Evaluation of Partial Nitritation/Anammox (PN/A) Process Performance and Microorganisms Community Composition under Different C/N Ratio
Round 1
Reviewer 1 Report
General comments:
In this study, authors mainly investigated the effect of C/N ratios and narrowing non-aeration time on removal efficiencies of nitrogen and COD, as well as the effects on changes of microbial community in a one-stage partial nitrification/anammox/denitritation process. The results detailed that by adjusting C/N ratios and aeration and non-aeration time periods, effective removal efficiencies on nitrogen and COD can be achieved, and manipulation on C/N ratios and aeration could be useful in practice. Figures and tables were fairly presented, results were well interpreted, and the manuscript was written in logic. Despite these interesting results, there are some improvements should be made to this manuscript.
In M&M section, concerning “Microbial analysis”, are the sequencing results standardized? If not, the relative abundance of corresponding communities may not be applicable for comparison in section 3.5. In “Results” section, the word “significant” appeared several times. Usually this comes with a p-value, and statistical analyses should be supplemented in “M&M” section. Section 3.1, the title seems not proper, as this section mainly concerns “nitrogen removal”. Additionally, please indicate the corresponding figures in the text when interpreting the results (e.g. Line 171-209). Section 3.4, The interpretation on NO2-N comes from nowhere, as the dynamic changes of NO2-N during the process were not included in the figures, tables or supplementary information. Please show or supplement the results on NO2-N concentrations. Section 3.5, how are the “dominant” communities defined? Figure 6 should be made more clearly to read. In “Conclusion” section, the last sentence, “It turns out therefore that ……inhibits NOB bacteria and allows the AOB and AAOB…….” This conclusion is not clearly and directly supported by current results. Please rephrase it. Proof read this manuscript to correct details, for example, make “AnAOB” and “AAOB” consistent.Author Response
Responses to the Reviewer nr 1
Dear Reviewer,
first of all, we would like to thank You, for crucial comments which enable us to improve our manuscript.
We upload a new version of our manuscript, with minor modifications labeled on yellow. Supplementary materials are added below the references.
Here is the list of the modifications provided to the text along to specified Reviewer Comments (RC):
RC In M&M section, concerning “Microbial analysis”, are the sequencing results standardized? If not, the relative abundance of corresponding communities may not be applicable for comparison in section 3.5.
RC: Figure 6 should be made more clearly to read.
RC Section 3.5, how are the “dominant” communities defined? Figure 6 should be made more clearly to read.
Standardization of the microbial analysis was provided at two levels:
sample preparation; described in the lines 136-140 which was mentioned in previous version of the manuscript, i.e. strict biomass sample weight was ensured before further steps of DNA extraction protocol, moreover before the sequencing step DNA matrix was concentrated to the similar concentration of 100 ng/μ (this information was added to the main text in the lines 140-141) DNA sequence data analysis; the same quality control parameters were applied during data processing, abundance of the particular microbial group, at the specified phylogenetic level, was estimated based on the absolute number of DNA sequence reads affiliated to the OTU in relation to the total number of the DNA sequence reads obtained from the sample, with a reservation that DNA sequences have passed QC step and were derived from the Bacteria domain (reads of the Archaea, Eukaryota as well unidentified at Domain level were rejected).Additionally to the Reviewer valuable comments statistical analysis of the microbial data was provided in order to validate significance of the shifts in microbial community after adaptation period to the carbon dosage. Description of the statistical step was added in M&M section lines 149 -151. While the results are presented on the Figure 1 (lines 208 -210). Use of the word “dominant” was limited in new version of the manuscript.
Due to text reorganization, microbial analysis are presented at the beginning of the combined Results – Discussion chapter (suggestion of the others Reviewers), Figure 6 now is Figure 1 and was modified to ensure better clarity, as well results of the statistical analysis were added.
RC: In “Results” section, the word “significant” appeared several times. Usually this comes with a p-value, and statistical analyses should be supplemented in “M&M” section.
