the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 27 Feb 2026
Continuous analysis of N2O isotopic composition during biological nitrogen removal in wastewater treatment to disentangle production and reduction processes
Abstract. Nitrous oxide (N2O) is a potent greenhouse gas, and emissions from wastewater treatment plants (WWTPs) represent a significant and highly variable source. Understanding the dynamics in microbial pathways of N2O formation and reduction during biological nitrogen removal is essential for targeted mitigation strategies. Stable isotope analysis of N2O (δ15Nα, δ15Nβ, δ18O, and 15N site preference) provides a powerful tool to disentangle and quantify N2O production and reduction processes, yet conventional analytical approaches lack temporal resolution. Here, we present the first long-term application of an off-axis integrated cavity output spectrometer for real-time N2O isotopic analysis at a pilot-scale WWTP over one year of operation. We developed a dynamic dilution system and implemented correction protocols for drift, N2O mole fraction dependence, and gas matrix effects on isotopic results, achieving uncertainties of 0.85 ‰ (δ15Nα), 1.08 ‰ (δ15Nβ), 0.81 ‰ (δ15Nbulk), 0.48 ‰ (δ18O) and 1.09 ‰ (15N site preference). Representative datasets demonstrate the system’s capability to (i) identify dominant N2O production pathways under standard operation, (ii) quantify N2O reduction in relation to dissolved oxygen concentration, and (iii) trace nitrogen transformation during low-level 15N-labelling experiments. Our results indicate nitrifier or heterotrophic denitrification as the main source of N2O, and that N2O reduction efficiency is strongly controlled by oxygen availability. This study highlights the potential of laser spectroscopy for continuous isotopic monitoring in real-world engineered systems and provides practical guidelines for uncertainty reduction and data interpretation. More specifically, our work forms a foundation for further investigations of the operational factors controlling N2O formation and N2O reduction in biological WWTPs and other complex anthropogenically-perturbed settings.
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Status: final response (author comments only)
- RC1: 'Comment on egusphere-2026-857', Anonymous Referee #1, 01 Apr 2026
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RC2: 'Comment on egusphere-2026-857', Anonymous Referee #2, 28 May 2026
The manuscript by Keck and co-authors describes the deployment of an optical instrument for the analysis of N2O isotopes at an experimental wastewater treatment plant. Despite such analysers being commercially available for over a decade, their operation is technically cumbersome and challenging, potentially limiting the use of these analysers. The fact that the authors have succeeded in operating such an analyser fully automated in the field with high performance is welcome and very newsworthy. The authors performed three experiments: 1) analysis of data from long-term deployment, 2) variation in dissolved oxygen, 3) addition of 15N-labelled compounds. The experiments provide conclusive results that demonstrate the ability of such measurements to deliver actionable data to wastewater treatment plant operators: isotope analysis in N2O, along with knowledge on the isotopic composition of local tab water, indicate the realised/or remaining potential in N2O emissions reduction. The study is comprehensive, very well thought out, and highly relevant. I therefore find it will be a very valuable contribution to AMT.
General comments:
In my view, this manuscript is likely to be of interest to spectroscopists, biogeochemists, but also to GHG emission-focussed wastewater treatment plant operators, who may not be the typical AMT audience. With this in mind, I recommend making the rationale behind the presented isotope technique more accessible to a broader audience interested in WWTP operations, which may be minor or major revisions. The authors are affiliated with a world-leading isotope laboratory, and different approaches to interpret isotope signals is common practice for the authors. However, this may not be the case for the wider audience potentially interested in this technique. I’d therefore suggest going through the manuscript and improve the story telling, guide the reader through critical thinking processes you present, take more time to spell out what is happening in the WWTP and how that relates to what you observe in the isotopes. How can you then use isotope data to infer what is happening in the WWTP and how to mitigate emissions? What isotope scenarios may a WWTP operator observe and what does this say about the processes? How are these processes reflected in your isotope data? I’d expect this will maximise the impact and interdisciplinary uptake of this valuable publication.
Specific comments
Line 39: maybe “…, countries and WWTP facilities still rely on…”?
Line 41: However, different emission factors…
Line 79: maybe “mass-dependent isotope effect”, instead of “normal isotope effect”?
Line 88: Here and everywhere else: use mole fraction or amount fraction instead of concentrations for gases. This differentiation is especially important as the manuscript includes concentrations of dissolved compounds. You could clarify that the term mole fraction is used for all gases within a gas matrix, concentrations for everything in a water matrix?
Line 89: maybe “matrix effect” instead of “bulk gas composition”?
