# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 3 Factors Influencing Dark Fermentation Reactions
### Substrates Used by the Dark Fermentation Bacteria  
Substrates are critical for H\({}_{2}\) production in dark fermentation. The choice of substrate depends on its availability, the type of microbial inoculum used, cost, composition, and the desired end product. In addition, pretreatment methods such as acid or alkali treatment, milling, and enzymatic hydrolysis can improve the accessibility and digestibility of complex molecules. In an integrated microbial electrolysis cell (MEC) and dark fermentation system, a twofold increase in H\({}_{2}\) yield was achieved, rising from 0.12 to 0.23 mol H\({}_{2}\)/mol substrate per day, and when 20 g/L corn stalk was used as the substrate in the same system, a simultaneous increase in H\({}_{2}\) production was observed [48]. In another H\({}_{2}\) production study, an MEC integrated with dark fermentation using cellulose as a feedstock resulted in a 42% increase in H\({}_{2}\) yield compared to dark fermentation alone [49]. [PERSON] et al. [50] used food waste from a treatment plant as a substrate in dark fermentation experiments. The concentration of the substrate was slowly increased from 10 to 120 g of volatile solids per liter. Silt from the bottom of a lake was used as the inoculum, and the fermentation was carried out for 10 days. An H\({}_{2}\) yield of less than 1.16-10\({}^{-4}\) mol H\({}_{2}\)/g of substrate was observed due to insufficient acidity. However, when a high substrate concentration was used, it facilitated rapid and strong acidification of the substrates, inhibiting only the non-hydrogen-producing bacteria, thus increasing the H\({}_{2}\) production capacity to 1.41\(\cdot\)10\({}^{-3}\) mol H\({}_{2}\)/g substrate. In the analysis of the dynamics of bacterial communities and substrate conversion, olive mill waste, lactate, and acetate were used as substrates. Clostridium and Bacillus were the most abundant microbes during H\({}_{2}\) generation. The inoculum used was obtained from an anaerobic reactor treating cattle effluent, and the analyses indicated that lactic acid fermentation was the main hydrogen-producing pathway, although the amount of H\({}_{2}\) produced was not mentioned in the article [51]. In a study with _Clostridium butylticum_, 3 g/L of glucose was used as a substrate in an anaerobic vessel flushed with nitrogen (N\({}_{2}\)). The experiments were performed in batch mode with an initial pH of 6.5 and a temperature of 37 \({}^{\circ}\)C. The conditions and substrate used allowed the production of 2.09 mol H\({}_{2}\)/mol hexose [23]. In H\({}_{2}\) production studies with _Clostridium beijerinclii_, the bacteria produced 3.58 mol H\({}_{2}\)/mol dextrose when 3 g/L of dextrose was used as a substrate in batch mode at 120 rpm and 37 \({}^{\circ}\)C [52]. In an analysis of thermophilic dark fermentation start-up for H\({}_{2}\) production, a bioreactor with a working volume of 5 L, 6 h HRT, and 55 \({}^{\circ}\)C temperature was set up. The results showed that a maximum of 1.64 mol H\({}_{2}\)/mol glucose was obtained at a glucose concentration of 6 g/L using _Thermoanaerobacterium thermosacharyticum_ strain TG57 [53]. In another study aiming at analyzing the influence of bisphenol A (BPA) and thermophilic bacteria, 20 g/L of food waste was used as a substrate in a 55 mL serum bottle. An experiment was conducted at 55 \({}^{\circ}\)C with a pH of 7, an inoculum ratio of 10% (\(\
u/\
u\)), and a stirring speed of 150 rpm. In the control experiment, without BPA, 1.36 mol H\({}_{2}\)/mol substrate was produced, while with the addition of 100 mg/L BPA, H\({}_{2}\) production was reduced to 1.30 mol H\({}_{2}\)/mol substrate and continued to decrease as the BPA concentration was further increased [54]. In a study with _Clostridium_ spp. YM1, 1.19 mol H\({}_{2}\)/mol sugar was produced when 27.73 g/L of rice straw hydrolysate, consisting of xylose and glucose, was used as a substrate, indicating that the butyric acid pathway was the main pathway followed by these bacteria.