The results not supported by statistical data, were described in accordance to the Reviewer suggestion without any ‘unnecessary’ words. Microbial data was subjected to the additional analysis with STAMP software, while basic statistics (average values, standard deviation) were calculated for the technological measurements and shown in supplementary materials.
RC: Section 3.1, the title seems not proper, as this section mainly concerns “nitrogen removal”.
RC: Additionally, please indicate the corresponding figures in the text when interpreting the results (e.g. Line 171-209).
RC: In “Conclusion” section, the last sentence, “It turns out therefore that ……inhibits NOB bacteria and allows the AOB and AAOB…….” This conclusion is not clearly and directly supported by current results. Please rephrase it. Proof read this manuscript to correct details, for example, make “AnAOB” and “AAOB” consistent.
In accordance to the You Sir or Madam, as well others Reviewers the manuscript structure was modified. We connected Results and Discussion section, rewrite some paragraphs. In overall we limited results description and focused on discussion part improvement. Moreover conclusion part was modified and clarified. More attention has been paid to connect microbial data with technological measurements. Abbreviations were uniformed, for instance in terms of bacteria responsible for anammox process AnAOB abbreviation was applied in all sections.
RC :Section 3.4, The interpretation on NO2-N comes from nowhere, as the dynamic changes of NO2-N during the process were not included in the figures, tables or supplementary information. Please show or supplement the results on NO2-N concentrations.
In accordance to the Reviewer suggestion, the N-NO2 and N-NO3 data were presented in the supplementary materials in table S2.
Author Response File: 
Author Response.docx
Reviewer 2 Report
no
Author Response
Dear Reviewer,
first of all, we would like to thank You, for some suggestions which enable us to improve our manuscript.
We upload new version of our manuscript, with minor modifications labeled on yellow.
Additionally to the other Reviewers valuable comments statistical analysis of the microbial data was provided in order to validate significance of the shifts in microbial community after adaptation period to the carbon dosage. Description of the statistical step was added in M&M section lines 149 -151. While the results are presented on the Figure 1 (lines 208 -210).
Due to text reorganization, microbial analysis are presented at the beginning of the combined Results – Discussion chapter (suggestion of the others Reviewers), Figure 6 now is Figure 1 and was modified to ensure better clarity, as well results of the statistical analysis were added.
The manuscript structure was modified. We connected Results and Discussion section, rewrite some paragraphs. In overall we limited results description and focused on discussion part improvement. Moreover conclusion part was modified and clarified. More attention has been paid to connect microbial data with technological measurements. Abbreviations were uniformed, for instance in terms of bacteria responsible for anammox process AnAOB abbreviation was applied in all sections.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript is reporting testing different operational parameters to increase treatment efficiencies via deammonification process. The language is mostly good and it's mostly clearly written. I have some questions concerning data reporting and discussion. I also recommend English and typo proofreading. Check also abbreviations and units for consistency.
Why are there no standard error or deviations reported, neither in methods nor on graphs? Have you not run replicate measurements of nutrient concentrations etc.?
The discussion should be re-written focusing only on discussing the results and their significance and providing less of background information. You don’t need to provide so many numbers in the discussion, then it becomes a repetition of the results section. You also don’t need to cite every paper and explain their findings. Only compare some very similar/different, most relevant that would help you understand your results. Focus more on processes behind the removal efficiency changes. And why weren’t microbial data discusses at all in context of efficiency of the treatments etc.? If they are not relevant, do not include them in the manuscript at all, otherwise you must put them in the context and connect all the pieces of information. Consider combining Results with Discussion.
Reconsider use of abbreviation so that it is consistent throughout the manuscript.
Abstract: AUR abbreviation is not explained; do not use excessive abbreviations in the abstract
Keywords: A typo in the word ‘deammonification’
Why would you have ‘16S 29 rRNA NGS’ in your keywords but not mention in the abstract? Most important information from findings about microbial community development is also worth mentioning in the abstract.
