The pretreated cornstalk (CS) was employed for biohydrogen production through combining dark-fermentative bacteria, cow dung, and photosynthetic bacteria, Rhodobacter capsulatus mutant in this work. In the first step, the cornstalk was pretreated with 0.75% NaOH, 12 IU/g-CS cellulase, and 2400 IU/g-CS hemicellulase under different hydrolysis time and temperature. The reducing sugar yields were very close under different conditions, and its maximum was 0.51 +/- 0.01 g/g-CS at hydrolysis 108 degrees C for 2.0hours. However, the H-2 yield of dark fermentation enhanced with the increasing hydrolysis time and temperature, and the maximum was 156.7 +/- 8.6 mL/g-CS at hydrolysis 126 degrees C for 2.0hours. The biogas was only H-2 and CO2, and no methane was found. In the second step, R. capsulatus ZYHAB3 with the mutation on both hvrA and hupAB genes was obtained from wild type (R. capsulatus SB1003) and the inactivation of both hvrA and hupAB genes remarkably enhanced nitrogenase activity. When the dark-fermentation effluent was employed as a substrate, the H-2 yield and maximum H-2 evolution rate of ZYHAB3 were 2827.5 +/- 283.5 mL/L and 40.9 +/- 2.3 mL/(Lh), which increased by 44.5 and 39.1% compared with those of wild type, respectively. The high H-2 yield of 439.4 mL/g-CS was obtained from pretreated cornstalk through the 2-step process, and the chemical oxygen demand removal rate achieved 90.6%. The results suggest that combining cow dung and R. capsulatus mutant (hvrA(-) hupAB(-)) could be a promising way to produce H-2 form agricultural wastes.