversity of culturable HNAD bacteria in the MBR sewage treatment system

PONE-D-23-17073R1

Dear Dr. Li,

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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Kind regards,

Catarina Leite Amorim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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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 #1: Yes

Reviewer #2: Yes

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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 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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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 #1: Yes

Reviewer #2: 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 #1: (No Response)

Reviewer #2: The revised manuscript is greatly improved, and the responses are also convincible. Now, it is acceptable.

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7. PLOS authors have the option to publish the peer review history of their article (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: No

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