BACKGROUND: Bacterial hydrogen evolution releases excess electrons or energy formed during the metabolic oxidations. Until now, few studies have investigated the global regulation of cellular energy flows, pertaining to bacterial hydrogen production for the improvement of hydrogen production. In this study, the cellular energy metabolism of Enterobacter aerogenes was regulated by the addition of pyrophosphate (PPi) and the overexpression of polyphosphate kinase (PPK), to improve hydrogen production. RESULTS: By overexpressing PPK at 10 mmol L-1 PPi, total hydrogen yields were increased by 32.7%. Metabolic flux analysis demonstrated that overexpression of PPK in E. aerogenes resulted in a higher cellular ATP level and a higher NADH consumption rate, which changed the cellular redox state and allowed more electrons to flow into the hydrogen production pathway. CONCLUSION: Alteration of the energy metabolism in E. aerogenes can affect hydrogen production. This implies that if one could accurately control the energy flow or the electron flow in the cells, hydrogen productivity would be further greatly improved. Copyright (c) 2012 Society of Chemical Industry