Acid treated rice straw hydrolysate was used as a substrate hydrogen production. Concentrated sulphuric acid (55%) was used to completely dissolve the rice straw cellulose at temperature of 40 degrees C. After hydrolysis of rice straw cellulose, calcium hydroxide was used to remove sulphate ions. Biohydrogen production was evaluated under different initial pH and substrate concentration conditions at a temperature of 37 degrees C in a batch mode. The maximum values of hydrogen yield and accumulation of hydrogen were 0.44 mol H-2/mol T-sugar and 97.30 +/- 0.17 mL with substrate utilization of 81.55% in a batch mode. A mixture of food industry wastewater with rice straw hydrolysate was used as substrate in a continuous mode for the biohydrogen production potential test in this study. The average hydrogen production yield of 0.69 mol H-2/mol T-sugar was 1.5 times higher than that in the batch mode while the hydrogen production rate was 10 +/- 1.17 L/d/L. The presence of bacteria Clostridium pasteurianum which favours the production of hydrogen from of acetic acid and butyric acid was determined by DGGE. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.