Effect of Sulfide on the Processes of Transformation of Nitrogen Compounds and the Microbial Community Composition in the Anammox Bioreactor
, Vladimir Grachev 1, Anna Kallistova 1, Alexander Dorofeev 1, Yuriy Litti 1, Evgeny Gruzdev 2, Alexey Beletsky 2, Nikolai Ravin 2 and Andrey Mardanov 2
Round 1
Reviewer 1 Report
Detailed comments:
1. The English of the text should be checked – minor mistakes, errors
2. The novelty of the manuscript is completely missing
3. Authors write: “Certain amounts of sulfide arrive into wastewater treatment bioreactors”. What means certain amounts of sulfide? The values must be indicated!
4. For all abbreviations or notation indicate the complete name
5. For unit of measure used the S.I., please check in all manuscript.
6. Authors write: “The experiments were carried out in two identical sequencing batch reactors operating in parallel”. A photo or a schematically diagram for the batch reactors must be included! It is not enough just to mention references. You have practically performed experimental analyzes on the same type of reactors, what is the novelty? So, you must mention very clearly how the reactors work in the case of these experiments, for a better understanding of the data and what you wanted to demonstrate!
7. Authors write: “5, 15, 25, 50 и 100 mg S/L”. Correct и with and! The text must be written in English!
8. For Figure 3, at axes, parameters and unit of measure must be indicated! What means C and S, at each column? For each Figure where are indicated C and S, must be write Control (C) and Sulfide (S), at legend, if C and S represent what I mentioned
9. What chemical reactions can take place in the reactor? You must mention them!
10. Comparison between the obtained results and measured in this study with other reported studies should be done and included for more clarity (indicate values not just number of reference).
11. The Discussions chapter must be separated from the Conclusions chapter. So, you must have a chapter with Conclusions, in which you should mention the best results obtained, but also potential other applications of bioreactors
12. “Challenges or Future Prospective” must be included
The English of the text should be checked – minor mistakes, errors
Author Response
Dear respectable Reviewer,
thank you very much for your job on improving our manuscript.
We have repaired the manuscript accordingly. Indeed it is much better now. Please find below our poit-to-point answer.
Sincerely yours,
On behalf of all co-authors,
Drs. A. Dorofeev and Yu.Nikolaev
Point 1: The English of the text should be checked – minor mistakes, errors.
Response 1: The text was checked by a professional interpreter and editor of scientific microbiological journal.
Point 2: The novelty of the manuscript is completely missing.
Response 2: Dear Reviewer, we can’t agree with “complete absence of novelty”. For example, in Abstract we wrote: “This is the first report on the different effect of sulfide on the growth rate on members of the nitrifying genus Nitrosomonas, increasing/decreasing or not affecting it for different phylotypes at elevated sulfide concentrations”.
Point 3: Authors write: “Certain amounts of sulfide arrive into wastewater treatment bioreactors”. What means certain amounts of sulfide? The values must be indicated!
Response 3: Dear Reviewer, according to your advice, in page 1 the sentence «Certain amounts of sulfide arrive into wastewater treatment bioreactors» was replaced by “At wastewater treatment plants (WWTP), sulfides enter the bioreactors with incoming wastewater, where their concentration can exceed 3 mg S/L [1]”.
Point 4: For all abbreviations or notation indicate the complete name.
Response 4: Dear Reviewer, thank you for being a very good observer! Indeed, couple of mistakes took place. We put one more decipher – UV as ultraviolet (page 3). Also AOБ (page 5) was replaced by AOB.
Point 5: For unit of measure used the S.I., please check in all manuscript.
Response 5: Dear Reviewer, we were not able to find any improper units except S.I. Could you please specify your remark.
Point 6. Authors write: “The experiments were carried out in two identical sequencing batch reactors operating in parallel”. A photo or a schematically diagram for the batch reactors must be included! It is not enough just to mention references. You have practically performed experimental analyzes on the same type of reactors, what is the novelty? So, you must mention very clearly how the reactors work in the case of these experiments, for a better understanding of the data and what you wanted to demonstrate!
Response 6: Dear Reviewer, again we can not agree with your demand. First of all, with this research tool (set of reactors) we regularly investigate different aspects of Anammox consortium (Kallistova et al. Frontiers in Microbiology. 2022. V. 12. P. 802201; Pimenov et al., Introduction of Exogenous Activated Sludge as a Way to Enhance the Efficiency of Nitrogen Removal in the Anammox Process Microbiology, 2022, Vol. 91, No. 4, pp. 356–363.) and we have one more paper under preparation. What for one need to show bioreactors’ photo each time? 2. Regimes of reactors’ operation were clearly described in sections 2. Materials and Methods. 2.1. Experimental setup and 2.2. Experiments on the effect of sulfide on nitrogen removal efficiency, abundance of bacteria, and activity of the specific genes.
Point 7. Authors write: “5, 15, 25, 50 и 100 mg S/L”. Correct и with and! The text must be written in English!
Response 7: Dear Reviewer, thank you for being a very good observer again! This mistake was repaired (page 3).
