The interactive effect of a hybrid pH and microwave pre-treatment on a mixed inoculum for biohydrogen production was investigated. Response surface methodology (RSM) was employed to obtain the optimum pre-treatment conditions of pH, microwave duration and microwave intensity for maximum hydrogen yield. The obtained model had a coefficient of correlation (R-2) of 0.87. The optimum inoculum pre-treatment conditions predicted were pH 11 and 2 min microwave treatment at 860 W and the validation experiments demonstrated a 32.41% increase on hydrogen yield. Two semi pilot scale-up processes were carried out in a 10 L bioreactor initiated in batch mode with 7 L working volume using the optimally pre-treated inoculum. In the absence of pH control, 46% of glucose was utilized corresponding to a molar hydrogen yield of 1.78 mol H-2/mol glucose and a maximum hydrogen fraction of 49.3% whereas under controlled pH environment, a twofold increase in glucose utilization was obtained which corresponds to a molar hydrogen yield of 2.07 mol H-2/mol glucose and a maximum hydrogen concentration of 56.4%. The controlled fermentation significantly improved biohydrogen production in the scale-up process and methane production was completely suppressed suggesting the effectiveness of the combined pre-treatment to enrich hydrogen producing bacteria. Viable counts and microscopical analysis indicated the presence of hydrogen producing endospore forming presumptive Clostridum species. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.