Is Anoxic Operation Effective to Control Nitrate Build-Up and Sludge Loss for the Combined Partial Nitritation and Anammox (CPNA) Process?
Round 1
Reviewer 1 Report
In line 221 add later nitrogens forms needs correction - i.e. N-NO3- not NO3--N
In statistical analysisi please explain why Authors use ony Pearson correlation coefficient. What about other statistical parameters (i.e. standard deviation, variation coefficient). Please also add information about p level and k level.
In unit decription please use approperiate symbols i.e. kg·m-3·d-1
Please also checked spaces in whole text - in some places they are unnecessary.
Author Response
Point 1: In line 221 add later nitrogen’s forms needs correction - i.e. N-NO3- not NO3--N

Response 1: The original formats for nitrate, nitrite and ammonia are widely described by NO3--N, NO2--N and NH4+-N in the published papers, so here we did not change them.
Point 2: In statistical analysis please explain why Authors use ony Pearson correlation coefficient. What about other statistical parameters (i.e. standard deviation, variation coefficient). Please also add information about p level and k level.
Response 2: Sorry for the mistake. Actually, we didn’t perform Pearson correlation analysis in the article. So the related content was deleted.
Point 3: In unit description please use appropriate symbols i.e. kg·m-3·d-1
Response 3: We have checked and changed all the inappropriate symbols in the unit description.
Point 4: Please also checked spaces in whole text - in some places they are unnecessary.
Response 4: According to the reviewer’s suggestion, we have checked all the unnecessary spaces and deleted them all.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear Authors,
Very interesting research and article, but to improve readership, corrections are needed:
- abstract: It is written in a chaotic manner, it should contain sentence describing the problem, then the methods adopted, results and the main achievement. For example, we have:.. first step, and the third stage appears at the end, and it is not known what was the second?
- I think the wording "three main challenges" is not appropriate.
- line 101-106- the sentence is too long.
- Information: realize fast start-up of PN for the CPNA by dosing 2 mg/L
105 NH2OH in these place is not needed. Should be described detail in point: methods. Here, the aim of the research should be given in relation to the problems described in Introduction. - line 123: The mineral elements and trace elements in the influent were listed in Table S1. In supplementary materials Table S1 is The operation mode of the CPNA process. There is no information about trace elements etc. I think that the composition of synthetic sewage should be given.
- line 150: (Error! Reference source not found.Table S2): There is not Table S2 in supplementary materials.
- What is the difference between Figure 1a (line 109) and Figure S1a (line 151). And the same: line 160 - Figure S1b? etc. There are not Figures with S.
- line 217 and 218, 333, 426: Table S3 and Table S4, Table S5, Table S6, where they are? In supplementary materials is only Table 1.
- what does it mean: The atlas of sludge concentration (line 329)
- the text of the discussion should be included in the conclusions.
Author Response
Point 1: abstract: It is written in a chaotic manner, it should contain sentence describing the problem, then the methods adopted, results and the main achievement. For example, we have:.. first step, and the third stage appears at the end, and it is not known what was the second?
Response 1: We have re-edit the abstract part according to the reviewer’s instruction.
Point 2: I think the wording "three main challenges" is not appropriate.
Response 2: We changed the “three main challenges” to “three main issues”
Point 3: line 101-106- the sentence is too long.
Response 3: We have re-written this sentence, and broken it to several short sentences.
Point 4: Information: realize fast start-up of PN for the CPNA by dosing 2 mg/L
105 NH2OH in this place is not needed. Should be described detail in point: methods. Here, the aim of the research should be given in relation to the problems described in Introduction.
Response 4: Here, to solve the fast start-up of PN, NH2OH addition was described here. We deleted the detailed amount of NH2OH.
Point 5: line 123: The mineral elements and trace elements in the influent were listed in Table S1. In supplementary materials Table S1 is The operation mode of the CPNA process. There is no information about trace elements etc. I think that the composition of synthetic sewage should be given.
