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

Environmental Microbiology

et al. 2021

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 .

Section: Introductionmentioning

confidence: 99%

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N₂ with a previously unknown pathway

Hou

Environmental Microbiology

et al. 2021

“…Y16 and Bacillus sp. K5 was 0.31 ± 0.06 and 0.045 ± 0.003 U/mg, respectively . And the activity of HAO purified from anammox bacterium was 9.6 U/mg .…”

Section: Discussionmentioning

confidence: 95%

“…K5 and Acinetobacter sp. Y16 is 71 and 61 kDa monomer, respectively . Therefore, purification and further research of HAO from different heterotrophic nitrifiers are necessary.…”

Section: Introductionmentioning

confidence: 99%

Optimization, purification, and characterization of hydroxylamine oxidoreductase from Acinetobacter sp. Y1

Yuan

Biotech and App Biochem

2019

Hydroxylamine oxidoreductase (HAO) is a key enzyme involved in ammonium removal pathway. To further study the enzyme, HAO was purified from heterotrophic nitrifier Acinetobacter sp. Y1 and its property was investigated. Results of single-factor experiments showed that the optimal carbon source, nitrogen source, and C/N ratio were trisodium citrate, ammonium sulfate, and 14, respectively, with incubation time of 16 H. DEAE Sefinose TM FF anion-exchange chromatography was used to purify HAO, followed by Sefinose TM CL-6B gel filtration chromatography. SDS-PAGE revealed that a 47 kDa enzyme was purified successfully, with a purification fold of 7.32 and a recovery rate of 19.40%. The optimized enzyme activity of purified HAO was tested at pH 8.0 and 30 • C. The results showed that the activity was increased by 43.78% and 25.64% in the presence of 1 mM Fe 2+ and Fe 3+ , respectively. HAO activity was increased with the increase of Na + and K + , Mn 2+ , Zn 2+ , Cu 2+ , Ca 2+ , Ba 2+ inhibited the HAO activity at three concentrations. In addition, HAO activity was activated by ethylenediaminetetraacetic acid at 0.4 mM, and a negative effect arose as the dose increased.

Environmental Engineering Research

“…The bacteria biomass was measured by determining the optical density at 600 nm (OD 600 ) using a spectrophotometer (TU-1800S, PERSEE, China) [32].…”

Section: Analysis Methodsmentioning

confidence: 99%

Biodegradation of omethoate by Bacillus sp. YB-10: optimization of culture conditions and degradation characteristics

Li²

2020

Omethoate is an acute organophosphorus pesticide which is widely used. It presents a high pollution potential and health risk nowadays. In this study, Bacillus sp. YB-10, which was isolated from the activated sludge of a pharmaceutical wastewater treatment plant, was used for degrading omethoate. The culture conditions and degradation characteristics were investigated. Results showed that the Bacillus sp. YB-10 could degrade omethoate through co-metabolism. The optimal carbon and nitrogen sources were glucose and NH4NO3, respectively. Response surface methodology (RSM) results showed that the most appropriate nutrition ratio of C:N:P was 3:1:1 when degrading 1,000 mg/L of omethoate. Omethoate degradation kinetics could be described by a first-order rate equation. The optimal degradation conditions were pH of 7.0, temperature of 30℃, initial bacteria concentration of 0.25%, rotation speed of 150 r/min. Under the optimal conditions, the degradation rate reached 77.24% within 5 d by the Bacillus sp. YB-10 when the initial omethoate concentration was 1,000 mg/L.