Effect of organic matters on anammox coupled denitrification system: when nitrite was sufficient

Anaerobic ammonia oxidation (anammox) and denitrification can work together to weaken the influence of organic matter on anaerobic ammonia oxidation bacteria (AAOB) and improve nitrogen removal performance. As the common substrate of anammox and denitrification, nitrite will also affect nitrogen removal performance when it is insufficient, which is not conducive to reflect the endurance of anammox reactor to organic matter. The UASB continuous flow experiment was carried out to investigate the effect of the concentration of glucose and sodium acetate on nitrogen removal performance of anammox reactor under the condition of sufficient nitrite. With glucose as the organic matter, when the chemical oxygen demand (COD) concentration increased to 200 mg l−1, nitrogen removal performance of the system began to deteriorate significantly, and the anammox activity was significantly inhibited. With sodium acetate as the organic substance, the anammox activity was affected when the COD was 20 mg l−1. Adequate nitrite could relieve the inhibition of the coupling system by a low concentration (COD < 200 mg l−1) of glucose organic matter. However, it could not relieve the inhibitory effect of sodium acetate. With the increase of organic concentration, the biological density of AAOB in granular sludge gradually decreased, while the biological density of denitrifying bacteria increased gradually.

3) In all the experiment hydraulic retention time (HRT) maintained was 0.96h.The author mention the justification for maintaining such a lower retention time. 4) References need to be updated.

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
With the development of water treatment processes, the technology of anammox coupled denitrification has become a research hotspot recently. In the present study, the UASB continuous flow experiment was carried out to investigate the effect of the concentration of glucose and sodium acetate on nitrogen removal performance of anammox reactor under the condition of sufficient nitrite nitrogen. Some novel and interesting results were presented. The manuscript could provide a practical guidance for applying in the process of anammox coupled denitrification. And it is appropriate for Royal Society Open Science. But the following questions need to be addressed before consideration of publication. 1) Introduction should be optimized further. More recently published references on the effect of the concentration of organics on nitrogen removal performance of anammox coupled denitrification should be cited and reviewed. And it would be better if the logical structure of introduction is adjusted and perfected.
2) The author need to explain why they choose UASB instead of other types of reactors. 3) When ensuring nitrite nitrogen is sufficient as a substrate, why did the author choose these nitrite nitrogen concentrations (100/110,77 mg/L)? 4) When investigating the effect of the concentration of glucose and sodium acetate on nitrogen removal performance of anammox coupled denitrification, I think the concentration gradient set by the authors is too few. 5) The annotation font of Figure 3a and Figure 3b is incorrect. Please modify it and all similar errors in this article should be re-examined and modified according to journal guide. 6) Line 276-278 of Page 10, please explain the reason why the biological density of AAOB and denitrifying bacteria has changed, and how to prove it. 7) Please explain which reactor and which stage the sludge of R1, R2 and R3 are taken from in "3.4 Analysis of microbial population structure of granular sludge". 8) All format of reference citation in the text should be re-examined and modified according to journal guide.

15-Aug-2019
Dear Miss Yang, The editors assigned to your paper ("Effect of organic matters on anammox coupled denitrification system: when nitrite nitrogen was sufficient") has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) The influence of organic matter has been investigated on ANAMMOX by authors. Please pay attention to adding: 1. recent citations 2. Clearly mention research gaps in existing studies. 3. Language should be improved. 4. organic matter may have some inhibitory effects on ANAMMOX discuss how these could be avoided? 5. In you methods, please mention clearly what was your plan to study effects of organic matter on anammox process.

Reviewer: 2
Comments to the Author(s) The author has described the effect of organic matter on the anammox bacteria and its inhibition effect through different forms of carbon sources. The concept seems to be novel and well explored in detail in terms of ammonia removal rate and growth of coupled denitrification system. However, following points should be addressed to enhance the quality of the manuscript: 1) The author should described more on how sodium acetate & glucose effect more on anammox bacteria rather their effect on denitrification is more pronounced. 2) If author can add biomass yield observed during the course of feeding sodium acetate and glucose, respectively, to arrive at their effect on their specific growth rate.
3) In all the experiment hydraulic retention time (HRT) maintained was 0.96h.The author mention the justification for maintaining such a lower retention time. 4) References need to be updated.

