Responses of Nitrogen Removal, Extracellular Polymeric Substances (EPSs), and Physicochemical Properties of Activated Sludge to Different Free Ammonia (FA) Concentrations

Round 1

Reviewer 1 Report

The inhibitory effect of free ammonia on wastewater treatment processes is well known, however, the attempt made in this paper to determine the relationship between FA concentration and the EPS production, physicochemical characteristics of activated sludge as well as microbial communities is valuable. The study was performed in four sequential batch reactors over a period of 244 days. In my opinion, the manuscript is suitable for publication in Water Journal, however some revisions need to be introduced.

The English language used is generally correct, although, I suggest checking the entire text for linguistic deficiencies.

Some of the examples:

in point 2.4.2 "Conventional analytical" - something is missing

page 4: "The FA concentration were"

page 5: "These results clearly shows"

page 5: "significant significant"

page 7: "typical cycles of four SBRs was monitored"

page 8: "The effect of FA concentration (...) as shown in Fig.4." - "is shown" rather

page 8: "significant difference for CST, SVI and SVI"

page 9: "SRF were was positively correlated"

page 9: "can used as indicator microorganisms"

page 9: the sentence starting from "Meanwhile..." should be corrected in terms of style and grammar

page 10: "above-mentions analysis"

Some issues related to the reference list:

"Mengistu et al., 2010" - a year does not match the references

"Sheng et al., 2010" two times placed in the references

"Wang 2017a" reference not present in the text

"Wei et al., 2017" not present in the text

Other minor issues:

TB-EPS is explained as "titled- bound EPS"? (page 2, third paragraph)

The first use of LB-EPS acronym is not explained (page 2, third paragraph)

In Figure 1 NO2-N content is indicated by squares (first two reactors) and by circles (last two reactors)?

Figure 2 caption says that the results refer to the end of nitrification. I think this information could also be mentioned in the text.

In Figure 4 no units are provided for CST, SRF and SVI.

The paper is generally well organized, with appropriate structure. The justification for undertaking the research has been clearly formulated. However, I have got several comments and some questions.

In the Methods section more details on the control of pH and temperature in SBRs could be provided.

In Figure 3 it can be seen that the carbon source is added at the beginning of denitrification phase. I think this information should be mentioned in the methods section.

Results presented in point 3.5 concerning the microbial communities are not mentioned in terms of the methods used (Material and methods section).

In the SBRs, the anoxic phase followed the aerobic phase. Could you please explain why such a sequence was chosen? Moreover, in accordance with Table 1, the four SBRs have different cycle times. What is the reason of this?

At the beginning of the Results and discussion section it was stated that "FA concentration range used in this work did not exert negative effects on the ammonia oxidization ability". Also cited is the study by Xu et al. (2018) who reported that FA pretreatment could enhance ammonia removal. However the study mentioned refers to the pretreatment of WAS. In my opinion other studies on the effect of FA concentrations on the nitrification process could have been pointed out.

On page 5 it was stated that TN removal (in mg/L) increased gradually from R0.5 to R10 and decreased in R15, which proves that sufficiently low FA concentration has a positive effect on TN removal efficiency. What about different ammonium concentrations in the influent, which were lower in R15 comparing to R5 and R10? And how to compare these results with the research concerning sludge pretreatment (the next sentence) with high levels of FA conducted outside the SBR (Wang et al. 2017b)?

Could you explain the first part of the conclusion number 1 ("The ammonia oxidation of the Nitrosomonas were affected by FA at a concentration of up to 15 mg/L")?

Author Response

Response to Reviewer 1 comments

Dear Editor and Reviewers:

Thank you for your letter and for the comments concerning our manuscript entitled “Responses of nitrogen removal, extracellular polymeric substances (EPS) and physicochemical properties of activated sludge to different free ammonia (FA) concentrations” (ID: Water-1440006). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have carefully considered these comments and have made corrections which we hope meet with approval. Revised portion are marked in yellow in the revised version. The main corrections in the paper and the responds to editor and reviewers’ comments are as following.

