Clumped isotope signatures of nitrous oxide formed by bacterial denitrification. (1st July 2022) - Kantnerová, Kristýna
Abstract: Multiply substituted isotopic species of nitrous oxide (N2 O), referred to as clumped isotopes, represent a promising new tool for distinguishing production pathways of this potent greenhouse gas. This work presents the first determination of enrichment factors of N2 O clumped isotopes during bacterial denitrification. Samples of N2 O obtained after 1-, 3-, and 7-day incubations of a pure culture of the denitrifier Pseudomonas aureofaciens at 20 °C and 30 °C were analysed by the recently developed quantum cascade laser absorption spectroscopy (QCLAS) method. Enrichment factors ε p/s of the cumulative product (p) relative to the substrate (s) were determined using a Rayleigh model for the seven most abundant isotopically substituted molecules (isotopocules) of N2 O. Values of the enrichment factors ε p/s (with uncertainty expressed as expanded standard uncertainty at the 95% confidence interval) at the two incubation temperatures (20 °C/30 °C) are: 14 N 15 N 16 O (456): ε 456 = (−40.3 ± 2.6)‰/(−35.1 ± 0.7)‰ 15 N 14 N 16 O (546): ε 546 = (−38.1 ± 3.4)‰/(−31.2 ± 0.6)‰ 14 N 14 N 17 O (447): ε 447 = (21.3 ± 1.2)‰/(24.5 ± 0.5)‰ 14 N 14 N 18 O (448): ε 448 = (38.8 ± 1.5)‰/(46.4 ± 1.2)‰ 14 N 15 N 18 O (458): ε 458 = (−8.9 ± 2.0)‰/(−11.7 ± 0.6)‰ 15 N 14 N 18 O (548): ε 548 = (−3.4 ± 1.1)‰/(−1.8 ± 0.5)‰ 15 N 15 N 16 O (556): ε 556 = (−85.9 ± 1.5)‰/(−63.9 ± 1.4)‰ Temporal evolutions of the abundances of singly substituted N2 O isotopocules during nitrate reduction agree with previously published experiments: there is normal isotope effect associated with the production of 14 N 15 N 16 O and 15 N 14 N 16 O; i.e., intermediates leading to 14 N 14 N 16 O react faster than intermediates leading to 14 N 15 N 16 O and 15 N 14 N 16 O. However, the production of 14 N 14 N 17 O and 14 N 14 N 18 O is associated with inverse isotope effect; i.e., intermediates leading to 14 N 14 N 16 O react slower than intermediates leading to 14 N 14 N 17 O and 14 N 14 N 18 O due to preferential cleavage of 16 O during nitrate reduction to N2 O. Isotopic fractionation at the incubation temperature of 30 °C was significantly lower compared to 20 °C. We observed a large kinetic isotope effect of the 15 N site preference (SP) and the 15 N– 18 O site preference (SP 18 ) at the onset of the reaction. SP 18 was found to be closer to 0‰ than SP, which is thought to arise from similar rates of breakage of the 15 N–O and 14 N–O bonds in the reaction intermediates. The 15 N– 18 O clumped isotope anomalies in two isotopic isomers (isotopomers) 14 N 15 N 18 O and 15 N 14 N 18 O (Δ 458+548 avg ) follow a temporal trend similar to those of SP and SP 18 . The 15 N– 15 N clumped isotope anomalies in 15 N 15 N 16 O are greater than 0‰ and show no clear temporal trend or influence of incubation temperature, suggesting no strong combinatorial effects involved during the N–N bond formation. Overall, our data illustrate that clumped N2 O isotopes may be used as independent tracers for reaction mechanisms of N2 O conversion and may establish themselves as a worthwhile tool to study the biogeochemical cycle of N2 O.