Line 32: introduce here the N abbreviation and be consistent throughout the text
Line 49: led => leads
Line 58: it => its
Line 61-63: At first it is said deammonification is applied to treat heavy ammonium loads, and in the next sentence, that is has a limitation of low ammonium concentrations? The same about C/N. It is confusing. Please clarify
Table 1: please add duration in days for every phase
Line 154-156: it is hard to understand how you divided phases. According to the Table 1 and Materials and Methods, that was done based on operational parameters (C/N ratio). Then, Figure titles “The division of the series of tests into phases based on ...” are not correct. Instead, it should be stating what removal efficiencies are shown and of what series.
Line 170 and Line 210: why are there two titles ‘Effect of C/N ratio on COD removal’? It should be only nitrogen in the first title, right?
Line 224: typo in the ‘nitrogen’
Line 304: it is very confusing that you were reporting results and also your objectives in the Introduction as to study deammonification and now you are talking about PN/A process. You should be very specific on what process you have studied throughout the manuscript.
Lines 305-316: this looks mostly like introduction. Discussion should be on discussion of your results; cite others and refer to some background information only when it is needed to explain your results.
Table 4: add ‘this study’ with your results so that it is easy to compare
Conclusions: re-write; write less or don’t write at all numbers; write the most important outcomes of your study
Author Response
Responses to the Reviewer nr 3
Dear Reviewer,
first of all, we would like to thank You, for crucial comments which enable us to improve our manuscript. Your suggestions after detailed review gave us lot of inspiration and ideas to improve manuscript itself and it composition. We uploaded a new version of our manuscript, with minor modifications labeled on yellow. Supplementary materials were added below the references.
Followed modifications are listed below and marked with marker in the new version of the manuscript.
Here is the list of the modifications provided to the text along to specified Reviewer Comments (RC):
ABSTRACT
RC: Abstract: AUR abbreviation is not explained; do not use excessive abbreviations in the abstract
AUR abbreviations is explained in lines 19-20, we limited use of the abbreviations into two most crucialRC: Why would you have ‘16S rRNA NGS’ in your keywords but not mention in the abstract? Most important information from findings about microbial community development is also worth mentioning in the abstract.
Abstract was shortened to meet 200 words limit, less important phrases were removed, microbial results are added to abstract in lines 22-25KEYWORDS
RC: Keywords: A typo in the word ‘deammonification’
Keywords were revised, typo in deammonifiaction was corrected, in accordance to further Reviewer comment, to ensure proper terminology all incorrect phrases “partial nitrification” were changed into “partial nitritation”, thus keywords were modified and unitary processes were mentionedINTRODUCITON
RC: Line 32: introduce here the N abbreviation and be consistent throughout the text
RC: Line 49: led => leads
RC: Line 58: it => its
Reviewer's suggestions have been included in the final version of the manuscript,RC: Line 61-63: At first it is said deammonification is applied to treat heavy ammonium loads, and in the next sentence, that is has a limitation of low ammonium concentrations? The same about C/N. It is confusing. Please clarify
Additional explanation has been provided to the text in lines 60 - 65, deammonifiaction is by default applied and designed for sidestream application, i.e. high strength ammonium reject water treatment from sludge dewatering. In such cases C/N ratio is relatively low or organic compounds are not biodegradable thus not inhibitory effect to AOB is observed. In mainstream application i.e. conventional domestic/municipal wastewater treatment, easy available for microorganisms carbon compounds are present, which are known (especially acetate) inhibitor of ammonium monooxygenase of AOB. Thus effective nitritation may occurs after reduction of organic compounds concentrations.MATERIALS AND METHODS
RC: Table 1: please add duration in days for every phase
Table 1 was modified in accordance to the Reviewer suggestions and phase duration were added,RC: Why are there no standard error or deviations reported, neither in methods nor on graphs? Have you not run replicate measurements of nutrient concentrations etc.?
