Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

Abstract

The kinetics of denitrification and the causes of NO2- and N2O accumulation were examined in resting cell suspensions of 3 denitrifiers. Alcaligenes sp. and a Pseudomonas fluorescens isolate characteristically accumulated NO2- when reducing NO32-; a Flavobacterium isolate did not. NO32- did not inhibit NO2--reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of NO2- seemed to depend on the relative rates of NO32- and NO2 even when NO32- or NO2- had been included in the incubation mixture. NO32- also did not inhibit N2O reduction in A. odorans, A. faecalis and is an organism incapable of NO32- reduction. Thus, added NO32- or NO2- does not always cause N2O accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of NO during denitrification in a pattern suggesting that NO was also under kinetic control similar to that of NO2- and N2O. Apparent Km values for NO32- and NO2 reduction were 15 .mu.M or less for each isolate. The Km value for N2O reduction by Flavobacterium sp. was 0.5 .mu.M. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of NO2-, NO and N2O accumulation. Addition of O2 inhibited gas production from 13NO3- by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. All 3 isolates produced higher ratios of N2O to N2 as the O2 tension increased. Inclusion of O2 in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas productoon but increased ratios of N2O to N2, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.