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  • The initial submission of this article was received on April 17th, 2019 and was peer-reviewed by 2 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on May 11th, 2019.
  • The first revision was submitted on June 22nd, 2019 and was reviewed by 2 reviewers and the Academic Editor.
  • A further revision was submitted on July 9th, 2019 and was reviewed by the Academic Editor.
  • The article was Accepted by the Academic Editor on July 16th, 2019.

Version 0.3 (accepted)

· Jul 16, 2019 · Academic Editor


Dear Dr. Payette and colleagues:

Thanks for revising your manuscript to PeerJ, and for addressing the concerns raised by the reviewers. I now believe that your manuscript is suitable for publication. Congratulations! I look forward to seeing this work in print, and I anticipate it being an important resource for research pertaining to methylotrophic denitrifying biofilms, and overall denitrifying bioprocessing.

Thanks again for choosing PeerJ to publish such important work.


Version 0.2

· Jun 24, 2019 · Academic Editor

Minor Revisions

Dear Dr. Payette and colleagues:

Thanks for revising your manuscript. The reviewers are very satisfied with your revision (as am I). Great! However, per reviewer 1, there are a few minor edits to make. Please address these ASAP so we may move towards acceptance of your work.




Basic reporting

no comment

Experimental design

no comment

Validity of the findings

no comment

Additional comments

Just few very minor things.

Lines 62-64. Citations are written twice.

Line 155. I think the correct form to write desimals is 51.2 g/L, not 51,2. (maybe check through the manuscript for this).

Figure 3. “Panel A” and “panel B” are not mentioned in the figure text.


Basic reporting

All changes were made as suggested and the manuscript was considerably improved.

Experimental design

All changes were made as suggested and the manuscript was considerably improved.

Validity of the findings

All changes were made as suggested and the manuscript was considerably improved.

Version 0.1 (original submission)

· May 11, 2019 · Academic Editor

Major Revisions

Dear Dr. Payette and colleagues:

Thanks for submitting your manuscript to PeerJ. I have now received two independent reviews of your work, and as you will see, the reviewers raised some concerns about the research. Despite this, these reviewers are optimistic about your work and the potential impact it will have on research pertaining to methylotrophic denitrifying biofilms, and overall denitrifying bioprocessing. Thus, I encourage you to revise your manuscript accordingly, taking into account all of the concerns raised by both reviewers.

While the concerns of the reviewers are relatively minor, this is a major revision to ensure that the original reviewers have a chance to evaluate your responses to their concerns.

I look forward to seeing your revision, and thanks again for submitting your work to PeerJ.

Good luck with your revision,



Basic reporting

This interesting study tests the effect of different levels of various physico-chemical parameters on the denitrification and the co-occurrence of the key taxons, Methylophaga and Hyphomicrobium, in marine methanol-fed denitrification system. It shows the plasticity of the marine methylotrophic biofilm in adapting to different conditions and gives also new insights into the factors controlling the abundance of the key taxons.

The manuscript is written in good English for most parts (see some comments in the general comments part).

Experimental design

The design is quite complex but it is nicely described in the text and in the tables and figures (incl. supplementary).

Research questions and aims are defined but they are mainly very technical. What would be your ultimate aim? Why would make this study? That information is lacking in the introduction. Is it because you want to know how the denitrification and community is controlled in order to benefit the development of optimal denitrifying bioprocess conditions (as stated in abstract)?

Validity of the findings

ANOVA-analysis is done only to test differences in denitrification rates. Why it is not done to test differences in other reported and discussed parameters like protein, growth and qPCR results?

Conclusions should not contain reference to the sister study which has now been also submitted to PeerJ (lines 501-504). Can you move this to discussion or to introduction?

You could also answer your ultimate aim in the conclusions. E.g. if the study is done to benefit the development of optimal denitrifying conditions, then please answer, how this benefits it, or at least if it benefits it? Thus, in general, please answer your ultimate aim in the conclusions.

Additional comments


Abstract´s results and conclusions: You report GP59 and NL23 results but not JAM1 results here. Should you include JAM1 here also?

