Inoculum pre-treatment is a crucial aspect of hydrogen fermentation processes to establish the required microbial community for hydrogen production. This paper models and optimizes two hybrid techniques of inoculum pre-treatment for fermentative hydrogen production: (1)pH and Autoclave (PHA); (2)pH and heat shock (PHS) using Response Surface Methodology (RSM). Coefficients of determination (R-2) of 0.93 and 0.90 were obtained for PHA and PHS respectively and the optimized pre-treatment conditions gave hydrogen yields up to 1.35 mol H-2/mol glucose and 0.75 mol H-2/mol glucose, thus a 37.75% and 15.38% improvement on model predictions for PHA and PHS respectively. Two semi pilot scale up processes were carried out in a 10 L bioreactor seeded with the optimally treated inocula. Maximum hydrogen yields of 1.19 mol H-2/mol glucose and 0.68 mol H-2/mol glucose were obtained for PHA and PHS respectively which further verifies the practicability of this technique to enrich hydrogen producing bacteria for fermentative biohydrogen production. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.