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

Highlights: • Pseudomonas aureofaciens can convert μM NO₃⁻ directly to N₂O. • This makes induction of denitrification with extraneous NO₃⁻ unnecessary. • Growth stage, O₂ and NH₄⁺ availability are the key factors for successful conversion. • A simplified denitrifier method is proposed for dual isotopic analysis of NO₃⁻. 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 - 1 M 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.