Abstract´s results: Inhibition occurred…. at pH10 or with 1.5% methanol. Should this be reworded in such way that reader understands that HIGH pH and HIGH methanol inhibited the process? Of course, everybody understands that pH 10 is high. But somebody unfamiliar with the subject may not know that 1.5% methanol is high.

Abstract´s conclusions and study´s conclusions: How these results could benefit in the development of optimal denitrifying process?


Line 67: Do you mean fluidized-BED?

Line 80: …bioprocess denitrifying activities (Reference lacking here).

Line 87: please correct “reducing NO3- to NO2- “ (not in)

Line 96: strain (not stain)


Line 391: were similar to that reported… (not than)


Line 429: stayed (not strayed)

Line 431-432: What factors specifically you suggest make the difference between batch mode system and the original continuous flow system?

Line 479 – 493: This is a matter of taste. However, I think that this part referencing Osaka et al 2008 and Rissanen et al. 2016 could be mentioned in shorter way in the introduction section together with references to your previous works just to show that the Methylophaga and Hyphomicrobium are generally the key taxa in marine/saline methanol-fed denitrification. This could add even more strength to justify your study.

Line 489-490: Please rephrase e.g. “The other denitrification system that is similar to the one that was used at the Biodome is a methanol-fed…”


Basic reporting

Payette et al. provide a manuscript in which they assess the influence of diverse physico-chemical parameters on the denitrifying activities in a methanol-fed marine biofilm. The main drivers in this marine denitrifying biofilm are Methylophaga nitratireducenticrescens and Hyphomicrobium nitrativorans. In this study, they are able to show, that this biofilm can adapt to varying conditions, while keeping it’s denitrifying capacity.

Payette et al. provide a clear introduction and background to the topic. However, two questions arise from the introduction:
1) To what purpose was the marine effluent treated with this biofilm and how was the marine effluent composed?
2) In line 108 the authors state that the genome of strain GP59 is highly similar to that of strain JAM1. To the authors mean the whole genome or just the genes in respect to denitrification? If the authors mean the whole genome, then a more in depth genome comparison should be provided. If they only mean in respect to certain denitrification genes, then this should clearly be stated and also shown, e.g. with a comparison of the genes, gene arrangement, gene neighbourhood.

Payette et al. provide clear and relevant figures of high-quality.

Following language changes are suggested:

Line 30: change here and elsewhere: ‘the Reference biofilm’ to ‘the reference biofilm’

Line 45: exposed to pH 10 or to 1.5% methanol

Line 56: development of an optimal denitrifying bioprocess

Line 92: H. nitrativorans NL23 have been sequenced previously

Line 94: ‘during growth of this strain’ or ‘during growth of strain JAM1’

Line 95: Do the authors mean cNor or is this a typo?

Line 96: Change stain to strain

Line 104: batch-mode and anoxic conditions

Line 129: trace elements

Line 131, 133: under ‘various concentrations’ a range should be provided or a reference to a Table indicating the range used

Line 132: Filter-sterilized methanol

Line 149: On average
Line 162: Figure S2 describes the protocol in detail.

Line 164: For the assays that were performed

Line 363: change ‘two-three’ to ‘two to three’

Line 370: ‘which is similar to that found…’

Line 375: ‘at similar level as in the OB’

Line 391: ‘were similar as reported in’

Line 397: ‘were the same as in the…’

Line 398/399: delete ‘important’

Line 403: ‘as in the OB’

Line 415: ‘similar to the ones in the…’

Experimental design

Payette et al. provide here original primary research. In addition, the authors identify how this research could benefit the development of an optimal denitrifying bioprocess.

In line 169 the authors state that “most assays were performed in triplicates”. This is a bit vague. Why have not all assays been carried out in triplicates. Which assays in particular have not been carried out in triplicates and how could that effect the results and conclusions?

Figure 1, Panel B: Was the same concentration of DNA loaded per DGGE lane? If yes this should be stated in Methods and also how much.

Validity of the findings

Payette et al. provide a clear and sound results section.

For the finding in line 279: ‘Absence of methanol showed very weak denitrifying activities as expected’ more explanation is needed for the reader in order to understand why this is expected.

In lines 435 to 441 the authors could elaborate on the pH optimum of the enzymes, since this might also be a possible explanation for these results.

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