# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 4 Bacteria Used for Hydrogen Production During Dark Fermentation
### Escherichia Coli  
_E. coli_ is a facultative anaerobic mesophilic bacterium that grows optimally around 37 \({}^{\circ}\)C [138]. During dark fermentation, in the absence of O\({}_{2}\), _E. coli_ must find an alternative terminal electron acceptor. Protons serve this role, with H\({}_{2}\) being produced when electrons are transferred to protons, while organic acids and ethanol are produced to maintain redox balance [139]. In _E. coli_, four [Ni-Fe]-hydrogenases are involved in H\({}_{2}\) metabolism. The activity and reversibility of these enzymes depend on various conditions, such as pH, substrate type and its concentration, O\({}_{2}\) availability, and oxidation-reduction potential [140]. The co-production of H\({}_{2}\) with ethanol is more profitable than separate fermentation stages [141], as it improves the energy balance of biorefinery designs [142]. Table 7 presents a list of _E. coli_ strains and their H\({}_{2}\) production abilities using different substrates and operational conditions.  
The analysis determined the efficiency of _E. coli_ in producing H\({}_{2}\) in monoculture and coculture. These bacteria can utilize a wide range of substrates, and the studies showed that they can produce H\({}_{2}\) at temperatures ranging from 31 to 37 \({}^{\circ}\)C. A minimum pH of 5.5 and a maximum pH of 8 were applied, with all studies performed in batch mode. As can be seen in Table 7, the maximum H\({}_{2}\) production of 2.82 mol H\({}_{2}\)/mol glucose was observed with _E. coli_ strain WDH-LF using glucose as a substrate at 31 \({}^{\circ}\)C, pH 8.2, and 400 rpm, proving optimal H\({}_{2}\) production under these certain conditions.  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Strain**} & \multicolumn{2}{c}{**Substrate**} & \multicolumn{2}{c}{**Conditions**} & \multicolumn{2}{c}{**Operation Mode**} & \multicolumn{2}{c}{**H\({}_{2}\) Yield as Mol**} \\  & \multicolumn{2}{c}{**(Concentration, g/L)**} & & & \multicolumn{2}{c}{**(Reactor Volume, mL)**} & \multicolumn{2}{c}{**H\({}_{2}\)/mol Substrate \({}^{*}\)**} & \multicolumn{1}{c}{**Ref.**} \\ \hline  & & **T** & & & & & \\  & & **(\({}^{\circ}\)C)** & **pH** & **Time (h)** & **More Details** & & & \\ \hline DSM 2026 & Glycerol (20.4) & 37 & 6.5 & 24 & 10\% /\(\
u\) inoculum & Batch (5000) & 0.80 & [127] \\ Glucose (10), & & & & 150 rpm, N\({}_{2}\) & Batch (1000) & 2.07 (glucose) & [128] \\ ECU-15 & Xylose (2), and & & & flushing & & & \\  & Celloose (1.5) & & & & & & \\ MGH 78578 & Brewery wastewater & 35 & 5.5 & 72 & 10\% /\(\
u\) inoculum, & Batch (250) & 0.80–1.67 (glucose) & [129] \\  & (3–4) & & & & 150 rpm, flushed & Batch (1000) & 1.22 & [130] \\ ECU-15 & Glucose (35.62) & 37 & 6.0 & & & 200 rpm & Batch (2500) & 0.34 & [131] \\ HE1 & Glycerol (50) & 35 & 6.0 & & & Batch (60) & 0.25 & [132] \\ TR17 & Glycerol (20) & 40 & 8.0 & 24 & & 20\% /\(\
u\) inoculum, & Batch (100) & 0.80 (xylose) & [133] \\  & Sucrose (3.588) & 37 & 5.5 & 48 & 850 rpm & & \\ TR17 & Glycerol (11.4) & 40 & 8.0 & & 10\% /\(\
u\) inoculum, & Batch (60) & 0.26 & [134] \\  & & & & & & Up-flow anaerobic & & \\ TR17 & Glycerol (10) & 40 & 8.0 & 12 & sludge blanket & Batch (1000) & 4.08 & [135] \\  & & & & & reactor & & \\ Y7-3 & Corn straw (50) & 37 & & 24 & 5\% /\(\
u\) inoculum, & Batch (100) & 0.18 & [136] \\ ABZ11 & Glucose (9.15) & 34 & 6.8 & 48 & 150 rpm & Batch (2000) & 2.71 & [137] \\ \hline \hline \end{tabular}
* In parentheses, the reference substrate for calculation when it differs from the cited one.  
\end{table}
Table 6: Hydrogen production by different _Klebsiella pneumoniae_ strains.