Additionally to the Reviewer valuable comments statistical analysis of the microbial data was provided in order to validate significance of the shifts in microbial community after adaptation period to the carbon dosage (M&M section lines 149 -151).
In terms of the nutrients concentrations we basically relied on the test accuracy ensured by the supplier. However as an additional control to the conventional nitrogen forms concentrations measurements, total N were measured by dedicated analyzer what is mentioned in M&M section (lines 156-159). In case of samples in which imbalance of nitrogen concentrations were detected, evaluation of the particular nitrogen forms was repeated.
Basic statistics (average values, standard deviation) were calculated in terms of AUR, COD and TN removal efficiencies and shown in supplementary materials.
RESULTS, DISCUSSION AND COCLUSIONS
RC: The discussion should be re-written focusing only on discussing the results and their significance and providing less of background information. You don’t need to provide so many numbers in the discussion, then it becomes a repetition of the results section. You also don’t need to cite every paper and explain their findings. Only compare some very similar/different, most relevant that would help you understand your results. Focus more on processes behind the removal efficiency changes. And why weren’t microbial data discusses at all in context of efficiency of the treatments etc.? If they are not relevant, do not include them in the manuscript at all, otherwise you must put them in the context and connect all the pieces of information. Consider combining Results with Discussion.
RC: Lines 305-316: this looks mostly like introduction. Discussion should be on discussion of your results; cite others and refer to some background information only when it is needed to explain your results.
RC: Conclusions: re-write; write less or don’t write at all numbers; write the most important outcomes of your study
RC: Line 170 and Line 210: why are there two titles ‘Effect of C/N ratio on COD removal’? It should be only nitrogen in the first title, right?
RC: Line 224: typo in the ‘nitrogen’
In accordance to the You Sir or Madam the manuscript structure was modified. We connected Results and Discussion section, rewrite some paragraphs. In overall we limited results description and focused on discussion part improvement. Moreover conclusion part was modified and clarified. More attention has been paid to connect microbial data with technological measurements. Abbreviations were uniformed, for instance in terms of bacteria responsible for anammox process AnAOB abbreviation was applied in all sections. Results and discussion section was divided into three main sections to ensure better clarity.
RC: Line 154-156: it is hard to understand how you divided phases. According to the Table 1 and Materials and Methods, that was done based on operational parameters (C/N ratio). Then, Figure titles “The division of the series of tests into phases based on ...” are not correct. Instead, it should be stating what removal efficiencies are shown and of what series.
In the lines 123-124 we emphasized the role of C/N ratio as a main criteria to establish phases, All figures titles were modified in accordance to Reviewer comments, Table 1 was modified in accordance to the Reviewer suggestions and phase duration were added,RC: Table 4: add ‘this study’ with your results so that it is easy to compare
Table 4 (in the current version of the manuscript Table 5) was modified in accordance to the Reviewer suggestion.
RC: Line 304: it is very confusing that you were reporting results and also your objectives in the Introduction as to study deammonification and now you are talking about PN/A process. You should be very specific on what process you have studied throughout the manuscript.
In the new version of the manuscript terminology was uniformed and to not provide potential misunderstanding, we emphasized that main topic was PN/A process. Due to this fact we decided to modify manuscript title and keep this terminology through the reaming parts of our paper.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors shall provide a reaction mechanism to explain their whole process in a single chart.
Author Response
Dear Reviewer,
thank You for valuable suggestion, to add chart which better explains mechanisms of the nitrogen removal during our experiment to the readers. We added additional figure to the last version of the manuscript (form the line 282, reference in the text is marked on yellow). Please find a new version attached to this message.
With regards
Authors
Author Response File: Author Response.docx
Reviewer 3 Report
Dear authors, thank you for work on your manuscript. It has been improved greatly.
Author Response
Dear Reviewer,
one more time we would like to thank You for the valuable comments which made it possible to improve the manuscript and ensured it better 'scientific' quality.
With regards
Authors
