Biogeochemical evidence of anaerobic methane oxidation and anaerobic ammonium oxidation in a stratified lake using stable isotopes

Einsiedl, Florian; Wunderlich, Anja; Sebilo, Mathieu; Coskun, Ömer K.; Orsi, William D.; Mayer, Bernhard

doi:https://doi.org/10.5194/bg-17-5149-2020

Articles | Volume 17, issue 20

https://doi.org/10.5194/bg-17-5149-2020

© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License.

https://doi.org/10.5194/bg-17-5149-2020

© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License.

Articles | Volume 17, issue 20

Research article

|

23 Oct 2020

Research article |

| 23 Oct 2020

Biogeochemical evidence of anaerobic methane oxidation and anaerobic ammonium oxidation in a stratified lake using stable isotopes

Florian Einsiedl, Anja Wunderlich, Mathieu Sebilo, Ömer K. Coskun, William D. Orsi, and Bernhard Mayer

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Final revised paper (published on 23 Oct 2020)
Preprint (discussion started on 06 Feb 2020)

Interactive discussion

Status: closed

AC: Author comment | RC: Referee comment | SC: Short comment | EC: Editor comment

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RC1: 'Review of Einsiedl et al.', Anonymous Referee #1, 25 Mar 2020
- AC1: 'Interactive comment on Biogeochemical evidence of anaerobic methane oxidation and anaerobic ammonium oxidation in a stratified lake using stable isotopes” by Florian Einsiedl et al.', Florian Einsiedl, 15 May 2020
RC2: 'Review of "Biogeochemical evidence of anaerobic methane oxidation and anaerobic ammonium oxidation in a stratified lake using stable isotopes" by Einsiedl et al.', Anonymous Referee #2, 23 Apr 2020
- AC2: 'Interactive comment on Biogeochemical evidence of anaerobic methane oxidation and anaerobic ammonium oxidation in a stratified lake using stable isotopes”', Florian Einsiedl, 15 May 2020

Peer-review completion

AR: Author's response | RR: Referee report | ED: Editor decision

ED: Reconsider after major revisions (24 May 2020) by Caroline P. Slomp

AR by Florian Einsiedl on behalf of the Authors (28 Jun 2020) Author's response Manuscript

ED: Referee Nomination & Report Request started (09 Jul 2020) by Caroline P. Slomp

RR by Anonymous Referee #2 (17 Jul 2020)

Suggestions for revision or reasons for rejection

The authors put a lot of efforts into addressing mine and the other reviewer’s concerns, and defending the original version of the MS. They did a good job in responding, and I think that the MS has benefitted from the changes made. I am still not convinced by the way they sell their data set (evidence for true AOM is weak), and I still think there are some issues, but I guess that this is part of the scientific exchange. We do not need to all agree. It is definitely a nice data set, certainly worth to be published.

A couple of things:

I am wondering about the fact that there was not an appropriate d18O_NO2 standard used. Coming then back to R1’s question: how were the nitrite isotope values corrected?

R1 argues correctly that the observed NO2 d18O data seem too low, assuming O isotope equilibrium with water. In their response, the authors write: “relatively constant δ18O values for nitrite close to -5‰ indicate that an isotopic exchange occurred between the oxygen of the water and the oxygen of nitrite and that the latter was predominately controlled by biotic reactions”. But doesn’t the d18O of nitrite argue against this?

I agree with R1 that the nitrite d18O data may be left out of the paper, as they do not really help.

The authors tone down in their response the value of incubation experiments. I do not agree. Experiments, even if not directly reflecting the natural conditions, would have helped to calibrate the observed isotopic signatures, and to gain confidence in their interpretation. I was not referring to isotope effect experiments, and I am aware that rate measurements will deliver potential rates only, but clearly, rate measurements would have helped to indicate the potential for the use of specific substrates, and thus would have helped to support (or not) the claimed links between different biogochemical reactions (anammox and AOM).

The authors state that there is barely any mixing during summer (“no mixing, no advection”); I assume they mean no advective mixing, because turbulent diffusive mixing, even if sluggish will take place.

As for the choice of a value for Kz: In my first review; I was not suggesting a value of 0.03 m2/d. I was referring to a paper by Oswald et al. from Lake La Cruz (Oswald et al. 2016), for which this value was reported. I mixed up the different studies/lakes investigated by Oswald et al., and values from Lake Rotsee, as well as values for Lake Lugano seem to be higher, indeed. But the broad range of Kz values observed for the different meromictic lakes highlights that there is not “a typical literature value”, and that just assuming a value for Kz is difficult.

I am still confused with regards to the d18O_NOx values. Again, in a sample that contains nitrite and nitrate, O isotope fractionation during the conversion to N2O (independent of the method of conversion) must be different for nitrite and nitrate (via nitrite), simply because one versus two O atoms will be plucked off during the fractionating transformation, respectively. Hence it remains unclear to me how the 18O of the combined NO3+NO2 sample is standardized. The problem is not the yield, the problem is that the N2O from the nitrate will likely have a much higher d18O than the d18O of the N2O generated from the NO2.

