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Peer-Review Record

Cultivation of Nitrifying and Nitrifying-Denitrifying Aerobic Granular Sludge for Sidestream Treatment of Anaerobically Digested Sludge Centrate

Processes 2022, 10(9), 1687; https://doi.org/10.3390/pr10091687

by Guillian Morgan and Rania Ahmed Hamza^*

Reviewer 1:

Nicoleta Ungureanu

Reviewer 2:

Jinxing Ma

Reviewer 3: Anonymous

Reviewer 4:

Loai Aljerf

Processes 2022, 10(9), 1687; https://doi.org/10.3390/pr10091687

Submission received: 28 June 2022 / Revised: 17 August 2022 / Accepted: 22 August 2022 / Published: 25 August 2022

(This article belongs to the Special Issue Environmental Protection by Aerobic Granular Sludge Process)

Round 1

Reviewer 1 Report

I strongly recommend the authors to use the Paper template specific to each journal when submitting an article for evaluation for publication.

Abstract: This section needs to be revised. It must contain a maximum of 200 words but contains 240. The authors present some results and conclude the abstract without considering that they should add the main conclusions or interpretations.

Keywords: It is preferable not to repeat words that are found in the title in this section.

Introduction:

According to the Paper template, bibliographic sources should be cited as numbers in the text, not by the names of the authors followed by the year of publication. Again, use Paper template!

Lines 39-40: “(or anaerobically digested sludge); which is a liquid slurry of microorganisms, substrates, and the carrier medium”. Delete the semicolon. Also, the digestate does not contain substrate (substrate = feedstock, raw materials) which is subjected to anaerobic digestion. After AD, the initial substrate becomes waste, so you should refer to as digestate or digested substrate.

Line 62: 600 mg/L

Line 84: kg N/m³·d

Line 166: 2 mg/L (leave space between the value and the unit of measurement, wherever it is missing).

The method and the results are presented in detail, and the conclusions are drafted according to the results obtained. The practical usefulness of the results or possible future research directions in the field could be added.

Author Response

Reviewer comment: I strongly recommend the authors to use the Paper template specific to each journal when submitting an article for evaluation for publication.

Authors response: Article arranged based on journal template. Changes reflected in manuscript.

Reviewer comment: Abstract: This section needs to be revised. It must contain a maximum of 200 words but contains 240. The authors present some results and conclude the abstract without considering that they should add the main conclusions or interpretations.

Authors response: Abstract shortened. Main conclusion added. Change reflected in manuscript.

Reviewer comment: Keywords: It is preferable not to repeat words that are found in the title in this section.

Authors response: The authors are aware of the preference, but the choice of words resonates well with the scope of the work.

Reviewer comment: Introduction:

Reviewer comment: According to the Paper template, bibliographic sources should be cited as numbers in the text, not by the names of the authors followed by the year of publication. Again, use Paper template!

Authors response: Bibliography and in-text citations revised to match template.

Reviewer comment: Lines 39-40: “(or anaerobically digested sludge); which is a liquid slurry of microorganisms, substrates, and the carrier medium”. Delete the semicolon. Also, the digestate does not contain substrate (substrate = feedstock, raw materials) which is subjected to anaerobic digestion. After AD, the initial substrate becomes waste, so you should refer to as digestate or digested substrate.

Authors response: Semicolon removed. Sentence revised to include the phrase “digested substrate.” Changes reflected in manuscript.

Reviewer comment: Line 62: 600 mg/L

Authors response: Unit revised. Change reflected in manuscript

Reviewer comment: Line 84: kg N/m³·d

Authors response: Unit revised. Change reflected in manuscript

Reviewer comment: Line 166: 2 mg/L (leave space between the value and the unit of measurement, wherever it is missing).

Authors response: Units revised. Changes reflected in manuscript

Reviewer comment: The method and the results are presented in detail, and the conclusions are drafted according to the results obtained. The practical usefulness of the results or possible future research directions in the field could be added.