Response 5: Here, Table S 1’s title was wrongly put. It should be “The compositions of the synthetic wastewater”, which was corrected by us. But the table’s content was exactly the mineral elements and trace elements in the influent.
Point 6: line 150: (Error! Reference source not found. Table S2): There is not Table S2 in supplementary materials.
Response 6: In our supplementary materials, there is Table S 2, whose title was “The operational mode of the CPNA process”. Please check again.
Point 7: What is the difference between Figure 1a (line 109) and Figure S1a (line 151). And the same: line 160 - Figure S1b? etc. There are not Figures with S.
Response 7: Figure 1 a is the set-up of CPNA, while Figure S 1 a is the operational control logic of CPNA. Figure S 1 a and Figure S 1 b are two different control modes for CPNA operation, one is intermittent aeration, and the other is continuous aeration. We can find the Figure S 1 a and Figure S 1 b in the supplementary materials.
Point 8: line 217 and 218, 333, 426: Table S3 and Table S4, Table S5, Table S6, where they are? In supplementary materials is only Table 1.
Response 8: These mentioned Tables are in the supplementary materials. We update our supplementary materials in the submitting system.
Point 9: what does it mean: The atlas of sludge concentration (line 329)
Response 9: We want to describe the variations of these parameters, so here we deleted “atlas of” to make it much clear for understanding.
Point 10: the text of the discussion should be included in the conclusions
Response 10: We changed this part to “3.5. The analysis of NH2OH’s effect and possible sludge loss reasons in the CPNA system”, making it the last part of “3. Results” analysis.
Author Response File: Author Response.docx
Reviewer 3 Report
General Comments
The study by Wang et al. is aimed to address long term startup and operational issues that typically hamper combined partial nitritation and annamox processes. A relatively quick startup was achieved by addition of hydroxylamine and using an intermittent aeration strategy; whereas, nitrate buildup and sludge loss were mitigated by changing reactor operating parameters, specifically by the addition of an anoxic phase without mixing between the feeding and aeration phases.The SBR reactor was operated for 300 consecutive days, using three different strategies, and a microbial community analysis was done at various points over the 300-days.
The approach taken, scientific methods, and scientific rigor all appear to be sound. Use of the English language is done reasonably well. However, in a few places there were grammatical/sentence structure issues that make the text difficult to interpret in multiple places. I have noted a few of them, but I think the whole paper needs to be gone through and tightened up. The scientific methods certainly appear sound, but in some places its hard to discern what findings were being taken from the results.
I also had problems with the supplementary Figures. Some of the information (such as operating regimes for the reactor) is critical to the hypothesis of the paper. As such, I feel that they need to be more prominent than put in a supplementary information. I would encourage the authors to re-evaluate this. Furthermore, the File that I downloaded as supplementary Figures appeared to be mostly the same as those appearing in the paper.
My recommendation is that moderate revision is required prior to being acceptable for publication: Specifically: 1) language use must be improved, currently it is too difficult in several key places to discern the intended meaning; 2) the figures themselves were generally fine; but their presentation in the text was a bit of a mess (I think Figure 4 showed up in three places) and I also feel that critical information to the paper got unjustly put in as supplementals.
Specific comments:
ABSTRACT
L26: proven instead of proved
L24-L28: this sentence is rather long. Consider revising.
INTRO:
L39: “skips of adjusting reactors” – doesn’t make sense to me. Please clarify?
L42 and L52: What is meant by the scarcity of anammox inoculation? Inoculum? Please clarify.
L50: ‘impractical’ or ‘not practical’ a better word choice over unpractical.
L51-L52: and then established CPNA process could also be….? This sentence doesn’t make much sense to me? It might be just my misinterpretation from poor grammar, but I think perhaps there needs to be a bit more background info before you can make this claim. For me, there is a logical gap here.
L56: the addition of 10 mg/L hydroxylamine? Also, the placement of reference 8 strikes me as odd.
