Effects of Hydraulic Retention Time and Influent Nitrate-N Concentration on Nitrogen Removal and the Microbial Community of an Aerobic Denitrification Reactor Treating Recirculating Marine Aquaculture System Effluent

Round 1

Reviewer 1 Report

In this work, authors have examined the effects of hydraulic retention time and influent nitrate concenation on nitrogen removal and microbial community composition in an aerobic denitrification reactor treating RAS effluent. The methods are suitable and up-to-date, and the manuscript is rather well written, although partly too detailed and not well focused. However, there are already quite extensive literature on the effect of HRT/nitrate on nitrogen removal and the microbiology of RAS that authors have mainly neglected, making this study less novel.

Authors do not define well the concept of “aerobic denitrification”. There is no such a process than denitrification existing under oxic conditions, since denitrifying microbes switch from nitrate to oxygen when available. Aerobic denitrification, to my knowledge, refers to simultaneous nitrification and denitrification processes, that in this case probably happen in different layer of the biofilm attached to carrier elements, being carried out by different bacterial groups. So authors should be very careful not to mix traditional heterotrophic denitrification and this aerobic “denitrification” in introduction and discussion.  For example, in lines 40-42, authors list variables affecting aerobic denitrification process. When I checked the references, they referred to nitrification+denitrification =(“aerobic denitrification”), denitrification and anammox (anaerobic ammonia oxidation). I would encourage authors to use coupled nitrification & denitrification or some other similar term instead of aerobic denitrification, since it is very uninformative and misleading, yet used, term.

The study setup is not ideal. Authors change the hydraulic retention time through changing the flow, which can affect the nitrate diffusion in the biofilm. Furthermore, the increasing the nitrate concentration does not increase denitrification rate too much, indicating that there is some disturbance e.g. decreased diffusion due to oxygen or changed flow to the denitrification layer of the biofilm in the carriers. Authors strongly argue that their study is applicable for industry, but the HRT is way too long for that, requiring very large bioreactor volumes, making the process less feasible and cost-efficient. Usually HRT of 1-2 hours is required, and improving of denitrification from the current standards requires short HRT, which has been achieved e.g. with using granules (Letelier-Gordo, C. O., & Herreros, M. M. (2019). Denitrifying granules in a marine Upflow Anoxic Sludge Bed (UASB) reactor. Aquacultural Engineering, 84, 42-49.)

On major issue is the interpretation and presentation of the sequencing data. Authors have done several analysis that partly overlap or provide very low amount of relevant information. Figure 3 is not relevant, it can be removed or transferred into supplementary material, since the information is provided in the text. Figure 4 is very repetitive, presenting the same data from different approaches. Panel a is very hard to read and understand. Panel b is ok. Panel c repeats panel b, and provides clustering but authors do not explain how the clustering is done or discuss about the results. I would suggest authors to keep panel b and discard others. Considering figure 5, PCoA is OK for this kind of data, RDA not, since the amount of explaining variables too high as compared to the amount of samples and RDA requires linear data which microbial data seldom is. In figure 5, authors state that they have done RDA at species level, which I hope is not true, since 16S rRNA gene sequencing allows reliable identification only until genus level. I do not understand figure 6. Have the data been combined from all sampling occasions? The same information can be gained from figure 4 in an easily understandable format, so I would forget this figure and analysis also. Altogether, I am not convinced that the authors have truly understood what they have done in the sequence data analysis, but just played around. I hope this is not the case, but it seems so. So, I strongly encourage authors to think what their hypothesis were and answer to those with the some well-defined tests.

Minor comments/questions:

Line 46: Sodium acetate is used in denitrification reactors yes, but methanol and even ethanol are more widely used in wastewater treatment and also in RAS effluent nutrient removal. There is plenty of literature on the topic, I encourage authors to read it well.

Lines 47-58: I do not find this part very relevant, since it focuses on denitrification using a specific material, not in general. And that material is not used in this study.

Lines 58-61: This is wrong. There are previous studies about the topic, even in RAS, that can be found using “denitrification salinity” or “denitrification aquaculture” in google scholar. Most of those references are currently lacking from the manuscript.

Lines 71-75: There are a lot of studies from Europe focused on aquaculture microbiomes. Authors should read them and integrate them here. It is not too relevant to focus only on some Chinese studies.

