Hydrogen is recognized as a clean fuel and useful substance; however, its biological production is still in the early stages of development. Today, the main application of hydrogen is to produce ammonia as an agricultural fertilizer. Agricultural residues are considered as one of the main sources of biomass that are usable for biological hydrogen production. In this study, the biological hydrogen production from organosolv pretreated rice straw using Enterobacter aerogenes as a facultative anaerobic bacterium was evaluated. The experimental design was conducted based on the operational parameters including ethanol concentration, temperature and time to achieve the best efficiency of hydrogen production using Design expert software and a central composite design (CCD) and the best fitted model with experimental data was obtained by ANOVA analysis. The operating conditions at a temperature of 180 ° C, concentration of 45% ethanol and 30 minutes were determined as the optimum pretreatment. For this operation, the yield was calculated to be 19.73 ml of hydrogen per each gram of straw. The effect of pretreatment severity on the solid and lignin recovery, and hydrogen yield was analyzed. By increasing the pretreatment severity, the solid recovery decreased and the lignin recovery increased; however, no significant trend was observed for the hydrogen yield at the different pretreatment severity that shows the considerable effect of ethanol concentration. The composition of the native and treated straw at various operating conditions was compared. The structure of straw was studied by FTIR analysis and scanning electron microscope images. Crystallinity index increased by enhancement in the concentration of ethanol. Although the crystallinity index increased at higher pretreatment severities, the surface area accessible to enzyme and the porosity of treated straw were improved. The influence of concentration of straw on the hydrogen production was investigated. By increasing the concentration of straw, the hydrogen amount and the ratio of carbon dioxide to hydrogen increased and the hydrogen yield decreased, which was in conformity to the experiment with glucose substrate. A reduction in the straw concentration from 50 to 3.33 g/l led to an increase in the hydrogen yield to 108.17 ml/gr. The higher concentration of substrate results in production of other metabolites. The inhibitory effect of ethanol produced by this microorganism was studied. Finally, based on the laboratory results of this study, an algorithm was proposed to estimate the large-scale hydrogen production and its use for the production of nitrogen fertilizer (ammonia) from 2013 to 2050. Based on the amount of rice produced in 2013, for the yield of 1.43 kg H 2 /tonne straw, the potential quantity of hydrogen was estimated to be 1.59 thousand tonnes that after conversion to ammonia, the nitrogen requirement for 12.5% ??of the rice cultivation area in Iran could be provided. This