Authors response: We would like to thank the reviewer for taking the time to review our manuscript and provide very encouraging and valuable feedback.

Author Response File: Author Response.docx

Reviewer 2 Report

The work by Morgan and Hamza investigated the cultivation of nitrifying and nitrifying-denitrifying aerobic granular sludge for sidestream treatment of anaerobically digested sludge centrate. In general, this paper is well organized and written. Long-term tests on different pilot-scale reactors could provide insights into the practical application of this technology for sidestream treatment. I would suggest its acceptance after minor revision.

Specific comments:

The current introduction part is too long. Some general information could be removed,

e.g., “Lines 29-40 Wastewater treatment plants (WWTPs) face the challenge of implementing sustainable unit processes that enhance nutrient removal and contribute to the recovery of energy and value-added…” “Lines 41-50 Dewatering anaerobically digested sludge generates a liquid sidestream – commonly referred to as centrate or reject water – with elevated concentrations of N (predominantly NH3-N) and P…” “Lines 51-71 Centrate can be adequately treated in a separate sidestream process using various wastewater technologies that belong to mainly two categories…” These three paragraphs could be combined into two paragraphs.

“Lines 72-82 Biological treatment is an inexpensive alternative compared to physical/chemical processes. Conventional activated sludge (CAS) is the most widely applied biological treatment technology…” This part is generally well established and could be largely removed.

“Lines 125-126 – aerobic granular sludge (AGS) – for separate sidestream treatment of centrate” should be “– aerobic granular sludge (AGS) – to separate sidestream treatment of centrate”.

Line 178. “2.1.2.Seed Sludge”. Some more information of the seed sludge should be provided, e.g. EPS contents and speciation.

Since there is only one subsection “2.1. Experimental Setup” in “2. Materials and Methods”, there is no need to use “2.1” and “2.1.1-2.1.4”. This reviewer would suggest using, for example, “2.1. Reaction Configuration and Operation”, “2.2.Seed Sludge”, “2.3.Wastewater Media Characteristics”, “2.4.Analytical Methods”.

Line 333. “In Figure 3(e), several small aggregates in the sample were observed to be > 100 ìm.” There was a typo. This assumes that it should be um? Similar typos occurred in the following lines.

Figure 3. Scale bars should be provided. IMHO, sludge from AGS-SBR-1 and AGS-SBR-2 was unlikely to be granular sludge.

Figure 4. The color used for lines in panels (a)-(c) should be the same, e.g., constant orange lines for influent ammonia.

3.4. Pollutants Degradation in SBR Cycle. While the relevant information may be not included in this paper, the authors should investigate the microbial diversity in future studies to investigate the mechanisms accounting for the different nitrification/denitrification processes.

Author Response

Reviewer comment: The work by Morgan and Hamza investigated the cultivation of nitrifying and nitrifying-denitrifying aerobic granular sludge for sidestream treatment of anaerobically digested sludge centrate. In general, this paper is well organized and written. Long-term tests on different pilot-scale reactors could provide insights into the practical application of this technology for sidestream treatment. I would suggest its acceptance after minor revision.

Authors response: We really appreciate the time and effort that you and the reviewer have dedicated to providing your valuable feedback on the manuscript. We are grateful for their insightful comments on the paper. We have been able to incorporate changes to reflect the suggestions provided by the reviewer. We have highlighted the changes within the manuscript.

Specific comments:

Reviewer comment: The current introduction part is too long. Some general information could be removed

Reviewer comment: e.g., “Lines 29-40 Wastewater treatment plants (WWTPs) face the challenge of implementing sustainable unit processes that enhance nutrient removal and contribute to the recovery of energy and value-added…” “Lines 41-50 Dewatering anaerobically digested sludge generates a liquid sidestream – commonly referred to as centrate or reject water – with elevated concentrations of N (predominantly NH3-N) and P…” “Lines 51-71 Centrate can be adequately treated in a separate sidestream process using various wastewater technologies that belong to mainly two categories…” These three paragraphs could be combined into two paragraphs.

