the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 28 May 2025
The isotopic signatures of nitrous oxide produced by eukaryotic and prokaryotic phototrophs
Abstract. Prokaryotic and eukaryotic microscopic phototrophs ('microalgae') can synthesize the potent greenhouse gas and ozone depleting pollutant nitrous oxide (N2O). However, we do not know how much microalgae contribute to aquatic N2O emissions because these organisms co-occur with prolific N2O producers like denitrifying and nitrifying bacteria. Here we demonstrate for the first time that microalgae produce distinct N2O isotopic signatures that will enable us to fill this knowledge gap. The eukaryotes Chlamydomonas reinhardtii and Chlorella vulgaris, and the prokaryote Microcystis aeruginosa synthesized N2O 265–755 nmol·g-DW-1·h-1 when in darkness and supplied with 10 mM nitrite (NO2-). The N2O isotopic composition (δ15N, δ18O, and site preference, SP) of each species was determined using a modified off-axis integrated-cavity-output spectroscopy analyser with an offline sample purification and homogenisation system. The SP values differed between eukaryotic and prokaryotic algae (25.8 ± 0.3 ‰ and 24.1 ± 0.2 ‰ for C. reinhardtii and C. vulgaris, respectively vs 2.1 ± 3.0 ‰ for M. aeruginosa), as did bulk isotope values. Both values differ from SP produced by denitrifiers. This first characterization of the N2O isotopic fingerprints of microscopic phototrophs suggests that SP-N2O could be used to untangle algal, bacterial, and fungal N2O production pathways. As the presence of microalgae could influence N2O dynamics in aquatic ecosystems, field monitoring is also needed to establish the occurrence and significance of microalgal N2O synthesis under relevant conditions.
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RC1: 'Comment on egusphere-2025-2337', Anonymous Referee #1, 08 Sep 2025
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The manuscript titled “The isotopic signatures of nitrous oxide produced by eukaryotic and prokaryotic phototrophs” by Plouviez et al. aimed to quantify the isotopic content of nitrous oxide (N2O) produced by phototrophs (two eukaryotic and one prokaryotic). The authors achieved that goal by culturing the eukaryotes Chlamydomonas reinhardtii and Chlorella vulgaris, and the prokaryote Microcystis aeruginosa, then measuring the bulk δ(15N) and δ(18O) of the N2O produced by each culture as well as the site-specific δ(15N) in N2O (isotopomers). As part of this work, the authors developed a method for measuring N2O isotopomers more carefully with laser-based analysis by removing gases with matrix effects and controlling the amount of N2O seen by the laser system, both of which have been shown to have profound effects on the resulting isotopomer measurements.
The authors found that the eukaryotes, C. reinhardtii and C. vulgaris, both produce N2O with a site preference of ~25 ‰, while the prokaryote M. aeruginosa produced N2O with a site preference closer to 0 ‰. This highly useful result will facilitate source partitioning of N2O produced by phototrophs, because it provides two distinct endmembers for eukaroytic and prokaryotic sources, respectively. These results also complicate the interpretation of N2O site preferences in nature, since nitrification tends to also produce N2O with a high positive site preference (~30 ‰), and denitrification tends to produce N2O with a site preference around 0 ‰.
Altogether, this is a concise, impactful study, and I highly recommend its publication. I only have a few major concerns and some suggestions for improving the manuscript.
Firstly, did the authors perform any kind of abiotic, illuminated control? Recent work has shown that sunlight can drive abiotic photochemical N2O production (Leon-Palmero et al., 2025), and it seems possible that this was occurring in the authors’ experiments.
Secondly, the authors added 10 mM NaNO2 to their cultures, which is orders of magnitude higher than the amount of nitrite in natural aquatic environments. Did the authors do any kind of experiment, feeding the cultures lower levels of nitrite to ascertain if the organisms would still produce N2O under less nutrient-laden conditions?
The authors provide the N2O site preference produced by each organism, but to incorporate this process into models, it is critical to also know the δ(15Nα) and δ(15Nβ) as well. What were the δ(15Nα) and δ(15Nβ) of the N2O produced by each organism, and what was the δ(15N) of the nitrite that they were supplied? This would allow us to calculate an isotope effect and thus incorporate this process into biogeochemical models.
The authors point to other studies showing how phototrophs produce N2O from NO within the cell, but the vastly different site preferences of the eukaryotic and prokaryotic N2O suggest different mechanisms. Could the authors speculate on possible different reaction mechanisms for the two kinds of organisms, even though the intermediate (NO) may be the same?
Line-by-line comments:
Line 157: It seems possible that there may have also been photochemical N2O production in the authors’ experiments.
Line 174: What is “instrument-grade” N2?
Line 233: What does “indicative” mean in this context?
Lines 283-284: Include the δ(15Nbulk) and δ(18O) from both gases in Table 3 to illustrate this.
Line 288: How does the uncertainty calculated this way compare to the standard deviation of replicate samples?
Line 290: The term UREF_span2 should be multiplied by the correction factor, squared.
Line 295: Not the standard error of the slope? Also, it would be highly useful to see a visual representation of these correction functions.
Line 455 and elsewhere: The formatting of the tables is confusing and difficult to read.
References
Leon-Palmero, E., Morales-Baquero, R., Thamdrup, B., Löscher, C., and Reche, I.: Sunlight drives the abiotic formation of nitrous oxide in fresh and marine waters, Science, 387, 1198–1203, https://doi.org/10.1126/science.adq0302, 2025.
Citation: https://doi.org/10.5194/egusphere-2025-2337-RC1
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