As for the interpretation of the NH4 concentration versus the d15NH4 profiles: I see a more or less steady decrease from 1 to <0.1 mgN/l between 22 and 12 m, without a strong overall 15N enrichment. From these patterns, I find it quite difficult to pinpoint anaerobic ammonium oxidation in the deep water column. Within the error of the d15NH4 analyses, the isotope profile looks almost straight to me….an observation that could most plausibly be explained by aerobic ammonium explanation at the redox transition zone, which serves as an efficient NH4 sink without much N isotope fractionation.

L915: established is misspelled

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RR by Anonymous Referee #1 (12 Aug 2020)

Suggestions for revision or reasons for rejection

Overall, the authors have done a commendable job addressing the majority of concerns raised by the two reviewers. The authors acknowledge the limitations of their data and are careful to place their findings into a reasonably tempered interpretation. While Reviewer #2 was concerned that the biogeochemical coupling of AOM, anammox and denitrification may not represent a substantial flux in the larger environmental system, at its core this study investigates a fundamentally important question with implications for the behavior of environmental systems across a broader range.

At this point I have only one remaining concern that has still gone unaddressed in the authors’ response to both the reviews. Both reviewer #2 and I independently pointed out concerns with how the oxygen isotope composition of nitrite was determined and reported. Simply put, there is no methodological approach for determining the δ18O of nitrite that does not explicitly involve the use of nitrite reference materials that have been calibrated for their oxygen isotopic composition (See Casciotti et al., 2007).

Whether using the denitrifier method (converting both nitrate and nitrite to N2O) or the azide method (converting nitrite to N2O), the reported approach by which the oxygen isotope values are referenced (to an ostensibly calibrated N2O reference tank), does not obviate the need for calibrated nitrite standards. Using nitrate oxygen isotope standards in the denitrifier method to report nitrite δ18O values will result in ‘over-correcting’ δ18O values of nitrite, since the branching effect during the O atom abstraction during nitrate reduction to nitrite (in the standards) cannot be independently accounted for. Similarly, by first reducing nitrate to nitrite using cadmium in the azide method, the O isotope effect imparted during this cadmium reduction step remains unknown. Using nitrate oxygen isotope standards to calculate and report nitrite oxygen isotope ratios is not a valid approach.

It should be noted however, that nitrogen isotope ratios, using this approach – including a “mass balance differing” method can be appropriately used – and all indications are that the N isotope data are reasonable and robust.

In Section 2.5.2 – the authors provide much detail about the isotopic measurements for NH4+, NO2- and NO3-. However, no information is offered about how oxygen isotope ratios of nitrite were calibrated (e.g., no oxygen isotope reference materials were mentioned). I believe the authors are using a mass-weighted isotope mass balance approach (which, as noted above, will work fine for N isotopes). For the reasons mentioned above, the oxygen mass balance is not as straightforward as the nitrogen mass balance. A simple mass balance approach cannot be used for disentangling analyses of oxygen isotopes of nitrate and nitrite combinations without nitrite reference materials (more directly, for every mole of nitrite there is a mole of O atoms missing from the O atom budget – and one cannot account for its isotopic composition – violating the mass balance approach). While the authors seem to rely on a previously published approach of theirs (Sebilo et al., 2019), for the reasons stated above, the approach outlined therein is also incorrect. This is also thoroughly discussed by Casciotti and McIlvin (2007).

As a final more relevant note – the nitrite oxygen isotope data as presented in the manuscript do not enter into the discussion or provide any avenue of reasoning as used by the authors. In other words, the data are never discussed and are never used to provide any support to any of the arguments laid out in the paper. As such, I must insist that the δ18O data for nitrite simply be removed from the manuscript – which will have no effect on the overall impact of the paper. I strongly feel this is the prudent decision, such that inappropriate methodologies are not perpetuated.

The paper presents a novel combination of interesting biogeochemical, isotopic and genomic data – that highlight a compelling link among some of the more cryptic processes involving N and C cycling. I look forward to learning more about how these processes are connected through the authors’ future research, building from the present work.

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ED: Publish subject to minor revisions (review by editor) (12 Aug 2020) by Caroline P. Slomp

AR by Florian Einsiedl on behalf of the Authors (24 Aug 2020) Author's response

ED: Publish as is (26 Aug 2020) by Caroline P. Slomp

AR by Florian Einsiedl on behalf of the Authors (01 Sep 2020) Author's response Manuscript

Short summary

Nitrate pollution of freshwaters and methane emissions into the atmosphere are crucial factors in deteriorating the quality of drinking water and in contributing to global climate change. Here, we report vertical concentration and stable isotope profiles of CH₄, NO₃^-, NO₂^-, and NH₄⁺ in the water column of Fohnsee (southern Bavaria, Germany) that may indicate linkages between nitrate-dependent anaerobic methane oxidation and the anaerobic oxidation of ammonium.