Authors response: Paragraphs revised. Change reflected in manuscript.

Reviewer comment: “Lines 72-82 Biological treatment is an inexpensive alternative compared to physical/chemical processes. Conventional activated sludge (CAS) is the most widely applied biological treatment technology…” This part is generally well established and could be largely removed.

Authors response: We would like to thank the reviewer for their suggestion. However, we think that this section would provide reader with understanding of scope and reaction pathways in this study.

Reviewer comment: “Lines 125-126 – aerobic granular sludge (AGS) – for separate sidestream treatment of centrate” should be “– aerobic granular sludge (AGS) – to separate sidestream treatment of centrate”.

Authors response: Sentence revised. Change reflected in manuscript.

Reviewer comment: Line 178. “2.1.2.Seed Sludge”. Some more information of the seed sludge should be provided, e.g. EPS contents and speciation.

Authors response: We agree that it would have been beneficial to have information on EPS, but this study focused nutrient removal after cultivating a bed of granular sludge. Due to the scope of the study, the extraction and characterization of EPS was not included.

Reviewer comment: Since there is only one subsection “2.1. Experimental Setup” in “2. Materials and Methods”, there is no need to use “2.1” and “2.1.1-2.1.4”. This reviewer would suggest using, for example, “2.1. Reaction Configuration and Operation”, “2.2.Seed Sludge”, “2.3.Wastewater Media Characteristics”, “2.4.Analytical Methods”.

Authors response: Suggestions included. Changes reflected in manuscript.

Reviewer comment: Line 333. “In Figure 3(e), several small aggregates in the sample were observed to be > 100 ìm.” There was a typo. This assumes that it should be um? Similar typos occurred in the following lines.

Authors response: We apologize for these typos. Units revised. Changes reflected in manuscript.

Reviewer comment: Figure 3. Scale bars should be provided. IMHO, sludge from AGS-SBR-1 and AGS-SBR-2 was unlikely to be granular sludge.

Authors response: We had some technical issues due to delays experienced during the pandemic to complete the calibration of the microscope to include the scale bars. Several images were captured to track the morphology of the sludge before the scale bar feature was available.

Reviewer comment: Figure 4. The color used for lines in panels (a)-(c) should be the same, e.g., constant orange lines for influent ammonia.

Authors response: Figure had been revised. Changes reflected in manuscript.

Authors response: We agree with the reviewer. This is very important work that we are considering in the future to understand the microbial community dynamics in these processes.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors did not use the journal template, neither the literature is arranged according to the instruction.

The paper is written in good English. My comments are quite minor:

1. The introduction section is too long.

2. Please indicate the practical meaning of your results. Can your findings be applied in WWTPs and what are the benefits?

3. Please emphasize the novelty and strength of your work, especially in the abstract and in the conclusion section.

Line 38: Please add "source" (biomethane - a renewable energy source)

Line 43: looks like the word "of" is missing

Lines 101, 106&107: SBR abbreviation appeared earlier in the text, so there is no need to explain this abbreviation here. It should be explained in the first line of its appearance.

Line 331: An error in the sentence.

Lines 333, 335 and other lines: Unit error. Please check all units throughout the manuscript.

At the beginning of the conclusion section please add one brief introduction sentence. The conclusion section should more or less stand alone since some readers focus only on this section.

Author Response

Reviewer comment: The authors did not use the journal template, neither the literature is arranged according to the instruction

Authors response: Literature arranged based on template. Changes reflected in manuscript.

Reviewer comment: The paper is written in good English. My comments are quite minor:

Authors response: We really appreciate the time and effort that the reviewer has dedicated to providing valuable feedback on the manuscript. We have been able to incorporate changes to reflect the suggestions provided by the reviewer. We have highlighted the changes within the manuscript.

Reviewer comment 1: The introduction section is too long.

Authors response: Introduction revised. Change reflected in manuscript.

Reviewer comment 2: Please indicate the practical meaning of your results. Can your findings be applied in WWTPs and what are the benefits?

