Journal of Microbial & Biochemical Technology

Abstract

Estimating Rates of Denitrification Enzyme Activity in Wetland Soils with Direct Simultaneous Quantification of Nitrogen and Nitrous Oxide by Membrane Inlet Mass Spectrometry

Fred J. Genthner, Dragoslav T. Marcovich and John C. Lehrter

The microbial mediated process of denitrification is a major pathway for the removal of reactive nitrogen a pollutant in natural environments and waste treatment facilities. Denitrification potential, measured as denitrification enzyme activity (DEA), was quantified in novel short-term (4 h) anaerobic assays using a more sensitive and precise technique, membrane inlet mass spectrometry (MIMS) rather than the traditional headspace electron capture gas chromatography (GC-ECD) method. Using MIMS modifications made to the instrument and sample handling allowed for the simultaneous and direct measurement of reaction products nitrous oxide (N2O), a potent greenhouse gas, and the chemically unreactive dinitrogen (N2). Rate determinations were made from the slope of a linear curve generated by plotting increasing concentrations of the reaction products with time. Strong evidence for the validity of MIMS measured DEA rates was provided by showing consistent, linear accumulations of N2O or N2 and close agreement in rates from replicate reactions. Reactions were performed using wetland soils and cultures of Pseudomonas aeruginosa and P. chloroaphis that generated denitrification end products of N2 and N2O, respectively. Under acetylene inhibition P. aeruginosa produced the N2O end product at a rate equivalent to the rate obtained in the uninhibited reaction that produced N2. No significant (p>0.05) difference was observed between MIMS or headspace with GC-ECD, determined DEA in wetland soil reactions under acetylene inhibition. Because of anoxic conditions in the reaction vessels used with MIMS, detectable rates of N2O accumulation were only observed in acetylene blocked reactions or in cultures of P. chloroaphis. This method has potential applications ranging from near realtime wastewater treatment process measurements to field studies of nitrogen cycling. The continued development and application of these types of methods are needed to improve our understanding of the mechanisms regulating denitrification and its benign, N2, and harmful, N2O, end-products.