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The isotopic composition of nitrous oxide (N2O) reveals valuable information on the biological production sources that contribute to N2O accumulation in the atmosphere, i.e. denitrification, nitrifier-denitrification and nitrification1. Isotopic fingerprints for each of these microbial pathways have been identified in past work, however, overlapping signatures of co-occurring N2O production processes2, and limitations in the robustness of associated fractionation factors under varying growth/environmental conditions3 still pose significant challenges.We will present data from the initial phase of our project, where we study N2O production and associated N and O isotopic fractionation by the denitrifier Pseudomonas aureofaciens, grown in the laboratory under different growth conditions and thus different reaction kinetics. N2O production was quantified on-line by Fourier-Transformation IR-spectroscopy (FTIR), and the isotopic composition of produced N2O was determined by quantum cascade-laser-absorption spectroscopy (QCLAS). The combination of N2O production and isotope data (continuously measured) allowed us to elucidate changes in N and O isotope fractionation in response to changing reaction kinetics.In a later phase of the project, we will expand our isotope-analytical capability by including also the doubly-substituted molecules of N2O, 15N15N16O (556), 14N15N18O (458) and 15N14N18O (548). More specifically, we will interrogate the symmetry of N – N bond formation, verify combinatorial effects during N2O production, and we will test whether Δ556, Δ458, and Δ548 (and the preference for 15N substitution in the central/terminal N-position) can be applied as proxies for reaction kinetics4, 5. 1 Yu, L., Harris, E., Lewicka Szczebak, D., Barthel, M., Blomberg, M.R., Harris, S.J., Johnson, M.S., Lehmann, M.F., Liisberg, J., Müller, C. and Ostrom, N.E., 2020. What can we learn from N2O isotope data?–Analytics, processes and modelling. Rapid Communications in Mass Spectrometry, 34(20), p.e8858.2 Kantnerová, K., Tuzson, B., Emmenegger, L., Bernasconi, S.M. and Mohn, J., 2019. Quantifying isotopic signatures of N2O using quantum cascade laser absorption spectroscopy. Chimia, 73(4), pp.232-232.3 Haslun, J.A., Ostrom, N.E., Hegg, E.L. and Ostrom, P.H., 2018. Estimation of isotope variation of N2O during denitrification by Pseudomonas aureofaciens and Pseudomonas chlororaphis: implications for N2O source apportionment. Biogeosciences, 15(12), pp.3873-3882.4 Yeung, L.Y., 2016. Combinatorial effects on clumped isotopes and their significance in biogeochemistry. Geochimica et Cosmochimica Acta, 172, pp.22-38.5 Kantnerová, K., Hattori, S., Toyoda, S., Yoshida, N., Emmenegger, L., Bernasconi, S.M. and Mohn, J., 2022. Clumped isotope signatures of nitrous oxide formed by bacterial denitrification. Geochimica et Cosmochimica Acta, 328, pp.120-129.

Nitrous oxide (N

Anaerobic ammonium oxidation (anammox) plays an important role in aquatic systems as a sink of bioavailable nitrogen (N), and in engineered processes by removing ammonium from wastewater. The isotope effects anammox imparts in the N isotope signatures (15N/14N) of ammonium, nitrite, and nitrate can be used to estimate its role in environmental settings, to describe physiological and ecological variations in the anammox process, and possibly to optimize anammox-based wastewater treatment. We measured the stable N-isotope composition of ammonium, nitrite, and nitrate in wastewater cultivations of anammox bacteria. We find that the N isotope enrichment factor 15ε for the reduction of nitrite to N2 is consistent across all experimental conditions (13.5‰ ± 3.7‰), suggesting it reflects the composition of the anammox bacteria community. Values of 15ε for the oxidation of nitrite to nitrate (inverse isotope effect, − 16 to − 43‰) and for the reduction of ammonium to N2 (normal isotope effect, 19–32‰) are more variable, and likely controlled by experimental conditions. We argue that the variations in the isotope effects can be tied to the metabolism and physiology of anammox bacteria, and that the broad range of isotope effects observed for anammox introduces complications for analyzing N-isotope mass balances in natural systems., Scientific Reports, 11 (1), ISSN:2045-2322

Bulk and position-specific stable isotope characterization of nitrous oxide represents one of the most powerful tools for identifying its environmental sources and sinks. Constraining (14) N(15) N(18) O and (15) N(14) N(18) O will add two new dimensions to our ability to uniquely fingerprint N2 O sources.We describe a technique to measure six singly and doubly substituted isotopic variants of N2 O, constraining the values of δ(15) N, δ(18) O, ∆(17) O, (15) N site preference, and the clumped isotopomers (14) N(15) N(18) O and (15) N(14) N(18) O. The technique uses a Thermo MAT 253 Ultra, a high-resolution multi-collector gas source isotope ratio mass spectrometer. It requires 8-10 hours per sample and ~10 micromoles or more of pure N2 O.We demonstrate the precision and accuracy of these measurements by analyzing N2 O brought to equilibrium in its position-specific and clumped isotopic composition by heating in the presence of a catalyst. Finally, an illustrative analysis of biogenic N2 O from a denitrifying bacterium suggests that its clumped isotopic composition is controlled by kinetic isotope effects in N2 O production.We developed a method for measuring six isotopic variants of N2 O and tested it with analyses of biogenic N2 O. The added isotopic constraints provided by these measurements will enhance our ability to apportion N2 O sources.