A series of batch tests was conducted to investigate the effect of a CO2 sparging pretreatment on hydrogen production from fermentation of the organic fraction of municipal solid waste. Tests were carried out in a stirred bioreactor without adding inoculum or supplementation solution, at 35 degrees C and with a pH regulated at 5.5. The influence of CO2 flowrate (30, 100 and 500 mL/min) was evaluated. Results showed a decrease in biological hydrogen production for a sparging flowrate of 500 mL(CO2)/min, similar performances for the control experiments and for a sparging flowrate of 100 mL(CO2)/min and an increase in biohydrogen production for a sparging flowrate of 30 mL(CO2)/min. For this latter operating condition, a substantial improvement in hydrogen production performances was obtained with an increase in the hydrogen yield by more than 20% and a decrease in the chemicals consumption used to maintain the pH by more than 20%. No shift in the metabolic pathways and in the bacterial community structure was observed but positive bacterial interactions occurred and enhanced the hydrogen production. Therefore, this study suggested that the growth of hydrogen producing bacteria could be either enhanced or slowed down by a CO2 sparging pretreatment depending on the level of CO2 flowrate, the shift being close to 100 mL(CO2)/min. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.