Hydrogen fermentation using glucose as a single substrate caused abrupt pH drops and the gradual losses of hydrogen producers, which in turn led to system failure. In this study the use of a proteinaceous substrate, peptone, avoided the abrupt pH drops in the reactive system and allowed for further exploration of volatile fatty acids (VFAs) and pH effects on the hydrogen fermentation process. Our results showed that: (1) during the hydrogen fermentation tests, the abrupt pH drops were avoided thus system stability increased due to the production of ammonia from the peptone fermented, (2) pH control was not necessary and the addition of acetate to the process had little effect on the hydrogen fermentation process, (3) at the extreme pHs the addition of acetate either lengthened the lag phase (pH < or = 6) or slowed the hydrogen production rate (pH > or = 8), and both situations were not desired, and (4) high VFA content in the system sped up the consumption of hydrogen gas. Results of this study suggested that the hydrogen fermentation using the protein-containing substances as substrate was beneficial in maintaining the system pH. As long as the pH was maintained around 6-8, system inhibition due to VFAs accumulation was minimized. Thus, the optimal operation of a hydrogen fermentation process would be achievable via the control of substrate composition at a certain carbohydrate-to-protein ratio.