Authors response: Practical meaning indicted in abstract. Change reflected in manuscript. “High nutrient removal efficiencies via the nitrification and SND pathways shows that AGS technology is a viable process for treating sidestreams generated in a WWTP.”

Reviewer comment 3: Please emphasize the novelty and strength of your work, especially in the abstract and in the conclusion section.

Authors response: Novelty and strength of work emphasized in abstract and conclusion. Change reflected in manuscript. “High nutrient removal efficiencies via the nitrification and SND pathways shows that AGS technology is a viable process for treating sidestreams generated in a WWTP.”

Reviewer comment: Line 38: Please add "source" (biomethane - a renewable energy source)

Authors response: Word added. Change reflected in manuscript.

Reviewer comment: Line 43: looks like the word "of" is missing

Authors response: Word added. Change reflected in manuscript.

Reviewer comment: Lines 101, 106&107: SBR abbreviation appeared earlier in the text, so there is no need to explain this abbreviation here. It should be explained in the first line of its appearance.

Authors response: Explanation of abbreviation removed. Changes reflected in manuscript.

Reviewer comment: Line 331: An error in the sentence.

Authors response: Sentence revised. Change reflected in manuscript.

Reviewer comment: Lines 333, 335 and other lines: Unit error. Please check all units throughout the manuscript.

Authors response: Units revised. Changes reflected in manuscript.

Reviewer comment: At the beginning of the conclusion section please add one brief introduction sentence. The conclusion section should more or less stand alone since some readers focus only on this section.

Authors response: Introductory sentence to beginning of conclusion. Change reflected in manuscript.

Author Response File: Author Response.docx

Reviewer 4 Report

Manuscript ID: processes-1815118

Type of manuscript: Article

Title: Cultivation of nitrifying and nitrifying-denitrifying aerobic granular sludge for sidestream treatment of anaerobically digested sludge centrate

Journal: Processes

The paper introduces some observations with no rigid argumentations or any justifications of the results (which are not proper for publishing), and no explanation of the kinetic model and no clear mechanism!!!!

Further, the Novelty is extremely weak and the report does not carry a significant value, so I suggest "Completely Reject".

Comments for author File: Comments.pdf

Author Response

We appreciate the time and effort the reviewer has dedicated to review our manuscript. We made the necessary revision to highlight novelty and explain results and the mechanisms.

Round 2

Reviewer 4 Report

Manuscript ID: processes-1815118R1

Title: Cultivation of nitrifying and nitrifying-denitrifying aerobic granular sludge for sidestream treatment of anaerobically digested sludge centrate

Comment 1: " The experimental study was performed in three column-type… to cultivate aerobic granules" That is not clear how you optimized these conditions.

Comment 2: "NO2-N, NO3-N and alkalinity analysis were performed using HCH test kits TNT plusTM839 & 840, TNT plusTM835 and TNT plusTM870, respectively" Analytically, it is hard to publish the results obtained by kits.

Comment 3: "Reaction Configuration and Operation" Study the reactor performance, P and N recoveries, and microbial community.

Comment 4: "This study shows that aerobic granular sludge systems are a viable treatment technology for liquid sidestreams generated in a WWTP" and "High nutrient removal efficiencies via the nitrification and SND pathways shows that AGS technology is a viable process for treating sidestreams generated in a WWTP " 1- Specify the sludge management strategies used in WWTP. 2- It is important to perform the LCA of the sidestream removal and NP recoveries from WWTP toward sustainable and energy efficient using real centrate and synthetic wastewater.

Note 1: "Returned centrate can result in shock loads, process instability and increased operation costs" You need to develop a modelling of the process for eliminating sidestreams impacts on full-scale WWTP.

Comment 5: "The application of AGS to centrate – containing high nutrient load and low biodegradable COD – is practical through the well-known nitrification-denitrification reaction pathway" Study in your work the nutrient loading rate effects and carbon, biomass growth, and lipid productions on energy reduction, nutrient release and removal based on intelligent control of alternating aeration cycles.

