# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 4 Bacteria Used for Hydrogen Production During Dark Fermentation
### Clostridium
#### 4.1.1. Clostridium butyricum  
C. butyricum was first isolated from pig intestines by [PERSON] in 1880 [66]. The substrates used by this species to produce H\({}_{2}\) include glucose, fructose, xylose, starch, and glycerol. In addition, complex organic compounds (biomass and organic waste) can be used. Certain strains of C. butyricum are tolerant to alkaline [67] and phenol [68]. Table 1 presents several hydrogen-producing C. butyricum strains used for H\({}_{2}\) production under different conditions.  
C. butyricum bacteria are widely used for H\({}_{2}\) production in pure culture as well as in mixed culture conditions. The studies indicated in Table 1 show that these bacteria are known to utilize a wide range of substrates, including glucose, sucrose, xylose, and even complex biomasses, and can work very efficiently for H\({}_{2}\) production around pH 7 and temperature of 30-37 \({}^{\circ}\)C in both batch and continuous mode of operation. C. butyricum TM-9A, when used with sugar molasses as a substrate at 37 \({}^{\circ}\)C and pH 7.5 for 24 h, produced  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Strain** & \begin{tabular}{c} **Substrate** \\ **(Concentration, g/L)** \\ \end{tabular} & \begin{tabular}{c} **Conditions** \\ **T (C)** \\ \end{tabular} & \begin{tabular}{c} **Operation Mode** \\ **(Time (h))** \\ \end{tabular} & \begin{tabular}{c} **H\({}_{2}\) Yield as Mol** \\ **(Reactor Volume, mL)** \\ \end{tabular} & \begin{tabular}{c} **Ref.** \\ **H\({}_{2}\)/mol Substrate \({}^{*}\)** \\ \end{tabular} \\ \hline DSM2478, & Glucose (3) & 37 & 6.5 & & & & & & \\ N\({}_{2}\) & & & & & & & & \\ TM-9A & Glucose (10) & 37 & 8.0 & 24 & 10.1 kPa & Batch (67) & 3.10 & [67] \\ RAK25832 & Glucose (10) & 30 & 8.0 & & & & & & \\  & & & & & & & & \\  & & & & & & & & \\ SP4 & \
\begin{tabular}{c} Hexose and pentose \\ (5) \\ \end{tabular} & 30 & & & & & & & & & \\  & & & & & & & & \\  & & & & & & & & \\ TM-9A & Sugarcane molasses & 37 & 7.5 & 24 & Serum bottles & Batch (120) &
\begin{tabular}{c} 3.34 (synthetic \\ analytical grade \\ glucose) \\ \end{tabular} \\ Synthetic food waste & & & & & & & & & & \\ TSTR 1032 & with volatile solids & 37 & 6.0 & & & 100 rpm & Semi-batch (5000) & 0.02 (volatile solids) & [72] \\ (28) & & & & & & & & \\  & Fruit waste with & & & & & & & \\  & & & & & & & \\  & & & & & & & & \\ COS2 & Glucose (5) & 37 & 6.8 & & & & & \\ CCT 7470 & Cellulose and glucose & 37 & & & & Cellulose & Batch (250) & 0.19 (cellulose), & [75] \\  & & & & & & & & \\  & & & & & & & & 0.58 (glucose) & \\  & & & & & & & \\ INET1 & Glucose (10) & 35 & 7.0 & & & & & \\  & & & & & & & & \\  & & & & & & & & \\  & & & & & & & & \\ NH-02 & & & & & & & & \\ (MT229351) & Malrose (10) & 30 & 7.0 & 23 & & & & & \\  & & & & & & & & \\ CWBI1009 & Glucose (5) & & & & & & & & \\ CWBI1009 & Glucose (5) & 30 & 7.3 & & & & & & \\  & & & & &  
a maximum of 3.34 mol H\({}_{2}\)/mol synthetic analytical-grade glucose. Therefore, these species are widely used in fermentation systems due to their high hydrogen-producing and substrate-degrading abilities.