Reviewer#1

The inhibitory effect of free ammonia on wastewater treatment processes is well known, however, the attempt made in this paper to determine the relationship between FA concentration and the EPS production, physicochemical characteristics of activated sludge as well as microbial communities is valuable. The study was performed in four sequential batch reactors over a period of 244 days. In my opinion, the manuscript is suitable for publication in Water Journal, however some revisions need to be introduced.

Comment (1): The English language used is generally correct, although, I suggest checking the entire text for linguistic deficiencies.

-----We accepted sincerely the advice of reviewer and checked the entire text for linguistic deficiencies.

• In point 2.4.2 "Conventional analytical" - something is missing.

----- “Conventional analytical” was revised to “Conventional analytical methods of wastewater parameters”.

• Page 4: "The FA concentration were".

-----"The FA concentration were" was revised to "The FA concentration was".

• Page 5: "These results clearly shows".

----- "These results clearly shows" was modified to "These results clearly showed".

• Page 5: "significant significant".

----- The second word “significant” was deleted.

• Page 7: "typical cycles of four SBRs was monitored".

----- "typical cycles of four SBRs was monitored" was revised to "typical cycles of four SBRs were monitored".

• Page 8: "The effect of FA concentration (...) as shown in Fig.4." - "is shown" rather.

----- "The effect of FA concentration (...) as shown in Fig.4." was revised to "The effect of FA concentration (...) was shown in Fig.4."

• Page 8: "significant difference for CST, SVI and SVI".

----- "significant difference for CST, SVI and SVI" was revised to "significant difference for CST, SVI and SRF".

• Page 9: "SRF were was positively correlated".

----- "SRF were was positively correlated" was revised to "SRF were positively correlated".

• Page 9: "can used as indicator microorganisms".

----- "can used as indicator microorganisms" was revised to "can be used as indicator microorganisms".

• Page 9: the sentence starting from "Meanwhile..." should be corrected in terms of style and grammar.

----- This sentence was rewritten.

 Meanwhile, there were significant correlations between PN and various abundant genera in the community. Specially, PN was significantly positively correlated with three genera, including Thauera (R²=0.752, P < 0.01), Lewinella (R²=0.814, P < 0.01) and Nitrosomonas (R²=0.839, P < 0.01), respectively, however, PN was significantly positively correlated with seven genera, including Zoogloea (R²=-0.962, P < 0.01), Nitrospira (R²=-0.873, P < 0.01), Azoarcus (R²=-0.894, P < 0.01), Denitratisoma (R²=-0.588, P < 0.05), Sulfuritalea (R²=-0.930, P < 0.01), Dechloromonas (R²=-0.876, P < 0.01) and Planctomyces (R²=-0.870 P < 0.01), respectively.

• Page 10: "above-mentions analysis".

----- "above-mentions analysis" was revised to "above analysis".

-----At the same time, we checked the whole manuscript, as details are as follows:

----- In abstract, “nitrogen consumption” was revised to “nitrogen removal”.

----- In introduction, “with a concentration controlled at 176.5 mg/L” was revised to “at 176.5 mg/L”.

----- In introduction, “For example” was added before “Basuvaraj et al. (2015)”.

----- In introduction, “protein rich” was revised to “rich protein”.

----- In revision, “between” was revised to “among”.

----- In section 2.1, the first sentence was written.

Four bench-scale sequencing batch reactors (SBRs) made of Plexiglas with a 5 L effective working volume, 15 cm diameter, and 40 cm effective height were used in the present study.

----- “varying” was revised to “various”.

----- “consisted of influent, aerobic reaction, anoxic reaction, settling, decanting, and idle” after “ SBRs (R0.5, R5, R10, and R15)” was deleted.

----- “In brief” was deleted in section 2.3.1.

----- The annotation of Eq. (5) was revised.

Where P is pump pressure (Pa), A is the filter area (m²), μ is dynamic viscosity of the filtrate (N·s/m²), C is the resistance of the filtrate per unit volume to the medium (kg/m³), b is the slope of the curve between t/V and V, t is filtrate time (s), and V is the filtrate volume (m³).

----- In section 3.1, “different” was added before the “FA stress”.

----- In section 3.1 “ammonium” was revised to “NH₄⁺-N”.

