This study aimed to explore the influences of single-chamber systems with different applied voltage on bio-hydrogen (H-2) production. The reactor used was the bio-electrochemically assisted microbial reactor (BEAMR) membrane-less (BEAMR-membrane-less, BML). The microbial dark fermentative H-2 production method was adopted. After the hot screening process and the DNA sequencing, the domesticated dominant microflora was Clostridium sp. This study discussed the influences of the cases with (continuous and intermittent) and without applied voltage separately. The results showed that, the H-2 production rate of the case with intermittent applied voltage (117 mL/h gVSS) of 0.24V was increased of 1.7 folds higher than the without applied voltage (69 mL/h gVSS) and 1.3 folds higher than the case with continuous applied voltage (88.2 mL/hgVSS) of 0.24V. The produced H-2 concentration with intermittent applied voltage was 18.9% (18.6-19.1%) higher than the without applied voltage, while there was no significant difference with continuous applied voltage. (C) 2015 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.