Impact of Chemical Oxygen Demand/Total Nitrogen Ratio on Shifting Autotrophic Partial Nitrification to Heterotrophic Nitrification and Aerobic Denitrification in High-Strength Ammonium Wastewater Treatment
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this paper the authors report the effect of changing the C/N ratio during high strength ammonia containing wastewater treatment as being the shifting of partial nitrification (PA) to heterotrophic nitrification- aerobic denitrification (HNAD). The effect of C/N ratio change in relatively low strength ammonia containing and also in high strength ammonia containing wastewater treatment was already shown by similar methods using oxygen uptake rates and/or mainly microbial community studies.
The authors mention that for high strength ammonia containing wastewater treatment and for low C/N ratios, the primary functional microorganism in the PA process is the ammonia oxidizing bacteria (AOB) but as the C/N ratio is increased, due to the occurrence of simultaneous nitrification and denitrification and since AOB could not compete with the heterotrophic nitrification, mainly heterotrophic denitrification (HNAD) bacteria appeared.
The authors in their discussion mention that: " Furthermore, previous study reported that the Amo and Hao genes in HNAD bacteria might complete the oxidation of ammonia through other unknown pathways [12].
Did the authors check the presence of Nitrospira and the Commamox microorganisms? The DNA of Nitrospira, is mentioned in the literature, to have genes encoding ammonia monooxygenase (Amo) and hydroxlyamine dehydrogenase (hao) enzymes.
In the introduction the authors may add another reference:
Friedman, L., Mamane, H., Avisar, D., Chandran, K. "The role of influent organic carbon-to-nitrogen (COD/N) ratio in removal rates and shaping microbial ecology in soil aquifer treatment (SAT)", Water Res. 2018, 146, 197–205.
Some comments:
1. Although each abbreviation is explained when it appears first in the text, since there are a lot of abbreviations, the authors can add a separate list for reference which will ease the lecture.
2. Still in the introduction, it is mentioned that: "The aim of this study is to provide a theoretical foundation, as well as useful reference for the practical utilization of PN process on the wastewater with different C/N ratios", but the performed work is mainly "empirical lab-scale or in vitro and not theoretical.
3. Materials and methods: Since the authors already mentioned the SBR working conditions, and they are also given in Table 1 there is no need to repeat it in the text again
4. Line 88 - If the authors meant Fig. S1 (Supplement - There was no supplement to the reviewed paper).
5. How the authors came out with the different operation times for the SBR? If this is based on previous work, please mention reference
6. Analytical methods: Line 119. Better to say that: "The COD demand for removing 1 gr of N=NO2 is 1.14 gr COD"
7. Equations Lines 128 to 130. Mention please the references for these equations. Specially the NAR equation.
8. PN -Partial Nitrification and Protein - PN have the same abbr. please change and as mentioned before add a separate Abbr. table to all these Abbrs.
9. For the SOUR test - Why NaOCL3 was added? The set up needs to be explained not just specified- what was the rational?
10. Results: line 229 -230. It is confusing to say that a TN removal of 7.2-39.3 % was obtained. Better to specify that the removal percentage increased from 7.2 to 39.3 from the start to the 4th day in Rb
11. Lines 247-249 What is the point to mention the TN for the first 4 days? The sharp decrease point is important
12. Typical single cycle in reactors: Is the reason to measure different N removal rates at day 42 only because the reactors were run till that time or is there another reason?
13. Does the difference in PH significant enough to mention that in lines 289-290?
14. Who are "them" in line 294?
15. Generally - Colors or different signs must be clearer on the graphs.
16. Fig. 3. - Specify in the title better all the abbreviations like ... LB-PS, PN-PS...
17. Line 340-341 Again no Fig. S2. No supplements were available.
18. Fig. 5 and line 396-408. As there are various genus in every phylum it is challenging to conclude on functionality, especially on denitrification. When there are variety of heterotrophs that can conduct part of the reaction only. It would be better to consider moving the phylum section to the supplementary material.
19. Line 425-6 " The relative abundances of denitrifiers at C/N ratios of 2 and 4 were lower than those of 1, but TN removal reached 69.9 and 75.4%, respectively". The difference after all, is not significant while C/N was increased from 2 to 4.
20. Line 430 - Elaborate. Why the authors believe ANAMMOX was not detected.
21. Line 460 - Can the authors explain why all genes, except napA and napB were observed?
22. Line 484-486 - Not necessarily true. It is species specific
23. Please refer to why the SOUR and NOB is much lower in C/N 0.5 but not in the other C/N ratios? Also please explain, why the total SOUR of the treatments at C/N of 2 and 0 are so similar.
24. The authors in their abstract wrote:" Were appeared" suggested to correct it. Also, it is strongly recommended to go through the whole paper for English language editing.
Comments on the Quality of English Language
Extensive editing of English language required
Author Response
1. The authors in their discussion mention that: “Furthermore, previous study reported that the Amo and Hao genes in HNAD bacteria might complete the oxidation of ammonia through other unknown pathways [12].” Did the authors check the presence of Nitrospira and the Commamox microorganisms? The DNA of Nitrospira, is mentioned in the literature, to have genes encoding ammonia monooxygenase (Amo) and hydroxlyamine dehydrogenase (hao) enzymes.
Reply: Thank you for your scientific suggestion. Although numerous studies have shown the presence of amo and hao genes in comammox. Nitrospira, the bacterial genus to which comammox typically belongs, has not been detected in this study. Therefore, we believe that the predicted amo and hao genes likely originate from AOB and HNAD bacteria.
2. In the introduction the authors may add another reference:
Friedman, L., Mamane, H., Avisar, D., Chandran, K. “The role of influent organic carbon-to-nitrogen (COD/N) ratio in removal rates and shaping microbial ecology in soil aquifer treatment (SAT)”, Water Res. 2018, 146, 197–205.
