The key factors influencing a co-digestion of the oil palm trunk (OPT) hydrolysate with a slaughterhouse wastewater (SHW) to produce hydrogen by Thermoanaerobacterium thermosaccharolyticum KKU19 were investigated. The OPT hydrolysate was obtained by the hydrolysis of OPT by microwave-H2SO4 method using 1.56% (w/v) H2SO4 and 7.50 min reaction time at 450 W. The Plackett Burman method was used to screen the key factors that influenced the hydrogen production potential (P-s). Results indicated that initial cell concentration, tCOD/TN (total COD/total nitrogen) ratio and CuSO4 concentration influenced the P-s. These factors were further optimized using response surface methodology (RSM) with central composite design (CCD). A maximum P-s of 2604 +/- 86 mL H-2/L substrate was achieved at an initial cell concentration of 224 mg dry cell/L, tCOD/TN ratio of 49.87 and CuSO4 concentration of 13.33 mg/L. The main soluble metabolite products were butyric and acetic acids. The P, obtained when the hydrolysate was supplemented with SHW (2604mL 86 mL H-2/L substrate) was comparable to the P, obtained when it was supplemented with yeast extract at the same tCOD/TN (2802 +/- 87 mL H-2/L substrate). This result suggests that SHW can be used to replace the costly nitrogen source. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.