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Estimation of isotope variation of N2O during denitrification by Pseudomonas aureofaciens and Pseudomonas chlororaphis: implications for N2O source apportionment

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 δ15N,
δ18O and site preference (SP; the intramolecular distribution of
15N in N2O) of N2O produced during
NO3- 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 N2. 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
δ15N or δ18O 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 δ15N and δ18O 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.