L57: 19 days compared to 20 days? Is that really significant? Also, “low DO conditions”
L61: greatly inhibit
L95: careful with use of the word “confronted” – here and several other instances. Also, the word “smart” may not be the best choice, either. Instead use plain language such as “these methods are hindered by technical challenges or incremental costs, and are often impractical”.
L97-L98: very awkward. Revise with “in the fields of software and online process monitoring equipment” or similar
L102: during ‘the’ CPNA life cycle
METHODS
L116: please specify type of impeller as well as the diameter, otherwise 150 rpm doesn’t mean much.
L136: did you mean: “the stirring” or “stirrer”? Not sure I like use of the word “remarkably” here: this is methods – you just describe what was done without commenting on the results.
L139-143: there are some sentence structure issues here that make it confusing – please revise.
L146: “and idle phases were the operational period?” Not sure what you are trying to say here.
L150: “error ref not found” needs to be fixed. Also – not sure why this operational information is in a supplementary table? Is this not critical to your overall hypothesis of reducing CPNA startup by changing operational parameters?
I am actually confused by the supplementary Figures – they seem to be the same as the ones already in the manuscript?
L155: combined “with”?
Comment: Figure 1: aeration appears to have been done via some kind of diffuser? Is this described in the methods? Also, I have not seen a description of the kind of DO probe you used or how DO was maintained? Increases to stirring or aeration rate?
This brings up another point that might potentially be of importance: difference operational strategies require different DO levels and presumably different aeration or mixing rates. Was there any attempt made to link shear forces with granulation/EPS production, which you have described for each stage later in the results?
Also, were the pH and the ORP probes specified?
L228: why is effluent represented as ‘e’ in equations 1-3 but as ‘eff’ in equations 4-5
Figure 2 comment: I find the fact the dual y-axes have a different scale in Figure 2a to be a bit misleading at first glance, and certainly make it more difficult to interpret.
L283: during the whole operational??? There is a missing word here.
L289: “benefited its increase” sounds awkward. Please revise.
L292: “was reduced to zero and recovered…”
L303-304: are you really confident to two significant figures on this?
L306-L307. Sentence needs to be revised.
L308: bacterial death.
L316: “…decreased SVI, and benefitted from….”
L329: Figure 3 caption needs some revision. Not sure what you mean by “atlas”.
L347: placement of ref [33] is odd.
L359: these are both genus names. They should be capitalized and italicized. Also “bacterial cells” or just “bacteria”
L364. Good. I was wondering what the role of EPS production might be in granulation/particle size. Was any attempt made to quantify EPS? You state that the concentrations in the CPNA sludge were 108-350 mg/L – is this from a referenced paper or was this your measurement? If the latter, how did you measure and where is this in your methods?
L383: do not capitalize species names.
Comment: Section 3.4: random words seem to be written in a different font? Also, the formatting for p. 11 appears to be a bit of a mess.
L429: have these indices been defined?
L434: I don’t’ know about the statement “a deep understanding of community function”. All it really tells us is who is all there and when – I think we can only speculate on community function.
L436-7: “microbes in WWTP was their origin”. Also “over time” should be separated into two words.
L437: SMP = soluble metabolic products? Has this been defined?
L442: did you mean cellular micro-aggregate carriers? I think this whole sentence needs some revision – it isn’t clear what you are saying.
L446: no figure caption for Figure on page 13? Which Figure is this? I think Figure 4 has actually been inserted into the text in three places? I can’t find any difference between these figures.
L500: “and used the remaining nitrate in the system as the electron donor”? This is unclear.
L502: helped
L512: what is a micro-aeration environment? Was a micro-bubbler used for aeration? Did you mean microaerophilic conditions?
L516: So, I am unclear if there is actually a net benefit/downside of adding hydroxylamine? Benefit only during startup, I suppose - is that correct? I think this needs to be better justified, perhaps in your initial hypothesis, introduction, and discussion. You comment on it being toxic, but in the intro you also discuss technical challenges and incremental costs – how does hydroxylamine addition contribute to those?
Author Response
ABSTRACT
Point 1: L26: proven instead of proved
Response 1: We had changed proved to proven.
