Understanding the role of nitrogen dissimilation in soil microorganisms

Uncertainty about the fate of nitrate in ecosystems has led to increased interest in soil nitrogen (N) transformations and microbial biogeochemistry of N. Microorganisms can utilize nitrate by either assimilatory or dissimilatory processes. The best studied dissimilatory processes are nitrate reduction to ammonium and denitrification, both of which are thought to occur under low O₂ conditions. While there is an appreciation that denitrifying bacteria are diverse, the activity of each enzyme in the pathway is viewed more uniformly, in that all are presumed to have activity that is inversely correlated with O₂ levels. However, the first step of denitrification, dissimilatory reduction of nitrate to nitrite, can occur at O₂ concentrations that are high enough to repress downstream reduction of nitrite to gaseous products. To explore this in more detail, we tested for aerobic nitrate reduction (ANR) activity in a range of agricultural, wetland and forest soils located near Ithaca, New York. ANR was found in some environments, as evidenced by nitrite production in samples provided with both nitrate and a carbon source but not in controls. We next undertook a screen to isolate bacteria capable of ANR on an oxidized carbon source, succinate. Bacteria capable of ANR were surprisingly easy to isolate, as this phenotype was present in 10-15% of the isolates. 16S rDNA sequencing showed that the isolates included both gram negative and gram positive bacteria, although the majority were proteobacteria. The ANR isolates were tested for anoxic growth and less then 20% were able to grow under anoxic conditions as denitrifiers. To confirm the ANR phenotype, we measured the level of O₂ present when nitrate reduction was first detected in two of the isolates using a robotic gas sampler. The O₂ levels detected during ANR were higher than levels associated with the onset of nitrite reduction, since nitrite production began between 84% to 22% of atmospheric O₂. Production of gaseous nitrogen oxides during an oxic to microoxic growth transition was also assessed for some of the isolates. Surprisingly, a wide phenotypic diversity was found, ranging from isolates stalling at NO production to others producing N₂ gas. Therefore, we have been able to observe that denitrification is not a single, tightly coupled process carried out by a physiologically similar group of organisms. Instead, denitrification should be modeled as consisting of two modules with complex regulation and a concomitant diverse phenotypic landscape.