----- In section 3.1, “Fig. 1-b” was revised to “Fig. 1b”.

----- In section 3.1, “reaction cycle” was revised to “whole experiment period”.

----- In the revision, “FA” behind “mg/L” was deleted.

----- In the revision, “during the end of nitrification” was revised to “at the end of nitrification”.

----- In section 3.2, “at” was revised to “with”.

----- In section 3.3, “between” was revised to “among”.

----- In section 3.3, “at” was revised to “with”.

----- In section 3.3, “are” was revised to “were”.

----- In section 3.4, “mg/L” was added behind “0.5” and “15”, respectively.

----- In section 3.4, “mgFA/L” was revised to “mg/L”.

----- In section 3.5, “interrelationships” was revised to “interrelation”.

----- In section 3.5, “R²=0.592 to 0.920,” was revised to “R² varied from 0.592 to 0.920”.

----- In section 3.5, “R² was -0.527 to -0.927,” was revised to “R² varied from -0.527 to -0.927”.

----- In section 3.5, “are” was revised to “were”.

----- In section 3.5, “is” was revised to “was”.

----- In section 3.5, “plays a much more” was revised to “played a more”.

----- In section 3.5, “actions” was revised to “action”.

----- In section 3.5, “were” was revised to “was”.

----- In Figure.5 title, “free ammonia” was revised to “FA”.

----- In the whole manuscript, “NH₄⁺-N, NO₃^--N, and NO₂^--N” were revised to “NH₄⁺-N, NO₃^--N, and NO₂^--N”, respectively.

Comment (2): Some issues related to the reference list.

• "Mengistu et al., 2010" - a year does not match the references.

----- "Mengistu et al., 2010" was revised to "Mengistu et al., 1994".

(2) "Sheng et al., 2010" two times placed in the references.

----- We have deleted the duplicate reference in reference list.

(3) "Wang 2017a" reference not present in the text.

----- "Wang 2017a" was deleted in reference list.

(4) "Wei et al., 2017" not present in the text.

----- "Wei et al., 2017" was deleted in reference list.

Comment (3): Other minor issues.

• TB-EPS is explained as "titled- bound EPS"? (page 2, third paragraph)

-----TB-EPS should be explained as “tightly bound EPS”. We have corrected this mistake in the revision.

• The first use of LB-EPS acronym is not explained (page 2, third paragraph)

----- LB-EPS is explained as “loosely bound EPS”. We have replenished the full name of the LB-EPS in the revision.

• In Figure 1 NO₂^--N content is indicated by squares (first two reactors) and by circles (last two reactors)?

----- We corrected this mistake in the Figure 1b. NO₂^--N marker matches the legend for FA 0.5,5,10 and 15mg/L using stars.

Figure 1. Long–term performance of SBR system under four kinds of FA concentrations. (a) NH₄⁺-N concentration and removal efficiency; (b) NO₂^--N concentration and NiAR values; (c) NO₃^--N concentration and NaAR values.

• Figure 2 caption says that the results refer to the end of nitrification. I think this information could also be mentioned in the text.

----- We replenished “at the end of nitrification” in the Figure 2 caption.

Figure 2. Effect of FA concentrations of the influent on the contents of three fractions of EPS and their components at the end of nitrification.

• In Figure 4 no units are provided for CST, SRF and SVI.

----- We replenished the units of CST (s), SVI (mL·g^-1) , and SRF (m·kg^-1) in the Figure 4.

Figure 4. Effect of FA concentration on the values of (a) CST; (b) SVI; (c) SRF.

Author Response File: Author Response.pdf

Reviewer 2 Report

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Author Response

Response to Reviewer 2 comments

Dear Editor and Reviewers:

Thank you for your letter and for the comments concerning our manuscript entitled “Responses of nitrogen removal, extracellular polymeric substances (EPS) and physicochemical properties of activated sludge to different free ammonia (FA) concentrations” (ID: Water-1440006). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have carefully considered these comments and have made corrections which we hope meet with approval.

