Rapid and complete inorganic nitrogen removal from real aquaculture wastewater via heterotrophic nitrification–aerobic denitrification by Pseudomonas putida 4-HYK

The accumulation of inorganic nitrogen in intensive aquaculture systems necessitates treatment strategies that combine rapid conversion kinetics with operational robustness. In this study, Pseudomonas putida 4-HYK was isolated and systematically evaluated for inorganic nitrogen removal under both synthetic media and real aquaculture wastewater conditions. Batch experiments demonstrated complete removal of ammonium, nitrite, and nitrate (20 mg L−1) within 15, 18, and 21 h, respectively, with maximum removal rates reaching 1.67, 1.33, and 1.04 mg L−1 h−1 and no detectable accumulation of intermediate nitrogen species. Under mixed-nitrogen conditions, the strain exhibited a consistent sequential utilisation pattern, preferentially assimilating ammonium prior to nitrite and nitrate reduction. Stable nitrogen removal was maintained across a broad operational window (C/N ratio 3–20, pH 7–9, and 25–35 °C), while measurable activity persisted at pH 4 and salinity up to 40‰, indicating strong environmental tolerance relevant to aquaculture settings. Dissolved oxygen availability was identified as a critical factor for achieving complete nitrogen removal. Process validation using real industrial aquaculture wastewater (NH₄⁺-N 8.36 mg L−1; NO₂−-N 5.12 mg L−1; NO₃−-N 2.85 mg L−1) showed that complete inorganic nitrogen removal could be achieved within 24 h at inoculation levels of 0.5–2% when brown sugar was applied as an economical external carbon source, supporting the technical feasibility of low-cost biological treatment. These results demonstrate that 4-HYK enables rapid and complete nitrogen removal with strong tolerance to variable environmental conditions, providing a practical biological option for nitrogen control in aquaculture wastewater treatment systems.