We evaluated the feasibility of improving the scale of hydrogen (H-2) production from sugar cane distillery effluent using co-cultures of Citrobacter freundii 01, Enterobacter aerogenes E10 and Rhodopseudomonas palustris P2 at 100 m(3) scale. The culture conditions at 100 ml and 2 L scales were optimized in minimal medium and we observed that the co-culture of the above three strains enhanced H-2 productivity significantly. Results at the 100 m(3) scale revealed a maximum of 21.38 kg of H-2, corresponding to 10692.6 mol, which was obtained through batch method at 40 h from reducing sugar (3862.3 mol) as glucose. The average yield of H-2 was 2.76 mol mol(-1) glucose, and the rate of H-2 production was estimated as 0.53 kg/100 m(3)/h. Our results demonstrate the utility of distillery effluent as a source of clean alternative energy and provide insights into treatment for industrial exploitation. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.