Some trace metals, organic compounds, nutrients and H+ concentration generally have a stimulating effect on the enzymatic activity pertaining to biochemical pro­cesses and might enhance process efficiency if added at optmial concentrations (Table 5). Hydrogenases that are able to catalyze the oxidation of H2 or the reduc­tion of H+ are classified into two major families: the [Ni-Fe] hydrogenases and the [Fe-Fe] hydrogenases, according to the metal content at their active site [130].

[Ni-Fe] hydrogenases have a higher substrate affinity [131]. During H2 production process catalyzed by the [Ni-Fe] hydrogenases, electrons are transported through an intra-molecular electron transfer chain from the redox partner such as NADH or NADPH to the active site, meanwhile, H+ are also transferred to the active site, and gets reduced by the e — to produce H2 [132, 133]. Since nickel is a fundamental com­ponent in [Ni-Fe] hydrogenases, it may influence the fermentative H2 production by influencing the activity of [Ni-Fe] hydrogenases and thus plays an important role in fermentative H2 production [91, 129]. A trace level of nickel is required for acti­vation or function of [Ni-Fe] hydrogenases and thus is conducive to fermentative H2 production [134]. Enhanced H2 production potential was observed with increas­ing Ni2+ concentration from 0 to 0.1 mg/l [91]. Trace metals such as magnesium, sodium, zinc and iron showed considerable affect on the fermentative H2 produc­tion with magnesium being the most significant one [134]. A nutrient formulation containing these four trace metals has shown a 66% enhanced H2 production rate as compared to the control. Iron is an important element which helps to mediate between hydrogenase and nicotinamide adenine dinucleotide (NADH)-ferredoxin reductase [135, 136]. Low iron concentration limits hydrogenase activity to effi­ciently mediate a reversible reaction between H2 and an electron donor such as reduced ferredoxin, thereby limiting H2 production [2]. About a 1.59-fold increase in H2 production and six-fold increase in hydrogenase activity was observed by increasing the FeSO4 concentration from 2.7 to 10.9 mg/l [137]. The role of metal ions (Mn+, Mg+2, Fe+3, etc.) as well as primary and secondary metabolites (adeno­sine mono phosphate, phosphoenolpyruvate, etc.) which have stimulation effects on the enzymatic activity pertaining to fermentative H2 production need to be studied to enumurate their specific function.