Influence of Season, Occupancy Pattern, and Technology on Structure and Composition of Nitrifying and Denitrifying Bacterial Communities in Advanced Nitrogen-Removal Onsite Wastewater Treatment Systems

Round 1

Reviewer 1 Report

The manuscript used high-throughput sequencing to evaluate the structure and composition of nitrifying and denitrifying bacterial communities in advanced N-removal OWTS, targeting the genes encoding ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) present in 44 advanced systems. The real system investigation is interesting and important for the overall understanding of this technology. However, they are few points need to be clarified.

• The introduction should be rewritten. It should be expanded to include a more detailed discussion of current problems and research gap.
• Would you explicitly specify the novelty of your work? What progress against the most recent state-of-the-art similar studies was made? If it’s just a report then it’s not fit well with the journal. The objective of the study should point out in a clear way.
• The literature review section should be improved. It should be dedicated to present critical analysis of state-of-the-art related work to justify the objective of the study. Also, critical comments should be made on the results of the cited works.
• The seasonal difference only based on two sampling action in June and September, which is not convincing to evaluate seasonal effect.
• The discussion statements are speculations. More detailed discussion of factors affecting the results should be added. Make every attempt to improve the discussion by critically analyzing your findings. At current form, the discussion part is a mess, the authors need to refine the main discussion point, and subtitles are appreciated for better understanding.
• The final conclusion is too long, the authors need to summarize the whole work, especially the contribution of the study to the research field.

Author Response

Thank you for a helpful and thoughtful review.  Changes to the document have been made using Track Changes and can be easily identified in the document.  Here are our responses to your comments:

 

Comment 1: The introduction should be rewritten. It should be expanded to include a more detailed discussion of current problems and research gap.

• Response: Resolved. We have amended the Introduction with language that discusses the research gap in this field and explains why the research is important. 

Comment 2: Would you explicitly specify the novelty of your work? What progress against the most recent state-of-the-art similar studies was made? If it’s just a report then it’s not fit well with the journal. The objective of the study should point out in a clear way.

• Response: We discussed the few other similar studies that have been done in the original draft of the manuscript and compared their results/design to ours. We did make sure to explicitly state that our study is one of the first to specifically target N-cycling microorganisms in advanced N-removal OWTS.

 

Comment 3: The literature review section should be improved. It should be dedicated to present critical analysis of state-of-the-art related work to justify the objective of the study. Also, critical comments should be made on the results of the cited works. 

• Response: Resolved. We have expanded the literature review to add several other studies targeting the microbial communities of BNR WTPs; however, as previously stated, to our knowledge we have discussed the only studies to be done on nitrifiers/denitrifiers in advanced N-removal OWTS.  We have also commented on the specific analyses/relationships analyzed in the WTP studies in the introduction; further comments on their findings are highlighted in the Results and Discussion.

 

Comment 4: The seasonal difference only based on two sampling action in June and September, which is not convincing to evaluate seasonal effect.

• Response: This study is a preliminary look at seasonal differences. We feel that this first look is important and highlights the need for additional research into more specific seasonal differences.

 

Comment 5: The discussion statements are speculations. More detailed discussion of factors affecting the results should be added. Make every attempt to improve the discussion by critically analyzing your findings. At current form, the discussion part is a mess, the authors need to refine the main discussion point, and subtitles are appreciated for better understanding.

• Response: We have added more specific subtitles to the results and discussion to improve the clarity of our sections. The comment about the discussion section is rather vague.  Discussion by nature involves speculation, but we believe that we support our claims with the results presented and have critically analyzed our findings.  The discussion is now even better supported by a concise and clear conclusion (see next comment/response).

 

Comment 6: The final conclusion is too long, the authors need to summarize the whole work, especially the contribution of the study to the research field.

• We have shortened and streamlined the conclusion so that it better represents the major takeaways from the study.

Reviewer 2 Report

This manuscript investigates nitrifying and denitrifying bacterial communities based on functional genes (amoA and nosZ genes) in advanced onsite wastewater treatment systems (OWTS).   OWTS is difficult to understand quickly. Should be described the system with the figure in an appendix.   Figure4 It is difficult to see the plots. Change the color or increase the size.   Figure 5,6 There are a lot of unassigned sequences to the database. The authors should make a polygenetic tree based on amoA and nosZ gene. And show the closest spices and these distances.   Conclusion It looks like a result and discussion. Should be emphasized specific results in conclusion.  

Author Response

Thank you for your helpful suggestions.  Changes to the document have been made using Track Changes and can be easily identified in the document.  Here are our responses to your comments:

 

• We have added a schematic of advanced N-removal OWTS to appendix to better illustrate treatment train/processes.
• We believe that a phylogenetic tree is not necessary given the focus of this paper (assessing trends in the diversity/composition of OWTS microbial communities). We made sure to emphasize the need for additional research on specific organisms in these communities in both the discussion and conclusion, given the large number of unassigned sequences.
• We have updated the colors in Figure 4 to improve clarity.
• We have shortened/streamlined the conclusion to emphasize specific results and takeaways.

Round 2

Reviewer 1 Report

The revised version improved a lot, I have no further comments.

Reviewer 2 Report

Responses were clearly, and this article is improved.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Ross et all analyzed the nitrogen cycle community from different onside waste water treatment systems. In general, these systems are understudied compared to wastewater treatment plants which clean municipal wastewater. The authors investigated the nitrogen cycle community by using two primer pairs; one targeting bacterial AmoA and one targeting nosZ. I have serious concerns about this approach. The primers used by the authors to investigate the nitrifiers do not target archaeal amo. There is no information about the absense of archaea in the paper, it can therefore be that these microorganisms are completely missed from the analysis. In addition, the primers used to not target the amo of comammox bacteria. By missing these two important groups of nitrifiers in the analysis (but also in the introduction and discussion, these other nitrifiers are not even mentioned), it is difficult to draw any conclusions on the ammonia oxidizing community of these systems and I therefore decided to not comment on this manuscript in detail. 

Reviewer 2 Report

The manuscript presents the influence of season, occupancy pattern, and technology on the structure and composition of nitrifying and denitrifying bacterial communities in advanced nitrogen-removal onsite wastewater treatment systems. The topic is interesting and important. In general, the work was designed well, the presentation of results and discussion were well organized. However, they are few points need to be clarified before it can be accepted.

• The novelty of the study, the authors pointed out their objectives clearly, but the novelty of the study is missing, which needs to be stressed in the revised version.
• The basic methodology is missing in the abstract.
• The conclusion need to be rewritten, the more concise and direct conclusion is appreciated, avoid citation in the conclusion section. Normally, the conclusion only summarizes the whole work and point out the future research.