Hydrogen production by Enterobacter aerogenes from glucose was enhanced by deleting the targeted ldhA and adh genes responsible for two NADH-consuming pathways which consume most NADH generated from glycolysis. Compared with the wild-type, the hydrogen yield of IAM1183-Delta ldhA increased 1.5 fold. Metabolic flux analysis showed both IAM1183-Delta ldhA and IAM1183-Delta adh exhibited significant changes in flux, including enhanced flux towards the hydrogen generation. The lactate production of IAM1183-Delta ldhA significantly decreased by 91.42%, while the alcohol yield of IAM1183-Delta adh decreased to 30%. The mutant IAM1183-Delta ldhA with better hydrogen-producing performance was selected for further investigation in a 5-L fermentor. The hydrogen production of IAM1183-Delta ldhA was 2.3 times higher than the wild-type. Further results from the fermentation process showed that the pH decreased to 5.39 levels, then gradually increased to 5.96, indicating that some acidic metabolites might be degraded or uptaken by cells. (C) 2015 Elsevier Ltd. All rights reserved.