# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 3 Factors Influencing Dark Fermentation Reactions
### Effect of Temperature  
Temperature directly affects the metabolic activity of bacteria and their ability to produce H2. The enzymatic reactions necessary to produce H2 occur under optimal temperature conditions. In addition, the dynamic growth of bacterial populations also depends on temperature. At higher temperatures within an optimal range, the growth of these bacterial populations can be accelerated, leading to an increase in biomass and potentially higher overall H2 production [42]. Hydrogen-producing microbes can operate in mesophilic, thermophilic, and even hyperthermophilic ranges. [PERSON] et al. [30] studied the effect of temperature on biohydrogen production using different mixtures of sewage sludge, vinasse, and poultry manure. In the biochemical H2 production (BHP) experiments, temperatures of 35, 55, and 70 \({}^{\circ}\)C were tested in two mixtures. One of them consisted of sewage sludge and vinasse in a ratio of 50:50, while the other consisted of sewage sludge, vinasse, and poultry manure in a ratio of 49:549.5:1. The results showed that the addition of poultry manure at a temperature of 55 \({}^{\circ}\)C was ideal for biohydrogen production, with a yield of 0.522 mol H2/mol volatile solids, which confirmed that H2 production efficiency through dark fermentation is favorable at thermophilic temperatures. The choice of the optimal temperature for fermentation depends entirely on the microbes used, whether they are from a pure culture or a mixture, and on the substrates [43]. Efficient substrate hydrolysis has been reported under thermophilic conditions, but the presence of chemical compounds in the fermentation substrate may be affected by higher temperatures, reducing the conversion efficiency of substrates [44].  
[PERSON] et al. [45] investigated the effect of temperature on the microbial community and biohydrogen production from lactate wastewater using dark fermentation. Temperatures of 35-45 \({}^{\circ}\)C were found to be favorable for H2 production, indicating that hydrogen-producing bacteria in anaerobic sludge can grow within a narrow temperature range and that the enzymes catalyzing lactate conversion are also temperature-specific. The maximum H2 yield of 0.85 mol H2/mol lactate was obtained at 45 \({}^{\circ}\)C and pH 8.5. Additionally, the fermentation batch cycle length and the acclimation time for H2 production are affected by temperature. The results showed that at 35 \({}^{\circ}\)C, the fermentation batch cycle length was about 65 h, while at 45 \({}^{\circ}\)C, it was 180 h. This suggests that different microbes are activated at different times due to variations in fermentation temperatures.  
Another study on dark fermentation reported that mesophilic temperatures (37 \({}^{\circ}\)C), an HRT of 72 h, and a pH close to 7 are recurrent parameters of biomass fermentation [16]. [PERSON] et al. [46] observed that H2 yield was maximized with thermophilic bacteria due to their slow proliferation and lower cell densities, although efficient biohydrogen volumetric productivity was achieved with intermediate thermophiles, indicating thermophiles as very efficient species for high H2 yield. In contrast, an analysis at temperatures of 50-55 \({}^{\circ}\)C showed 5-10 times higher H2 production compared to 30-45 \({}^{\circ}\)C. A comparative analysis between mesophilic and thermophilic temperatures for H2 production using cheese whey wastewater showed that 0.25 mol H2/mol cheese whey was produced at 55 \({}^{\circ}\)C, while 0.29 mol H2/mol cheese whey was produced at 35 \({}^{\circ}\)C [47]. These studies show that temperature alone is not responsible for high H2 production; other parameters, such as substrate, pH, and other environmental factors, determine which bacteria are best suited for a particular fermentation process.