Organic acid especially butyric acid accumulation can inhibit fermentative hydrogen production. However acid tolerance could be increased when mixed cultures adapted to butyric acid stress in an intentional and continuous operation. As compared to the original cultures, the yield and productivity of hydrogen were enhanced by 56.5% with more acid production and a higher HAc/HBu (acetic acid/butyric acid) of 1.33 by the evolved cultures. The enhancement was due to the increase of acid tolerance with more acid tolerance response (ATR) induction. Later the representative enzymatic ATR systems were investigated in this study. It was found that two ATR systems including H+-ATPase and hydrogenase played important roles in hydrogen evolution and the evolved cultures had a higher overall induction. Furthermore, the dynamics of the ATR systems indicated that hydrogen-producing microorganisms with acid tolerance could prepare themselves better against self-produced acid stress and the evolved cultures had a better stress anticipation possibly due to the bacterial communities change via adaptive evolution. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.