In order to enhance anaerobic hydrogen production from yeast waste, a series of 120-mL batch co-cultures of Clostridium beijerinckii L9, Clostridium butyricum M1, and Bacillus thermoamylovorans B5 under mesophilic conditions were established according to full factorial design (FFD) and mixture design (MD). The experimental results were subjected to multivariate and response surface analyses to determine the relationships between bacteria converting yeast waste into hydrogen. The results indicated clearly that C. beijerinckii L9 and C. butyricum M1 had significant potential to convert yeast waste into hydrogen. There was no significant hydrogen generation when B. thermoamylovorancs B5 alone was cultured with yeast waste. However, B. thermoamylovorancs B5 could significantly shorten the co-culture's hydrogen-producing lag phase. Response surface analyses demonstrate that B. thermoamylovorancs B5 can stimulate the specific hydrogen production rate of C. beijerinckii L9 and C. butyricum M1, greater in the case of the former than of the latter. An ultimate hydrogen yield of 46 mL H-2/g COD added yeast waste was obtained with an optimal volumetric ratio C. beijerinckii L9: C. butyricum M1: B. thermoamylovoranc B5 of 8.9:4.8:10.3. Highly reproducible co-culture results confirm that FFD and MD, via response surface analysis, are applicable to assess the roles of the individual microorganisms in the defined co-culture. Crown Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.