Fractionation of nitrogen and oxygen isotopic composition in N2O produced by bacterial denitrification

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 kinetics^{4, 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.