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

Abstract

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 N₂ 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 N₂ and N₂ O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N₂ 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 N₂ from (¹⁵ NH₄ )₂ SO₄ . 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 direct ammonia oxidation (Dirammox) to N₂ .