Massive utilization of petroleum and natural gas caused fossil fuel shortages. Consequently, a large amount of carbon dioxide and other pollutants are produced and induced environmental impact. Hydrogen is considered a clean and alternative energy source. It contains relatively high amount of energy compared with other fuels and by-product is water. In this study, the combination of ultrasonic mechanical and biological effects is utilized to increase biohydrogen production from dark fermentation bacteria. The hydrogen production is affected by many process conditions. For obtaining the optimal result, experimental design is planned using the Taguchi Method. Four controlling factors, the ultrasonic frequency, energy, exposure time, and starch concentration, are considered to calculate the highest hydrogen production by the Taguchi Method. Under the best operating conditions, the biohydrogen production efficiency of dark fermentation increases by 19.11%. Results have shown that the combination of ultrasound and biological reactors for dark fermentation hydrogen production outperforms the traditional biohydrogen production method. The ultrasonic mechanical effects in this research always own different significances on biohydrogen production.
