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 (R2) 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 H2/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 H2/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.