Controlled induction of denitrification in Pseudomonas aureofaciens: A simplified denitrifier method for dual isotope analysis in NO3−

Abstract

Dual isotope signatures (δ15N and δ18O) of nitrate (NO3−) are invaluable to constrain nitrogen transformation processes in the biosphere. Biological conversion to nitrous oxide (N2O) by denitrifiers lacking the gene coding for N2O reductase has become the method of choice for isolating NO3− from natural samples for 15N and 18O analysis (“denitrifier method”). The success of the method depends on the ability of the culture to rapidly and quantitatively convert NO3− to N2O with as little as possible contamination from non-sample NO3− or interference with water 18O. We studied oxic-anoxic transition in Pseudomonas chlororaphis ss. aureofaciens grown oxically in complex medium. When letting the culture turn anoxic starting with ample O2 and mM NO3−, nitric oxide accumulated to toxic levels, impairing denitrification. With initially low O2 availability and μM NO3−, efficient conversion to N2O depended on the presence of mM ammonium. At higher cell densities, conversion efficiencies decreased, suggesting that the ability to induce balanced denitrification is growth dependent. Our results show that P. aureofaciens can induce denitrification successfully in the presence of μM NO3−, making anoxic pre-culturing with extraneous NO3− obsolete. We devise a simplified denitrifier method, which is fast and robust and does not require concentration of cells or removal of extraneous NO3− or N2O. Background NO3− in the medium can be removed as N2 by anoxic pre-incubation with P. denitrificans. Repeatability at natural abundance ranged from 0.1–0.3‰ for δ15N and 0.2–0.7‰ for δ18O. We successfully tested the method for various sample matrices including low pH (pH=4) and high salinity (0.25 - 1M KCl). Analyzing NO3− isotopes in the presence of highly 15N-labelled NH4+ or amino acids showed no cross-contamination. We conclude that the simplified denitrifier method is well suited for analyzing dual NO3− isotopes in a wide range of natural samples.