The hydrogen over-producing Escherichia coli HD701, a hydrogenase up-regulated strain, has the potential for industrial-scale hydrogen production; however, this strain is unable to metabolize sucrose which is a major constituent of many waste organic materials that can be used as feedstock for industrial hydrogen production. Invertase from Sacharomyces cervacea (yeast) was partially purified and characterized where its apparent optimum temperature when using short reaction period (15 min) was 55 degrees C; however the enzyme couldn't continue active due to its short half-life time at such high temperature. In contrast, a lower optimum temperature (35 degrees C) was recorded when using long reaction period (5 h) where the enzyme showed long half-life time and stability at such degree of temperature. Consequently, a concomitant hydrolysis of sucrose by yeast invertase and hydrogen production by E. coil at 35 degrees C was conducted and showed a high potency for industrial application with a hydrogen yield of 0.48 mole hydrogen/mole reducing sugars using batch fermentation at optimum sucrose concentration of 10 g/L. The described approach might be applicable for biotechnologies of other bio-products by E. coli from sucrose as a carbon source. (C) 2016 Elsevier Ltd. All rights reserved.