Reply: Thank you for your scientific suggestion. The reference have been added in the revised manuscript, as shown below, “Friedman et al. found that the genus Nitrospira predominated over AOB and the genus Nitrobacter in oil aquifer treatment of synthetic secondary effluent when COD/N ratios were 3 and 5, strongly indicating the activity of complete ammonia-oxidizing bacteria”. (Lines 51 to 54 )
Some comments:
(1) Although each abbreviation is explained when it appears first in the text, since there are a lot of abbreviations, the authors can add a separate list for reference which will ease the lecture.
Reply: Thanks for your suggestion. We have compiled all abbreviations into a table and placed it at the beginning of the article.
(2) Still in the introduction, it is mentioned that: “The aim of this study is to provide a theoretical foundation, as well as useful reference for the practical utilization of PN process on the wastewater with different C/N ratios”, but the performed work is mainly "empirical lab-scale or in vitro and not theoretical.
Reply: Thanks for your suggestion. We have made modifications to the revised manuscript, as shown below “The aim of this study is to offer practical insights for applying the PN process in wastewater treatment with varying COD/N ratios”. (Lines 76 to78)
(3) Materials and methods: Since the authors already mentioned the SBR working conditions, and they are also given in Table 1 there is no need to repeat it in the text again.
Reply: Thanks for your suggestion. We have removed duplicate sections and added more details about the SBR experiment in the revised manuscript based on the suggestions from other reviewers, as shown blew, “Five SBR reactors were used in the experiment, each with a working volume of 1 L. The reactors were equipped with internal stirring, aeration, and temperature control devices, maintaining a temperature of 26 ± 1°C. The SBR volume exchange ratio was 50%, and the hydraulic retention time was 12 hours. Every single cycle of the SBRs had a total duration of 6 hours, which included 0.25 hours of filling, 5 hours of aerobic reaction, 0.5 hours of settling, and 0.25 hours of decanting. To suppress the growth of nitrite-oxidizing bacteria (NOB), the sludge retention time and DO concentration was controlled at 13.5 days and 0.5-1 mg/L, respectively. Meanwhile, due to the different COD concentrations in each reactor, the aeration rates required to maintain a consistent DO concentration varied, as shown in Table 1”. (Lines 82 to 92)
(5) How the authors came out with the different operation times for the SBR? If this is based on previous work, please mention reference.
Reply: We used a time controller to regulate the power supply to various devices, enabling different operation times. During the filling stage, the peristaltic pump was activated for 15 minutes to complete the wastewater intake. During the aeration stage, the air pump and magnetic stirrer operated to facilitate the aerobic reaction. When the air pump and magnetic stirrer were deactivated, the reactor entered the settling stage. Finally, the effluent was discharged from the reactor using the peristaltic pump.
(6) Analytical methods: Line 119. Better to say that: “The COD demand for removing 1 gr of N=NO2 is 1.14 gr COD”.
Reply: Thanks for your suggestion. We have made modifications to the revised manuscript.
(7) Equations Lines 128 to 130. Mention please the references for these equations. Specially the NAR equation.
Reply: Thank you for your reminder. The reference have added to the revised manuscript.
(8) PN -Partial Nitrification and Protein - PN have the same abbr. please change and as mentioned before add a separate Abbr. table to all these Abbrs.
Reply: We were really sorry for our careless mistake. In the revised manuscript, “PN” refers exclusively to partial nitrification, and “protein” is no longer abbreviated.
(9) For the SOUR test - Why NaOCl3 was added? The set up needs to be explained not just specified- what was the rational?
Reply: Thank you for your suggestion. NaOCl3 was used to inhabited the NOB activity, and the arylthiourea was added as an inhibitor of AOB. The reasons for adding the two substances mentioned above have been included in the revised manuscript.
(10) Results: line 229 -230. It is confusing to say that a TN removal of 7.2-39.3 % was obtained. Better to specify that the removal percentage increased from 7.2 to 39.3 from the start to the 4th day in Rb.
Reply: Thank you for your suggestion. After 4 days, the TN removal percentage of Rb fluctuated and did not directly increase from 7.2% to 39.3%. To make it easier to understand, change this sentence to “The TN removal percentage fluctuated between 7.2% and 39.3% in Rb starting from day 4”. (Lines 208 to 209)
(11) Lines 247-249 What is the point to mention the TN for the first 4 days? The sharp decrease point is important.
Reply: The change in TN indicated the presence of denitrification capability within the reactor. In the initial stages of Rd and Re, their TN removal percentage were not high, suggesting that PN was still occurring. However, as the operation progressed, a significant decrease in effluent TN indicated an increase in the denitrification capacity of these two reactors.
(12) Typical single cycle in reactors: Is the reason to measure different N removal rates at day 42 only because the reactors were run till that time or is there another reason?
Reply: As the reviewer noted, the typical single cycle experiment was conducted on day 42 primarily because the reactor operation concluded on that day.
(13) Does the difference in PH significant enough to mention that in lines 289-290?
Reply: Thank you for your suggestion. pH is a key indicator for distinguishing between acid production during partial nitrification and alkali production during denitrification. The continuous decrease in pH in Ra indicates that the PN process was consistently active in this reactor. In both Rb and Rc, the pH first decreased and then increased, suggesting that denitrification begins after an extended PN process. In Rd and Re, the continuous increase in pH corresponded to the ongoing denitrification process.
(14) Who are "them" in line 294?
Reply: “Them” refer to AOB and NOB, respectively.
(15) Generally - Colors or different signs must be clearer on the graphs.
Reply: Thank you for your suggestion. We have modified the colors and signs in the graphs to enhance differentiation.
(16) Fig. 3. - Specify in the title better all the abbreviations like ... LB-PS, PN-PS...
Reply: Thank you for your reminder. We have updated the abbreviations in Fig. 3 and included all abbreviations at the beginning of the article.
(17) Line 340-341 Again no Fig. S2. No supplements were available.
