An absolute biological microbial electrolysis cell (MEC) was operated for a prolonged period under different applied potentials (E-app, -0.2 V to -1.0 V) and hydrogen (H-2) production was observed using acid-rich effluent. Among these potentials, an optimal voltage of -0.6 V influenced the biocathode by which maximum H-2 production of 120 +/- 9 ml was noticed. This finding was corroborated with dehydrogenase activity (1.8 +/- 0.1 mu g/ml) which is the key enzyme for H-2 production. The in situ biocathode regulated buffer overpotentials which was remarkably observed by the change in peak heights of dissociation value (pKa) from the titration curve. Substrate degradation analysis gave an estimate of coulombic efficiency of about 72 +/- 5% when operated at optimal voltage. Evidently, the electron transfer from solid carbon electrode to biocathode was analyzed by cyclic voltammetry and its derivatives showed the involvement of redox mediators. Despite, the MEC endures certain activation overpotentials which were estimated from the Tafel slope analysis. (C) 2015 Elsevier Ltd. All rights reserved.