Highly Efficient Nitrogen Removal by Stutzerimonas stutzeri Strain MJ20: Metabolic Pathways and Potential for Biofloc Systems and Low C/N Ratio Aquaculture Wastewater

Although numerous studies have focused on the potential application of heterotrophic nitrification–aerobic denitrification (HNAD) bacteria in wastewater treatment, research exploring their potential in aquaculture biofloc systems remains limited. In this study, a promising HNAD strain, identified as Stutzerimonas stutzeri MJ20, was isolated from mature biofloc. This strain efficiently utilized low-cost carbon sources (e.g., glucose) and small-molecule carbon sources (e.g., sodium acetate and sodium succinate). Under conditions with glucose as the carbon source, a carbon-to-nitrogen (C/N) ratio of 15, pH 6–9, temperature 25–35 °C, salinity 0–35‰, and shaker speed of 0–150 rpm, it achieved removal rates of 95–100% for NH4+-N, NO2−-N, and NO3−-N at initial concentrations of 100 mg/L each. Even at higher concentrations (up to 200 mg/L NH4+-N and 500 mg/L for both NO2−-N and NO3−-N), removal rates exceeded 99%. Under mixed nitrogen sources, strain MJ20 demonstrated efficient nitrogen removal, preferentially utilizing NH4+-N, with only minimal and transient accumulation of nitrite and nitrate. Genomic analysis revealed that MJ20 carries key denitrification genes, including napA, nirS, norB and nosZ, and possesses complete pathways for nitrate reduction to nitrogen gas and ammonia assimilation, although typical autotrophic nitrification genes were not detected. Combined genomic data and autotrophic culture experiments indicated that, in addition to utilizing various organic carbon sources, the strain also exhibited certain autotrophic growth capabilities. Furthermore, MJ20 showed strong flocculation ability (flocculation rate > 96% within 16 h), sensitivity to multiple common antibiotics, and no toxicity to zebrafish, demonstrating favorable biosafety. In simulated seawater aquaculture wastewater with a C/N ratio of 5, it achieved a total nitrogen removal rate exceeding 94% within 72 h. These results indicate that strain MJ20 possesses comprehensive advantages, including efficient nitrogen removal, broad carbon source adaptability, strong environmental resilience, minimal accumulation of intermediate nitrogen products, excellent flocculation ability, and high biosafety. These traits highlight its potential for application in biofloc systems and in treating aquaculture tail water with a low C/N ratio. This study provides theoretical insights and practical guidance for screening HNAD bacteria suitable for biofloc systems.