Lines 78-79. Anammox has nothing to do with the “aerobic” denitrification. Its coupled nitrification-denitrification. It happens, because there are nitrifiers in the outer oxic layers of the biofilm that produce nitrate which is rapidly diffused into the deeper layers where denitrifying microbes are. Or it can be also nitrifier denitrication. I strongly encourage authors to read more about nitrogen transformation processes and modify these parts to be clear.

Line 81: Correlation does not mean that they are denitrifiers. They can be aerobic heterotrophs. Or did they do metagenomics or transcriptomics?

Line 126: Did you manage to remove all the biofilm using this technique? Did you control that somehow?

Line 152: Which version of Silva?

Lines 154-155: This belongs under title 2.5, when you describe the sequence quality control (see my later comment on lines 213-220)

Line 153: This is statistical analysis not bioinformatics. Bioinformatics you do for the sequence data, here you already have OUT tables etc

Lines 210-213: Repetition, not needed. Focus on the biological results.

Lines 213-220: This information is more quality analysis of the sequencing results and belongs to M&M under title 2.5

Figure 3: Supplements, not important for the manuscript aims.

Table 3: Not so many decimals needed for coverage, ACE and Chao. And could you choose one richness, diversity and evenness index?

Lines 226-238: You already have values in the table. Please do not repeat them, makes the text very hard to read. Just state when richness/diversity lowest, highest etc.

Lines 249-251: Not needed to repeat methods.

Lines 255-6: These phyla include also many other members that  ones doing nitrogen removal. Bacteroidetes and Firmicutes are common fermentative phyla. And Proteobacteria is a very large group, including sulfate reducers, sulfur oxidizers, denitrifiers, nitrifiers, dnra microbes, all kinds of heterotrophs etc. So I would not conclude anything at phylum level.

Lines 285-7: This means that Denitrovibrio does not remove nitrogen. It removes nitrate yes, but converts it into ammonia, meaning that its doing DNRA process, not denitrification. This should be acknowledged. I think the final sentence is wrong then?

Line 307: A more descriptive title would be better, not focused on the method used

Lines 309-337: Maybe it would be more useful to do correlation analysis without microbial data, to look into the correlation between removal rates/efficiencies and operating conditions. Figure 5 does not give too much information, especially when the amount of explaining variables is so high compared to number of samples.

Line 362: I would avoid subtitles in the discussion.

Lines 364-365: See my previous comments on introduction, I do not agree with this statement.

Lines 405-406: I see no point of repeating this again.

Lines 407-408: probably it is other way around. Changes in the microbial community composition are related to operating conditions?

Line 412: But also others!

Line 426: Arcobacter is common SOB and also DNRA bacteria

Line 425: I would like to see more conclusive discussion on the microbial data. Now, its more listing of the potential functions. There is no discussion on the diversities and richnesses. Is more diverse community better etc?

Line 453: The conclusions are rather vague, maybe authors could think them a bit more and focus on they key findings in relation to the current literature. Also my comments on the applicability of the research should be taken into account.

Author Response

Thanks for the constuctive comments for our manuscript “Effects of Hydraulic Retention Time and Influent Nitrate-N Concentration on Nitrogen Removal and Microbial Community of an Aerobic Denitrification Reactor Treating Recirculating Marine Aquaculture System Effluent”.  Please see the point-by-point response at the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors of the manuscript 'Effects of hydraulic retention time and influent nitrate-N concentration on nitrogen removal and mcirobial community of an aerobic denitrification reactor treating recirculating marine aquaculture system effluent' deals about the treatment of saline wastewater with nitrate. The study in a compact laboratory reactor with synthetic wastewater is about different hydraulic retention times and different nitrate concentrations. A lot of attention has been given to the microbial composition in the different experimental set-ups.

The manuscript is well written and results, including discussion and conclusions, appear clear and logical.

Only on page 8 line 227 it appears that the number of species observed is not 117, but should be 215.

Very minor text additions are needed:

Page 5, line 161:...was analyzed based on the…; remove analyzed with e.g. 'used'.

Page 12, title contains an abbreviation: PCoA. Is that Principal Component Analysis?

Page 12 line 312: replace 'that'with 'than'?

Page 12 line 333: remove spaces between the sentences.

Page 12 line 334: HRTS with small 's' (HRTs)?

Page 15 line 377: remove 'the': … an appropriate hydraulic retention time...

Page 16 line 459: remove 'Illumina'. DNA sequencing analyses revealed….

