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The present study investigated the nitrogen removal characteristics and metabolic pathway of bacteria in aquatic ecosystem, with a focus on heterotrophic nitrification and aerobic denitrification. The bacteria demonstrated significant heterotrophic nitrification and aerobic denitrification capacity. The highest ammonium-N, nitrate-N, and nitrite-N removal efficiencies were 95.31 ± 0.11%, 98.91 ± 0.05%, and 98.79 ± 0.09%, respectively. The Monod model was used to estimate the maximum rate of substrate utilization (R mo) and the half-saturation concentration (K s) for the two substrates, i.e., ammonium and nitrate. The kinetic coefficients were 3.34 mg/L/d (R mo) and 30.59 mg/L (K s) for ammonium-N, respectively, and 14.23 mg/L/d (R mo) and 215.24 mg/L (K s) for nitrate-N, respectively. The effects of initial nitrogen (ammonium-N or nitrate-N) concentration, temperature, and dissolved oxygen (DO) on nitrogen removal rate were investigated using response surface methodology (RSM), and the optimal conditions for nitrogen removal were determined. The principal nitrogen removal pathway of the bacteria was proposed as complete heterotrophic nitrification and aerobic denitrification, which was performed by six key genera: Arthrobacter , Pseudomonas , Rhodococcus , Bacillus , Massilia , and Rhizobium. Chryseobacterium and other denitrifying species may also reduce nitrification products (NO X −) via aerobic denitrification. • Heterotrophic nitrification and aerobic denitrification were established. • Monod kinetic coefficients indicated great application potential of the well-cultured sediment. • RSM was a reliable method for determining the optimum point for nitrogen removal. • Complete heterotrophic nitrification and aerobic denitrification was the principle nitrogen removal pathway. [ABSTRACT FROM AUTHOR]