Reply: We were really sorry for our careless mistake. Thank you for your reminder. Supplementary materials will be added to the revised content.
(18) Fig. 5 and line 396-408. As there are various genus in every phylum it is challenging to conclude on functionality, especially on denitrification. When there are variety of heterotrophs that can conduct part of the reaction only. It would be better to consider moving the phylum section to the supplementary material.
Reply: We think this is an excellent suggestion. At the phylum level, there is significant commonality among microorganisms, making it challenging to clarify the roles of functional microorganisms. We have included this information in the supplementary materials.
(19) Line 425-6 “The relative abundances of denitrifiers at C/N ratios of 2 and 4 were lower than those of 1, but TN removal reached 69.9 and 75.4%, respectively”. The difference after all, is not significant while C/N was increased from 2 to 4.
Reply: We apologize for any lack of clarity in our writing and have revised this section accordingly in the revised manuscript, as shown below, “The relative abundances of the denitrifiers at COD/N ratios of 2 and 4 were lower than those of 1, but TN removal reached 69.9 and 75.4%, respectively, which are higher than the 53.6% observed at a COD/N of 1”. (Lines 336 to 339)
(20) Line 430 - Elaborate. Why the authors believe ANAMMOX was not detected.
Reply: Thank you for your scientific suggestion. In this study, the absence of the anammox process in the reactor was determined by the lack of anammox-related bacterial genera in the results of high throughput 16S rRNA sequencing. Although the 338F-806R primer used for high throughput sequencing is a universal bacterial primer, it was highly efficient in detecting all anammox bacteria except Candidatus Scalindua [1]. Candidatus Scalindua is a marine anammox bacteria that rarely appears in low-salinity wastewater treatment [2]. Therefore, the absence of anammox bacteria detected using this method strongly indicates that anammox process were not present in any reactor. Additionally, in previous studies using this primer for 16S rRNA sequencing high throughput, the absence of anammox genera in the results corresponded with the absence of the anammox activity [3-5]. Thus, we believe it is reasonable to confirm the occurrence of the anammox process by examining the presence of anammox bacterial genera. However, in future research, we plan to employ activity testing and qPCR to confirm the presence of anammox bacteria and processes from multiple perspectives, thereby enhancing the credibility of the study.
(21) Line 460 - Can the authors explain why all genes, except napA and napB were observed?
Reply: Thank you for your scientific suggestion. Although both the nap and nar genes can encode nitrate reductase, their locations within microorganisms differ. The periplasmic nitrate reductase encoded by nap is located in the cytoplasm, whereas the cytosolic nitrate reductase encoded by nar is bound to the cell membrane. Due to their different locations, periplasmic nitrate reductase has a higher tolerance to dissolved oxygen and is more likely to function under aerobic conditions [6]. Consequently, the relative abundance of other denitrification genes aligned with that of denitrifiers, while the relative abundance of napA and napB aligned with that of HNAD.
(22) Line 484-486 - Not necessarily true. It is species specific.
Reply: Thank you for your scientific suggestion. Due to the incomplete literature review, there are indeed omissions in this section. Both AOB and NOB include bacteria with k-strategy and r-strategy. Notably, k-strategy NOB have a higher affinity for oxygen compared to AOB, with the Nitrospira genus being the primary representative of this strategy [7, 8]. The inhibitory concentrations of free ammonia (FA) and free nitrous acid (FNA) for Nitrospira are relatively low, ranging from 0.04-0.08 mg/L and 0.02 mg/L, respectively [9, 10]. When the COD/N ratios was 0 and 1, at least one of the concentrations of FA or FNA exceeded the inhibitory threshold, leading to the inhibition of Nitrospira. This inhibitory effect was further supported by the absence of Nitrospira detected in the bacterial community structure. We have revised this section accordingly in the revised manuscript, as shown below, “Even though the PN process could also be achieved under high DO, the upholding of low DO was still an essential factor that affected the PN. However, maintaining a low DO concentration did not necessarily guarantee a stable PN process. Low DO (0.1-1 mg/L) concentrations did not inhibit the activity of NOB, and the relative abundance of NOB was greater than that of AOB during the treatment of rural wastewater. In a long-term activated sludge system operating under low DO conditions, Liu and Wang demonstrated that the system ultimately achieved complete nitrification rather than PN at DO levels of 0.37 mg/L and 0.16 mg/L. These findings are related to the types of NOB, NOB can employ either r-strategy or k-strategy survival strategies. In this study, the r-strategist bacterium Nitrobacter exhibited a higher oxygen half-saturation constant (1.98 mgO2/L) compared to Nitrosomonas (0.22-0.56 mgO2/L), which was the only AOB genus detected. Consequently, Nitrobacter could be eliminated at low DO concentration. In contrast, the k-strategist NOB Nitrospira, with a lower oxygen half-saturation constant (0.13 mgO2/L), was not effectively limited by low DO concentrations. The inhibitory concentrations of FA and FNA for Nitrospira are relatively low, ranging from 0.04-0.08 mg/L and 0.02 mg/L, respectively. At COD/N ratios of 0 and 1, at least one of the concentrations of FA or FNA could reach the inhibitory threshold, thereby inhibiting the growth of Nitrospira” (Lines 412 to 431)
(23) Please refer to why the SOUR and NOB is much lower in C/N 0.5 but not in the other C/N ratios? Also please explain, why the total SOUR of the treatments at C/N of 2 and 0 are so similar.
Reply: Thank you for your scientific suggestion We observed that the SOUR of NOB was significantly lower when the COD/N ratio was 0.5 compared to other COD/N ratios. Although we have thoroughly analyzed the factors of DO, FA, FNA, and the abundance of functional genes from multiple perspectives, none of these factors, whether through their inhibitory effects on AOB or the abundance of NOB-related genes during nitrification, can fully account for the reduced SOUR of NOB at a COD/N ratio of 0.5. Consequently, this finding prompts the need for more in-depth research in future studies to elucidate the underlying mechanisms. Additionally, the similarity in the SOUR of total bacteria at COD/N ratios of 0 and 2 may be attributed to the comparable microbial biomass present in these two reactors.
