Accumulation of acid intermediates during fermentation causes feedback inhibition to the substrate/glucose flux through hydrogen (H-2) metabolism, especially at higher organic loads. Partial substrate replacement was studied in detail as a strategy to overcome this and the efficiency was compared among three experiments studied including control. After H-2 production stopped at 41st h, new substrate was replaced at a rate of 25% and 50% in two experiments, keeping the third as control. Substrate replacement resulted in dilution of accumulated acid intermediates causing reversal of feedback inhibition that helped in regaining H-2 production. However, the efficiency varied with the fraction of replacement, wherein 50% replacement showed higher H-2 production. Increased system buffering capacity after each replacement assured decrement of cytosolic pH stress on biocatalyst. Regain in the dehydrogenase activity to certain extent after each replacement supported the regain of H-2 production and were correlated well with the bio-electro kinetics studied. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.