Point 2: L24-L28: this sentence is rather long. Consider revising.
Response 2: We break it to three short sentences without changing the meaning.
INTRO:
Point 3: L39: “skips of adjusting reactors” – doesn’t make sense to me. Please clarify?
Response 3: It means that if we use two reactors for partial nitritation and anammox separately, we need to adjust these two reactors to make them suitably connected and operated harmoniously. Here skips of adjusting reactors means that we don’t need to adjust the operational parameters like DO or temperature to make the two reactors suitable for anammox reaction if we take the single-stage anammox systems.
Point 4: L42 and L52: What is meant by the scarcity of anammox inoculation? Inoculum? Please clarify.
Response 4: The pure anammox sludge (anammox inocula) is still hard to get. So, it is scarce. Here we changed the “anammox inoculation” to “anammox inocula”.
Point 5: L50: ‘impractical’ or ‘not practical’ a better word choice over unpractical.
Response 5: Thanks for the advice. We changed the “unpractical” to “impractical”.
Point 6: L51-L52: and then established CPNA process could also be….? This sentence doesn’t make much sense to me? It might be just my misinterpretation from poor grammar, but I think perhaps there needs to be a bit more background info before you can make this claim. For me, there is a logical gap here.
Response 6: For the start-up of CPNA, we have two choices. One is to inoculate suitable partial nitritation sludge and anammox sludge (inocula) and then operated the reactor under right conditions. The other choice was to break it into two steps. It means that, firstly we try to start up partial nitritation. And then, by reduce the air supply and the length of aeration and other conditions adjustment, we try to develop anammox from the previous partial nitritation process. If this trial is successful, then the start-up of CPNA is done. If this trial failed, then we can inoculate small amount anammox to help start up the CPNA process. For the second choice to start up CPNA, to shorten the period of partial nitritation means to cut the length of the whole CPNA start-up period.
Point 7: L56: the addition of 10 mg/L hydroxylamine? Also, the placement of reference 8 strikes me as odd.
Response 7: Yes, here in the study of Reference 8, they achieved stably PN with 20 days by the addition of 10 mg/L hydroxylamine.
Point 8: L57: 19 days compared to 20 days? Is that really significant? Also, “low DO conditions”
Response 8: Here we don’t mean to compare the time they spend to achieve PN. We want to compare the DO conditions, one is under high DO concentrations of 5 mg/L, and the other is under low DO conditions of 1 mg/L.
Point 9: L61: greatly inhibit
Response 9: We had changed “inhibited” to “inhibit”.
Point 10: L95: careful with use of the word “confronted” – here and several other instances. Also, the word “smart” may not be the best choice, either. Instead use plain language such as “these methods are hindered by technical challenges or incremental costs, and are often impractical”.
Response 10: Thanks for the instruction. We had changed this unsuitable sentence.
Point 11: L97-L98: very awkward. Revise with “in the fields of software and online process monitoring equipment” or similar
Response 11: We had changed “As the technological advances in software and online equipment’s fields” to the recommend “In the fields of software and online process monitoring equipment”.
Point 12: L102: during ‘the’ CPNA life cycle
Response 12: Since this sentence is too long, we break it into several short sentences. Here we changed it to “during the CPNA life cycle”.
METHODS
Point 13: L116: please specify type of impeller as well as the diameter, otherwise 150 rpm doesn’t mean much.
Response 13: We supplied with “The stirrer was consisted of three steel impellers with diameter of 5 cm.” in the article.
Point 14: L136: did you mean: “the stirring” or “stirrer”? Not sure I like use of the word “remarkably” here: this is methods – you just describe what was done without commenting on the results.
Response 14: It is stirrer. Here, we deleted the word “remarkably”. This belongs to “Experimental operation”, one of the parts of materials and methods.
Point 15: L139-143: there are some sentence structure issues here that make it confusing – please revise.
Response 15: We had revised these sentences.
Point 16: L146: “and idle phases were the operational period?” Not sure what you are trying to say here.
