In this process, metabolically engineered microorganisms such as bacteria convert ethanol to hydrogen under the facilitation of hydrogenase enzymes which are metalloproteins, containing complicated metal active centres that catalyze the interconversion of protons and electrons with dihydrogen. According to literature reporting [3-5], two major classes of hydrogenases are recognized based on their metal active sites: [FeFe] and [NiFe]. Depending on whether light will be involved, this biological hydrogen production process can be simply classified as photo — and dark-fermentation processes [6].

During the photo-fermentation process, the hydrogenase enzyme synthesized and activated under dark anaerobic condition is used to convert ethanol to biohydrogen under light anaerobic condition. Since the light acts as the energy source, the consumption rate of substrate is less than that required for dark fermentation. However, the hydrogen efficiency will be dramatically reduced in the presence of oxygen concurrently produced through photosynthesis by bacteria, which has been evidenced by many researchers [7]. Furthermore, the ultra-violet wavelength radiation requirement and relatively slower production rate limit its industrial application at large scale.

Under the dark operation environment, there is no risk for hydrogenases exposed to oxygen, which makes the hydrogenase enzymes remain active throughout the whole process, leading to more efficient hydrogen production. Compared to photo-fermentation, the inherent continuous and fast production feature makes dark anaerobic digestion economically promising for industrial scale practice. In recent years, many publications have reported their efforts spent on optimization of operation parameters, development of genetically modified microorganism, metabolic engineering, improvement of reactor designs, use of different solid matrices for cell immobilization, etc. to maximize hydrogen yield. Among many considerations, the blockage of methanogenesis in the anaerobic pathway is crucial to improve hydrogen selectivity through the inhibition of methane formation.