Line 97: “We demonstrate the first…”
Line 102: “…practical recommendation on how to…”
Line 116: “sections, optimal”
Line 117: “approaches, as”
Line 124: Can you comment on a potential N2O blank?
Line 133: “From every 10-minute measurement interval, the first…”?
Line 133: Why is the number of discarded minutes variable (3-5), and what does it depend on?
Line 138: Can the SA gas be added to Table 1?
Line 140: “isotopic composition analysed by EMPA”, can you give a reference for the technique?
Line 154: Here and everywhere else: it seems to me that the uncertainty might be within 1 significant digit? Suggest simplifying by rounding, i.e., 0.3, 0.9, 0.5, 0.2 and 0.9 permille?
Line 157: Can you comment on potential offsets between EMPA and Science Tokyo from previous studies/publications?
Line 174: You show in Figure 2 that the N2O mole fraction effect is variable with time. The magnitude of the time-dependent variation is massive in some examples. Can you provide a comment on your certainty that this effect is sufficiently captured across your experimental period of 1 year, even though it was only determined on 4 days? Also, I wonder if it’s worth focussing on the time-dependent variation within the 11-13 ppm band, which is somewhat acceptable. The variation over the entire N2O range is very interesting but could also be shown as an Appendix figure without the insert, which covers this for 3 out of 5 isotope ratios.
Line 180: The authors describe data processing details before explaining the analytical setup, described in Figure 4. I wonder if describing the setup first would help the logical flow? Otherwise, when you talk about CO2 removal here, can you give a reference to Figure 4?
Line 182: If this is pure N2, wouldn’t this experiment vary O2 as well as Ar at the same time, causing this to show the effect of simultaneously varying both gases? You speculate about the Ar effect in line 157, does it matter here?
Line 186: Maybe “…was linear over the entire…”?
Line 189: Maybe “was measured using a PG…”?
Line 206: The typesetting of the letters in the bracket seems a bit off.
Line 220: It is not clear to me if all isotope measurements of the entire year are corrected for the same O2 difference, or each 5 minute average isotope value for the O2 difference corresponding to those 5 minutes?
Line 229: I do not understand this section. Please clarify.
Line 235: What is the lower-left region? Does this correspond to a figure?
Line 236: What are the 5 lowest-ranked points? Are they shown in Figure A1, if so, can this be visualised/described with more clarity?
Line 239: I’d suggest moving Figure A1 into the main text.
Line 248: How is the repeatability of the instrument determined?
Line 269: How many measurements fell outside the 11-13 ppm band, what percentage was discarded?
Line 282: Could “retained” be a better word to describe what this filter does?
Line 286: Does the CO2 sensor not have a fixed flow rate?
Line 294: “The reactors received subsamples of the local municipal wastewater”?
Line 303: Maybe “The following representative datasets were obtained under three different experimental conditions and are used to demonstrate the usability and applicability of our analytical; setup.”?
Line 305: I suggest be more clear: “Experiment 1: To determine…” and then refer to this in the results/discussion section.
Line 306: Here and elsewhere, the text talks about “standard operation”, but it is not always clear if this refers to the measurement setup of the WWTP. Could this say “standard WWTP operation”?
Line 319: “different operational conditions of the WWTP.”?
Line 335: “11 to 13 ppm N2O target range.”
Line 348: “…the isotopic measurement presented here will be capable…”
Line 349: It might be worth mentioning that these data are in fact “actionable”, therefore high impact and potential to influence decisions by WWTP operators.
Line 351: I wonder why Figure 5 does not include SP?
Line 357: “Experiment 1: Constraining…”? For a reader who is not familiar with the experiments, relating this section directly to the experiment description would make it a lot easier to follow. In my view, this is the text location from where onwards it could be very beneficial to get more guidance on the assumptions and what has been done. Why do you do what you do, why are several data important.
Line 361: I am not sure if “corrected” is the right concept here. The goal is to observe the difference between H2O and N2O. Why is that important, what does Dd18O tell us? Could this be described with readers in mind who are not isotope specialists?
Line 363: It took me a while to understand that the slope of 0.37 is the slope you show in Fig 6 from your data of Experiment 1, and that it is not the slope reported by Yu 2020. Can this be clarified in the text, and maybe also included in the figure itself? At a later point, you mention the range in slopes reported by Yu 2020. Maybe the range reported by Yu 2020 can be presented here, when Yu 2020 is mentioned first to discuss your slope in the context of their findings?
“From Fig 6, we can infer nitrifier denitrification (nD) or heterotrophic denitrification (hD)…”
Line 369: Can you explain more broadly why you think your data show nD and hD as the most dominant sources, even though your data are outside the corresponding shaded areas in Fig 6?