(24) The authors in their abstract wrote:" Were appeared" suggested to correct it. Also, it is strongly recommended to go through the whole paper for English language editing.
Reply: Thank you for your suggestion. The content has been modified in the abstract. According to your advice, this manuscript has been edited for proper English language, grammar, punctuation, spelling, and overall style by the highly qualified native English speaking editors at MDPI, and the certificate has been provided as follow.
Reference:
[1] Y. Yang, M. Li, H. Li, X.-Y. Li, J.-G. Lin, M. Denecke, J.-D. Gu, Specific and effective detection of anammox bacteria using PCR primers targeting the 16S rRNA gene and functional genes, Science of The Total Environment, 734 (2020) 139387.
[2] S. Yin, J. Li, H. Dong, Z. Qiang, Unraveling the nitrogen removal properties and microbial characterization of “Candidatus Scalindua”-dominated consortia treating seawater-based wastewater, Science of The Total Environment, 786 (2021) 147470.
[3] H. Wang, Q. Dan, R. Du, J. Li, S. Wang, X. Li, Q. Zhang, Y. Peng, Enhanced nitrogen removal in partial nitrification-anammox (PNA) suspended sludge system for real municipal wastewater treatment at extremely low carbon to nitrogen ratio, Chemical Engineering Journal, 452 (2023) 139256.
[4] Q. Huang, W.-L. Du, L.-L. Miao, Y. Liu, Z.-P. Liu, Microbial community dynamics in an ANAMMOX reactor for piggery wastewater treatment with startup, raising nitrogen load, and stable performance, AMB Express, 8 (2018) 1-15.
[5] Y. Zhao, J. Li, Q. Liu, Z. Qi, X. Li, Q. Zhang, J. Sui, C. Wang, Y. Peng, Fast start-up and stable operation of mainstream anammox without inoculation in an A2/O process treating low COD/N real municipal wastewater, Water Research, 231 (2023) 119598.
[6] R. Gupta, B. Poddar, S. Nakhate, A. Chavan, A. Singh, H. Purohit, A. Khardenavis, Role of heterotrophic nitrifiers and aerobic denitrifiers in simultaneous nitrification and denitrification process: a nonconventional nitrogen removal pathway in wastewater treatment, Letters in applied microbiology, 74 (2022) 159-184.
[7] R. Nogueira, L.F. Melo, Competition between Nitrospira spp. and Nitrobacter spp. in nitrite‐oxidizing bioreactors, Biotechnology and bioengineering, 95 (2006) 169-175.
[8] E. Martinez‐Rabert, C.J. Smith, W.T. Sloan, R. González‐Cabaleiro, Biochemistry shapes growth kinetics of nitrifiers and defines their activity under specific environmental conditions, Biotechnology and Bioengineering, 119 (2022) 1290-1300.
[9] D.-J. Kim, D.-I. Lee, G.-C. Cha, J. Keller, Analysis of free ammonia inhibition of nitrite oxidizing bacteria using a dissolved oxygen respirometer, Environmental Engineering Research, 13 (2008) 125-130.
[10] V. Vadivelu, J. Keller, Z. Yuan, Free ammonia and free nitrous acid inhibition on the anabolic and catabolic processes of Nitrosomonas and Nitrobacter, Water science and technology, 56 (2007) 89-97.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsGood day! look at the file
Comments for author File: Comments.pdf
Author Response
1. It is necessary to check the design of the article, for example: various formats (templates) up to section 3.6; spelling of the authors’ surnames with reference to the University. The entire text must be carefully checked: L157 – The word "with" is used twice; L534 – No Space; L 224 – The abbreviation TN (total nitrogen?) should be given here for a better understanding by readers; L144, 145, 177 – The same unit of measurement is presented differently here (I think it’s better to do it in the same style).
Reply: Thank you for your rigorous comment. We have addressed the formatting issues in the revised manuscript and removed the duplicate content. TN is indeed an abbreviation for total nitrogen. Additionally, all abbreviations have been compiled into a table, which has been placed at the beginning of the article. The units on Lines 144, 145, and 177 have also been standardized.
2. L 232 – The phrase “to denitrifiers grow” should be checked. Maybe we are talking about the growth of denitrifying bacteria?
Reply: Thank you for your suggestion. We have revised this section accordingly in the revised manuscript, as shown below, “which was higher than that of Ra and was probably caused by the presence of organic carbon sources that helped the denitrifiers to grow.” (Lines 209 to 210)
3. L92-93: Why was such a high degree of oxygen saturation adopted? What is aeration rate? In aeration tanks, the required concentration in aerobic areas is usually no more than 2-4 mg/L. Or is this an error in the unit of measurement? It is necessary to check the data carefully!
Reply: Thank you for your suggestion. The DO concentration in each reactor was maintained at 0.5-1 mg/L. Due to the varying COD concentrations in each reactor, different aeration rates were applied. The aeration rates in reactors Ra, Rb, Rc, Rd, and Re were 600, 800, 1200, 1800, and 2000 mL/min, respectively.
4. The quality of Figure 1 needs to be improved: the upper letters (a-J) are superimposed on the graphs; in the legend, it is not entirely clear what the words "Inffluent" and "Enffluent" mean in a particular case.
Reply: Thank you for your suggestion. The numbering of the Fig. 1 has been changed to uppercase and placed above the graphs. "Influent" and "Effluent" respectively represent the inlet and outlet wastewater in the reactor.
5. The quality of Figure 2 also needs to be improved: the upper letters are superimposed on the graphs.
Reply: Thank you for your suggestion. The numbering of the Fig. 2 has been changed to uppercase and placed above the graphs.
