The pretreatment of environmental microbial consortia by five methods (acid, base, heat shock, dry heat and desiccation, freezing and thawing) was conducted in order to evaluate their applicability for the selection of hydrogen-producing bacteria capable of using residual glycerol from biodiesel synthesis as substrate. Results showed that substrate degradation rates of consortia pretreated with dry heat and desiccation and heat shock were higher compared with controls during the fermentation using glycerol, with degradation rates as high as 65%. The maximal hydrogen and biomass productions were obtained by dry heat and desiccation: 34.19% mol and 4340 mg/L, respectively. Dry heat and desiccation followed by heat shock are simple pretreatments methods that can be used to improve the biotechnological production of hydrogen. DNA sequencing performed to identify the bacteria strains present in the consortium showed that they belonged to the genus Klebsiella and Pantoea. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.