Potential of Aerobic Denitrification by Pseudomonas stutzeri TR2 To Reduce Nitrous Oxide Emissions from Wastewater Treatment Plants

In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N₂O) even under aerobic conditions. We compared the denitrification activity of strain TR2 with those of various denitrifiers in an artificial medium that was derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and produced little N₂O under all conditions tested. Its growth rate under denitrifying conditions was near comparable to that under aerobic conditions, showing a sharp contrast to the lower growth rates of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to toxic nitrite, even utilizing it as a good denitrification substrate. When both nitrite and N₂O were present, strain TR2 reduced N₂O in preference to nitrite as the denitrification substrate. This bacterial strain was readily able to adapt to denitrifying conditions by expressing the denitrification genes for cytochrome cd₁ nitrite reductase (NiR) (nirS) and nitrous oxide reductase (NoS) (nosZ). Interestingly, nosZ was constitutively expressed even under nondenitrifying, aerobic conditions, consistent with our finding that strain TR2 preferred N₂O to nitrite. These properties of strain TR2 concerning denitrification are in sharp contrast to those of well-characterized denitrifiers. These results demonstrate that some bacterial species, such as strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen respiration. The bacterium was also shown to contain the potential to reduce N₂O emissions when applied to sewage disposal fields.Wastewater treatment processes produce one of the major greenhouse effect gases, nitrous oxide (N₂O) (7, 25, 30). The global warming potential of N₂O relative to that of carbon dioxide (CO₂) is 298 for a 100-year time horizon, and its concentration in the atmosphere continues to increase by about 0.26% per year (9). Nitrogen removal in wastewater treatment plants is essentially based on the activities of nitrifying and denitrifying microorganisms, both of which are inhabitants of activated sludge. Nitrifying bacteria aerobically oxidize ammonium to nitrite (NO₂⁻) and nitrate (NO₃⁻), which are then reduced anaerobically by denitrifying bacteria to gaseous nitrogen forms, such as N₂O and dinitrogen (N₂). It has long been known that N₂O can be produced during both nitrification and denitrification processes of wastewater treatment (3, 19, 23), but the cause of N₂O emission during the nitrification process was not clear. We recently showed, however, using activated sludge grown under conditions that mimicked a piggery wastewater disposal, that N₂O emission during the nitrification process depends on denitrification by ammonia-oxidizing bacteria (Nitrosomonas) (18). On the other hand, it is believed that denitrifying bacteria produce N₂O as a by-product when anaerobiosis is insufficient during the denitrification process, because N₂O reductase is the enzyme that is most sensitive to oxygen (6). Piggery wastewater, in particular, contains a high concentration of ammonia, and N₂O emission tends to take place during the nitrogen removal process (5, 10). Experiments on the removal of ammonia and organic carbon by the aerobic denitrifier Pseudomonas stutzeri SU2 (24) and the heterotrophic nitrifier-aerobic denitrifier Alcaligenes faecalis no. 4 (16, 17) have been reported as examples of bioaugmentation in piggery wastewater treatment. Reduction of N₂O emissions from pig manure compost by addition of nitrite-oxidizing bacteria has also been reported (11). However, there have been no reports of methods for reducing N₂O emissions by bioaugmentation using aerobic denitrifying bacteria.Takaya et al. isolated the aerobic denitrifying bacterium Pseudomonas stutzeri TR2 (26). The denitrification activity of strain TR2 was monitored in batch and continuous cultures, using denitrification and artificial wastewater media, and the strain was found to keep a distinct activity (producing N₂ from NO₃⁻) and to produce a very low level of N₂O at a dissolved oxygen (O₂) concentration of 1.25 mg liter⁻¹. Therefore, strain TR2 should be useful in the future for reducing N₂O emissions from wastewater treatment plants by bioaugmentation. To investigate the feasibility of using strain TR2 for future application to wastewater treatment processes, we examined its denitrification activity, N₂O production, growth rate, and expression of denitrifying genes in batch cultures, using a medium that mimics the composition found in nitrogen removal wastewater plants. Comparison of the properties of strain TR2 with those of well-characterized denitrifying bacteria revealed characteristics of the strain that favor denitrification, although it can also respire oxygen.