Comparison of the Efficiency of Deammonification under Different DO Concentrations in a Laboratory-Scale Sequencing Batch Reactor

Round 1

Reviewer 1 Report

I´m delighted by an opportunity to review an interesting manuscript for your journal. My suggestions, comments and recommendations can be found below in bullet points.
- The title of the manuscript sounds informative and attractive.
- The introduction, which serves as the theoretical background to the paper, is coherently done.
- The methodology of the experiment seems to be correctly developed. 
However, in Figure 1 there is an error when describing the bioractor, or more precisely the probes. Instead of pH it is written Ph. Please correct.  
- Also puzzling is the fact that the intermittent aeration off/on (12/3 min)  is used in phase I and completely different ones in subsequent phases.  No reference or explanation. 
- The article lacks an explanation of the abbreviation COD. There is a reference to the designation in Figure 5, among others. 
- Please improve the quality of figure 6, it is unclear. 
- Please make editorial corrections in the line 216 - 0 mg O2.L

Let me thank authors for the work on the manuscript they did so far. In the current version, the manuscript is not ready for publishing and a revision has to be done. I hope that the authors find my comments useful.

Author Response

The authors thank the Editor and the Reviewer for their time and effort in peer-reviewing the manuscript, their valuable feedback and constructive comments. We have carefully revised the manuscript to improve the readability and technical content of the paper.

Please see below our responses to each of the comments and suggestions. The exact comments from the Reviewers are in black italic type. The responses to the Reviewer’s comments are indented and in Red normal font. The appropriate changes, made in the revised manuscript, have been marked in Track Changes

Reviewer (I)

Comment for water-1541501is listed as follows,

There are some misses been named or error typing.

1. In Figure 1 there is an error when describing the bioreactor, or more precisely the probes. Instead of pH it is written Ph. Please correct.  

Response 1: Figure 1 was corrected as suggested by the Reviewer

2. Also, puzzling is the fact that the intermittent aeration off/on (12/3 min) is used in phase I and completely different ones in subsequent phases. No reference or explanation.

Response 2: The deammonification process needs start-up and gradually adapted with a partial nitritation step to produce NO₂-N which is consumed with the remaining NH₄-N by the anammox bacteria. After adapting the process, the activities of AOB and anammox bacteria increased with stable NOB suppression. Therefore, higher frequencies of intermittent aeration regimes led to stabile produce of NO₂-N that is, NH₄-H oxidation (aerated phase) by AOB versus stabile consume NO₂-N by autotrophic anammox bacteria (non-aerated phase) (Feng et al., 2017; Al-Hazmi et al., 2020, 2021)

3. The article lacks an explanation of the abbreviation COD. There is a reference to the designation in Figure 5, among others.

Response 3: As the Reviewer suggested, in the caption of Figure 5, the COD has been clarified.

4. Please improve the quality of figure 6, it is unclear.

Response 4: The drawing quality of Figure 6 has been improved in the revised manuscript.

5. Please make editorial corrections in the line 216 - 0 mg O2.L

Response 5: Manuscript was corrected as the Reviewer mentioned

References

Al-Hazmi, H.; Lu, X.; Majtacz, J.; Kowal, P.; Xie, L.; Makinia J. Optimization of the Aeration Strategies in a Deammonification Sequencing Batch Reactor for Efficient Nitrogen Removal and Mitigation of N2O Production. Environ. Sci. Technol 2021, 55(2), 1218-1230. doi/10.1021/acs.est.0c04229.

Al-Hazmi, H., Lu, X., Grubba, D., Majtacz, J., Kowal, P., Mąkinia, J., 2021. Achieving Efficient and Stable Deammonification at LowTemperatures—Experimental and Modeling Studies. Energies 14, 3961. https://doi.org/10.3390/en14133961

Feng, Y.; Lu, X.; Al-Hazmi, H.; Mąkinia, J. An overview of the strategies for the deammonification process start-up and recovery after accidental operational failures. Reviews in Environmental Science and Biotechnology 2017, 16(3), 541-568. doi:10.1007/s11157-017-9441-2

Author Response File: Author Response.pdf

Reviewer 2 Report

This article demonstrates the feasibility and importance of dissolved oxygen (DO) as the realization of Deammonification, and obtains the optimal concentration and the best stop-exposure ratio of DO. The experimental design is scientific, starting from a single-cycle batch test. It also verifies the reliability after long-term operation. In addition, it has not only actual experimental data, but also corresponding verification from the perspective of the ASM model.

However, several main questions need to be answered.

