# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 6 Discussion
### C. beijerinckii
beijerincli_ in batch mode showed a maximum H\({}_{2}\) yield of 9.39 mol H\({}_{2}\)/mol sucrose when 7.5 g/L of sucrose was used at 37 \({}^{\circ}\)C and pH 6-7 [93], followed by dextrose. When using 3 g/L of dextrose with the same strain, it produced 3.58 mol H\({}_{2}\)/mol dextrose in batch mode at 37 \({}^{\circ}\)C and 120 rpm [52]. Glucose resulted in an H\({}_{2}\) productivity of 2.7 \(\pm\) 0.2 mol/mol glucose at 37 \({}^{\circ}\)C and 100 rpm with the _C. beijerincli_ strain DSM 1820. The _C. beijerincli_ NICIMB-8052 strain produced an H\({}_{2}\) yield of 2.47 mol H\({}_{2}\)/mol glucose using 10% \(v/v\) inoculum, at pH 6.5, 32 \(\pm\) 1 \({}^{\circ}\)C, 120 rpm, 120 h, and light illumination [24]. When using a mixture of glucose and xylose as the substrate at 30 \({}^{\circ}\)C, the same strain produced a lower H\({}_{2}\) yield of 0.086 mol H\({}_{2}\)/mol substrate [95], indicating that the use of glucose alone as the substrate provides higher H\({}_{2}\) yields. _C. pasteurianum_ was analyzed using glucose, glycerol, and xylose as substrates. The DSM525 strain produced the highest H\({}_{2}\) yield of 3.55 mol H\({}_{2}\)/mol using glucose as substrate at 37 \({}^{\circ}\)C in batch mode [106]. When _C. pasteurianum_ MTCC 116 was used with glycerol as the substrate at 37 \({}^{\circ}\)C, pH 6.7, and 150 rpm, following the acetate pathway for metabolism, a maximum H\({}_{2}\) yield of 3 mol H\({}_{2}\)/mol glycerol [108]. In contrast, when the same strain was used with glucose as the substrate at 37 \({}^{\circ}\)C, pH 6.6, and 192 rpm, the H\({}_{2}\) yield decreased to 0.0036 mol H\({}_{2}\)/mol glucose, indicating that glycerol is a better substrate for H\({}_{2}\) production by this strain. _C. pasteurianum_ CH5 was studied with xylose and glucose. When xylose was used as the substrate, a maximum H\({}_{2}\) yield of 1.46 mol H\({}_{2}\)/mol xylose was obtained at 35 \({}^{\circ}\)C and 120 rpm [110], whereas a higher H\({}_{2}\) yield (1.61 mol H\({}_{2}\)/mol glucose) was achieved using glucose at 35 \({}^{\circ}\)C, pH 7, and 120 rpm [104], indicating glucose as a better substrate for H\({}_{2}\) production by this strain. Finally, _C. thermocellular_ was also considered for the production of H\({}_{2}\). The DSM 1313 strain of _C. thermocellular_ used cellulose (Avicel) as a substrate. In continuous mode at 60 \({}^{\circ}\)C, pH 7, with argon flushing and 60 rpm, it produced 0.76-1.21 mol H\({}_{2}\)/mol hexose [115], whereas when the studies were performed in a batch mode at 55 \({}^{\circ}\)C, with 8 days of incubation and 150 rpm agitation, 1.15 mol H\({}_{2}\)/mol hexose was produced [118]. The highest H\({}_{2}\) production rate of 1.92 mol H\({}_{2}\)/mol hexose was observed with _C. thermocell_ strain 27405, using 5 g/L of cellulose in batch mode at 55 \({}^{\circ}\)C, pH 7, flushed with N\({}_{2}\), 150 rpm for 168 h [116]. Various conditions, such as an optimum temperature of 55 \({}^{\circ}\)C and 150 rpm agitation, were used in all studies, proving this strain to be efficient for the production of H\({}_{2}\). When 5 g/L of sucrose was used as a substrate with an HRT of 168 h,the H\({}_{2}\) production rate was 1.92 mol H\({}_{2}\)/mol hexose [116], whereas when the substrate concentration was increased to 18 g/L with an HRT of 10 days, 11.19 mol H\({}_{2}\)/mol cellulose was produced [160], indicating the effect of substrate and its concentration, as well as HRT, on H\({}_{2}\) yields. _Clostridium_ spp. has shown great potential, as most of the dark fermentation studies have used strains from these species to produce H\({}_{2}\). In addition to these bacteria, some other facultative anaerobic bacteria have shown the potential to produce H\({}_{2}\) through dark fermentation. _E. aerogenes_, _K. pneumoniae_, and _E. coli_ are facultative anaerobes used to produce H\({}_{2}\) via dark fermentation. _E. aerogenes_ requires the addition of nanoparticles to increase H\({}_{2}\) production.