A step toward environmental sustainability of recirculat aquaculture systems (RAS) is implementation ofsingle-sludge denitrification, a process eliminating nitrate from the aqueous environment while reduc-ing the organic matter discharge simultaneously. Two 1700 L pilot-scale RAS systems each with a 85 Ldenitrification (DN) reactor treating discharged water and hydrolyzed solid waste were setup to testthe kinetics of nitrate and COD removal. Nitrate removal and COD reduction efficiency was measured attwo different DN-reactor sludge ages (high X: 33–42 days and low X: 17–23 days). Nitrate and total N(NO3−+ NO2−+ NH4+) removal of the treated effluent water ranged from 73–99% and 60–95% during theperiods, respectively, corresponding to an overall maximum RAS nitrate removal of approximately 75%.The specific nitrate removal rate increased from 17 to 23 mg NO3−-N (g TVS d)−1and the maximal poten-tial DN rate (measured at laboratory ideal conditions) increased correspondingly from 64–68 mg NO3−-N(g TVS d)−1to 247–294 mg NO3−-N (g TVS d)−1at high and low X, respectively. Quantification of denitri-fiers in the DN-reactors by qPCR showed only minor differences upon the altered sludge removal practice.The hydrolysis unit improved the biodegradability of the solid waste by increasing volatile fatty acid CODcontent 74–76%. COD reductions in the DN-reactors were 64–70%. In conclusion, this study showed thatsingle-sludge denitrification was a feasible way to reduce nitrate discharge from RAS, and higher DN rateswere induced at lower sludge age/increased sludge removal regime. Improved control and optimizationof reactor DN-activity may be achieved by further modifying reactor design and management scheme asindicated by the variation in and between the two DN-reactors.