6. The quality of Figure 5 also needs to be improved: the upper letters are superimposed on the graphs; there are small and fuzzy fonts.
Reply: Thank you for your suggestion. The numbering of the Fig. 5 has been changed to uppercase and placed above the graphs, and the font within the image has been enlarged for easier viewing.
7. Not all terms of Equations 1-4 are described below.
Reply: Thank you for your suggestion. We have revised this section accordingly in the revised manuscript, as shown below, “where CInf-NH4+ and CEff-NH4+are the ammonium concentrations in the influent and effluent, and CInf-NO2- and CEff-NO2- are the nitrite and nitrate concentrations in the effluent. CNH4+ and CNO2- are the ammonium and nitrite concentrations. T indicates the temperature (oC).” (Lines 120 to 123)
8. Here are references to Fig. S.1 (L88) and Fig. S.2 (L341), which I did not find. Maybe there are no Supplementary materials here? Or is it a typo?
Reply: We were really sorry for our careless mistake. Thank you for your reminder. Supplementary materials will be added to the revised content.
9. Section 2.1: Information on oxygen concentration and water temperature, which were maintained by during nitrification and denitrification in SBR reactors should be added.
Reply: Thank you for your scientific suggestion. The temperature and DO concentration inside the reactor were controlled at 26 ± 1°C and 0.5-1 mg/L, respectively.
10. References must be designed according to the requirements of Water magazine.
Reply: Thank you for your suggestion. References have been formatted according to the guidelines of Water magazine.
11. Water temperature is the most important indicator of biological treatment, along with other equally important ones. It is necessary to explain why data on temperature changes in reactors in nitrification-denitrification processes are not provided.
Reply: Thank you for your scientific suggestion. During the experiment, we maintained the temperature inside the reactor at 26 ± 1°C using a temperature control device. This was because previous studies have shown that the growth rate of AOB is significantly higher than that of NOB at temperatures above 25°C [1].
12. It is necessary to present here a diagram of the mechanism of processes in reactors for a better understanding of the content of the article.
Reply: Thank you for your scientific suggestion. The reaction mechanism diagram is shown in Fig. 1. When COD/N ratios were 0, 0.5, and 1, the growth of NOB was inhibited by low dissolved oxygen and high concentrations of FA and FNA, allowing the PN process to be completed by AOB. Simultaneously, the denitrification process was carried out by denitrifying bacteria present in the reactor. As the COD/N ratio increases to 2 and 4, the denitrification process in the reactor became dominated by HNAD bacteria.
13. In my opinion, the Conclusion needs to be rewritten in a slightly more detailed and understandable way, for example, some general conclusions from sections 3.Results and 4.Discussion should be transferred here.
Reply: Thank you for your scientific suggestion. Based on the suggestions from other reviewers, we have revised the conclusion as follows: “When treating high-ammonia wastewater, an increase in the COD/N ratio could shift the process from autotrophic PN to HNAD. At COD/N ratios of 0 and 0.5, the growth of NOB was inhibited by low dissolved oxygen and high concentrations of FA and FNA, leading to a significant proliferation of AOB. Consequently, the relative abundance of Nitrosomonas was 15.5% at COD/N = 2 and 9.7% at COD/N = 4. However, the PN process was disrupted at a COD/N ratio of 1 and completely destroyed at COD/N ratios of 2 and 4, with HNAD becoming the dominant process. The proliferation of HB at C/N ≥ 1 inhibited the autotrophic AOB, allowing HNAD bacteria to become the predominant denitrifying microorganisms. Thauera, Pseudomonas, Flavobacterium, Hydrogenophaga, Acinetobacter, Microbacterium, Corynebacterium, Brevundimonas and Comamonas—all HNAD bacteria—emerged at COD/N ratios of 2 and 4, with their relative abundance increasing as the total COD/N ratio rose, coinciding with the disappearance of Nitrosomonas. Therefore, in the treatment of high-ammonia wastewater, when COD/N ≤ 1, the PN process can be combined with other processes, such as partial denitrification and anammox, for biological denitrification. When COD/N > 1, HNAD should be used as the primary denitrification process.” (Lines 498 to 541)
14. In general, in my opinion, the article is written in a very complex scientific language. There is a high saturation of special terms without explanations. This reduces the ability to understand the essence of the text by a wide range of readers.
Reply: We have reduced some unnecessary abbreviations and added more detailed content in the Materials and Methods section based on your and other reviewers’ comments to facilitate readers’ understanding. Additionally, we have polished the language through MDPI to enhance readability, and the certificate has been provided as follow.
Reference:
[1] J. Guo, Y. Peng, H. Huang, S. Wang, S. Ge, J. Zhang, Z. Wang, Short-and long-term effects of temperature on partial nitrification in a sequencing batch reactor treating domestic wastewater, Journal of Hazardous Materials, 179 (2010) 471-479.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsToo many unnecessary abbreviations make it hard to read and review.
1. Grammatical errors check and English polishing are very necessary.
2. Paper title is redundant and hard to understand the work.
3. Note the scientific meanings of C/N = 0.
4. L92-93: Wrong unit for “the aeration rate” (2000 mg/L).
5. L147: Proteins (PN)??
6. Too many unnecessary abbreviations make it hard to read.
7. L104-105: Give the actual volume of seed for each reactor.
8. L210-212, 229-232: Give data or citations.
9. Parameters EPS, SOUR and microbial community were determined. They should be discussed simultaneously.
10. Section Conclusion was weak. Its content was of only experimental results. Give more information about the conclusion drawn from the experimental result.
Comments on the Quality of English LanguageGrammatical errors check and English polishing are very necessary.
Author Response
1. Please explain the abbreviations when they appear for the first time, e.g., C/N and COD. In particular, C/N in the manuscript refers to COD/total nitrogen, but it can easily be mistaken for total nitrogen/total carbon, after all, this is a more common representation except in the field of sewage treatment.
