# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 6 Discussion
### C. beijerinckii  
at the highest H\({}_{2}\) production yield, 9.39 mol H\({}_{2}\)/mol sucrose, at 37 \({}^{\circ}\)C and pH 6-7 [93], demonstrating that this species is very efficient for H\({}_{2}\) production. _Chemocellum_ strain 27,405 follows _C. beijerinckii_ with an H\({}_{2}\) production yield of 5.87 mol H\({}_{2}\)/mol hexose, using cellulose as a substrate, at 55 \({}^{\circ}\)C for 168 h, with the addition of 20 mM CaCO\({}_{3}\)[119]. _C. pasteurium_ DSM525 achieved a maximum H\({}_{2}\) production of \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Bacteria** & \multicolumn{2}{c}{**Substrate**} & \multicolumn{2}{c}{**Conditions**} & \multicolumn{2}{c}{**Operation Mode**} & \multicolumn{2}{c}{**H\({}_{2}\) Yield as Mol**} & \multicolumn{2}{c}{**Ref.**} \\ \hline  & & \multicolumn{2}{c}{**T**} & \multicolumn{2}{c}{**} & \multicolumn{2}{c}{**} & \multicolumn{2}{c}{**} & \multicolumn{2}{c}{**} \\  & & \multicolumn{2}{c}{**C**O**} & \multicolumn{2}{c}{**pH**} & \multicolumn{2}{c}{**Time (h)**} & \multicolumn{2}{c}{**More Details**} & & \\ \hline _C. butylicum_ & & & & & & & & 3.34 (synthetic & \\ TM-9A & & & & & & & & & \\ _C. beijerinckii_ & & Sucrose (7) & 37 & 6.0–7.0 & & 1 mL inoculum & & & \\ _C._ & & & & & & & & \\ _C._ & & & & & & & & \\ _pasteurium_ & Glucose & 37 & & & & & & \\ DSM525 & & & & & & & & \\ _C. thermocellum_ & & & & & & & & \\ ATCC 27405 & Cellulose (3) & 55 & & 168 & & & & \\ _E. aerogenes_ & & Fermentable sugar & 37 & & 72 & & & \\ _Kkbsiella_ & & & & & & & & \\ species TR17 & & Glycerol (10) & 40 & 8.0 & 12 & & & \\ _E. coli_ & & & & & & & & \\ WDH-LF & & Glucose (16) & 31 & 8.2 & & 400 rpm & Batch (10,000) & 2.82 & [148] \\ \hline \hline \end{tabular}
\end{table} 
Table 9: Maximum hydrogen production by different species. 3.55 mol H\({}_{2}\)/mol glucose at 37 \({}^{\circ}\)C [106], whereas _C. butyricum_ TM-9A attained 3.34 mol H\({}_{2}\)/mol synthetic analytical-grade glucose, using sugarcane molasses, also at 37 \({}^{\circ}\)C [71]. _E. aerogenes_, using fermentable sugar, had a maximum H\({}_{2}\) production of 2.6 mol H\({}_{2}\)/mol sugar at 37 \({}^{\circ}\)C with an HRT of 172 h [122]. The only drawback observed with this species was the need for the addition of nanoparticles in all studies. _Klebsiella_ spp. TR17 provided a maximum H\({}_{2}\) production of 4.08 mol H\({}_{2}\)/mol using glycerol at 40 \({}^{\circ}\)C and 12 h HRT [122], whereas the _E. coli_ WDH-LF strain produced up to 2.82 mol H\({}_{2}\)/mol glucose [148]. These results allowed the identification of the most suitable strain from each species. In addition, the analysis of each bacterial species identified several conditions and factors essential to obtain maximum H\({}_{2}\) production. In a study with _C. butyricum_ TM-9A, using sugarcane molasses as the substrate at 37 \({}^{\circ}\)C, pH 7.5, and an HRT of 24, 3.335 mol H\({}_{2}\)/mol synthetic analytical-grade glucose was produced [71]. However, when the same strain with 10 g/L of glucose as the substrate at 37 \({}^{\circ}\)C at a pH 8, it produced 3.1 mol H\({}_{2}\)/mol glucose [67], indicating no significant differences between the results. Another strain of _C. butyricum_, TISTR 1032, using sucrose at a pH of 6.5 in a batch mode, produced 1.34 mol H\({}_{2}\)/mol hexose [81]. However, when sugarcane juice was used as the substrate at 37 \({}^{\circ}\)C, pH 6, in continuous mode, only 1.0 mol H\({}_{2}\)/mol hexose was produced [85], confirming that pure sucrose is a better substrate for this strain to produce H\({}_{2}\). _C. butyricum_ CWBI1009 was studied using glucose monohydrate as the substrate under different operational conditions. When this strain was used in batch mode at 400 rpm and pH 7, 3.1 mol H\({}_{2}\)/mol glucose was obtained [78], while at 30 \({}^{\circ}\)C and pH 7.3, the production was 1.43 mol H\({}_{2}\)/mol glucose [76], proving that the latter conditions are better for higher H\({}_{2}\) production. The analysis with _C.