Point 8. For Figure 3, at axes, parameters and unit of measure must be indicated! What means C and S, at each column? For each Figure where are indicated C and S, must be write Control (C) and Sulfide (S), at legend, if C and S represent what I mentioned.
Response 8: Dear Reviewer, as we understood you, we have replaced “Figure 3. Relative abundance of bacterial phyla in the activated sludge communities of the control (C) and experimental (S) bioreactors.” by “Figure 3. Relative abundance of bacterial phyla in the activated sludge communities of the control (C) and experimental with sulfides (S) bioreactors.”
Point 9. What chemical reactions can take place in the reactor? You must mention them!
Response 9: Dear Reviewer, we specified the phrase “… sulfide was not blown out and/or oxidized abiotically». Now it looks like “…sulfide was not blown out or oxidized abiotically by oxygen to S0 “.
Point 10. Comparison between the obtained results and measured in this study with other reported studies should be done and included for more clarity (indicate values not just number of reference).
Response 10: Dear Reviewer, in all places we compare our data with somebody else’s numbers are provided now.
Point 11. The Discussions chapter must be separated from the Conclusions chapter. So, you must have a chapter with Conclusions, in which you should mention the best results obtained, but also potential other applications of bioreactors.
Response 11: Dear Reviewer, we created an additional chapter (chapter 5. Conclusions), thank you.
Point 12. Challenges or Future Prospective” must be included.
Response 12: Dear Reviewer, we included few points about future works and perspectives in the chapter 5. Conclusions. We decided not to destroy smooth canvas of it, as chapter “Future Prospectives/challenge” is not mandatory according MDPI rules. Please, see chapter 5. Conclusions below:
- Conclusions
In this study, the effect of sulfide on the transformation of nitrogen compounds by the anammox community has been studied by the use of a laboratory sequencing batch bioreactor with stationary biomass carrier. The 16S rRNA gene profiling of the community of the anammox activated sludge revealed members of the key physiological groups: ammonium oxidizers of the genus Nitrosomonas, nitrite oxidizers of the genus Nitrosospira, anammox bacteria of the genera Candidatus Brocadia and Ca. Jettenia, as well as members of other bacterial genera. Nitrate removal was not sensitive to sulfide at concentrations up to 50 mg S/L and decreased by 17% at 100 mg/L. The threshold of sulfide sensitivity for group I nitrifiers was ~50 mg/L, while anammox bacteria were resistant to sulfide concentrations of up to 100 mg S/L in the incoming water. Group II nitrifiers were obviously the most sulfide-sensitive components of the community. A practice-wise consequence of this research is that Anammox technologies can be applied towards waste waters with high sulfides load. A drastic increase in abundance of members of the family Hydrogenophilaceae at elevated sulfide concentrations, together with precipitation of elemental sulfur was detected. This may indicate sulfide oxidation either by molecular oxygen or by nitrate; this finding requires further investigation. This is the first report on the different effect of sulfide on the growth rate on members of the nitrifying genus Nitrosomonas, increasing/decreasing or not affecting it for different phylotypes at elevated sulfide concentrations. Further investigation are necessary to cultivate and to isolate unique sulfide-tolerant and “sulfide-loving” Nitrosomonas.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear authors of the manuscript Effect of Sulfide on the Processes of Transformation of Nitrogen Compounds and the Microbial Community Composition in and Anammox Bioreactor, have a good paper that they present for consideration for publication in this journal. I am sending you some observations and comments on some details of your work
1. Improve the writing and use of English with a native of the language
2. Place error bars in Figures 1 and 2
3. Improve the "symbols" NO2- and NO3- in line 327
4. And where is the conclusion? Since after the discussion, this section is not presented. Please, if the conclusion is within the discussion, separate both sections
Author Response
Dear respectable Reviewer,
thank you very much for your job on improving our manuscript.
We have repaired the manuscript accordingly. Indeed it is much better now. Please find below our poit-to-point answer.
Sincerely yours,
On behalf of all co-authors,
Drs. A. Dorofeev and Yu.Nikolaev
Point 1: Improve the writing and use of English with a native of the language
Response 1: The text was checked by a professional interpreter and editor of scientific microbiological journal.
Point 2: Place error bars in Figures 1 and 2.
Response 2: Dear Reviewer, we have placed error bars in Figures 1 and 2.
Point 3: Improve the "symbols" NO2- and NO3- in line 327.
Response 3: Dear Reviewer, thank you for being a very good observer! We have improved the symbols.
Point 4: And where is the conclusion? Since after the discussion, this section is not presented. Please, if the conclusion is within the discussion, separate both sections.
Response 4: Dear Reviewer, we created an additional chapter (chapter 5. Conclusions), thank you. Please see it below.