Comment 6: Table 5 – The studied characteristics are based on the ammonia volatilization kinetics and affected by the temperature, pH, TSS, and TDS content.

Comment 7: " Dissolved oxygen (DO) was considered >2mg2 mg/L 244 or non-limiting since air flow was controlled by a flow meter" Combine the DO results with the aeration time, oxygen half-saturation index, oxygen transfer into activated sludge, temperature, F/M, HRT, MCRT, MLVSS, sludge age, SVI, conversion rates, phosphorous and ammonia concentrations, ammonia-oxidizing activity, and the feasibility of mainstream deamonification.

Note 2: "Biomass in AGS-SBR-1 and AGS-SBR-2 was augmented with RAS during periods of low sludge concentration or following a process upset" Perform a critical analysis of biomass retention in AGS-SBR-1 and AGS-SBR-2.

Note 3: "The relationship between influent NH3-N and effluent NO3-N in AGS-SBR-2 between days 31 and 72, proved that the use of synthetic wastewater positively affected impacted nitrogen balance in the system" Why you ignored the relationships with COVID-19 and SARS-CoV2 viral load in WWTP influent?

Note 4: "AGS-SBR-1 displayed evidence of nitrification through the production of NOx-N [NO2-N + NO3-N]" Elucidate the specific reaction pathway for NOx reduction involving NO+NO2 and link the obtained results to the outcomes of DFT and atomistic thermodynamic study which must be performed.

Comment 8: "The presence of AOBs in the mixed liquor allowed for efficient oxidation of NH3-N to NO2-N, resulting in partial nitrification" Why you missed the emission of nitrous oxide?

Comment 9: "Further oxidation of NO2-N to NO3-N was impacted due to the absence of NOBs coupled with the increased pH resulting from the addition of NaHCO3 to supplement the alkalinity in centrate" 1- Link these observations to the O2 reduction rates and the inorganic nutrients as PO4, NH4, and SiOH4. 2- The model of N and PH must be simulated for a low-alkalinity stream.

Comment 10: "During the anoxic feeding period, NO3-N is biologically used as an oxidizer to remove organics –representing denitrification" Study the effects of TC, TOC, and COD/NO3-N with the accumulations of nitrite and nitrate under constant temperature in anoxic denitrification process.

Note 5: Differentiate between the autotrophic and heterotrophic nitrification-anoxic denitrification which dominated the anoxic/oxic sewage treatment process during optimization for higher loading rate and energy savings.

Note 6: "COD increases during the anaerobic feed phase before it is assimilated under aerobic conditions" Estimate and compare of the rate of slowly biodegradable COD (sbCOD) hydrolysis under anaerobic, anoxic and aerobic conditions.

Comment 11: There are such grammatical mistakes.

Comment 12: References:

Note 7: "Evidently, a lower MLVSS/MLSS concentration ratio indicated a change in the biomass component such as a decrease in the concentration of viable sludge" Add the following reference:

2015. Prediction of the effect of fine grit on the MLVSS/MLSS ratio of activated sludge. Bioresource Technology 190, 51-56.

Note 8: "Nitrification reduces alkalinity and subsequently the pH of the system through oxidation of NH3-N" Add the following book chapter:

2003. Alkalinity and pH. Nitrification and Denitrification in the Activated Sludge Process, pp.109-114.

Note 9: "A fraction of the COD is assimilated under anaerobic conditions for denitrification and phosphorus release" Add the following references:

2001. Removal of organic carbon, nitrogen and phosphorus in sequential batch reactors integrating the aerobic/anaerobic processes. Water Science and Technology 44(4), 263-270.

2022.Enhancing biological nitrogen and phosphorus removal performance in aerobic granular sludge sequencing batch reactors by activated carbon particles. Journal of Environmental Management 303, 114134.

Note 10: "Wastewater treatment plants (WWTPs) face the challenge of implementing sustainable unit processes that enhance nutrient removal and contribute to the recovery of energy and value-added products" Add the following book chapter:

2020.Novel biological processes for nutrient removal and energy recovery from wastewater. Wastewater and Biosolids Management, pp.27-42.

