Fermentation is the process of deriving energy from the oxidation of organic com­pounds using an endogenous electron (e-) acceptor, which is usually an organic compound [14]. This is in contrast to cellular respiration, where e — are donated to an exogenous e- acceptor, such as oxygen, via an electron transport chain (ETC). Considering glucose as substrate, fermentative H2 production starts with the con­version of glucose to pyruvate through glycolysis by both obligate and facultative anaerobic bacteria. In facultative anaerobes, pyruvate is converted to acetyl-CoA and formate, which is catalysed by pyruvate formate lyase (PFL) [3] and H2 is produced from formate by the formate hydrogen lyase (FHL) complex. In obligate anaerobes, pyruvate is converted to acetyl-CoA and CO2 through pyruvate ferre — doxin oxidoreductase (PFOR) and this oxidation requires reduction of ferredoxin (Fd) [3, 15]. The fate of pyruvate in the case of anaerobic operation depends on the operating pH. Under acidic condition pyruvate is converted into volatile fatty acids along with H2 by acidogenic bacteria. Neutral operation leads to the formation of CH4 and CO2 by methanogenic bacteria. Under basic pH, anaerobic digestion leads to solventogenesis. At all the pH conditions, H+ shuttling takes place between metabolic intermediates with the help of various redox mediators under anaerobic operation. The H+ from the redox mediator is detached by a specific dehydrogenase (NADH-dehydrogenase) and combined with the e- from oxidized ferredoxin to gen­erate H2 in presence of the hydrogenase enzyme (Fig. 1b). Hydrogenase activity is higher at acidic pH but with increase in pH, metabolic pathway might proceed to the next step of anaerobic digestion where H+ get reduced to CH4 (methanogenesis) or ethanol (solventogenesis).

Biodegradation of substrate is always accompanied by the release of protons (H+) and electrons (e-) associated with various redox reactions and enzymes. Dehydrogenase is one of the important enzymes involved in the inter-conversion of metabolites and the transfer of protons (H+) between metabolic intermediates through redox reactions using several mediators (NAD+, FAD+, etc.). Redox medi­ators are capable of carrying H+ and e-, otherwise known as energy carriers as they are involved in biological energy (ATP) generation [16]. Generally, in the anaerobic microenvironment, inter-conversion of substrates takes place through degradation that increases the availability of H+ in the cell. The protons associated with redox mediators are the main source of fermentative H2 production. The protons from redox mediators are detached in presence of NADH-dehydrogenase and reduced to H2 in presence of the hydrogenase enzyme with the help of e — donated by oxidized ferredoxin (co-factor) [3]. Hydrogenases are complex metalloenzymes that can be classified into three groups based on the number and identity of the metals in their active sites: [NiFe]-, [FeFe]- and [Fe]-hydrogenases [17]. These enzymes are also responsible for the reversible conversion of molecular H2 into two H+ and two e — [H2 ^ 2H+ + 2e-] [3]. The dehydrogenase activity is crucial along with the hydro­genase activity as it maintains H+ equilibrium in the cell through redox reactions and inter-conversion of metabolic intermediates. Nitrogenase enzymes are also involved in H2 production along with nitrogen-fixation. Nitrogenases irreversibly catalyze
the reduction of molecular nitrogen to ammonium by consuming reducing power (e- mediated by ferredoxin, NAD+ etc.) and ATP. Nitrogenase catalyzes H+ reduc­tion in the absence of nitrogen gas. Even in nitrogen atmosphere, H2 production is catalyzed by nitrogenase as a side reaction at a rate of one-third to one-fourth that of nitrogen-fixation. Nitrogen-fixing cyanobacteria are potential candidates for H2 production by nitrogenase but it is an energy-consuming process due to breakdown of many ATP molecules.