Author Response File: Author Response.pdf

Reviewer 3 Report

Title: Responses of nitrogen removal, extracellular polymeric substances (EPS) and physicochemical properties of activated sludge to different free ammonia (FA) concentrations

General Comments:

This paper examines a wide range of parameters and has novelty. However, the materials and methods section must be expanded, and there needs to be more through discussion of the results and comparisons to existing literature. This paper also needs further editing for grammar and clarity (particularly the introduction) as several sections are hard to follow.

Specific Comments:

Page 2 - Basuvaraj et al. (2015) demonstrated that titled- bound EPS should be tightly-bound? Also, the acronym LB-EPS should be explained at its first use.

How was FA added? What chemical was used? How did you ensure that the ammonium present was in the FA form? Was the pH adjusted and why was it different in the first run?

Why was the SBR operated for different times between scenarios?

Table 1 does not present well.

EPS extraction – There are many different EPS extraction methods, each of which tend to produce different results. Please cite where you obtained these methods.

Conventional analytical parameters. Where were these formulas obtained from (eqns 1-4)? How many replicates were analyzed per sample and how often was sampling done?

How was the analysis of the microbial communities performed? This needs to be stated in the materials and methods section (or at a minimum, refer to an existing paper).

Results and discussion

Section 3.1: How do your Ni/NaAR% compare to previous studies?

Figure 1: The NO2 marker doesn’t match the legend for FA 10&15 (circles vs. squares). Also, the figure caption is repeated in the text at the top of the next page.

Figure 3: Having the legend split across two graphs is confusing and doesn’t present well. Perhaps make one legend for all on the side of bottom of the figure. Also, there are no values for LB-EPS on the figures.

The graphs indicate that “add ethonal” as done at 5 hours, should this be “ethanol”? If something is being added during the cycle it needs to be stated in the materials and methods.

Dewaterability – how do your values compare to existing studies?

Caption for figure 5 is also repeated in the text body.

VPA acronym should be explained at first use

Author Response

Response to Reviewer 3 comments

Dear Editor and Reviewers:

Thank you for your letter and for the comments concerning our manuscript entitled “Responses of nitrogen removal, extracellular polymeric substances (EPS) and physicochemical properties of activated sludge to different free ammonia (FA) concentrations” (ID: Water-1440006). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have carefully considered these comments and have made corrections which we hope meet with approval. Revised portion are marked in yellow in the revised version. The main corrections in the paper and the responds to editor and reviewers’ comments are as following.

Reviewer#3

Comments and suggestions for authors, Title: Responses of nitrogen removal, extracellular polymeric substances (EPS) and physicochemical properties of activated sludge to different free ammonia (FA) concentrations.

General Comments:

Comment (1): This paper examines a wide range of parameters and has novelty. However, the materials and methods section must be expanded, and there needs to be more through discussion of the results and comparisons to existing literature. This paper also needs further editing for grammar and clarity (particularly the introduction) as several sections are hard to follow.

----- We replenished the corresponding content in materials and methods (section 2.1).

The temperature control system and heating water jacket were used to control the experimental temperature in the range of 20-35℃. The pH was controlled at 7.5-8.0 by adding 0.1 M HCl and 0.1 M NaOH. The DO level was kept within the range of 1.0-2.5 mg/L using an air compressor during the aeration period.

----- In material and methods (section 2.1). We replenished “Ethanol as external carbon source was added into the reactors at the beginning of denitrification.”

-----At the same time, we checked the whole manuscript, as details are as follows:

----- In abstract, “nitrogen consumption” was revised to “nitrogen removal”.

----- In introduction, “with a concentration controlled at 176.5 mg/L” was revised to “at 176.5 mg/L”.

----- In introduction, “For example” was added before “Basuvaraj et al. (2015)”.

----- In introduction, “protein rich” was revised to “rich protein”.

----- In revision, “between” was revised to “among”.

----- In section 2.1, the first sentence was written.

Four bench-scale sequencing batch reactors (SBRs) made of Plexiglas with a 5 L effective working volume, 15 cm diameter, and 40 cm effective height were used in the present study.

----- “varying” was revised to “various”.

----- “consisted of influent, aerobic reaction, anoxic reaction, settling, decanting, and idle” after “ SBRs (R0.5, R5, R10, and R15)” was deleted.

----- “In brief” was deleted in section 2.3.1.

