# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 4 Bacteria Used for Hydrogen Production During Dark Fermentation
### Clostridium
#### 4.1.3 Clostridium pasteurianum  
_C. pasteurianum_ follows a similar pathway to other _Clostridium_ spp. It is known as an iron-reducing bacterium used for biofuel production via fermentation. Discovered in 1890 by [PERSON], this bacterium can survive in mesophilic conditions and can fix atmospheric N\({}_{2}\). _C. pasteurianum_ can utilize various substrates [98], release various products, and show rigorous growth in basic media under non-sterile conditions. The C. pasteurianum strains DSM525, CH5, H4 (DSM 525), and MTCC 116 produce H\({}_{2}\) with greater \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Strain**} & \multicolumn{2}{c}{**Substrate**} & \multicolumn{2}{c}{**Operation Mode**} & \multicolumn{2}{c}{**H\({}_{2}\) yield as Mol**} & \multirow{2}{*}{**Ref.**} \\  & \multicolumn{2}{c}{**(Concentration, g/L)**} & & & & & **(Reactor Volume, mL)** & \multicolumn{1}{c}{**H\({}_{2}\)/mol Substrate**} & \\ \hline  & & **T** & & & & & \\  & & \(C\) & **pH** & **Time (h)** & **More Details** & & \\ \hline DSM 1820 & Glucose (2) & 37 & & & 10\% \(\pm\)/o, flushed with N\({}_{2}\), 100 rpm & Batch (180) & 2.70 \(\pm\) 0.20 & [24] \\ Not specified & Dextrose (3) & 37 & 7.5 & 96 & Flushed with N\({}_{2}\), 120 rpm & Batch & 3.58 & [52] \\ DSM 791 & Glycerol (11) & 37 & 7.5 & 96 & Batch (120) & 1.21 & [90] \\ G117 & Glycerol (20–80) & 39 & 6.8 & 12–72 & Purged with N\({}_{2}\), 150 rpm & Batch (120) & 1.18–1.45 & [91] \\  & & & & & & Cathodic electror- & & \\  & & & & & & & \\ NCIMB 8052 & Glucose (20) & 37 & 6.5 & & & & \\  & & & & & & 130 rpm, sprayed & & \\ ATCC 8260 & Sucrose (7.5) & 37 & 6.0– & & 1 mL inoculum & Batch (15) & 9.39 & [93] \\  & & & & & & Sparged with N\({}_{2}\), & \\ NCIMB-8052 & Glucose (3) & 32 & 6.5 & 120 & 10\% \(v/v\) inoculum, & Batch (60) & 2.47 & [94] \\  & & & & & & illumination & \\ NCIMB 8052 & Glucose\({}_{1}\)5 & 30 & & & & Fushed with N\({}_{2}\), & \\  & & & & & & 3\% \(v/v\) inoculum & Batch (26) & 0.09 & [95] \\  & & & & & & Fushed with N\({}_{2}\), & \\  & & & & & & & \\ RZF-118 & Glucose (9) & 35 & 7.0 & 20 & 8\% \(v/v\) inoculum, & Batch (100) & 1.97 & [96] \\  & & & & & & 140 rpm & \\ ATCC 8260 & Glucose (3) & 30 & 6.3 & & & 3\% \(v/v\) inoculum, & Batch (100) & 0.191 & [97] \\  & & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Hydrogen production by different _Clostridium beijerinckii_ strains. efficiency in the presence of iron oxides [99]. This bacterium is highly efficient in anaerobic H\({}_{2}\) production. Strict anaerobic bacteria of this species contain Fe-Fe hydrogenases in their cytoplasm, and when they use organic matter as a substrate, electrons are released. These electrons are used by the Fe-Fe hydrogenase to form H\({}_{2}\) with the concomitant reoxidation of reduced ferredoxin produced during degradation [100]. In a study, the effect of ZnFe\({}_{2}\)O\({}_{4}\) nanoparticles on H\({}_{2}\) production by _C. pasteurianum_ DSM 525 was investigated [101]. Optimal operational conditions were determined using the response surface method, which provided 2.14 mol H\({}_{2}\)/mol glucose, corresponding to a 116% increase in H\({}_{2}\) production compared to the control after 48 h of batch culture. The metabolic pathway for H\({}_{2}\) production shifted from the butyric acid type to the ethanol type. These bacteria were also used as a coculture with _Geobacter sulfurreducens_ in glucose fermentation [102]. This study showed that the maximum rate and yield of H\({}_{2}\) production in the coculture increased by 122.2% and 28.92%, respectively, compared to monoculture, using 50 mL serum vials at 37 \({}^{\circ}\)C, without agitation. Table 3 presents a list of _C. pasteurianum_ strains used to produce H\({}_{2}\) with different substrates and operational conditions. Most of these bacteria were used at 35-37 \({}^{\circ}\)C since they are mesophilic, with the maximum H\({}_{2}\) production rate observed at pH 6.6-6.8. _C. pasteurianum_ has been studied using glucose, glycerol, and xylose as substrates.