A long-term performance of a packed-bed bioreactor containing sulfur and limestone was evaluated for the denitrification of drinking water. Autotrophic denitrification rate was limited by the slow dissolution rate of sulfur and limestone. Dissolution of limestone for alkalinity supplementation increased hardness due to release of Ca(2+). Sulfate production is the main disadvantage of the sulfur autotrophic denitrification process. The effluent sulfate concentration was reduced to values below drinking water guidelines by stimulating the simultaneous heterotrophic and autotrophic denitrification with methanol supplementation. Complete removal of 75 mg/L NO(3)-N with effluent sulfate concentration of around 225 mg/L was achieved when methanol was supplemented at methanol/NO(3)-N ratio of 1.67 (mg/mg), which was much lower than the theoretical value of 2.47 for heterotrophic denitrification. Batch studies showed that sulfur-based autotrophic NO(2)-N reduction rate was around three times lower than the reduction rate of NO(3)-N, which led to NO(2)-N accumulation at high loadings. (C) 2011 Elsevier Ltd. All rights reserved.