Fossil fuels as primary sources of energy have been shown various disasters during last decades including dominant source of air pollution and greenhouse gas emission and global climate change, and therefore negative impacts on human and ecosystem. Hydrogen with the highest energy density and as a clean without any pollutant generation has been considered as a proper alternative of energy. Biomass is the most abundant renewable energy resource in the world. Rice straw with 600-900 million tons annually is one of the abundant agricultural biomass residues in the world that can be used to produce hydrogen by the biological process. In this study, rice straw was collected from a farm located in Lenjan, Isfahan, Iran. Determination of structural carbohydrates and lignin showed that this rice straw was contained 40.51, 20.26, 7.33 % (w/w) glucan, xylan, and lignin, respectively. It was ball-milled and screened by 20 and 80 meshes, then hydrolyzed using two different dilute-sulfuric acid stages. In the first hydrolysis stage, 10 g rice straw was mixed with 100 ml sulfuric acid (1% w/v) at 120 °C for 30 min. Afterward, the solid residues were used for the second hydrolysis stage with 0.5% sulfuric acid at 180 °C for 30 min. The results show the ability of first stage hydrolysis to depolymerize xylan to xylose with a yield of 58.49% and second stage hydrolysis with a yield of 19.28% for glucose. The reduced sugars from two hydrolysis stages were fed to the continuous biofilm fixed bed reactor for the production of biohydrogen via dark fermentation. The microbial mixture used for biofilm formation was anaerobic sludge from north-east sewage treatment plant of Isfahan, which was pre-treated in a thermal shock at 90 °C for 30 min and acid shock at pH 4.8 for 12 h. The effect of three different HRTs (17, 8.2, 2 h) on hydrogen production rate and hydrogen yield was investigated at pH 5.3 and 37°C. Reduction of HRT resulted in the reduction of methane percentage on total biogas. Further by shifting from high to low HRTs, output COD was decreased (from 3119.9 to 2972.9 mg / L). On the other hand, acetic acid and butyric acid production reduction were slower than other soluble metabolites like ethanol. The highest gas production rate of 0.252 L H 2 / L.h and hydrogen yield of 1 mol H 2 / mol sugar were reported at 2 h HTR. Keywords: Biohydrogen, Rice straw, Dark fermentation, Bioreactor, Continues reactor, Biofilm reactor, Hydraulic retention time.
