Defined co-cultures of hydrogen (H2) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H-2 production. Out of 11 co-cultures consisting of 2-4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H-2 yield up to 3.0 mol mol(-1) of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H-2 production with an actual evolution of 7.44 L of H-2. It constituted 58.2% of the total biogas. Continuous culture evolution of H-2 by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H-2 yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H-2 production than undefined or mixed cultures. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.