This study evaluated the elimination of alkalinity need and excess sulfate generation of sulfur-based autotrophic denitrification process by stimulating simultaneous autotrophic and heterotrophic (mixotrophic) denitrification process in a column bioreactor by methanol supplementation. Also, denitrification performances of sulfur-based autotrophic and mixotrophic processes were compared. In autotrophic process, acidity produced by denitrifying sulfur-oxidizing bacteria was neutralized by the external NaHCO3 supplementation. After stimulating mixotrophic denitrification process, the alkalinity need of the autotrophic process was satisfied by the alkalinity produced by heterotrophic denitrifiers. Decreasing and lastly eliminating the external alkalinity supplementation did not adversely affect the process performance. Complete denitrification of 75 mg L-1 NO3-N under mixotrophic conditions at 4 h hydraulic retention time was achieved without external alkalinity supplementation and with effluent sulfate concentration lower than the drinking water guideline value of 250 mg L-1. The denitrification rate of mixotrophic process (0.45 g NO3-N L-1 d(-1)) was higher than that of autotrophic one (0.3 g NO3-N L-1 d(-1)). Batch studies showed that the sulfur-based autotrophic nitrate reduction rate increased with increasing initial nitrate concentration and transient accumulation of nitrite was observed. (c) 2012 Elsevier Ltd. All rights reserved.