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 <SUP>o</SUP>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 <SUP>o</SUP>C in a batch mode. The maximum values of hydrogen yield and accumulation of hydrogen were 0.44 mol H<SUB>2</SUB>/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<SUB>2</SUB>/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.