Reply: Thank you for your scientific suggestion. We have compiled all abbreviations into a table and placed it at the beginning of the article. Based on your suggestion, we will replace C/N with COD/N in the article.
2. What was added to increase COD in the experiment?
Reply: Sodium acetate was added as a carbon source to increase COD in the simulated wastewater, as described in Section 2.2.
3.Explain why the aeration rates were different for different treatments.
Reply: Thank you for your scientific suggestion. By controlling the aeration rate, the DO concentration in each reactor was maintained at 0.5-1 mg/L to suppress the growth of NOB. Due to the different COD concentrations in each reactor, different aeration rates were adopted. We have added corresponding content in the revised manuscript, as shown below “Meanwhile, due to the different COD concentrations in each reactor, the aeration rates required to maintain a consistent DO concentration varied, as shown in Table 1”. (Lines 90 to 91)
4. Specific information on microbial sampling.
Reply: Thank you for your suggestion. Fifty milliliters of sludge samples was collected from each reactor and centrifuged at 10000 rpm for 15 minutes at 4oC on day 42. We have added corresponding content in the revised manuscript, as shown below “The SBRs was located in Kunming, Yunnan Provence, Chian at 24°50′55″N, −24°51′45″E. On day 42 (June 17, 2021), fifty milliliters of suspended sludge samples were collected from each reactor and centrifuged at 10,000 rpm for 15 minutes at 4°C”. (Lines 159 to 161)
5. The physicochemical parameters were intensively sampled and analyzed, but the analysis of the microbial community was not very reliable. The 16S universal primers detected only bacteria, not microbial communities. After all, microbial communities are more than just bacteria. The manuscript should avoid inaccurate statements. The manuscript only has one sample of a bacterial community in each reactor. What was the timing and location of this sampling in the reactor, and what were the sampling and processing methods?
Reply: Thank you for your scientific suggestion. We apologize for the inappropriate vocabulary used in the article, which led to imprecise results. The 16S universal primer is capable of detecting bacterial communities, not the broader microbial communities. Therefore, to ensure accuracy, we have used “bacterial community structure” instead of “microbial community” in the conclusion.
Additionally, we regret that bacterial community detection was only conducted at the final stage of the reactor during the experimental process, leaving a gap in understanding the changes in bacterial community structure throughout reactor operation. This omission has limited our insight into the evolution of bacterial community structure. In future research, we plan to collect samples throughout the reactor’s operation to gain a more comprehensive understanding of these changes.
We have included a more detailed description of the sampling time, location, and process in the revised manuscript, as shown in “The SBRs was located in Kunming, Yunnan Provence, Chian at 24°50′55″N, −24°51′45″E. On day 42 (June 17, 2021), fifty milliliters of suspended sludge samples were collected from each reactor and centrifuged at 10,000 rpm for 15 minutes at 4°C”. (Lines 159 to 161)
6. There is only one variable in the experiment, i.e., C/N, so it is meaningless to do CCA analysis, and the results are completely predictable. Even the PCoA analysis is of little use because number of samples is too small.
Reply: Thank you for your scientific suggestion. Due to the limited scope of variables, specifically C/N, the application of CCA and PCoA analyses was not appropriate. Therefore, we conducted correlation analysis on reactor performance, sludge characteristics, and bacterial abundance, followed by a comprehensive summary of the results. This section has been incorporated into the revised manuscript as follows: “To quantitatively assess the relationship between the bacterial communities and the performance/sludge data at different COD/N ratios, Pearson's correlation coefficient was computed, and the degree of association was statistically evaluated, as shown in Fig. 2B. COD/N was found to be significantly positively correlated with Acholeplasma (r=0.88, p<0.1), Luteimonas (r=0.95, p<0.1), Acinetobacter (r=0.90, p<0.1), Flavobacterium (r=0.99), and Corynebacterium (r=0.89), all of which are classified as heterotrophic, denitrifying, and HNAD bacteria. An increase in COD/N is beneficial for their growth. Additionally, Nitrosomonas showed a significant positive correlation with ARR, which is consistent with its role as the only detected AOB bacterial genus capable of converting ammonia nitrogen. There is no significant correlation between it and SOUR-AOB (r=0.58). However, a significant positive correlation existed between EPS content and Moheibacter (r = 0.90, p < 0.1), as EPS contained substantial amounts of organic matter that could support Moheibacter metabolism and growth. Additionally, no correlation was found between the SOUR of total bacteria and any specific bacterial genus, indicating that no single bacterial species predominantly influences the increase in COD/N. Instead, various bacterial genera are involved in oxygen consumption at different stages”. (Lines 358 to 374)
7. The PICRUSt for predicting microbial community function is now in debate, and it is believed that the power of such predictions is very limited, especially for complex communities. Because the 16S sequencing length is too short, using it to predict other functional genes will have very limited accuracy. I retain my doubts about this analysis, although the authors obtained very good results.
Reply: Thank you for your scientific suggestion. Although PICRUSt technology is controversial in predicting bacterial functional genes, it is still widely used for bacterial community functional analysis due to its cost-effectiveness and convenience. PICRUSt has been applied in various fields, including soil [1], gut microbiomes [2], marine microorganism [3], digestion of animal manures [4], and wastewater treatment [5]. In a study involving 16S rRNA sequencing and metagenomic sequencing of bacteria in the South Pacific, the correlation coefficients revealed strong associations between the predicted KO ranks and the MGS KO abundances [3]. Additionally, the latitudinal trends in metabolic pathways predicted by PICRUSt were consistent with the observed physicochemical parameters [3]. The trends in the relative abundance of the narG and nosZ genes predicted by PICRUSt align with the results obtained from qPCR analysis [7, 8]. When examining the effect of phenacetin on the PN process, the abundance trends of amoA, amoB, and amoC genes analyzed by PICRUSt were also consistent with qPCR results [9]. Similarly, the study by Liu et al. [8] demonstrated that the nitrogen cycle genes analyzed by PICRUSt correlated well with qPCR results. Therefore, using PICRUSt to predict trends in genes related to the nitrogen cycle is considered reliable.
