Nuclear irradiation was used for the first time to generate efficient mutants of hydrogen-producing bacteria Enterobacter aerogenes, which were screened with larger colour circles of more fermentative acid byproducts. E. aerogenes cells were mutated by nuclear irradiation of Co-60 gamma-rays. The screened E. aerogenes ZJU1 mutant with larger colour circles enhanced the hydrogenase activity from 89.8 of the wild strain to 157.4 mL H-2/(g DW h). The hereditary stability of the E. aerogenes ZJU1 mutant was certified after over ten generations of cultivation. The hydrogen yield of 301 mL H-2/g glucose with the mutant was higher by 81.8% than that of 166 mL/g glucose with the wild strain. The peak hydrogen production rate of 27.2 mL/(L.h) with the mutant was higher by 40.9% compared with that of 19.3 mL/(L.h) with the wild strain. The mutant produced more acetate and butyrate but less ethanol compared with the wild strain during hydrogen fermentation. (C) 2016 Elsevier Ltd. All rights reserved.