----- The annotation of Eq. (5) was revised.

Where P is pump pressure (Pa), A is the filter area (m²), μ is dynamic viscosity of the filtrate (N·s/m²), C is the resistance of the filtrate per unit volume to the medium (kg/m³), b is the slope of the curve between t/V and V, t is filtrate time (s), and V is the filtrate volume (m³).

----- In section 3.1, “different” was added before the “FA stress”.

----- In section 3.1 “ammonium” was revised to “NH₄⁺-N”.

----- In section 3.1, “Fig. 1-b” was revised to “Fig. 1b”.

----- In section 3.1, “reaction cycle” was revised to “whole experiment period”.

----- In the revision, “FA” behind “mg/L” was deleted.

----- In the revision, “during the end of nitrification” was revised to “at the end of nitrification”.

----- In section 3.2, “at” was revised to “with”.

----- In section 3.3, “between” was revised to “among”.

----- In section 3.3, “at” was revised to “with”.

----- In section 3.3, “are” was revised to “were”.

----- In section 3.4, “mg/L” was added behind “0.5” and “15”, respectively.

----- In section 3.4, “mgFA/L” was revised to “mg/L”.

----- In section 3.5, “interrelationships” was revised to “interrelation”.

----- In section 3.5, “R²=0.592 to 0.920,” was revised to “R² varied from 0.592 to 0.920”.

----- In section 3.5, “R² was -0.527 to -0.927,” was revised to “R² varied from -0.527 to -0.927”.

----- In section 3.5, “are” was revised to “were”.

----- In section 3.5, “is” was revised to “was”.

----- In section 3.5, “plays a much more” was revised to “played a more”.

----- In section 3.5, “actions” was revised to “action”.

----- In section 3.5, “were” was revised to “was”.

----- In Figure.5 title, “free ammonia” was revised to “FA”.

----- In the whole manuscript, “NH₄⁺-N, NO₃^--N, and NO₂^--N” were revised to “NH₄⁺-N, NO₃^--N, and NO₂^--N”, respectively.

Comment (2) Results presented in point 3.5 concerning the microbial communities are not mentioned in terms of the methods used (Material and methods section).

----- We replenished the corresponding content in section 2.4.4.

2.4.4 DNA extraction, PCR amplification, Illumina MiSeq sequencing and microbial diversity analysis

According to the manufactur’s instructions, DNA was extracted from activated sludge sample (2.5 mL) using the FastDNA® SPIN extraction kits (MP, biomedicals, CA, USA), and stored at -20℃ prior to the further analysis. the extracted DNA were quantified using a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis.

PCR amplification and Illumina MiSeq sequencing were adopted to analyze the microorganism’s diversity. For 16S rRNA PCR amplification, the V3-V4 region of 16S rRNA was amplified using the primer set 338F (5’-ACTCCTACGGGAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The PCR reaction conditions were as follows: preheated at 95◦ C for 2 min; 25 cycles of amplification (denatured at 95 ℃  for 30 s; annealed at 55℃  for 30 s; elongated at 72 ℃  for 40 s); and finally extended at 72 ℃  for 10 min. Illumina Miseq high-throughput sequencing was conducted at Shanghai Personalbio Technology Co. Ltd (Shanghai, China).

The MOTHUR software was used for quality control and filtering of the raw sequence. The main steps were discarding short sequences less than 50 bp, removing repetitive sequences, and discarding chimeric sequences. The effective sequences were quantified and classified using QIMME software and a 97% sequence similarity threshold of was used to classify reads into operational taxonomic units (OTUs).

-----In section 2.1, we replenished following content about the detailed SBR operation.

The filling (5 min), aerobic reaction (240-360 min), anoxic reaction (300-420 min), decanting (25-455 min) and idling period (variable time) consisted a cycle of each SBR (Table 1). The aerobic and anoxic reaction durations varied with initial substrate concentrations with the purpose of achieving complete nitrification and denitrification. The different setting time was caused due to various settleability of AS in each SBR. Idle periods of the SBR at four FA levels were also different for maintaining same total each cycle time (1 day) for the four SBRs in this present work. In order to reflect the accurate biochemical reaction time, the one cycle time in the table 1 was the sum of filling, aerobic reaction, anoxic reaction and decanting period, excluded the idle period.