1. Dissolved oxygen, as a key control parameter of the Deammonification process, has been a consensus confirmed by a large number of relevant literatures. What are the main points of innovation or contribution to the industry in this article?
2. The inhibition of NOB is indeed one of the biggest bottlenecks of mainstream Anammox. It is difficult for FA and FNA to inhibit NOB in mainstream Anammox, but it is worth discussing whether the DO strategy is as effective as discussed in the article (lines 50 and 51). Several articles have proved that excessive aeration is prone to occur in mainstream Anammox. Please consider it.
3. The stable operation of mainstream Anammox is the current research hotspot and difficult point, which is also involved in the introduction. However, the 900 ± 100 mg/L is adopted for the setting of influent ammonia nitrogen concentration (Table 1), which belongs to a relatively mature high ammonia nitrogen wastewater. In this field, the value of research has been greatly reduced.
4. Under the conditions of 900 ± 100 mg/L influent ammonia nitrogen concentration, 40% exchange ratio, and pH = 7.3-7.9, there must be a FA inhibitory effect. Although DO and the exposure-stop ratio are experimental variables, the entire 180-day experiment can maintain a stable denitrification effect, not only the role of DO, but the role of FA should not be ignored. The authors need to be explained.
5. In the paper, granular sludge is used in SBR. Whether the stirring effect cause the sludge crushing? Will the settlement performance deteriorate? Is the sludge loss serious? How to maintain the sludge concentration in the system?
6. The article mentioned the stable operation of the Deammonification process many times, which essentially lies in the proliferation and maintenance of high activity of AOB, while inhibiting NOB activity or eliminating NOB in the system. Besides, it also cited the microbial community abundance data and qPCR data in other articles for many times. However, in this paper, only the simulated values of microbial biomass of each function (Fig. 5) are not sufficient. Can you provide more direct data support?

Author Response

The authors thank the Editor and the Reviewer for their time and effort in peer-reviewing the manuscript, their valuable feedback and constructive comments. We have carefully revised the manuscript to improve the readability and technical content of the paper.

Please see below our responses to each of the comments and suggestions. The exact comments from the Reviewers are in black italic type. The responses to the Reviewer’s comments are indented and in Red normal font. The appropriate changes, made in the revised manuscript, have been marked in Track Changes

Reviewer (II)

Comment for water-1541501is listed as follows,

1. Dissolved oxygen, as a key control parameter of the Deammonification process, has been a consensus confirmed by a large number of relevant literatures. What are the main points of innovation or contribution to the industry in this article?

Response 1: It is important to have a good understanding of the optimum dissolved oxygen (DO) value in the aeration period and optimize non- aeration time used during the reaction phase. The impact of the DO value and the intermittent aeration frequencies requires further observation and research at the same time. There are many studies that focus only on the right DO setting, or only on the on off aeration mode. It is important to understand both factors at the same time. In this article, we comparison between low DO concentration 0.4-0.7 mg O₂/L and high DO concentration 1-1.5 mg O₂/L which are studied in long-term deammonification operation for 6 months. The results confirmed that the feasible DO setpoint 0.7 and appropriate of intermittent aeration mode off/on (6/3 min) were especially suitable for the optimal balance between the NOB suppression and keeping high activities of AOB and anammox in the system.

2. The inhibition of NOB is indeed one of the biggest bottlenecks of mainstream Anammox. It is difficult for FA and FNA to inhibit NOB in mainstream Anammox, but it is worth discussing whether the DO strategy is as effective as discussed in the article (lines 50 and 51). Several articles have proved that excessive aeration is prone to occur in mainstream Anammox. Please consider it.

Response 2: Thank you for mentioning FA and FNA impact on doammonofication in mainstream wastewater

Actually, we are preparing an article that addresses 'the Inhibition effect of free ammonia on deammonification process under different intermittent aeration strategies in sequencing batch reactor' which was also partially published in Conference: 1st Polish IWA Young Water Professionals Conference “Water, Wastewater and Energy in Smart Cities” Cracow 2017At: Cracow University of Technology, Warszawska St. 24) Cracow, Poland and we will consider that information with a wide explanation

3. The stable operation of mainstream Anammox is the current research hotspot and difficult point, which is also involved in the introduction. However, the 900 ± 100 mg/L is adopted for the setting of influent ammonia nitrogen concentration (Table 1), which belongs to a relatively mature high ammonia nitrogen wastewater. In this field, the value of research has been greatly reduced.

Response 3: Thank you for your note. Partial nitritation/Anammox is studied in mainstream wastewater by Al-Hazmi et al. (2019; 2021b) and in sidestream wastewater by Al-Hazmi et al. (2021a).

