Isotopomer Site Preference Associated with Nitrous Oxide Production and Consumption During Nitrification and Denitrification

Pitt, A.J., R.L. Sutka, N.E. Ostrom, P.H. Ostrom, H. Gandhi, T.T. Bergsma, and J.A. Breznak

Presented at the All Scientist Meeting (2002-10-04 )

Agricultural ecosystems are an important source of nitrous oxide to the atmosphere, however, determination of the precise microbial processes contributing to nitrous oxide fluxes from soils has been very difficult.  We investigated the isotopomer site preference in pure microbial culture and in soil incubations to determine if nitrous oxide flux can be attributed to a specific microbial process.  The term isotopomer refers to not only the abundance of nitrogen isotopes in nitrous oxide but specifically to isotope abundance within each nitrogen atom within the nitrous oxide molecule.  Thus the isotopomer site preference is the difference in ¹⁵N between the central and outer nitrogen atoms in nitrous oxide.  In pure microbial culture of Nitrosomonas europaea, we observed a site preference during nitrous oxide production of –1.4 /_- 1.7 and –7.7 /_- 3.1  per mil with hydroxylamine and nitrite as the exclusive substrates, respectively.  This result indicates that isotopomers can be used to separate nitrous oxide produced during nitrification from nitrite and hydroxylamine.  Furthermore, in cultures of a methane oxidizer, Methylococcus capsulatus Bath, we observed a site preference of 5.5 /_- 3.1 per mil that was markedly distinct from nitrous oxide produced by Nitrosomonas.  During the reduction of nitrite by Pseudomonas chloroaphis, a denitrifying bacteria incapable of reducing nitrous oxide, we observed a site preference in nitrous oxide produced of –7.3 /_- 3.0 per mil.  Thus the site preferences in nitrous oxide produced during denitrification by Pseudomonas and during nitrification by Nitrosomonas are nearly identical.  This observation is consistent with expectation that nitrous oxide production in nitrifiers proceeds by a similar chemical pathway as that used by denitrifiers.  In anaerobic incubations of soils from the KBS LTER site, we observed no change in site preference during the consumption of nitrous oxide.  Thus we conclude that reduction of nitrous oxide during denitrification has no effect on the site preference.  Our work is an important step toward understanding the response of specific microbial processes and associated nitrous oxide fluxes to management activities.