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. Soil microbial processes, stimulated by agricultural fertilization, accountfor 90 % of anthropogenic nitrous oxide ( N2O ), the leadingsource of ozone depletion and a potent greenhouse gas. Efforts to reduce N2O flux commonly focus on reducing fertilization rates. Managementof microbial processes responsible for N2O production may also beused to reduce N2O emissions, but this requires knowledge of theprevailing process. To this end, stable isotopes of N2O have beenapplied to differentiate N2O produced by nitrification anddenitrification. To better understand the factors contributing to isotopicvariation 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 ofdenitrification for these two species is N2O because they lack thegene nitrous oxide reductase, which is responsible for the reduction of N2O to N 2 . In addition to species, treatments includedelectron donor (citrate and succinate) and electron donor concentration(0.01, 0.1, 1 and 10 mM) as factors. In contrast to the expectation of aRayleigh model, all treatments exhibited curvilinear behaviour between δ15 N or δ18 O and the extent of the reaction. Thecurvilinear behaviour indicates that the fractionation factor changed overthe course of the reaction, something that is not unexpected for a multi-stepprocess such as denitrification. Using the derivative of the equation, weestimated that the net isotope effects ( η ) vary by as much as100 ‰ over the course of a single reaction, presenting challengesfor using δ15 N and δ18 O as apportionment tools. Incontrast, SP for denitrification was not affected by the extent of thereaction, the electron donor source or concentration, although the mean SP of N2O produced by each species differed. Therefore, SP remains arobust indicator of the origin of N2O . To improve apportionmentestimates with SP, future studies could evaluate other factors thatcontribute to the variation in SP.

biogeosciences

Estimation of isotope variation of N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O during denitrification by &amp;lt;i&amp;gt;Pseudomonas aureofaciens&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;Pseudomonas chlororaphis&amp;lt;/i&amp;gt;: implications for N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O source apportionment