European Journal of Soil Science, June 2010, 61, 364–374 doi: 10.1111/j.1365-2389.2010.01233.x Dual isotope and isotopomer measurements for the understanding of N 2 O production and consumption during denitrification in an arable soil A. Meijide a,∗ , L. M. Cardenas b , R. Bol b , A. Bergstermann c , K. Goulding d , R. Well e , A. Vallejo a & D. Scholefield b a Technical University of Madrid, Departmento Qu´ ımica y An´ alisis Agr´ ıcola, ETSI Agr´ onomos, C/Ciudad Universitaria s/n, 28040 Madrid, Spain, b North Wyke Research, North Wyke, Okehampton, Devon, EX20 2SB, UK, c Institute of Soil Science and Forest Nutrition, University of G¨ ottingen, B¨ usgenweg 2, 37077 G¨ ottingen, Germany, d Rothamsted Research, Centre for Soils and Ecosystem Function, Harpenden, Hertfordshire AL5 2JQ, UK, e Johann Heinrich von Th¨ unen-Institut, Federal Research Institute for Rural Areas, Forestry and Fisheries Institute of Agricultural Climate Research Bundesallee 50, 38116 Braunschweig, Germany Summary The aim of our research was to obtain information on the isotopic fingerprint of nitrous oxide (N 2 O) associated with its production and consumption during denitrification. An arable soil was preincubated at high moisture content and subsequently amended with glucose (400 kg C ha −1 ) and KNO 3 (80 kg N ha −1 ) and kept at 85% water-filled pore space. Twelve replicate samples of the soil were incubated for 13 days under a helium-oxygen atmosphere, simultaneously measuring gas fluxes (N 2 O, N 2 and CO 2 ) and isotope signatures (δ 18 O-N 2 O, δ 15 N bulk -N 2 O, δ 15 N α , δ 15 N β and 15 N site preference) of emitted N 2 O. The maximum N 2 O flux (6.9 ± 1.8 kg N ha −1 day −1 ) occurred 3 days after amendment application, followed by the maximum N 2 flux on day 4 (6.6 ± 3.0 kg N ha −1 day −1 ). The δ 15 N bulk was initially −34.4‰ and increased to +4.5‰ during the periods of maximum N 2 flux, demonstrating fractionation during N 2 O reduction, and then decreased. The δ 18 O-N 2 O also increased, peaking with the maximum N 2 flux and remaining stable afterwards. The site preference (SP) decreased from the initial +7.5 to −2.1‰ when the N 2 O flux peaked, and then simultaneously increased with the appearance of the N 2 peak to +8.6‰ and remained stable thereafter, even when the O 2 supply was removed. We suggest that this results from a non-homogenous distribution of NO − 3 in the soil, possibly linked to the KNO 3 amendments to the soil, causing the creation of several NO − 3 pools, which affected differently the isotopic signature of N 2 O and the N 2 O and N 2 fluxes during the various stages of the process. The N 2 O isotopologue values reflected the temporal patterns observed in N 2 O and N 2 fluxes. A concurrent increase in 15 N site preference and δ 18 O of N 2 O was found to be indicative of N 2 O reduction to N 2 . Introduction Nitrous oxide (N 2 O) is an important greenhouse gas that also contributes to the depletion of the ozone layer (Prather et al., 2003). Nitrogen fertilizers applied to agricultural land are the major source of N 2 O from terrestrial ecosystems (Nevison & Holland, 1997; Mosier & Kroeze, 1998). Nitrous oxide is produced predominantly by microbial processes, as by-products of nitrification and products of denitrification (Firestone & Davidson, ∗ Present address: European Commission - DG Joint Research Centre, Institute for Environment and Sustainability, Climate Change Unit, Via E. Fermi, 2749, I-21027 Ispra, VA, Italy Correspondence: L. M. Cardenas. E-mail: laura.cardenas@bbsrc.ac.uk Recieved 25 August 2009; revised version accepted 26 January 2010 1989). Under denitrifying conditions, which mainly occur under low oxygen (O 2 ) concentrations in soil, the associated increases in N 2 O tend to be short-lived, lasting from a few days to several weeks (Scholefield et al., 1997a). During denitrification, both production and consumption of N 2 O can take place simultaneously and significant amounts of N may also be lost to the atmosphere as dinitrogen (N 2 ). Natural abundance stable isotopic signatures, such as δ 15 N and δ 18 O of N 2 O, have increasingly been used to identify N 2 O source processes in the soil (Baggs, 2008; Well et al., 2008b), both in the field (P´ erez et al., 2001; Yamulki et al., 2001) and in laboratory- based studies (Bol et al., 2003; Cardenas et al., 2007). Generally, N 2 O emitted from denitrification in soils has higher δ 15 N and δ 18 O signatures than when derived from nitrification (Baggs, 2008). Recent studies have also provided information on isotope  2010 The Authors 364 Journal compilation  2010 British Society of Soil Science