Response 16: Normally, the idle phase is included in the operational period. It is a part of the whole operational cycle.
Point 17: L150: “error ref not found” needs to be fixed. Also – not sure why this operational information is in a supplementary table? Is this not critical to your overall hypothesis of reducing CPNA startup by changing operational parameters?
I am actually confused by the supplementary Figures – they seem to be the same as the ones already in the manuscript?
Response 17: We didn’t see the “error ref not found” in L 150.
The operational information in Table S2 is very important. It was too long, so we put it in the supplementary materials for typographic convenience.
Point 18: L155: combined “with”?
Response 18: Yes, it should be combined with. We had revised it.
Point 19: Comment: Figure 1: aeration appears to have been done via some kind of diffuser? Is this described in the methods? Also, I have not seen a description of the kind of DO probe you used or how DO was maintained? Increases to stirring or aeration rate?
This brings up another point that might potentially be of importance: difference operational strategies require different DO levels and presumably different aeration or mixing rates. Was there any attempt made to link shear forces with granulation/EPS production, which you have described for each stage later in the results?
Also, were the pH and the ORP probes specified?
Response 19: The aeration is done by aeration pump (HAILEA® V-10, Guangdong, China) (Line 141). The aeration pump was connected with the diffuser immersed in the reactor by a glass rotor flowmeter (air). The DO control is done by adjust the glass rotor flowmeter.
We did not try to link the shear forces with granulation/EPS production here.
The DO, pH and ORP probes are all bought from HACH (USA). All of them are connected to the PLC system. We had added this to Line 118-119.
Point 20: L228: why is effluent represented as ‘e’ in equations 1-3 but as ‘eff’ in equations 4-5
Response 20: To keep uniformity, we changed i to inf and e to eff in equations 1-3.
Point 21: Figure 2 comment: I find the fact the dual y-axes have a different scale in Figure 2a to be a bit misleading at first glance, and certainly make it more difficult to interpret.
Response 21: In order to clearly show the variations of effluent nitrate, nitrite and ammonia, we have to make their y-axes less than that of influent nitrogen parameter. If we make them same, it is hard to find any change of the effluent nitrogen parameter for they are all very low.
Point 22: L283: during the whole operational??? There is a missing word here.
Response 22: It should be “during the whole operational stages of SBR”
Point 23: L289: “benefited its increase” sounds awkward. Please revise.
Response 23: We changed to “facilitated its increase”
Point 24: L292: “was reduced to zero and recovered…”
Response 24: We had changed the relative part according to the reviewer’s instruction.
Point 25: L303-304: are you really confident to two significant figures on this?
Response 25: Yes, these data are calculated according to the operational data.
Point 26: L306-L307. Sentence needs to be revised.
Response 26: We revised “used” to “performed”.
Point 27: L308: bacterial death.
Response 27: We changed “bacteria death” to “bacterial death”.
Point 28: L316: “…decreased SVI, and benefitted from….”
Response 28: We had changed according to the reviewer’s instruction.
Point 29: L329: Figure 3 caption needs some revision. Not sure what you mean by “atlas”.
Response 29: We deleted the “atlas of" without changing the whole sentence’s meaning.
Point 30: L347: placement of ref [33] is odd.
Response 30: We moved ref [33] to “Hao et al. [33]’’.
Point 31: L359: these are both genus names. They should be capitalized and italicized. Also “bacterial cells” or just “bacteria”
Response 31: We changed the relative contents to “Bacilli and Brevibacterium. Bacterial cells”.
Point 32: L364. Good. I was wondering what the role of EPS production might be in granulation/particle size. Was any attempt made to quantify EPS? You state that the concentrations in the CPNA sludge were 108-350 mg/L – is this from a referenced paper or was this your measurement? If the latter, how did you measure and where is this in your methods?
Response 32: The EPS concentrations here were measured by ourselves. The detailed methods were supplied in the supplementary materials.
Point 33: L383: do not capitalize species names.
Response 33: In previous Point 31, the reviewer said the genus name should be capitalized and italicized.
