Effects of different microaerophilic conditions on cell growth, glucose consumption, hydrogen production and cellular metabolism of wild Enterobacter aerogenes strain and polyphosphate kinase (PPK) overexpressing strain were systematically studied in this paper, using NaH2PO4 as the phosphate sources. Under different microaerophilic conditions, PPK-overexpressing strain showed better cell growth, glucose consumption and hydrogen production than the wild strain. In the presence of limited oxygen (2.1%) and by PPK overexpression, the hydrogen production per liter of culture, the hydrogen production per cell and the hydrogen yield per mol of glucose increased by 20.1%, 12.3% and 10.8%, respectively, compared with the wild strain under strict anaerobic conditions. Metabolic analysis showed that the increase of the total hydrogen yield was attributed to the improvement of NADH pathway. The result of more reductive cellular oxidation state balance also further demonstrated that, under proper initial microaerophilic conditions and by PPK overexpression, the cell could adjust the cellular redox states and make more energy flow into hydrogen production pathways. (C) 2010 Elsevier Inc. All rights reserved.