Rice straw, a low-cost lignocellulosic biomass was used as feedstock for thermophilic hydrogen fermentation by Thermotoga neapolitana. Hydrogen production, the growth and cellulose digestibility of the hyperthermophile in batch mode from untreated as well as chemically pretreated (ammonia and dilute sulfuric acid) Korean rice straws were investigated. Pretreatment method using combination of 10% ammonia and 1.0% dilute sulfuric acid was developed to increase the digestibility of rice straw for the hyperthermophilic H(2) fermentation and to decrease the time consumption. In a typical fermentation using raw rice straw, 29% of the substrate was digested and 2.3 mmol H(2)/g straw of hydrogen yield was consistently obtained. Compared with the pretreatments using only ammonia or dilute sulfuric acid, the combined pretreatment method using both chemical agents significantly increases the digestibility of rice straw with 85.4% of substrate consumption. H(2) production on rice straw from this combined pretreatment showed the highest yield (2.7 mmol H(2)/g straw) and the highest sugar conversions (72.9% of glucose and 95.7% of xylose). (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.