Treatment of anaerobic granules with heat and two chemical treatments, contacting with 2-bromoethane-sulfonate (BES) and with BES+Chloroform, were applied to suppress hydrogen-consuming microorganisms. Three mesophilic expanded granular sludge bed (EGSB) reactors-R-Heat, R-BES, and RBES+Chlo-were inoculated with the treated sludges and fed with synthetic sugar-based wastewater (5 g(COD) L-1, HRT 20-12 h). Morphological integrity of granules and bacterial communities were assessed by quantitative image analysis and 16S rRNA gene based techniques, respectively. Hydrogen production in R-Heat was under 300 mL H-2 L-1 day(-1), with a transient peak of 1,000 mL H-2 L-1 day(-1) after decreasing HRT. In RBES+Chlo hydrogen production rate did not exceed 300 mL H-2 L-1 day(-1) and there was granule fragmentation, release of free filaments from aggregates, and decrease of granule density. In R-BES, there was an initial period with unstable hydrogen production, but a pulse of BES triggered its production rate to 700 +/-200 mL H-2 L-1 day(-1). This strategy did not affect granules structure significantly. Bacteria branching within Clostridiaceae and Ruminococcaceae were present in this sludge. This work demonstrates that, methods applied to suppress H-2-consuming microorganisms can cause changes in the macro-and microstructure of granular sludge, which can be incompatible with the operation of high-rate reactors. Biotechnol. Bioeng. 2011;108: 1766-1775. (C) 2011 Wiley Periodicals, Inc.