Acclimation of autohydrogenotrophic denitrifying bacteria using inorganic carbon source (CO2 and bicarbonate) and hydrogen gas as electron donor was performed in this study. in this regard, activated sludge was used as the seed source and sequencing batch reactor (SBR) technique was applied for accomplishing the acclimatization, Three distinct strategies in feeding of carbon sources were applied: (I) continuous sparging Of CO2, (II) bicarbonate plus continuous sparging Of CO2, and (III) only bicarbonate. The pH-reducing nature Of CO2 showed an unfavorable impact on denitrification rate; however bicarbonate resulted in a buffered environment in the mixed liquor and provided a suitable mean to maintain the pH in the desirable range of 7-8.2. As a result, bicarbonate as the only carbon source showed a faster adaptation, while carbon dioxide as the only carbon source as well as a complementary carbon source added to bicarbonate resulted in longer acclimation period. Adapted hydrogenotrophic denitrifying bacteria, using bicarbonate and hydrogen gas in the aforementioned pH range, caused denitrification at a rate of 13.33 mg NO3--N/g MLVSS/h for degrading 20 and 30 mg NO3--N/L and 9.09 mg NO3--N/g MLVSS/h for degrading 50 mg NO3--N/L. (C) 2008 Elsevier B.V. All rights reserved.