----- Moreover, we replenished the following content for detailed description about statistical analysis.

2.4.5 Statistical analysis

The rates of ammonia oxidation, nitrite accumulation and nitrate production were calculated by using Microsoft Excel 2016. Moreover, one-way analysis of variance (ANOVA) was applied to evaluate the relationship and significant difference among microorganisms, EPS and sludge properties. Results was considered statistically significant when P < 0.05 and was considered highly significant when P < 0.01. One-way ANOVA and Pearson’s correlation analysis were conducted by using SPSS software (version 20, SPSS Inc., Chicago, U.S.A.). Furthermore, the network analysis was performed by using Cytoscape3.3.0 to construct the network visualization to identify the correlation among FA, EPS, sludge property and microbial community. Variation partitioning analysis (VPA) was clarified to quantify the contributions of FA, EPS and sludge property to microbial community using R software 2.15.3.

----- We further edited for grammar and clarity (particularly the introduction) as several sections as the Reviewer #1 comment (1).

Specific Comments:

Comment (1): Page 2 - Basuvaraj et al. (2015) demonstrated that titled- bound EPS should be tightly-bound? Also, the acronym LB-EPS should be explained at its first use.

-----We corrected this mistake in the revision. “titled- bound EPS” was revised to “tightly bound EPS”. The acronym LB-EPS was explained as “loosely bound EPS” at its first use in the revision.

Comment (2): How was FA added? What chemical was used? How did you ensure that the ammonium present was in the FA form? Was the pH adjusted and why was it different in the first run?

-----We replenished the corresponding content in section 2.4.2.

The FA concentrations were calculated according to the following equation Eq. (4) which was reported by Anthonisen et al.(1976):

F

where NH₄⁺-N is the ammonia concentration (mg/L); T is the temperature (°C); pH is the pH level.

Based on this equation, FA depended on the ammonia nitrogen concentration, pH value and temperature. Different FA concentration could be achieved by adjusting the initial NH₄⁺-N concentration, pH value and temperature. Various NH₄⁺-N concentration was achieved by adding different amount of ammonium chloride (NH₄Cl) (1mol/L) as the energy and nitrogen source. COD concentration was obtained by adding ethanol (CH₃CH₂OH) as the carbon source. The temperature control system and heating water jacket were used to control the experimental temperature in the range of 20-35℃. The pH value was controlled at 7.5-8.0 by adding 0.1 M HCl and 0.1 M NaOH.

Comment (3): Why was the SBR operated for different times between scenarios?

-----Thanks for the comment. We also tried to response to this query as previously mentioned (Response to Reviewer#4 (Comment (4)). The following content was added in 2.1section.

The filling (5 min), aerobic reaction (240-360 min), anoxic reaction (300-420 min), decanting (25-455 min) and idling period (variable time) consisted a cycle of each SBR (Table 1). The aerobic and anoxic reaction durations varied with initial substrate concentrations with the purpose of achieving complete nitrification and denitrification. The different setting time was caused due to various settleability of AS in each SBR. It is point out that idle periods of the SBR at four FA levels were also different for maintaining same total each cycle time (1 day) for the four SBRs in this present work. In order to reflect the accurate biochemical reaction time, the one cycle time in the table 1 was the sum of filling, aerobic reaction, anoxic reaction and decanting period, excluded the idle period.

Comment (4): Table 1 does not present well.

----- Table 1 presents operational conditions of the SBRs at four FA levels during the entire experimental period, which includes influent concentration (COD and NH₄⁺-N), phase time (filling, aeration, anoxic, decantation) and operational parameters (FA, MLSS, temperature, pH and DO). We also refereed reported literatures Sun et al., (2020). Therefore, Table1 presents overall operational parameters for better description of operational conditions.

Sun et al., (2020), Bioresource Technology.

[1] Sun, H. W., Shi, W. Y., Cai, C. J., Ge, S. J., Ma, B., Li, X.Q., Ding, J. 2020. Responses of microbial structures, functions, metabolic pathways and community interactions to different C/N ratios in aerobic nitrification. Bioresour Technol, 31,123422.