As you mentioned the 900 ± 100 mg/L is adopted for the setting of influent ammonia nitrogen concentration (Table 1) also please check in the same table, we calculated the TN loading rate 108-185 mg N/g VSS/d and initial NH₄-N concentration in the reactor 400 ± 30 mg N/L. We indicated that the article focused on sidestream wastewater.

4. Under the conditions of 900 ± 100 mg/L influent ammonia nitrogen concentration, 40% exchange ratio, and pH = 7.3-7.9, there must be a FA inhibitory effect. Although DO and the exposure-stop ratio are experimental variables, the entire 180-day experiment can maintain a stable denitrification effect, not only the role of DO, but the role of FA should not be ignored. The authors need to be explained.

Response 4: Thank you for this indicate.  

Feng et al. (2017) reported that FA can inhibit the activities of nitrifying bacteria at particular NOB. Therefore, the determination of suitable concentration of FA may be effective strategy to enrich AOB population with simultaneous washout of the NOB from the system. Thus, FA can inhibit the activities of NOB more than AOB. Therefore, the suitable concentration of FA may be effective to enrich AOB and washout NOB.

FA concentration was calculated by Anthonisen et al. (1976) Eq. (1) and (2)

Where:

K_b is the ionization constant of the ammonia equilibrium, (at the 25 ℃, K_b=1.8×10^-5)

K_w is the ionization constant of water (at the 25℃ K_w=1.0×10^-14)

TAN (Total Ammonium Nitrogen), T (Temperature) and pH

In our study, the FA concentration was reached to 12.5 mg FA/L. This value does have positively effect on deammonification performance as discussed before in previous numerous studies. According to (Anthonisen et al., 1976) both AOB and NOB are effected by FA. The inhibition threshold for AOB was 10–150 mg FA L^-1 and NOB was begun to inhibit 0.1–1.0 mg FA L^-1 while, NOB was washed out in the range 1.0-10 mg FA L^-1. The optimal FA range stimulating AOB was 5-10 mg FA L^-1 in a shortcut biological nitrogen removal reactor (SBNR) with the NH₄-N concentration of 1000 mg N L^-1 (Chung et al., 2006). Therefore, these characteristics can be utilized to washout NOB from the deammonificatin system obtaining an enriched AOB population. In order to inhibit NOB activities and enrich AOB, the important point is to keep simultaneously the most different specific growth rates between AOB and NOB, thus eliminating the NOB from the deammonificaiton reactor. Therefore, Eq. (1) and (2) show that FA inhibition coupled with other main factors such as TAN (Torà et al., 2010) temperature (Sun et al., 2015) and pH (Zhang et al., 2015) was a possible operational strategy for NOB suppression.

FA has a significant impact on the anammox process. Fernández et al. (2012) supposed that 50% inhibition of the Anammox activity was observed at a FA level of 38 mg FA/L, and 80% inhibition at 100 mg FA/L. In contrast, Aktan et al. (2012) mentioned that the suppression of AnAOB activity was shown only when FA concentration reached 150 mg FA/L. However, AnAOB was completely suppressed when the FA level increased over 150 mg FA/L.

5. In the paper, granular sludge is used in SBR. Whether the stirring effect cause the sludge crushing? Will the settlement performance deteriorate? Is the sludge loss serious? How to maintain the sludge concentration in the system?

Response 5:

In this research, the stirring does not affect the sludge crushing because the stirring is fixed in minimum value at 40-45 rpm. It is a soft move mixing without any effect the settlement performance does not deteriorate the sludge in the system. Moreover, the reactor was operated in cyclic modes (8-24 h), including feeding (30 min, 4 L of the working volume), reaction (7-23 h), sedimentation (2 min) and decantation (28 min, 4 L of the working volume) with that systematic control could be maintained the sludge concentration in the system.

6. The article mentioned the stable operation of the Deammonification process many times, which essentially lies in the proliferation and maintenance of high activity of AOB, while inhibiting NOB activity or eliminating NOB in the system. Besides, it also cited the microbial community abundance data and qPCR data in other articles for many times. However, in this paper, only the simulated values of microbial biomass of each function (Fig. 5) are not sufficient. Can you provide more direct data support?

Response 6: You are completely right, but this work focused on experiments and mathematical modeling. As you know, the microbial community structure analysis needs a long time to get results and specialist analyses. Thus, it is difficult to provide more microbial communities data in this work. In our next publication, we will pay more attention to this and do our best to enrich the article with such data and analysis.

