This study aims to produce hydrogen from sludge of poultry slaughterhouse wastewater treatment plant (5% total solid) by anaerobic batch fermentation with Enterobactor aerogenes or mixed cultures from hot spring sediment as the inoculums. Sludge was heated in microwave at 850 W for 3 min. Results indicated that a soluble chemical oxygen demand (sCOD) of pretreated sludge was higher than that of raw sludge. Pretreated sludge inoculated with E. aerogenes and supplemented with the Endo nutrient had a higher hydrogen yield (12.77 mL H(2)/g tCOD) than the raw sludge (0.18 mL H(2)/g tCOD). When considered the hydrogen yield, the optimum initial pH for hydrogen production from microwave pretreated sludge was 5.5 giving the maximum value of 12.77 mL H(2)/g tCOD. However, when considered the hydrogen production rate (R(m)), the optimum pH for hydrogen production would be 9.0 with the maximum R(m) of 22.80 mL H(2)/L sludge.h. Copyright (C) 2010, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.