Comment (5): EPS extraction–There are many different EPS extraction methods, each of which tend to produce different results. Please cite where you obtained these methods.

----- We sincerely accepted the comment of Reviewer and replenished the two relevant references about the methods of EPS extraction in section 2.3.

Sheng et al., 2010; Sun et al., 2020.

[1] Sun, H, W., Cai, C. J., Chen, J. X., Liu, C,Y.,  Wang, G. J., Li, X. Q., Zhao, H. N., 2020. Effect of temperatures and alternating anoxic/oxic sequencing batch reactor (SBR) operating modes on extracellular polymeric substances in activated sludge. Water Sci Technol. 82 (1), 120-130.

[2] Sheng, G. P., Yu, H. Q., Li, X. Y., 2010., Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28, 882–894.

Comment (6): Conventional analytical parameters. Where were these formulas obtained from (eqns 1-4)? How many replicates were analyzed per sample and how often was sampling done?

----- We replenished the relevant reference.

The ammonia removal rate (ARE), nitrite accumulation ratio (NiAR) and nitrate accumulation ratio (NaAR) were calculated according to the following equation (Eq. (1), (2) and (3)) (Sun et al., 2021):

[1] Sun, H. W., Jiang, T. T., Zhang, F., Zhang, P., Zhang, H., Yang, Hao., Lu, J. B., Ge, S. J., Ma, B., Ding, J., Zhang, W., 2021. Understanding the effect of free ammonia on microbial nitrification mechanisms in suspended activated sludge bioreactors. Environ Res, 200, 111737.

----- We replenished this explanation in the section 2.4.

For long-term performance detection, sample was taken from each SBRs at the begin and end of nitrification and denitrification, respectively, for every operational cycle. Sample was taken from each SBRs every hour to analyze typical variation of nitrogen, COD, EPS in a SBR cycle. Each sample was measured parallelly three times and average was applied.

Comment (7): How was the analysis of the microbial communities performed? This needs to be stated in the materials and methods section (or at a minimum, refer to an existing paper).

----- We replenished the analysis of the microbial communities in the section 2.4.4.

2.4.4 DNA extraction, PCR amplification, Illumina MiSeq sequencing and microbial diversity analysis

According to the manufactur’s instructions, DNA was extracted from activated sludge sample (2.5 mL) using the FastDNA® SPIN extraction kits (MP, biomedicals, CA, USA), and stored at -20℃ prior to the further analysis. the extracted DNA were quantified using a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis.

PCR amplification and Illumina MiSeq sequencing were adopted to analyze the microorganism’s diversity. For 16S rRNA PCR amplification, the V3-V4 region of 16S rRNA was amplified using the primer set 338F (5’-ACTCCTACGGGAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The PCR reaction conditions were as follows: preheated at 95◦ C for 2 min; 25 cycles of amplification (denatured at 95 ℃  for 30 s; annealed at 55℃  for 30 s; elongated at 72 ℃  for 40 s); and finally extended at 72 ℃  for 10 min. Illumina Miseq high-throughput sequencing was conducted at Shanghai Personalbio Technology Co. Ltd (Shanghai, China).

The MOTHUR software was used for quality control and filtering of the raw sequence. The main steps were discarding short sequences less than 50 bp, removing repetitive sequences, and discarding chimeric sequences. The effective sequences were quantified and classified using QIMME software and a 97% sequence similarity threshold of was used to classify reads into operational taxonomic units (OTUs).

Comment (8): Section 3.1: How do your Ni/NaAR% compare to previous studies?

-----We accepted the reviewer’s advice and compared our NiAR/NaAR with previous studies in section 3.1. As follows:

Sun et al., (2015) reported that the FA concentration at 3.0-33.5mg/L initiated and maintained a stable nitrite pathway with NiAR of 91.3-95.5% in a SBR treating real municipal landfill leachate, corresponding, NaAR gradually decreased from 98.2% to 4.7%. Comparing this result with our work, NiAR in Sun et al., (2015) was obviously higher that (0.8% at 0.5 mg/L and 1.5% at 5 mg/L), however, was slightly slower than that (98.4% at 10 mg/L and 96.7% at 15 mg/L) in our work. For this biological mechanism, a high concentration of FA can promote nitrite accumulation by selectively inhibiting the activity of nitrite oxidation bacteria (NOB) but not ammonia oxidation bacteria (AOB) (Sun et al., 2021; Anthonisen et al., 1976).These results clearly showed that a higher FA concentration of 10 and 15 mg/L was conducive to establishment of the partial nitrification due to the stronger inhibition of FA on NOB than AOB (Liu et al., 2019).

