# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 3 Factors Influencing Dark Fermentation Reactions
### Hydraulic Retention Time (HRT)  
HRT strongly influences microbial growth, substrate utilization, and H\({}_{2}\) yield [56]. Longer HRT allows for more substrate utilization by the microbes, influences the growth rate and metabolic activity of the bacteria, and provides sufficient acclimation and adaptation time for the bacteria to optimize their metabolic pathways for efficient substratedegradation and biogas production. In addition, long HRT helps dominant species establish themselves and suppress less competitive species. It contributes to process stability by ensuring adequate substrate utilization rates, high efficiency, and improved microbial performance. However, on the other hand, in a continuous process, extended HRT can cause biomass washout, making longer HRTs not very adequate to achieve high H\({}_{2}\) yields [57]. For H\({}_{2}\) fermentation, shorter HRTs are preferred to reduce H\({}_{2}\) consumption, and in smaller reactors, they help reduce costs as well as wash out competing species [58].  
In a study using molasses as a substrate in a vertical continuous stirred tank reactor with dark fermentation, an initial HRT of 8 h was gradually decreased to 2 h. The maximum H\({}_{2}\) yield was 0.6 mol H\({}_{2}\)/mol substrate at 5 h HRT [59]. [PERSON] et al. [60] utilized 15 g/L glucose as a substrate in a mesophilic fixed-bed reactor with anaerobic digester sludge as the inoculum, gradually decreasing HRT from 12 to 1.5 h. Butyrate and acetate were the metabolic products during fermentation, with the highest H\({}_{2}\) yield of 2.3 mol H\({}_{2}\)/mol glucose produced by _Clostidium butyticum_ as the dominant species at all the HRTs analyzed. Another analysis using galactose and glucose in a continuously stirred tank reactor with inoculum from heat-treated digester sludge was performed for HRTs ranging from 6 to 24 h. The peak H\({}_{2}\) yield of 1.62 mol H\({}_{2}\)/mol glucose was obtained using glucose as the feedstock at HRTs of 6 and 18 h, whereas with galactose, the H\({}_{2}\) yield was 1.00 mol H\({}_{2}\)/mol galactose at HRTs of 12 and 24 h. In addition, in a mixture of galactose and glucose in a ratio of 8:2, an H\({}_{2}\) yield of 0.48 mol H\({}_{2}\)/mol carbohydrate added was observed at HRTs of 6 and 18 h, respectively [61].  
Hydrogen production in a continuously stirred tank reactor is most often studied because it provides more interaction between the substrate and the microbes. In a continuously stirred bioreactor at a temperature of 32 \(\pm\) 1 \({}^{\circ}\)C and stirring at 60 rpm, the HRT was studied from 24 to 3 h under dark fermentation conditions. A mixture of 4 g/L each of cellobiose, xylose, and arabinose was used as the substrate to obtain the highest average H\({}_{2}\) yield of 7.84 mol H\({}_{2}\)/mol substrate mixture at 3 h and pH 6, indicating the influence of HRT and pH on H\({}_{2}\) yield [62]. In another study using xylose in a dynamic membrane module bioreactor with a 444 \(\upmu\)m pore polyester mesh, the H\({}_{2}\) production rate was analyzed from 12 to 3 h HRT at 37 \({}^{\circ}\)C. The results showed that the maximum yield of 1.40 mol H\({}_{2}\)/mol xylose consumed was observed at 3 h HRT, indicating that lower HRTs are better for H\({}_{2}\) production because the bacterial population can shift from non-H\({}_{2}\) producers like _Lactobacillus_ and _Sporolactobacillus_ spp. to H\({}_{2}\) producers such as _Clostidium_ spp. [63].