References

Al-Hazmi H.; Grubba D.; Majtacz J.; Kowal P.; Makinia J. Evaluation of Partial Nitritation/Anammox (PN/A) Process Performance and Microorganisms Community Composition under Different C/N Ratio. Water 2019, 11(11), 1-17. doi: 10.3390/w11112270

Al-Hazmi, H.; Lu, X.; Majtacz, J.; Kowal, P.; Xie, L.; Makinia J. Optimization of the Aeration Strategies in a Deammonification Sequencing Batch Reactor for Efficient Nitrogen Removal and Mitigation of N2O Production. Environ. Sci. Technol 2021a, 55(2), 1218-1230. doi/10.1021/acs.est.0c04229.

Al-Hazmi, H., Lu, X., Grubba, D., Majtacz, J., Kowal, P., Mąkinia, J., 2021b. Achieving Efficient and Stable Deammonification at LowTemperatures—Experimental and Modeling Studies. Energies 14, 3961. https://doi.org/10.3390/en14133961

Feng, Y.; Lu, X.; Al-Hazmi, H.; Mąkinia, J. An overview of the strategies for the deammonification process start-up and recovery after accidental operational failures. Reviews in Environmental Science and Biotechnology 2017, 16(3), 541-568. doi:10.1007/s11157-017-9441-2

Aktan, C.K., Yapsakli, K., Mertoglu, B., 2012. Inhibitory effects of free ammonia on Anammox bacteria. Biodegradation 23, 751–762. https://doi.org/10.1007/s10532-012-9550-0

Anthonisen,  a C., Srinath, E.G., Loehr, R.C., Prakasam, T.B.S., 1976. Inhibition of nitrification and nitrous acid compounds. J. Water Pollut. Control Fed. 48, 835–852. https://doi.org/10.2307/25038971

Chung, J., Shim, H., Park, S.J., Kim, S.J., Bae, W., 2006. Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process. Bioprocess Biosyst. Eng. 28, 275–282. https://doi.org/10.1007/s00449-005-0035-y

Fernández, I., Dosta, J., Fajardo, C., Campos, J.L., Mosquera-Corral, A., Méndez, R., 2012. Short- and long-term effects of ammonium and nitrite on the Anammox process. J. Environ. Manage. 95, S170–S174. https://doi.org/10.1016/j.jenvman.2010.10.044

Sun, H., Peng, Y., Wang, S., Ma, J., 2015. Achieving nitritation at low temperatures using free ammonia inhibition on Nitrobacter and real-time control in an SBR treating landfill leachate. J. Environ. Sci. China 30, 157–163. https://doi.org/10.1016/j.jes.2014.09.029

Torà, J.A., Lafuente, J., Baeza, J.A., Carrera, J., 2010. Combined effect of inorganic carbon limitation and inhibition by free ammonia and free nitrous acid on ammonia oxidizing bacteria. Bioresour. Technol. 101, 6051–6058. https://doi.org/10.1016/j.biortech.2010.03.005

Zhang, C., Zhang, S., Zhang, L., Rong, H., Zhang, K., 2015. Effects of constant pH and unsteady pH at different free ammonia concentrations on shortcut nitrification for landfill leachate treatment. Appl. Microbiol. Biotechnol. 99, 3707–3713. https://doi.org/10.1007/s00253-014-6340-0

Author Response File: Author Response.pdf

Reviewer 3 Report

Please see the attached review file.

Comments for author File: Comments.pdf

Author Response

The authors thank the Editor and the Reviewer for their time and effort in peer-reviewing the manuscript, their valuable feedback and constructive comments. We have carefully revised the manuscript to improve the readability and technical content of the paper.

Please see below our responses to each of the comments and suggestions. The exact comments from the Reviewers are in black italic type. The responses to the Reviewer’s comments are indented and in Red normal font. The appropriate changes, made in the revised manuscript, have been marked in Track Changes

Reviewer (III)

Review of manuscript water-1541501

Abstract:

Lines 17-22: These sentences do not read well. Please improve the wording.

Response: The sentences in the manuscript have been corrected (lines 17-20 in the revised manuscript).

Lines 24-26: Specify the values of N removal efficiency, NPR and AUR.

Response: The values of N removal efficiency, NPR and AUR added to the manuscript (lines 22-25 in the revised manuscript).

Introduction:

Lines 46-52: It should be noted, that NOB can be inhibited/outcompeted also by applying spike concentrations of FA at certain points in the SBR cycle (see Zekker et al 2013).