Line 377: Can you add the slope value or the equation for the linear fit to the figure?
Line 381: “…with progressive heterotrophic N2O reduction.”?
Line 383: “Experiment 2: Assessing…”?
Line 387: “The slope of our reduction line in Experiment 2 (0.8…)”, or can it be clarified further that this slope is based on different data, excluding Experiment 1? Also, can you comment on why the slopes are different between Experiment 1 and 2? What does this tell someone who is not an expert in isotope techniques?
Line 389: Here and everywhere else, consistent use of “DO concentrations” would help clarity.
Line 391: I am not sure I understand this section properly and suggest this to be clarified further. How do those redN2O percentages relate to DO and isotopes? Can you guide the reader by simply explaining something general along the lines of “if this goes up, that goes down because of that and that.” And then “what we see in our data is… suggesting that… Therefore, isotopes can highlight times when DO can be optimised to mitigate N2O emissions. “
Line 397: Can “decreasing O2 concentration” be changed to “decreasing DO concentration”, or is this referring to O2 mole fractions in the gas phase?
Line 398: Maybe “In contrast, Rees….”?
Line 401: What species are you thinking of, “related to microbial species”?
Line 405: I can’t see the colour/time gradient in the plotted symbols in A and B. Can the slope be written in the figure? The caption talks about % uncertainty, the axis is in fractions of 1, which seems inconsistent. Can all be in %?
Line 413: “Experiment 3: 15N…”?
Line 421: Can you expand the explanation of your thinking about the stagnation, i.e., 15N bulk initially increases because of that, then stagnates when NH4 is depleted, while Dd18O still increases. What is happening when Dd18O still changes, but not 15N bulk? Why is NO2 still changing for a bit, and why is NO3 changing throughout the entire experiment? I can’t see much difference in the colours of the symbols in panel A. Is there a better way to show the length of tie that has passed for each data point?
Line 423: Isotope conventions are -27 should be shown as –27, delta symbols are italicised.
Line 430: “standard WWTP operation”?
Line 436: This is an unconventional way to refer to site preference. Why not use SP?
Line 452: “…greenhouse gas emission reduction.”?
Citation: https://doi.org/10.5194/egusphere-2026-857-RC2
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- 1
General comments
Keck et al. report methodological advances in monitoring isotope ratios of N2O emitted from waste water treatment processes. They conducted a detailed investigation of the dependence of N₂O isotope ratios on N₂O and oxygen concentrations and long-term stability of the measurements, and established an automated method for obtaining accurate analytical values using infrared absorption spectroscopy for sources with high variability.
The strength of this paper is that the developed system allows for frequent measurement of N₂O isotope ratios at the source site. This is particularly useful for assessing anthropogenic sources that fluctuate significantly over time. I hope that this method will provide information on the hour-by-hour processes of N₂O formation and dissipation, thereby contributing to proposals aimed at reducing N₂O emissions from various N2O sources including WWTP.
However, I am concerned that the authors have made little mention of the shortcomings and limitations of this method compared to conventional mass-spectroscopic or infrared-absorption-spectroscopic measurements using an intermittent gas sampling method. The following disadvantages can be cited. First, the system requires a large amount of dilution gases and calibration gases to maintain constant N2O concentration of the analyte gas fed to the laser spectrometer and to correct for temporal drift of the spectrometer responses, respectively. Although the cost of synthetic air to dilute raw sample gases may be low, it is necessary to install pure air cylinders or pure air generators at the site, and one has to consider logistics. Preparation of calibration standards will likely require both money and effort, as some of the authors have already reported. Second, as stated in conclusion, the developed system actually allows three 5-minute averaged measurements per hour. I expect the authors’ further efforts to improve the measurement frequency, but at this stage, I think it would be an exaggeration to state that this method allows “continuous analysis” of N2O in source gases.
In summary, this paper is worth publishing in Atmospheric Measurement Techniques after revision regarding above points and minor comments below.
Specific comments
Title. “continuous” would be better replaced with “high-frequency” or “on-line”.
L62. There is a paper (Toyoda et al., 2011) published earlier than Wunderlin et al. (2013) that reported isotope ratios of N2O emitted from WWTP.
L74 & L80. “resN2O” and “redN2O” look very similar and are easy to confuse. I suggest the authors to use a single character (with sub/super scripts, if any) to represent a variable. Please also refer to this journal’s submission guidelines.
