# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 4 Bacteria Used for Hydrogen Production During Dark Fermentation
### Enterobacter
#### 4.2.2 Klebsiella pneumoniae  
_K. pneumoniae_ has been studied for its growth rate, growth conditions, and production of valuable byproducts. This bacterium produces H\({}_{2}\) through a mixed-acid fermentation pathway, converting glucose or other substrates into various end products. Formate, produced via the formate H\({}_{2}\) lyase (FHL) pathway, is broken down by the FHL complex through the activity of formate dehydrogenase and hydrogenase to produce H\({}_{2}\) and CO\({}_{2}\). In a study with biodiesel waste using _K. pneumoniae_ DSM2026, an H\({}_{2}\) production rate of 0.532 mol H\({}_{2}\)/mol glycerol was obtained by optimizing the medium components. Using Plackett-Burman and uniform design methods, the optimized medium contained 20.4 g/L glycerol, 5.7 g/L KCl, 13.8 g/L, NH\({}_{4}\)Cl, 1.5 g/L CaCl\({}_{2}\), and 3 g/L yeast extract, resulting in a 5-fold increase in H\({}_{2}\) levels [127]. Table 6 presents a list of _K. pneumoniae_ strains used to produce H\({}_{2}\) under different substrates and operational conditions.  
As shown in Table 6, _K. pneumoniae_ can utilize different substrates, such as glucose and glycerol. Studies were performed in batch mode over a temperature range of 33-40 \({}^{\circ}\)C, between 12 and 72 h HRT, with a main pH range of 5.5 to 8. In a study, strain TR17 produced an H\({}_{2}\) yield of 0.26 mol H\({}_{2}\)/mol glycerol using 11.14 g/L crude glycerol as a substrate at 40 \({}^{\circ}\)C and pH 8 in batch mode. However, when an up-flow anaerobic sludge blanket reactor was used with this same strain TR17 and 10 g/L glycerol as the substrate at 40 \({}^{\circ}\)C, pH 8 for 12 h in batch mode, a H\({}_{2}\) production of 4.08 mol H\({}_{2}\)/mol glycerol was observed, indicating that this reactor is an optimal setup for achieving higher H\({}_{2}\) yields with this strain of _K. pneumoniae._  
\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  & & **T** & **pH** & **Time (h)** & **More Details** & & \\ \hline  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\ ATCC13408 & Glucose (3) & 37 & 6 & & & & & \\  & & & & & & & \\  & Mahogany wood & & & & & & \\  & & & & & & & \\ ATCC 13048 & hydrolysate and & & & & & & \\  & preculture medium & 37 & 48 & 120 rpm, 15\% o/o & & & \\  & & & & & & & \\  & (45) & & & & & & \\ IAM1183 & Glucose (15) & 37 & 20 & 200 rpm, purging & Batch (100) & 1.34 \(\pm\) 0.21 & [125] \\  & & & & & & & \\  & & & & & & 250 rpm, 10\% o/o & & \\
2822 & Cheese whey (32.5) & 31 & 6.5 & 104 & inoculum, flushing & Batch (2000) & 0.26 (lactose) & [126] \\  & & & & & with argon & & \\ \hline \hline \end{tabular}
* In parentheses, the reference substrate for calculation when it differs from the cited one.  
\end{table}
Table 5: Hydrogen production by different _Enterobacter aerogenes_ strains.