Note 11: "WWTPs predominantly return centrate to the mainstream process for treatment, but the additional nutrient loading negatively impacts removal efficiency" Add the following reference:

2018.Approaches and processes for ammonia removal from side-streams of municipal effluent treatment plants. Bioresource Technology 268, 797-810.

Note 12: "Ion exchange is another physical/chemical treatment technology that can achieve high ammonia removal from a liquid waste stream" Add the following reference:

2018. Advanced highly polluted rainwater treatment process. Journal of Urban and Environmental Engineering 12(1), 50-58.

Note 13: "In CAS systems, nitrogen is removed from wastewater via biological nitrification and denitrification" Add the following reference:

2019.Performance and Bacterial Communities in Conventional Activated Sludge and Membrane Bioreactor Systems with Low C/N Ratio Wastewater for Nitrogen Removal. Environmental Engineering Science 36(9), 1112-1126.

Note 14: "The first step, nitrification, occurs under aerobic conditions, where NH3-N is oxidized to NO2-N by ammonia oxidizing bacteria (AOB) and further to NO3-N by nitrite oxidizing bacteria (NOB)" Add the following reference:

2015. Is Nitrite-Shunt Happening in the System? Are Nob Repressed? Proceedings of the Water Environment Federation 2015(13), 1360-1374.

Note 15: "Typically, processes that utilize a shortcut route to nitrogen removal require less oxygen and biodegradable carbon" Add the following references:

2007. Organic matter and concentrated nitrogen removal by shortcut nitrification and denitrification from mature municipal landfill leachate. Journal of Environmental Sciences 19(6), 647-651.

2020.Full‐scale N removal from centrate using a sidestream process with a mainstream carbon source. Water Environment Research 92(11), 1922-1934.

Author Response

Comment 1: " The experimental study was performed in three column-type… to cultivate aerobic granules" That is not clear how you optimiWzed these conditions.

Authors response: We have revised the sentence to remove the last 4 words (i.e., to cultivate aerobic granules). Essentially, the entire paragraph discusses reactor operation and steps taken to optimize conditions to cultivate aerobic granules. Change reflected in manuscript.

Authors response: We would like to thank the reviewer for their suggestion. However, the kits used in this study follow the standard methods, EPA approved, and are commonly used in laboratories. Additionally, sample collection and processing were key to the use of test kits.

Comment 3: “Reaction Configuration and Operation” Study the reactor performance, P and N recoveries, and microbial community.

Authors response: We would like to thank the reviewer for their suggestion. For future it is very important that we consider nutrient recovery and the microbial communities involved in that process.

Authors response: We would like to thank the reviewer for their suggestions. The authors are aware of several relatable and very important topics that can be researched, but the scope of work for this project focused on granule cultivation and nutrient removal in lab scale reactors as a proof-of-concept for the feasibility of using AGS for a sidestream treatment of centrate. “ABTP utilizes the conventional activated sludge process for biological treatment of wastewater and anaerobic digestion for sludge stabilization followed by the addition of a polymer compound prior to dewatering.”

Authors response: We would like to thank the reviewer for their comment. A model would definitely be a step ahead for the plant to identify which alternative to choose in terms of sludge management. Development of a model would have extended the scope of the work. The authors are prepared to perform process modeling on the impact of eliminating centrate through sidestream treatment in future work along with resource recovery options.

Authors response: We would like to thank the reviewer for their suggestions. This study focused on nutrient removal after cultivating a bed of granular sludge.

Comment 6: Table 5 – The studied characteristics are based on the ammonia volatilization kinetics and affected by the temperature, pH, TSS, and TDS content.

Authors response: Table 5 is showing the characteristics of real versus synthetic centrate used as feed in this study.

