LAUSR

Abstract. Soil microbial processes, stimulated by agricultural fertilization, account for 90 % of anthropogenic nitrous oxide ( N2O ), the leading source of ozone depletion and a potent greenhouse gas. Efforts to reduce N2O flux commonly focus on reducing fertilization rates. Management of microbial processes responsible for N2O production may also be used to reduce N2O emissions, but this requires knowledge of the prevailing process. To this end, stable isotopes of N2O have been applied to differentiate N2O produced by nitrification and denitrification. To better understand the factors contributing to isotopic variation during denitrification, we characterized the δ15 N, δ18 O and site preference (SP; the intramolecular distribution of 15N in N2O ) of N2O produced during NO 3 - reduction by Pseudomonas chlororaphis subsp. aureofaciens and P. c. subsp. chlororaphis. The terminal product of denitrification for these two species is N2O because they lack the gene nitrous oxide reductase, which is responsible for the reduction of N2O to N 2 . In addition to species, treatments included electron donor (citrate and succinate) and electron donor concentration (0.01, 0.1, 1 and 10 mM) as factors. In contrast to the expectation of a Rayleigh model, all treatments exhibited curvilinear behaviour between δ15 N or δ18 O and the extent of the reaction. The curvilinear behaviour indicates that the fractionation factor changed over the course of the reaction, something that is not unexpected for a multi-step process such as denitrification. Using the derivative of the equation, we estimated that the net isotope effects ( η ) vary by as much as 100 ‰ over the course of a single reaction, presenting challenges for using δ15 N and δ18 O as apportionment tools. In contrast, SP for denitrification was not affected by the extent of the reaction, the electron donor source or concentration, although the mean SP of N2O produced by each species differed. Therefore, SP remains a robust indicator of the origin of N2O . To improve apportionment estimates with SP, future studies could evaluate other factors that contribute to the variation in SP.