Background: Due to depletion of fossil fuel reserves, global warming, and the need for energy efficiency, the new energy resource is required. For this purpose, hydrogen can be considered as clean and efficient energy carrier. In this study, biohydrogen production from physical pretreated mixed culture was investigated. Materials and Methods: In this study, the optimal conditions for physical pretreatment of mixed culture were investigated for the enrichment of hydrogen-producing bacteria (HPB). Three individual pretreatment processes, including thermal, ultraviolet (UV) radiation, and ultrasonication (US) were conducted in batch tests. The batch tests were carried out via 500 mL glass flasks with working volume 400 mL. Glucose was used as sole substrate, and other essential nutrient and trace elements were added. To agitation of substrate with HPB inoculum, the magnet stirrer was used at 60 rpm. Results: The results showed the production of H2gas could be increased by appropriate pretreatment methods including thermal pretreatment at 100°C. At 100°C as a pretreatment, the volume of H2gas was 112.25 mL and followed by US (97.46 mL), UV (89.11 mL), 80°C (72.72 mL), 90°C (53.95 mL), and 70°C (26.01 mL). In the pretreatment methods, the solution pH and effluent soluble chemical oxygen demand was 6.3–6.7 and 5100–8300 mg/L, respectively. Conclusions: The Gompertz model depicts that all R2 values were more than 0.93, indicating that the fitted curves were best fitted with the experimental points.
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