Authors response: We would like to thank the reviewer for their suggestions. For cultivation of granular sludge, this work focused on superficial upflow velocity (SAV), which correlates with non-limiting DO. Providing sufficient SAV to provide shear forces is also key to AGS formation and stability not only the DO level.

Authors response: We thank the reviewer for their comment. We have included the SRTs in the supplementary material along with the biomass concentrations in the bioreactors and made reference to that. Also, the sentence below has been updated in the manuscript:

“Biomass in AGS-SBR-1 and AGS-SBR-2 was augmented with RAS during periods of low sludge concentration or following a process upset. Additionally, biomass augmentation with RAS was performed to maintain mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) concentrations above 2 g/L and 1.5 g/L, respectively.”

Additional information on the SRT is available in the supplementary documents.

Authors response: We agree that incorporating the relationship between WWTP influent and COVID-19 and SARS-CoV2 would have been beneficial, but the scope of work was limited to granule cultivation and nutrient removal. Also, AGS-SBR-2 was fed with synthetic centrate for the duration of its operation. The sentence below is included in the manuscript:

“Synthetic centrate prepared in the laboratory contained considerably less impurities and was void of polymer compounds in comparison to real centrate collected from ABTP.”

Authors response: We would like to thank the reviewer for their suggestion. The authors are in agreement that further research can be performed, but this study focused on nutrient removal after cultivating a bed of granular sludge.

Comment 8: "The presence of AOBs in the mixed liquor allowed for efficient oxidation of NH3-N to NO2-N, resulting in partial nitrification" Why you missed the emission of nitrous oxide?

Authors response: The reactors were open-top, therefore gas emissions were not included in the scope of work.

Authors response: We would like to thank the reviewer for their suggestions. The authors are in agreement that further research can be performed, but this study focused on nutrient removal after cultivating a bed of granular sludge.

Authors response: We would like to thank the reviewer for their suggestions. The effects of TC and TOC ware not analyzed in this study, but the relationship between COD/N ratio and nitrite and nitrate accumulation was studied in AGS-SBR-3. This is covered in section 3.3.3 in the manuscript, where the authors observed that nitrite and nitrate accumulation occurred when COD/N ratio was as low as 4. Note: Experimental work was carried out at room temperature.

Authors response: We would like to thank the reviewer for their suggestions. The authors observed that simultaneous nitrification and denitrification (SND) was prevalent during high COD/N ratio - therefore nitrite and nitrate accumulation did not occur (i.e., essentially zero). Once the COD/N ratio was lowered, autotrophic nitrification and heterotrophic denitrification become prevalent, and nitrite and nitrate accumulation were evident in the system.

Excerpt from manuscript: “An increase in NH₃-N concentration in the feed stock with reduced COD resulted in excess NO₃-N in the effluent as shown in Figure 4(c). NO₃-N accumulation is shown to moderately increase between days 100 and 114 with an average COD/N ratio of 13 ± 1. A tremendous increase in effluent NO3-N is seen between days 114 and 135; the latter representing the peak value of 134.8 mg/L. During this period, the average COD/N ratio was 5 ± 0.4. Effluent NO3-N concentrations of 105.2 mg/L and 102.4 mg/L were recorded on days 145 and 156, respectively. NO3-N concentration >100 mg/L is considered high in comparison to recorded values prior to the first substantial spike on day 114. The COD/N ratio averaged 4 ± 0.5 for day 145 and 156. Overall, the average COD/N ratio for days 114 to 156 was 5 ± 0.5. NOx-N accumulation at a low COD/N highlights the dependence on readily biodegradable in the feed for adequate SND.”

Comment 11: There are such grammatical mistakes.

Authors response: We apologize for grammatical mistakes. The authors have worked diligently to correct grammatical mistakes and proofread the manuscript.

Comment 12: References:

Note 7: "Evidently, a lower MLVSS/MLSS concentration ratio indicated a change in the biomass component such as a decrease in the concentration of viable sludge" Add the following reference:

Prediction of the effect of fine grit on the MLVSS/MLSS ratio of activated sludge. Bioresource Technology 190, 51-56.