# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 2 Hydrogen Production Pathways Utilized by Dark Fermentation Bacteria  
Dark fermentation is considered a promising alternative method for H\({}_{2}\) production. The general outline of the pathway followed by dark fermentation bacteria involves the breakdown of organic matter into simpler compounds and the release of H\({}_{2}\) as a byproduct under anaerobic conditions. The microbes involved in this process are either strict or facultative anaerbes. This fermentation does not require light energy and is mainly carried out by bacteria from the genus _Clostridium_ and the family Enterobacteriaceae with the ability to produce H\({}_{2}\)[15]. The most suitable pathway for H\({}_{2}\) production depends on the bacterial species, the substrate type and concentration, pH, temperature, HRT, and other environmental factors used during the process. Figure 2 shows the general outline of the dark fermentation pathway, along with the key parameters related to dark fermentation, which will be discussed in detail in the following sections. The pathway involves the uptake of organic substrates such as carbohydrates, organic acids, and other biodegradable compounds. These substrates undergo glycolysis in the cytoplasm of the bacterial cell, where glucose is converted to pyruvate, producing adenosine triphosphate (ATP) and reduced cofactors such as nicotinamide adenine dinucleotide (NADH). Pyruvate also undergoes acidogenesis, where it is converted into organic acids through the activity of enzymes such as pyruvate decarboxylase and formate hydrogenylase to produce H\({}^{+}\) ions. Electrons are generated through the reduction of NADH, which is produced during glycolysis. Hydrogenase enzyme facilitates the release of molecular H\({}_{2}\) from the protons and electrons produced [22]. The fermentation pathways include the formate and NADH pathways, which can be further classified based on the bacteria involved. For example, pathways followed by _Clostritium_ and _Enterobacter_ spp. are classified as Clostrtial-type and Enterobacterial-type fermentations based on specificity. In both Clostrtial-type and Enterobacterial-type fermentations, H\({}_{2}\) is produced during the glycolytic pathways mentioned above. In Clostrtial-type fermentation, pyruvate:ferredoxin oxidoreductase (PFOR) oxidizes pyruvate to acetyl coenzyme A (acetyl-CoA) in the presence of ferredoxin, which is simultaneously reduced. On the other hand, NADH:ferredoxin oxidoreductase (NFOR) catalyzes the reduction of ferredoxin by NADH. Electrons are released from reduced ferredoxin and are used in proton reduction by hydrogenases to produce H\({}_{2}\). In Enterobacteriaceae-type fermentation, NADH oxidation by NFOR forms H\({}_{2}\), similar to the process in Clostrtial-type fermentation. In mixed-acid fermentation, how  
Figure 2: General outline of dark fermentation pathways and the parameters concerning dark fermentation. Adapted and reprinted with permission from [21]_Renewable Energy Conversion Systems_, Academic Press, [PERSON], Chapter 8—Bioenergy, Copyright (2021), Elsevier, and [16]_Chemical Engineering Journal_, 481, [PERSON] [PERSON], [PERSON], [PERSON], [PERSON], [PERSON], Advances in the biomass valorization in dark fermentation systems: A sustainable approach for biohydrogen production, 148444, Copyright (2024), Elsevier. ever, pyruvate is converted to acetyl-CoA and formic acid by the activity of pyruvate formate lyase (PFL). Formate hydrogenlyase degrades formic acid into H2 and CO2[23; 24]. Hydrogen production by dark fermentative bacteria from biomass mainly occurs via the acetate and butyrate pathways [25]. The maximum H2 yield through the acetate pathway is 4 mol H2/mol glucose consumed, while the butyrate pathway offers a maximum theoretical H2 yield of 2 mol H2/mol glucose [26]. According to these studies, the type of bacteria, the environmental conditions, and the process parameters are critical in determining the optimal conditions for maximizing H2 yield.