Response: thank for this indicate, Feng et al. (2017) reported that FA can inhibit the activities of nitrifying bacteria at particular NOB. Therefore, the determination of suitable concentration of FA may be effective strategy to enrich AOB population with simultaneous washout of the NOB from the system. Thus, FA can inhibit the activities of NOB more than AOB. Therefore, the suitable concentration of FA may be effective to enrich AOB and washout NOB. In our study, the FA concentration was reached to 12.5 mg FA/L. This value does have positively effect on deammonification performance as discussed before in previous study.

Lines 93-98: The novelty of the current study in comparison with earlier studies should be specified and stressed in a more convincing way.

Response: The reviewer is right, we have expanded the last two paragraphs of the introduction to indicate in detail what's new in the publication (lines 89-91, 98-99 in the revised manuscript).

Materials and methods:

Lines 103-104: I guess that the synthetic medium was free of organic carbon. Correct?

Response: Yes, it is a synthetic medium free of organic carbon.

Results: 

Please provide standard deviations for each measured/calculated value.

We have provided standard deviations for the measured or calculated value for the influent quality and operating parameters, as shown in Figure 6. But for the batch tests or continuous tests, there are no standard deviations for the effluent quality, as each effluent sample was measured only once.

Subchapters 3.1 and 3.2 have been written in a repetitive manner. The organisation of the text can be improved. It is not easy to guess the trends of output parameters resulting from the variation of certain input parameter.

Show error bars in Figures 2, 3 and 6.

Response: For the batch tests or continuous tests, there are no standard deviations for the effluent quality, as each effluent sample was measured only once. So no error bar would be shown in the Figures.

Lines 288-290: Speaking of amounts of different microorganisms changed, do you mean the modelcalculated amounts? 

Response: Exactly. This work focused on experiments and mathematical modeling. As you know, the microbial community structure analysis needs a long time to get results and specialist analyses. Thus, it is difficult to provide more microbial communities data in this work. In our next publication, we will pay more attention to this and do our best to enrich the article with such data and analysis.

Line 429: So, the anoxic period was longer than aerobic?

Response: Yes, all the experiments were carried out under an aeration phase constant of 3 min and the non-aeration was changed (12, 9, 6, 3 min).

Lines 432-434: This in in contradiction with lines 428-429.

Response: Thank you for pointing out this error in the text. This has been corrected in the revised manuscript as follows: off/on instead of on/off (line 438 in the revised manuscript).

Conclusions:

Line 459: Again, the same contradiction as above.

Response: This has been corrected in the revised manuscript as follows: off/on instead of on/off (line 468 in the revised manuscript).

Author contributions are not provided as well as funding.

Author Contributions: added to the manuscript.

Funding: Not applicable.

Supplementary materials

Figure S1: Please explain the symbols (R1-R5, meaning of arrows) in the figure caption.

Response: We added the description in the figure caption, they are:  

R1: Aerobic growth of AOB; R2: Aerobic growth of NOB; R4-R5: Anoxic growth of HDB; R6:        Anaerobic growth of AnAOB, which were in accordance with Table S2.

Figure S2 (also the body text referencing to this figure): The figure shows composition of seeding sludge and predicted microbial dynamics during the operation(right?), not the layout of reactor. How the initial composition was determined? Did you measure the COD of unit mass of wet sludge? If so, how did you obtain the per centages of different bacteria? Sludge also contains extracellular polymers, remnants of dead cells and matter adsorbed from the environment, in addition to living cells. Later on, as I understood, its microbial composition was predicted by the model. Metagenomic analysis was mentioned in the body text. Please add a reference, if it has published, else add a desciption of it to the main text or supplementary materials.

Response: The initial composition was firstly estimated considering the results of our previous study (Al-Hazmi et al., 2020), metagenomic analysis and the measured MLVSS, and then the composition was slightly adjusted in during the preliminary model simulations.

Tables S4 and S5: Add references.

Response: The references have been added in the supplementary files.

References

Feng, Y.; Lu, X.; Al-Hazmi, H.; Mąkinia, J. An overview of the strategies for the deammonification process start-up and recovery after accidental operational failures. Reviews in Environmental Science and Biotechnology 2017, 16(3), 541-568. doi:10.1007/s11157-017-9441-2

Al-Hazmi, H. E., Lu, X., Majtacz, J., et al. Optimization of the Aeration Strategies in a Deammonification Sequencing Batch Reactor for Efficient Nitrogen Removal and Mitigation of N₂O Production[J]. Environmental Science & Technology, 2020, 55(2): 1218-1230.

Author Response File: Author Response.pdf