Characterization of denitrification and net N2O-reduction properties of novel aerobically N2O-reducing bacteria

Nitrous oxide, one of the earth-warming and ozone-destructing gases, is produced through either nitrification or denitrification depending on the O-2 availability in soil. Aerobically denitrifying bacteria express denitrification tract even under the gas phase containing O-2 at the ambient air level. The net reduction of exogenous N2O by novel aerobically denitrifying bacteria were studied. We carried out two different isolation strategies in the primary screening. One was to select isolates of interest out of periplasmic nitrate reductase-dependent denitrifying bacteria in a eutrophic condition. The other was to use diluted nutrient agar to allow the formation of colonies of diverse bacteria. Among aerobically denitrifying bacteria, those which showed net aerobic N2O reduction were only minor populations. As a result, eight isolates belonging to Proteobacteria were obtained from soil and cow manure. The denitrification and net N2O reduction properties of the three representative isolates, Pseudomonas sp. CM1, Thauera sp. PM2 and Paracoccus denitrificans 96, were determined separately by the acetylene inhibition method after exposure to aerobic or low O-2 conditions, a 24h starvation prior to the determination of the aerobic activity and inoculation to a cow manure-amended sterile soil. The phenotype inversion from net N2O-reducing to N2O-emitting, and vice versa, attested to the fact that activity of the N2O-producing and -reducing steps changed in different intensities to each other. The activity values and the direction of activity changes varied among the isolates. When they were inoculated in a sterilized soil microcosm at 40% maximum water holding capacity, the denitrification and the N2O-reducing activities were comparable with or, in some cases, facilitated more than those determined under the low-O-2 condition. It is possible that these isolates sensed the O-2 deficiency even in such a relatively dry condition. Pseudomonas sp. CM1 was unique because it lacked nitrate reducing activity and acted as a net aerobic N2O reducer.