8. Appropriate conclusions should be added. For example, Since both PN and HNAD can treat high-nitrogen wastewater, what guidance does the shift between them provide for the selection of processes in actual water treatment?
Reply: Thank you for your scientific suggestion. Based on the suggestions from other reviewers, we have revised the conclusion as follows: “When treating high-ammonia wastewater, an increase in the COD/N ratio could shift the process from autotrophic PN to HNAD. At COD/N ratios of 0 and 0.5, the growth of NOB was inhibited by low dissolved oxygen and high concentrations of FA and FNA, leading to a significant proliferation of AOB. Consequently, the relative abundance of Nitrosomonas was 15.5% at COD/N = 2 and 9.7% at COD/N = 4. However, the PN process was disrupted at a COD/N ratio of 1 and completely destroyed at COD/N ratios of 2 and 4, with HNAD becoming the dominant process. The proliferation of HB at C/N ≥ 1 inhibited the autotrophic AOB, allowing HNAD bacteria to become the predominant denitrifying microorganisms. Thauera, Pseudomonas, Flavobacterium, Hydrogenophaga, Acinetobacter, Microbacterium, Corynebacterium, Brevundimonas and Comamonas—all HNAD bacteria—emerged at COD/N ratios of 2 and 4, with their relative abundance increasing as the total COD/N ratio rose, coinciding with the disappearance of Nitrosomonas. Therefore, in the treatment of high-ammonia wastewater, when COD/N ≤ 1, the PN process can be combined with other processes, such as partial denitrification and anammox, for biological denitrification. When COD/N > 1, HNAD should be used as the primary denitrification process.” (Lines 498 to 541)
Reference:
[1] D.F. Cleary, N.J. de Voogd, A.R. Polónia, R. Freitas, N.C. Gomes, Composition and predictive functional analysis of bacterial communities in seawater, sediment and sponges in the Spermonde Archipelago, Indonesia, Microbial ecology, 70 (2015) 889-903.
[2] J.K. Goodrich, J.L. Waters, A.C. Poole, J.L. Sutter, O. Koren, R. Blekhman, M. Beaumont, W. Van Treuren, R. Knight, J.T. Bell, Human genetics shape the gut microbiome, Cell, 159 (2014) 789-799.
[3] E. Raes, K. Karsh, S. Sow, M. Ostrowski, M. Brown, J. van de Kamp, R. Franco-Santos, L. Bodrossy, A. Waite, Metabolic pathways inferred from a bacterial marker gene illuminate ecological changes across South Pacific frontal boundaries. Nature Communication 12: 2213, 2021.
[4] G.N. Ijoma, R. Nkuna, A. Mutungwazi, C. Rashama, T.S. Matambo, Applying PICRUSt and 16S rRNA functional characterisation to predicting co-digestion strategies of various animal manures for biogas production, Scientific Reports, 11 (2021) 19913.
[5] Y. Zhang, Y. Qiao, Z. Fu, Shifts of bacterial community and predictive functional profiling of denitrifying phosphorus removal–Partial nitrification–Anammox three-stage nitrogen and phosphorus removal before and after coupling for treating simulated wastewater with low C/N, Chemical Engineering Journal, 451 (2023) 138601.
[6] S. Agrawal, C.T. Kinh, T. Schwartz, M. Hosomi, A. Terada, S. Lackner, Determining uncertainties in PICRUSt analysis–An easy approach for autotrophic nitrogen removal, Biochemical Engineering Journal, 152 (2019) 107328.
[7] J. Ni, X. Li, F. Chen, H. Wu, M. Xu, Community structure and potential nitrogen metabolisms of subtropical aquaculture pond microbiota, Applied Ecology & Environmental Research, 16 (2018).
[8] F. Wang, S. Chen, Y. Wang, Y. Zhang, C. Hu, B. Liu, Long-term nitrogen fertilization elevates the activity and abundance of nitrifying and denitrifying microbial communities in an upland soil: implications for nitrogen loss from intensive agricultural systems, Frontiers in microbiology, 9 (2018) 2424.
[9] Y. Zhang, Z. Wu, Y. Gao, J. Gao, F. Bao, Y. Zhao, Y. Guo, Y. Liu, Phenacetin promoted the rapid start-up and stable maintenance of partial nitrification: Responses of nitrifiers and antibiotic resistance genes, Science of The Total Environment, 933 (2024) 173190.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe paper deals with the autotrophic partial nitrification and heterotrophic nitrification and aerobic denitrification in high-strength ammonium wastewater treatment. Efficient removal of nitrogen from ammonia-rich wastewater is an important issue. The authors conducted high-density sampling on five groups of treatment experiments and obtained rich data.
The subject matter is interesting and within the scope of the Water. I list hereafter my main concerns with some propositions to alleviate them:
1. Please explain the abbreviations when they appear for the first time, e.g., C/N and COD. In particular, C/N in the manuscript refers to COD/total nitrogen, but it can easily be mistaken for total nitrogen/total carbon, after all, this is a more common representation except in the field of sewage treatment.
2. What was added to increase COD in the experiment?
3. Explain why the aeration rates were different for different treatments.
4. Specific information on microbial sampling.
5. The physicochemical parameters were intensively sampled and analyzed, but the analysis of the microbial community was not very reliable. The 16S universal primers detected only bacteria, not microbial communities. After all, microbial communities are more than just bacteria. The manuscript should avoid inaccurate statements. The manuscript only has one sample of a bacterial community in each reactor. What was the timing and location of this sampling in the reactor, and what were the sampling and processing methods?
6. There is only one variable in the experiment, i.e., C/N, so it is meaningless to do CCA analysis, and the results are completely predictable. Even the PCoA analysis is of little use because number of samples is too small.