L120. From this point onward, measurements have been conducted with the N₂O concentration set to 12 ppm. Please explain why this value was chosen—was it set to match the lowest expected N₂O concentration in WWTP emissions, or is there another reason?
L137. Although two-point calibration was conducted, neither the isotopic values of N2O in target gas nor sample gas fall within the range of the two standard gas values. This means the authors are extrapolating; please explain whether this is acceptable and justify the validity of this approach.
L150. Do “blocks” mean the four dates described in the next line?
L159. It would be helpful for readers unfamiliar with this method if the authors explain following points in the introduction or discussion section: the reasons and principles behind why isotope ratio measurements using infrared absorption spectroscopy depend so heavily on concentration of target gas and coexisting gases.
L168–169. It is unclear how the authors are using the term “strong”. Does it mean that the slope of delta value versus N2O (d(delta)/d(N2O)) is large? If so, it would be better to show the concentration ranges lower than 6 ppm more closely, as in the case of 8-16 ppm range. The horizontal scale of Figure 2 is too coarse, making it difficult to read the data. Simply adding a secondary scale would help. Also, because the inset figure of 8-16 ppm range hides the plots of d15Nbulk, d18O, and SP at >50 ppm, one cannot examine the relationship for high concentration ranges.
L178. I think air or oxygen is injected not only to oxidize ammonium to nitrite, but also oxidize organics using microorganisms.
L213–214. Notation of variables are not fully explained and are partly inconsistent or confusing. For example, is the integral sign attached to delta_cal1 in line 213 the same as “Int” attached to the parameters in equation 3? Moreover, eq. 3 itself is not correct if I follow the explanation described in L211–213. It seems that the coefficients of the time-difference terms in the denominator are reversed.
L215. I think subscript “sa” in equation 4 denotes sample, but the authors have already used “SA” to mean synthetic air. To avoid confusion, replace with other expression.
L237–238. I think estimating the isotope ratios at the time of formation based on the distribution of observed values is a second-best approach, but it is not clearly explained how the reduction line was estimated. To calculate the “perpendicular distance” of the data point and the reduction line, the latter must be estimated independently with the observation. While the slope can be determined using literature values, how was it decided which points on the graph the line passes through (or what the y-intercept should be)?
L242. I agree that d18O of N2O is related to d18O of water, but do the authors argue that N2O and water is under full isotopic equilibrium? I wonder the changes in relative contributions from Hy and nD/hD or the changes in dissolved oxygen isotope ratios due to partial consumption might affect d18O of N2O or precursors like NO2-.
L248. Does “repeatability of the instrument” include uncertainty of drift correction?
L260. It would be helpful if the authors explain derivation of equation 9. What does “n” mean?
Figure 4. I would like to confirm the accuracy of the description in this figure regarding the gas sampling method from the reactors. A portion of the water’s surface is covered, and something resembling a chimney is depicted. Is the gas being actively drawn from the water surface, or is it passively carried by the airflow generated by aeration? This question concerns whether the representativeness of the collected gas is ensured.
L380–382. It seems the authors assume that isotopic signature of produced N2O is constant over time and that the variation in dO and SP is simply caused by difference in the progress of N2O reduction. This assumption should be clearly stated along with its basis, together with the reason why the slope of the line in Figure 6 is different from that in Figure 7 or Figure A1. To my eyes, variation around the regression line seems to indicate variation in the produced N2O.
L391–393. This statement is difficult to confirm looking at Figure 7B, mainly because the color gradients corresponding to the time are difficult to distinguish.
L417–421. Although the caption of Figure 8 mentions grey dots representing the results from standard operation, I cannot see the data points. If I estimate the d15Nbulk values for the standard operation of ca. -50‰ from Figure 5, d15Nbulk obtained for Cycle 1 here is higher by 40-60‰. Assuming the d15N of NH4 during the standard operation of 6-24‰ based on literature (Toyoda et al. 2011), the enhancement of d15N of N2O due to the labeled NH4 is significantly smaller than the difference in d15N of NH4+ (100 – 6 or 24 = 94 to 76‰). If the measured d15N of N2O here is correct, what would be the reason for the discrepancy?
P28. Fix the citation of Morley et al..
Figure A1. I can see only a single “blue area”. If the authors really plotted both light blue and dark blue areas, please differentiate them by improving the color contrast.
References
Toyoda, S., Suzuki, Y., Hattori, S., Yamada, K., Fujii, A., Yoshida, N., et al. (2011). Isotopomer analysis of production and consumption mechanisms of N2O and CH4 in an advanced wastewater treatment system. Environmental Science and Technology, 45, 917–922. https://doi.org/10.1021/es102985u