# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 4 Bacteria Used for Hydrogen Production During Dark Fermentation
### Clostridium
#### 4.1.4 Clostridium thermocellum  
_C. thermocellum_ is a thermophilic anaerobe first isolated in 1926. This bacterium is known to grow at 60-65 \({}^{\circ}\)C and pH 6.5-7 and to form cellulosomes on its cytomembrane. _C. thermocellum_ is attracting attention for H\({}_{2}\) production as it can convert cellulose and hemicellulose directly into ethanol and H\({}_{2}\)[111]. This bacterium is widely used for H\({}_{2}\) production from lignocellulosic biomass and paper waste [112]. In a study to determine the cellulose-degrading ability of these bacteria, cellobiose was used at three different substrate concentrations. When the substrate concentration was low at 0.1-4.5 g/L, H\({}_{2}\) yields of 1-1.5 mol H\({}_{2}\)/mol glucose were obtained at 60 \({}^{\circ}\)C, pH 7.3 in batch mode with  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Strain**} & \multicolumn{2}{c}{**Substrate**} & \multirow{2}{*}{**Conditions**} & \multicolumn{2}{c}{**Operation Mode**} & \multicolumn{2}{c}{**H\({}_{2}\) Yield as Mol**} \\  & **(Concentration, g/L)** & & & & **(Reactor Volume, mL)** & **H\({}_{2}\)/mol Substrate** & **Ref.** \\ \hline \multirow{5}{*}{CH5} & Glucose (0.12) & 30 & 7.0 & 60 & \multirow{5}{*}{120 rpm} & \multirow{5}{*}{Batch (120)} & \multirow{5}{*}{1.61} & \multirow{5}{*}{1.61} \\  & Glucose & 35 & 7.0 & & & 120 rpm & & 1.61 \\ \cline{1-1} \cline{6-7} MTCC16 & Glucose (54.18) & 37 & 6.6 & 96 & 10\% & 1/\(\circ\) in vacuum, & \\ \cline{1-1} \cline{6-7}  & Glucose & & & & 192 rpm & & & 3.60-10\({}^{-3}\) \\ \cline{1-1} \cline{6-7}  & Glucose & 37 & & & 5\% & 1/\(\circ\) in vacuum, & \\ \cline{1-1} \cline{6-7} DSM52 & Glucose & 37 & & & & ferrylhydrite & & \\ \cline{1-1} \cline{6-7}  & Glycerol (7.4) & 36 & 6.7 & & 10\% & 1/\(\circ\) in vacuum, & \\ \cline{1-1} \cline{6-7} MTCC 116 & Glycerol (7.4) & 36 & 6.7 & & 10\% & 1/\(\circ\) in vacuum, & \\ \cline{1-1} \cline{6-7}  & Glycerol (7.4) & 36 & 6.7 & & flushing with N\({}_{2}\), & & (100) & \\ \cline{1-1} \cline{6-7}  & & & & & 150 rpm & & & \\ \cline{1-1} \cline{6-7}  & & & & & & & & \\ \cline{1-1} \cline{6-7} CH5 & Xylose (40) & 35 & 6.8 & & & & & \\ \cline{1-1} \cline{6-7}  & & & & & & & & \\ \cline{1-1} \cline{6-7}  & & & & & & & & \\ \cline{1-1} \cline{6-7}  & & & & & & & & \\ \cline{1-1} \cline{6-7} CH5 & Xylose (40) & 35 & & & & & & \\ \cline{1-1} \cline{6-7}  & & & & & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 3: Hydrogen production by different _Clostridium pasteurianum_ strains.  
10 mL working volume [113]. Table 4 shows a list of _C. thermocell_ strains with their H\({}_{2}\) production ability using different substrates and operational conditions.  
Studies have shown that these anaerobic bacteria are very capable of degrading cellulose. Most of the studies have used a temperature of either 55 or 60 \({}^{\circ}\)C, which is optimal for the activity of _C. thermocellum_. The most commonly used strains include _C. thermocellum_ ATCC 27405 and DSM1313. The maximum H\({}_{2}\) production of 5.87 mol H\({}_{2}\)/mol hexose was obtained with 3 g/L of cellulose at 55 \({}^{\circ}\)C, 168 h, 20 mM CaCO\({}_{3}\), and 150 rpm, indicating the influence of CaCO\({}_{3}\) in obtaining higher H\({}_{2}\) yields. In addition, it was observed that these bacteria have been used at higher HRTs, indicating that their metabolic activity and growth can be maintained for longer periods compared to other species to produce H\({}_{2}\).