7. The PICRUSt for predicting microbial community function is now in debate, and it is believed that the power of such predictions is very limited, especially for complex communities. Because the 16S sequencing length is too short, using it to predict other functional genes will have very limited accuracy. I retain my doubts about this analysis, although the authors obtained very good results.
8. Appropriate conclusions should be added. For example, Since both PN and HNAD can treat high-nitrogen wastewater, what guidance does the shift between them provide for the selection of processes in actual water treatment?
Author Response
Too many unnecessary abbreviations make it hard to read and review.
Reply: Thank you for your suggestion. We have eliminated unnecessary abbreviations from the article and added an abbreviation table at the beginning to enhance readability.
1. Grammatical errors check and English polishing are very necessary.
Reply: Thank you for your suggestion. In accordance with your advice, this manuscript has been edited for proper English language, grammar, punctuation, spelling, and overall style by highly qualified native English-speaking editors at MDPI, and the certificate is provided below.
2. Paper title is redundant and hard to understand the work.
Reply: Thank you for your scientific suggestion. We have changed the title of the article to “Impact of COD/N Ratio on Shifting Autotrophic Partial Nitrification to Heterotrophic Nitrification and Aerobic Denitrification in High-Strength Ammonium Wastewater Treatment”.
3. Note the scientific meanings of C/N = 0.
Reply: Thank you for your suggestion. In response to other reviewers’ suggestions, we have changed C/N to COD/N, as C refers to the content of COD. When COD/N = 0, it indicates that the COD concentration in the wastewater is zero, which is a reasonable definition.
4. L92-93: Wrong unit for “the aeration rate” (2000 mg/L).
Reply: We were really sorry for our careless mistake. The unit “mL/min” has been correctly revised in the manuscript.
5. L147: Proteins (PN)??
Reply: We were really sorry for our careless mistake. In the revised manuscript, “PN” refers exclusively to partial nitrification, and “protein” is no longer abbreviated.
6. Too many unnecessary abbreviations make it hard to read.
Reply: Thank you for your suggestion. We have eliminated unnecessary abbreviations from the article and added an abbreviation table at the beginning to enhance readability.
7. L104-105: Give the actual volume of seed for each reactor.
Reply: Thank you for your suggestion. 1 L of seeding sludge was added to each reactor, and this revision has been reflected in the manuscript.
8. L210-212, 229-232: Give data or citations.
Reply: Thank you for your suggestion. Corresponding citations have been provided in both instances.
9. Parameters EPS, SOUR and microbial community were determined. They should be discussed simultaneously.
Reply: Thank you for your scientific suggestion. We conducted a correlation analysis between the properties of the sludge and the bacterial community structure, as shown in Fig. 5B. The revised manuscript is as follows: “To quantitatively assess the relationship between the bacterial communities and the performance/sludge data at different COD/N ratios, Pearson’s correlation coefficient was computed, and the degree of association was statistically evaluated, as shown in Fig. 2B. COD/N was found to be significantly positively correlated with Acholeplasma (r=0.88, p<0.1), Luteimonas (r=0.95, p<0.1), Acinetobacter (r=0.90, p<0.1), Flavobacterium (r=0.99), and Corynebacterium (r=0.89), all of which are classified as heterotrophic, denitrifying, and HNAD bacteria. An increase in COD/N is beneficial for their growth. Additionally, Nitrosomonas showed a significant positive correlation with ARR, which is consistent with its role as the only detected AOB bacterial genus capable of converting ammonia nitrogen. There is no significant correlation between it and SOUR-AOB (r=0.58). However, a significant positive correlation existed between EPS content and Moheibacter (r = 0.90, p < 0.1), as EPS contained substantial amounts of organic matter that could support Moheibacter metabolism and growth. Additionally, no correlation was found between the SOUR of total bacteria and any specific bacterial genus, indicating that no single bacterial species predominantly influences the increase in COD/N. Instead, various bacterial genera are involved in oxygen consumption at different stages”. (Lines 358 to 374)
10. Section Conclusion was weak. Its content was of only experimental results. Give more information about the conclusion drawn from the experimental result.
Reply: Thank you for your scientific suggestion. Based on the suggestions from other reviewers, we have revised the conclusion as follows: “When treating high-ammonia wastewater, an increase in the COD/N ratio could shift the process from autotrophic PN to HNAD. At COD/N ratios of 0 and 0.5, the growth of NOB was inhibited by low dissolved oxygen and high concentrations of FA and FNA, leading to a significant proliferation of AOB. Consequently, the relative abundance of Nitrosomonas was 15.5% at COD/N = 2 and 9.7% at COD/N = 4. However, the PN process was disrupted at a COD/N ratio of 1 and completely destroyed at COD/N ratios of 2 and 4, with HNAD becoming the dominant process. The proliferation of HB at C/N ≥ 1 inhibited the autotrophic AOB, allowing HNAD bacteria to become the predominant denitrifying microorganisms. Thauera, Pseudomonas, Flavobacterium, Hydrogenophaga, Acinetobacter, Microbacterium, Corynebacterium, Brevundimonas and Comamonas—all HNAD bacteria—emerged at COD/N ratios of 2 and 4, with their relative abundance increasing as the total COD/N ratio rose, coinciding with the disappearance of Nitrosomonas. Therefore, in the treatment of high-ammonia wastewater, when COD/N ≤ 1, the PN process can be combined with other processes, such as partial denitrification and anammox, for biological denitrification. When COD/N > 1, HNAD should be used as the primary denitrification process.” (Line 498 to 541)
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe author's answers are satisfying and their assumptions according to the literature are reasonable, although, the authors could mention in the text and make reservations that if you do not find COMMAMOX it be assumed that these genes are from AOB.
Reviewer 2 Report
Comments and Suggestions for AuthorsGood day! The comments have been corrected.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe review comments have been ressponded.
Comments on the Quality of English LanguageIt is better to E
