# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 6 Discussion
### C. beijerinckii
The maximum H\({}_{2}\) yield of 2.6 mol H\({}_{2}\)/mol sugar was observed with fermentable sugar as the substrate at 37 \({}^{\circ}\)C for 72 h, along with the addition of ferric oxide nanoparticles [120]. This was followed by _E. aerogenes_ strain ATCC13408 using glucose as a substrate at 37 \({}^{\circ}\)C, pH 6, 220 rpm, and ferric oxide nanoparticles, which yielded H\({}_{2}\) in the range of 1.32-1.55 mol H\({}_{2}\)/mol glucose [123]. In the case of _K. pneumoniae_, the ECU-15 and TR17 strains were mainly used to produce H\({}_{2}\). The ECU-15 strain, when used with a mixture of glucose, xylose, and cellobiose at 150 rpm in batch mode, produced up to 2.07 mol H\({}_{2}\)/mol substrate [128], which was the maximum H\({}_{2}\) produced by this strain. When the same strain was used with only glucose as the substrate at 37 \({}^{\circ}\)C, pH 6, and 150 rpm, it produced 1.22 mol H\({}_{2}\)/mol glucose [130], indicating that the mixture of glucose, xylose, and cellobiose is a better substrate for high yields of H\({}_{2}\). In another analysis with _K. pneumoniae_ strain TR17, lower H\({}_{2}\) yields were observed with crude glycerol. At 40 \({}^{\circ}\)C, pH 8.0 for 24 h in batch mode, 0.25 mol H\({}_{2}\)/mol glycerol was produced [132]. In a similar study with crude glycerol at 40 \({}^{\circ}\)C and pH 8 in batch mode, an H\({}_{2}\) yield of 0.26 mol H\({}_{2}\)/mol glycerol was observed [134], but when an up-flow anaerobic sludge blanket reactor was used with strain TR17 and glycerol as the substrate at 40 \({}^{\circ}\)C, pH 8 for 12 h in batch mode, an H\({}_{2}\) yield of 4.08 mol H\({}_{2}\)/mol glycerol was observed [135], indicating that this reactor is optimal for higher H\({}_{2}\) yield with this strain of _K. pneumoniae_. Finally, in the analysis of _E. coli_, a maximum H\({}_{2}\) production of 2.82 mol H\({}_{2}\)/mol glucose was observed with strain WDH-LF, using glucose as a substrate at 31 \({}^{\circ}\)C, pH 8.2, and 400 rpm [148]. For the _E. coli_ strain W3110, hemicellulose hydrolysates were found to be a better substrate than acetate. When hemicellulose hydrolysates were used at 31 \({}^{\circ}\)C, pH 8.2, and 200 rpm, H\({}_{2}\) production of 1.15-1.73 mol H\({}_{2}\)/mol substrate was obtained [142], but when acetate was used as the substrate at 33 \({}^{\circ}\)C and pH 6.3, H\({}_{2}\) production of 0.21 mol H\({}_{2}\)/mol acetate was achieved [144]. When _E. coli_ strain WDHL was analyzed with different substrates, the highest H\({}_{2}\) production of 1.12 mol H\({}_{2}\)/mol was observed with galactose at 37 \({}^{\circ}\)C, pH 6, and 175 rpm, followed by glucose, which produced 0.30 mol H\({}_{2}\)/mol glucose produced under the same conditions, proving that galactose is a better substrate for this strain [146]. This comparative analysis allowed for the determination of the most suitable bacterial strain and the advantages and disadvantages of using each strain for dark fermentation studies to produce H\({}_{2}\) under specific conditions available in the particular laboratory or industry at that particular time of the year since temperature plays a major role in H\({}_{2}\) production efficiency, along with other conditions, such as bioreactor design and material, type of bioprocess, and the substrate used.