Author Response

Thanks for your comments for our manuscript “Effects of Hydraulic Retention Time and Influent Nitrate-N Concentration on Nitrogen Removal and Microbial Community of an Aerobic Denitrification Reactor Treating Recirculating Marine Aquaculture System Effluent”.  Please see the point-by-point response at the attachment. Thanks

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript entitle

Comments for author File: Comments.pdf

Author Response

Thanks for your comments for our manuscript “Effects of Hydraulic Retention Time and Influent Nitrate-N Concentration on Nitrogen Removal and Microbial Community of an Aerobic Denitrification Reactor Treating Recirculating Marine Aquaculture System Effluent”.  Please see the point-by-point response at the attachment. Thanks

Author Response File: Author Response.docx

Reviewer 4 Report

Overall a well-written manuscript. However, minor modifications needed to improve the readability of the manuscript.

Author Response

Thanks for your comments for our manuscript “Effects of Hydraulic Retention Time and Influent Nitrate-N Concentration on Nitrogen Removal and Microbial Community of an Aerobic Denitrification Reactor Treating Recirculating Marine Aquaculture System Effluent”.  Please see the point-by-point response at the attachment. Thanks.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I have read already the previous version of the manuscript and am happy to see that authors have done their best to improve it and answer to my questions. Lines 395-402: I am happy to see this discussion part, thank you

However, I am still not convinced about the concept of “aerobic denitrification”. I have gone through an extensive literature review, where aerobic denitrification is used to indicate simultaneous nitrification&denitrification, and this is what probably happens in the reactor, because there are nitrifiers in the outer oxic layers of the biofilm that produce nitrate which is rapidly diffused into the deeper layers where denitrifying microbes are. Authors provide yet unpublished reference, so I can only trust the previous published knowledge. I think it is interesting to know that oxygenation can improve nitrate removal when using this kind of a reactor, and not stick into the concept of aerobic denitrification that they cannot prove to exist.

Minor things:

Line 114: space between “and” and “1” missing

Lines 261-263: Among or between? I also think authors could simply focus on describing the differences between phases without saying that communities were distinctive or varied dramatically, I found these sentences vague.

Line 382: I did not provide reference for this, since I though authors would look for it, so I strongly recommend them to add reference for Arcobacter &SOB&DNRA statement

Author Response

Dear Reviewer,

I have read already the previous version of the manuscript and am happy to see that authors have done their best to improve it and answer to my questions. Lines 395-402: I am happy to see this discussion part, thank you.

REPSONSE: We are pleased for the positive comment.

However, I am still not convinced about the concept of “aerobic denitrification”. I have gone through an extensive literature review, where aerobic denitrification is used to indicate simultaneous nitrification & denitrification, and this is what probably happens in the reactor, because there are nitrifiers in the outer oxic layers of the biofilm that produce nitrate which is rapidly diffused into the deeper layers where denitrifying microbes are. Authors provide yet unpublished reference, so I can only trust the previous published knowledge. I think it is interesting to know that oxygenation can improve nitrate removal when using this kind of a reactor, and not stick into the concept of aerobic denitrification that they cannot prove to exist.

RESPONSE: At line 42, we provide four citations [15-18], all published, regarding aerobic denitrification. Reference 19 discusses the issue of oxic or anoxic denitrification. We have added more supporting citations at line 79. A Google Scholar search on “aerobic denitrification bacteria” yielded scores of papers discussing the concept. We feel our point is well supported by a number of recent literature reports.  

Minor things:

Line 114: space between “and” and “1” missing.

RESPONSE: This has been fixed. Other minor issues caused by use of Track Changes were fixed. The manuscript was closely read and a few prose glitches were fixed.

Lines 261-263: Among or between? I also think authors could simply focus on describing the differences between phases without saying that communities were distinctive or varied dramatically, I found these sentences vague.

RESPONSE: We take the reviewer’s point. Rather than relying on the reader to make the interpretations of the heat map, we now lay out a more detailed description of the respective communities, noting that the differences among communities were mostly among phase H4_N50 and other phases. We fee that the passage was indeed strengthened by attention to this comment.  

Line 382: I did not provide reference for this, since I though authors would look for it, so I strongly recommend them to add reference for Arcobacter &SOB&DNRA statement

RESPONSE:  We have added two supporting citations to this passage. In addition, having removed Figure 6, we removed mention of it and the network that had been depicted.                                                                                                                 

Summary comment

We hope that with these revisions, that our manuscript becomes acceptable for publication in Water.

                                                               Thank you,

                                                               Zhitao Huang

Author Response File: Author Response.docx