*2.2. Dark Fermentation*

Dark fermentation (DF) is a process involving fermentation by dark-adapted microbes of carbohydrates under anoxic conditions to give H2 as a resulting product along with acids, as illustrated in Figure 2 [28]. Studies have shown that *Clostridium* spp. can use a wide variety of sugars, making it a possible candidate to form H2 from wastewater at a large scale [31]. The lowered pH due to acid output hampers the generation of H2.

**Figure 2.** Diagrammatic representation of dark and photo fermentation (hybrid process).

A major economic emphasis is on biological H2 production from wastewater using dark fermentation. The H2 production system has many similarities with methanogenic anaerobic digestion, notably, the two gaseous compounds that can be separated from treated wastewater. The mixed microbial population present in both bioprocesses have the same properties but exhibit one major difference in biological H2 production: bacteria such as homo-acetogens and methanogens inhibit hydrogen formation. To destroy these microbes, heat treatment is required without affecting spore-forming fermenting bacteria. Other approaches include high dilution rates or low pH for higher activity of the reactor and optimized operating environmental conditions to maximize the output of hydrogen. Unfortunately, the production of bio-H2 depends on a comparatively limited volume of the overall equal H2 present in wastewater. We cannot estimate recovery efficiency in high-carbohydrate wastewater to surpass 15% of the electron equivalent, under optimized conditions. Therefore, various researchers have performed two-step processes that involve the development of bio-H2 via methanogenic anaerobic digestion to increase the overall energy output of the process. Methanogenic anaerobic digestion, as described later, is a useful and convenient process. Another promising technique is the conversion of methane

to H2 via a catalytic method. Therefore, H2 production through the direct method is somewhat limited to the pretreatment step in large-scale energy production, while the discharge of H2 gas through plastic enclosures and thin metal sheets represents another limitation because of the high diffuse rate of H2 [32]. To increase the overall hydrogen yield, hybrid processes are also suggested, whereby organic acids produced in dark fermentation can be used as a feedstock in photo fermentation to produce hydrogen (Figure 2).
