Degradation characteristics and mechanisms of ammonium and phenol by the direct ammonia oxidation bacterium Alcaligenes faecalis AP3-14.

Direct ammonia oxidation (Dirammox) is an emerging nitrogen removal process that exhibits strong environmental adaptability, a simple flow, and can directly convert ammonium into N₂. However, no Dirammox bacterium has been reported for efficient treatment of antibiotic pharmaceutical wastewater co-contaminated with phenol and nitrogen. In this study, a simultaneous nitrogen- and phenol-removing strain, Alcaligenes faecalis AP3-14, was isolated from activated sludge. The strain harbors the key Dirammox gene cluster dnfABC, suggesting its direct ammonia oxidation potential. With sodium acetate as the carbon source, C/N = 12 resulted in efficient nitrogen removal. When phenol served as the sole carbon source (C/N 2-8), complete phenol degradation was achieved, with the highest soluble total nitrogen (TDN) removal of 55.42 ± 1.61% at C/N = 8. Strain AP3-14 exhibited excellent simultaneous nitrogen and phenol removal at pH 6-9, 120 rpm, and 30 °C (NH₄⁺-N removal ≥ 83.72%, phenol removal ≥ 82.75%). The strain showed a clear preference for ammonium, with transient hydroxylamine accumulation, but could not utilize nitrate or nitrite. Genomic and transcriptomic analyses indicated that nitrogen removal mainly relies on synergy between ammonia assimilation and Dirammox, while phenol is mainly degraded via the ortho-cleavage pathway, supported by enhanced TCA cycle activity. Biosafety assessment indicated no hemolytic activity or broad-spectrum antibiotic resistance. In actual pharmaceutical wastewater, inoculation with AP3-14 achieved ammonium and TDN removal efficiencies of 89.64 ± 0.26% and 71.65 ± 3.22%, respectively, far exceeding the non-inoculated control (13.02 ± 1.28% and 11.20 ± 5.05%). In conclusion, Alcaligenes faecalis AP3-14 possesses excellent antibiotic tolerance and simultaneous nitrogen/phenol removal capability, offering a promising microbial resource for Dirammox-based treatment of phenol-laden pharmaceutical wastewater.