- Conclusions
In this study, the effect of sulfide on the transformation of nitrogen compounds by the anammox community has been studied by the use of a laboratory sequencing batch bioreactor with stationary biomass carrier. The 16S rRNA gene profiling of the community of the anammox activated sludge revealed members of the key physiological groups: ammonium oxidizers of the genus Nitrosomonas, nitrite oxidizers of the genus Nitrosospira, anammox bacteria of the genera Candidatus Brocadia and Ca. Jettenia, as well as members of other bacterial genera. Nitrate removal was not sensitive to sulfide at concentrations up to 50 mg S/L and decreased by 17% at 100 mg/L. The threshold of sulfide sensitivity for group I nitrifiers was ~50 mg/L, while anammox bacteria were resistant to sulfide concentrations of up to 100 mg S/L in the incoming water. Group II nitrifiers were obviously the most sulfide-sensitive components of the community. A practice-wise consequence of this research is that Anammox technologies can be applied towards waste waters with high sulfides load. A drastic increase in abundance of members of the family Hydrogenophilaceae at elevated sulfide concentrations, together with precipitation of elemental sulfur was detected. This may indicate sulfide oxidation either by molecular oxygen or by nitrate; this finding requires further investigation. This is the first report on the different effect of sulfide on the growth rate on members of the nitrifying genus Nitrosomonas, increasing/decreasing or not affecting it for different phylotypes at elevated sulfide concentrations. Further investigation are necessary to cultivate and to isolate unique sulfide-tolerant and “sulfide-loving” Nitrosomonas.
Reviewer 3 Report
Manuscript ID water-2504688 Type Article Title Effect of Sulfide on the Processes of Transformation of Nitro-gen Compounds and the Microbial Community Composition in and Anammox Bioreactor The manuscript has been describing the effect of the presence of sulfide in wastewater on the transformation of nitrogen compounds by the anammox community has been insufficiently studied. Also, the present work dealt with the effect of sulfide on the nitrogen removal efficiency and dynamics of nitrogen species in a laboratory sequencing batch bioreactor modeling the functioning of the anammox community carrying out ammonium oxidation via nitritation and anammox and nitrite oxidation. Some important actions need to be done before the acceptance of the manuscript. 1) All chemicals and instruments used in the study should mention the suppliers and manufacturers. 2) Section 2.3 needs to be written again because it looks like copy and paste from previous studies. 3) section of the conclusion needs to be added to the manuscript.just review som spelling of some words
Author Response
Dear respectable Reviewer,
thank you very much for your job on improving our manuscript.
We have repaired the manuscript accordingly. Indeed it is much better now. Please find below our poit-to-point answer.
Sincerely yours,
On behalf of all co-authors,
Drs. A. Dorofeev and Yu.Nikolaev
Point 1: All chemicals and instruments used in the study should mention the suppliers and manufacturers.
Response 1: Dear Reviewer, all necessary changes were made according your advice. Manufacturers and suppliers of all chemicals and instruments used in the study were mentioned.
Point 2: Section 2.3 needs to be written again because it looks like copy and paste from previous studies.
Response 2: Dear Reviewer, we rewrote section 2.3.
Point 3: Section of the conclusion needs to be added to the manuscript.
Response 3: Dear Reviewer, we created an additional chapter (chapter 5. Conclusions), thank you. Please see it below.
- Conclusions
In this study, the effect of sulfide on the transformation of nitrogen compounds by the anammox community has been studied by the use of a laboratory sequencing batch bioreactor with stationary biomass carrier. The 16S rRNA gene profiling of the community of the anammox activated sludge revealed members of the key physiological groups: ammonium oxidizers of the genus Nitrosomonas, nitrite oxidizers of the genus Nitrosospira, anammox bacteria of the genera Candidatus Brocadia and Ca. Jettenia, as well as members of other bacterial genera. Nitrate removal was not sensitive to sulfide at concentrations up to 50 mg S/L and decreased by 17% at 100 mg/L. The threshold of sulfide sensitivity for group I nitrifiers was ~50 mg/L, while anammox bacteria were resistant to sulfide concentrations of up to 100 mg S/L in the incoming water. Group II nitrifiers were obviously the most sulfide-sensitive components of the community. A practice-wise consequence of this research is that Anammox technologies can be applied towards waste waters with high sulfides load. A drastic increase in abundance of members of the family Hydrogenophilaceae at elevated sulfide concentrations, together with precipitation of elemental sulfur was detected. This may indicate sulfide oxidation either by molecular oxygen or by nitrate; this finding requires further investigation. This is the first report on the different effect of sulfide on the growth rate on members of the nitrifying genus Nitrosomonas, increasing/decreasing or not affecting it for different phylotypes at elevated sulfide concentrations. Further investigation are necessary to cultivate and to isolate unique sulfide-tolerant and “sulfide-loving” Nitrosomonas.
Round 2
Reviewer 1 Report
The recommendation for this manuscript is Accept in present form.
Reviewer 2 Report
Dear authors of the manuscript "Nikolai Pimenov, Yuri Nikolaev, Vladimir Grachev, Anna Kallistova, Alexander Dorofeev, Yuriy Litti, Evgeny Gruzdev, Alexey Beletsky, Nikolai Ravin, Andrey Mardanov of the manuscript "Effect of Sulfide on the Processes of Transformation of Nitro-gen Compounds and the Microbial Community Composition in an Anammox Bioreactor" I thank you for considering the observations and comments towards your work. When reviewing the new version of the manuscript, I want to mention that if the editor finds it, it can be accepted in its current form.
Good luck
