Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N₂ 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 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 direct ammonia oxidation (Dirammox) to N 2 .

Dosage-Dependent Toxicity of Universal Solvent Dimethyl Sulfoxide to the Partial Nitrification Wastewater Treatment Process

“…The application of the traditional biological nitrogen removal process in actual wastewater treatment plants is not recommended due to high capital costs and the need for external carbon sources, aeration, and other conditions that raise treatment costs, coupled with long hydraulic retention time and high energy consumption. An innovative nitrogen removal process with high efficiency and low treatment cost has become a research hotspot for eco-environmental researchers. , The emergence of nitrogen removal reactions such as simultaneous nitrification and denitrification, anammox, , and direct ammonia oxidation (Dirammox) provides more low-cost and low-energy pathways for removing nitrogen compounds. Anammox is valued by scholars for its ability to convert NH 4 + –N as an electron donor and NO 2 – –N as an electron acceptor through anaerobic ammonia-oxidizing bacteria (AOB) into N 2 and rare NO 3 – –N under anaerobic conditions without an organic carbon source. , The partial nitrification (PN) process oxidizes NH 4 + –N to NO 2 – –N by AOB, which can be used as a pretreatment process for anammox .…”

“…An innovative nitrogen removal process with high efficiency and low treatment cost has become a research hotspot for eco-environmental researchers. 6,7 The emergence of nitrogen removal reactions such as simultaneous nitrification and denitrification, 8 anammox, 9,10 and direct ammonia oxidation (Dirammox) 11 provides more low-cost and low-energy pathways for removing nitrogen compounds. Anammox is valued by scholars for its ability to convert NH nitrification (PN) process oxidizes NH 4 + −N to NO 2 − −N by AOB, which can be used as a pretreatment process for anammox.…”

Direct ammonium oxidation to nitrogen gas (Dirammox) in Alcaligenes strain HO-1: The electrode role

“…Initial testing suggested that Alcaligenes used a two-step process involving the oxidation of NH 4 + to nitrite/nitrate, followed by a reduction to N 2 [ 12 , 13 ]. However, it has been recently suggested that Alcaligenes use a previously unknown pathway to convert ammonium into N 2 (Dirammox route) [ 14 , 15 ]. According to whole genome annotation data, Alcaligenes strain HO-1 encodes a complete denitrification pathway, but it lacks genes coding for ammonia monooxygenases or hydroxylamine oxidoreductases homologs.…”

“…However, a combination of inhibition experiments and isotopic labelling suggested that strain HO-1 had a novel genetic cluster for ammonia oxidation, encoding for a previously unknown pathway able to shortcut the conventional nitrogen cycle. In the Dirammox route, NH 4 + is firstly oxidised to hydroxylamine (NH 2 OH) and then to N 2 [ 14 ]. This enzyme (encoded by the DnfA) has finally been identified and characterised by isotopic experiments.…”

Fungus Pichia kudriavzevii XTY1 and heterotrophic nitrifying bacterium Enterobacter asburiae GS2 cannot efficiently transform organic nitrogen via hydroxylamine and nitrite

“…LAD9 isolated by Chen and Ni (2012) and Alcaligenes ammonioxydans sp. HO-1 isolated by Wu et al (2021) denitrified to N 2 after transformation of NH 4 + to NO 2 − . In contrast, Pseudomonas putida Y-9 directly transformed NH 4 + to nitric oxide (NO) and then to N 2 O, and no other intermediates were found ( Huang et al, 2019 ).…”