Point 34: Comment: Section 3.4: random words seem to be written in a different font? Also, the formatting for p. 11 appears to be a bit of a mess.
Response 34: The words in this section were uniformly changed to Palatino. The formatting for page 11 was revised.
Point 35: L429: have these indices been defined?
Response 35: These indices for describing the microbial community diversity are clearly defined in the microbial analysis reports. These were done by the biological analysis company. But the calculated of these indices are the same among different testing companies.
Point 36: L434: I don’t’ know about the statement “a deep understanding of community function”. All it really tells us is who is all there and when – I think we can only speculate on community function.
Response 36: The Microbial community analysis tells us who is all there and when. While the microbes’ functions in the activated sludge system were reported by previous studies. So combined our findings and the previous reports, we can speculate on community function. It is stricter. So, we changed the sentence to “Microbial community analysis at the genus level provided helped speculate deep understanding of the community function”.
Point 37: L436-7: “microbes in WWTP was their origin”. Also “over time” should be separated into two words.
Response 37: We had revised the relative contents according to the reviewer’s instruction.
Point 38: L437: SMP = soluble metabolic products? Has this been defined?
Response 38: It is soluble microbial products (SMP). We supplied this in Line 446.
Point 39: L442: did you mean cellular micro-aggregate carriers? I think this whole sentence needs some revision – it isn’t clear what you are saying.
Response 39: Yes, we mean cellular micro-aggregate carriers. We had revised the whole sentence.
Point 40: L446: no figure caption for Figure on page 13? Which Figure is this? I think Figure 4 has actually been inserted into the text in three places? I can’t find any difference between these figures.
Response 40: It has caption (Figure 6). It was put on page 14. So here we adjust it to make it appear just below the figure. All the figures are different.
Point 41: L500: “and used the remaining nitrate in the system as the electron donor”? This is unclear.
Response 41: The CPNA is operated as an SBR mode. And nitrate was the product of anammox. So, for each cycle of SBR, some amount of nitrate was left in the system. With this nitrate as electronic acceptor, the denitrification could happen. The acceptor and donor were wrongly placed in the text. We had revised them.
Point 42: L502: helped
Response 42: We revised help with helped. Thanks for the correction.
Point 43: L512: what is a micro-aeration environment? Was a micro-bubbler used for aeration? Did you mean microaerophilic conditions?
Response 43: The micro-aeration environment here means the aeration condition with low DO concentration (0.05~0.3 mg/L). It was also controlled by the adjustment of glass rotor flowmeter to control the air supply.
Point 44: L516: So, I am unclear if there is actually a net benefit/downside of adding hydroxylamine? Benefit only during startup, I suppose - is that correct? I think this needs to be better justified, perhaps in your initial hypothesis, introduction, and discussion. You comment on it being toxic, but in the intro you also discuss technical challenges and incremental costs – how does hydroxylamine addition contribute to those?
Response 44: Actually, if we add too much hydroxylamine, like 30 mg/L, it will be toxic to the dominant microorganisms like AOB and anammox bacteria. Actually, we had performed studies on its effect on nitrate build-up control. The addition of NH2OH combined with SRT control was showed to be very effective to control nitrate build-up[1].
For the three operational challenges of CPNA, fast start-up of PN was just one aspect. By adding 2 mg/L NH2OH, we could successfully start up PN within short periods. While for the other two challenges of nitrate build up and sludge loss, normal control methods like re-inoculation or install assistant devices are both money-consuming and hard to enforce. But by operational procedures adjustment (in our study here), we could omit the re-inoculation or installation of assistant devices to control nitrate build-up and sludge loss. So the method we use here are cost-effective.
- Wang, Y.; Wang, Y.; Wei, Y.; Chen, M. In-situ restoring nitrogen removal for the combined partial nitritation-anammox process deteriorated by nitrate build-up. BIOCHEM ENG J/2.463 2015, 98, 127-136, doi:http://dx.doi.org/10.1016/j.bej.2015.02.028.
Author Response File: Author Response.docx
