In this study, granular and flocculated anaerobic mixed cultures were pretreated using heat, shock loading, acid, alkali, linoleic acid (LA) and 2-bromoethane sulphonic acid (BESA). Under mesophilic conditions (37 degrees C) and an initial pH value of 6.0, higher H-2 yields were observed for the flocculated cultures when compared to the granular cultures. The maximum yield for granular cultures treated acid, BESA or LA were statistically the same. Butyric acid fermentation was dominant in a majority of the treated cultures. The maximum hydrogenase evolution specific activity (ESA) (124 +/- 8 U-e mg VSS-1) at 37 degrees C correlated with the maximum H-2 yield for the LA treated flocculated cultures (1.69 +/- 0.18 mol mol(-1) glucose). The microbial diversity data clearly showed that the low H-2 yield in the granular cultures was due to the lower proportion of H-2 producers. A principle component analysis (PCA) revealed that the LA treated flocculated and granular cultures were grouped together and showed more diversity in comparison to other pretreatment methods. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.