Heterotrophic nitrogen removal in Bacillus sp K5: involvement of a novel hydroxylamine oxidase

An aerobic denitrifying bacterium isolated from a bio-trickling filter treating NOx, Bacillus sp. K5, is able to convert ammonium to nitrite, in which hydroxylamine oxidase (HAO) plays a critical role. In the present study, the performance for simultaneous nitrification and denitrification was investigated with batch experiments and an HAO was purified by an anion-exchange and gel-filtration chromatography from strain K5. The purified HAO's molecular mass was determined by SDS-PAGE and its activity by measuring the change in the concentration of ferricyanide, the electron acceptor. Results showed that as much as 87.8 mg L-1 ammonium-N was removed without nitrite accumulation within 24 hours in the sodium citrate medium at C/N of 15. The HAO isolated from the strain K5 was approximately 71 KDa. With hydroxylamine (NH2OH) as a substrate and potassium ferricyanide as an electron acceptor, the enzyme was capable of oxidizing NH2OH to nitrite in vitro when the pH varied from 7 to 9 and temperature ranged from 25 degrees C to 40 degrees C. This is the first time that an HAO has been purified from the Bacillus genus, and the findings revealed that it is distinctive in its molecular mass and enzyme properties.