Reviewer: 3
Comments to the Author(s) With the development of water treatment processes, the technology of anammox coupled denitrification has become a research hotspot recently. In the present study, the UASB continuous flow experiment was carried out to investigate the effect of the concentration of glucose and sodium acetate on nitrogen removal performance of anammox reactor under the condition of sufficient nitrite nitrogen. Some novel and interesting results were presented. The manuscript could provide a practical guidance for applying in the process of anammox coupled denitrification. And it is appropriate for Royal Society Open Science. But the following questions need to be addressed before consideration of publication. 1) Introduction should be optimized further. More recently published references on the effect of the concentration of organics on nitrogen removal performance of anammox coupled denitrification should be cited and reviewed. And it would be better if the logical structure of introduction is adjusted and perfected.
2) The author need to explain why they choose UASB instead of other types of reactors. 3) When ensuring nitrite nitrogen is sufficient as a substrate, why did the author choose these nitrite nitrogen concentrations (100/110,77 mg/L)? 4) When investigating the effect of the concentration of glucose and sodium acetate on nitrogen removal performance of anammox coupled denitrification, I think the concentration gradient set by the authors is too few. 5) The annotation font of Figure 3a and Figure 3b is incorrect. Please modify it and all similar errors in this article should be re-examined and modified according to journal guide. 6) Line 276-278 of Page 10, please explain the reason why the biological density of AAOB and denitrifying bacteria has changed, and how to prove it. 7) Please explain which reactor and which stage the sludge of R1, R2 and R3 are taken from in "3.4 Analysis of microbial population structure of granular sludge". 8) All format of reference citation in the text should be re-examined and modified according to journal guide.
Author's Response to Decision Letter for (RSOS-190771.R0) See Appendix A.

Comments to the Author(s) Not required
Review form: Reviewer 3

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation? Accept as is
Comments to the Author(s) All comment has already been responed.

23-Sep-2019
Dear Miss Yang, I am pleased to inform you that your manuscript entitled "Effect of organic matters on anammox coupled denitrification system: when nitrite was sufficient" is now accepted for publication in Royal Society Open Science.
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On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal.
Kind regards, Andrew Dunn Royal Society Open Science Editorial Office Royal Society Open Science openscience@royalsociety.org The reply of "Effect of organic matters on anammox coupled denitrification system: when nitrite was sufficient" Thank you very much for your email with which you sent us the reviewer's report on our paper. We also wish to take this opportunity to thank the reviewer for his constructive comments and valuable recommendations. We have carefully revised the manuscript according to reviewer's suggestion. Our responses to the comments are listed below:

Reviewer: 1
Comments to the Author(s) The influence of organic matter has been investigated on ANAMMOX by authors. Please pay attention to adding: Comment 1: Please pay attention to adding recent citations. Reply: Thanks for your comments. Recent citations has been added as required. Such as: Reference 11,21,22,23,24,25,43. Comment 2: Clearly mention research gaps in existing studies. Reply: Thanks for your comments. Line 54, we insert: "Moreover, in existing studies, there was no systematic comparison on the effects of different organics on anammox coupled denitrification system when nitrite was sufficient." Comment 3: Language should be improved. Reply: Thanks for your comments. Language has been improved as required. Such as: We replace "nitrite nitrogen" with "nitrite" in the revised manuscript. And other modifications have been noted in the manuscript.
Comment 4: organic matter may have some inhibitory effects on ANAMMOX discuss how these could be avoided? Reply: The inhibition of anammox by organic substances is divided into toxicity inhibition and non-toxic inhibition. In this experiment, non-toxic organic substances were used, so there was only non-toxic inhibition. And the mainly non-toxic inhibition was competitive inhibition on substrates, which could be avoided by keeping nitrite sufficient in the experiment. Line 60, we insert "In the rector, nitrite nitrogen was maintained sufficient as substrate to avoid competitive inhibition." Comment 5: In you methods, please mention clearly what was your plan to study effects of organic matter on anammox process. Reply: Thanks for your comments. Line 56, we replace "In this study, the effect of glucose and sodium acetate concentration…" with "In this study, to explore the influence threshold of organic matters on anammox under the condition of sufficient nitrite, and to analyze the competitive inhibition characteristics on substrates of anammox coupled denitrification system, the effect of glucose and sodium acetate concentration…"