Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N 2 with a previously unknown pathway

Summary Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen‐rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO‐1, oxidizing ammonia to hydroxylamine and ending in the production of N 2 gas. Genome analysis revealed that strain HO‐1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO‐1 denitrified nitrite (not nitrate) to N 2 and N 2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N 2 production. A gene cluster ( dnfT1RT2ABCD ) was cloned from strain HO‐1 and enabled E . coli accumulated hydroxylamine. Sub‐cloning showed that genetic cluster dnfAB or dnfABC already enabled E . coli cells to produce hydroxylamine and further to 15 N 2 from ( 15 NH 4 ) 2 SO 4 . Transcriptome analysis revealed these three genes dnfA , dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO‐1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of di rect amm onia ox idation (Dirammox) to N 2 .