To obtain a clean hydrogen production system, we have developed an inorganic-bio hybrid photocatalyst system based on the combination of anatase TiO2, methylviologen (MV) as an electron mediator, and a whole-cell biocatalyst consisting of [FeFe]-hydrogenase and maturase gene-harboring recombinant Escherichia coli; however, the apparent quantum yield at 300 nm (AQY(300)) for hydrogen production was low (0.3%). The system consists of a two-step reaction: (1) photocatalytic MV reduction by Ti02, and (2) hydrogen production with reduced MV using a biocatalyst. The enhancement of step 1 under biocatalyst-friendly conditions was investigated in an attempt to further improve the reaction efficiency. Among the condition tested, the use of 100mM Tris-HCl (pH 7), 150mM NaCl, and 5% (v/v) glycerol with P-25 TiO2 especially enhanced the step 1 reaction by a 300-fold increase in the MV reduction rate compared with previously tested reaction condition (100 mM Tris-HCl( pH 7), 150 mM NaCl, 5% (v/v) glycerol, and 100 mM ascorbate with anatase Ti02). Under the enhanced step 1 reaction, AQY(300) and AQY350 for photocatalytic MV reduction reached 60.8% and 52.2%, respectively. The enhanced step 1 reaction thus significantly improved the overall photocatalytic hydrogen productivity of the hybrid system and AQY(300) and AQY(350) reached 26.4% and 31.2%, respectively. The inorganic-whole-cell biohybrid system can therefore provide noble metal-free, efficient, and clean hydrogen production. (C) 2017 Elsevier B.V. All rights reserved.