Sequential dark-photo fermentations (SDPF) was used for hydrogen production from bagasse, an acetaldehyde dehydrogenase (adhE) gene inactivated Klebsiella oxytoca HP1 (Delta adhE HP1) mutant was used to reduce the alcohol content in dark fermentation (DF) broths and to further enhance the hydrogen yield during the photo fermentation (PF) stage. Compared with that of the wild strain, the ethanol concentration in OF broths of Delta adhE HP1 decreased 69.4%, which resulted in a hydrogen yield in the PF stage and the total hydrogen yield over the two steps increased by 54.7% and 23.5%, respectively. The culture conditions for hydrogen production from acid pretreated bagasse by SDPF were optimized as culture temperature 37.5 degrees C, initial pH 7.0, and cellulase loading 20 FPA/g in the OF stage, with initial pH 6.5, temperature 30 degrees C and photo intensity 5000 lux in the PF stage. Under optimum conditions, by using Delta adhE HP1 and wild type strain, the H-2 yields were 107.8 +/- 5.3 mL H-2/g-bagasse, 96.2 +/- 4.4 mL H-2/g-bagasse in OF and 54.3 +/- 2.2 ml, H-2/g-bagasse, 35.1 +/- 2.0 mL H-2/g-bagasse in PF, respectively. The special hydrogen production rate (SHPR) were 5.51 +/- 0.34 mL H-2/g-bagasse h, 4.95 +/- 0.22 mL H-2/g-bagasse h in OF and 0.93 +/- 0.12 mL H-2/g-bagasse h, 0.59 +/- 0.07 mL H-2/g-bagasse h in PF, respectively. The total hydrogen yield from bagasse over two steps was 162.1 +/- 7.5 mL H-2/g-bagasse by using Delta adhE HP1, which was 50.4% higher than that from dark fermentation only. These results indicate that reducing ethanol content during dark fermentation by using an adhE inactivated strain can significantly enhance hydrogen production from bagasse in the SDPF system. This work also proved that SDPF was an effective way to improve hydrogen production from bagasse. (C) 2010 Elsevier Ltd. All rights reserved.