----- Three references were added in the reference list.

[1] Sun, H. W., Peng, Y. Z, Shi, X. N., 2015. Advanced treatment of landfill leachate using anaerobic–aerobic process: Organic removal by simultaneous denitritation and methanogenesis and nitrogen removal via nitrite. Bioresour Technol, 2015, 177: 337-345.

[2] Sun, H. W., Jiang, T. T., Zhang, F., Zhang, P., Zhang ,H., Yang, Hao., Lu, J.B., Ge, S. J., Ma, B., Ding, J., Zhang, W., 2021. Understanding the effect of free ammonia on microbial nitrification mechanisms in suspended activated sludge bioreactors. Environ Res, 200, 111737.

[3] Anthonisen, A. C., Loehr, R. C., Prakasam, T. B. S., Srinath, E. G., 1976. Inhibition of nitrification by ammonia and nitrous acid. J. Wat. Pollut. Control Fed. 48 (5), 835–852.

Comment (9): Figure 1: The NO₂^--N marker doesn’t match the legend for FA 10&15 (circles vs. squares). Also, the figure caption is repeated in the text at the top of the next page.

----- We corrected this problem in the Figure 1. NO₂^--N marker matches the legend for FA 0.5,5,10 and 15mg/L using stars. Moreover, we deleted the repeatable figure caption in the text at the top of the next page.

Figure 1. Long–term performance of SBR system under four kinds of FA concentrations. (a) NH₄⁺-N concentration and removal efficiency; (b) NO₂^--N concentration and NiAR value; (c) NO₃^--N concentration and NaAR value.

Comment (10): Figure 3: Having the legend split across two graphs is confusing and doesn’t present well. Perhaps make one legend for all on the side of bottom of the figure. Also, there are no values for LB-EPS on the figures.

----- We corrected this mistake and made the legend to the bottom of the figures. In addition, the value of LB-EPS was originally presented in the figures.

Comment (12): The graphs indicate that “add ethonal” as done at 5 hours, should this be “ethanol”? If something is being added during the cycle it needs to be stated in the materials and methods.

----- We revised “ethonal” to “ethanol” and replenished the corresponding content in the materials and methods.

It is noted that ethanol as external carbon source was added into the reactors at the beginning of denitrification.

Comment (12): Dewaterability – how do your values compare to existing studies?

-----The authors have supplemented this explanation in the 3.4 section.

This result was consistent with Liu et al. (2021), who found that the dewaterability of the digested sludge with FA pretreatment was improved by 9.2% (from 12.0 to 13.1% in solids content of the dewatered digested sludge). The possible reason mainly lies in the following two aspects: First, the destruction of EPS by FA could weak the hydrophilicity of the sludge and reduce the bound water content, thereby improving the dewaterability of the sludge. Secondly, FA can reduce th1e number of negative charges, indicating that the electrostatic repulsion interaction is reduced, and the flocculation of sludge particles is promoted, resulting in better dewatering performance of the sludge.

Comment (13): Caption for figure 5 is also repeated in the text body.

-----We deleted the repeatable caption in the revision.

Comment (14): VPA acronym should be explained at first use

----- We revised “2.4.5 Statistical analysis” to “2.5 Statistical analysis”, and replenished the acronym of VPA (variance partitioning analysis) at first use in section 2.5.

Variation partitioning analysis (VPA) was clarified to quantify the contributions of FA, EPS and sludge property to microbial community using R software 2.15.3.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The manuscript has improved significantly since the last version. From a readability point, some minor formatting issues in Table 1 and perhaps an increase in the size (or present them in landscape) of Figs. 1 & 3) would make them much easier to read.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.