Hydrogen production has the simplest chemical reaction, catalyzed by enzymes. During this reaction, a reversible reduction of protons to molecular hydrogen occurs (compare reaction equation of molecular hydrogen production).
The necessary enzymes include complex metal clusters as active centres, which consist of several subunits, being very susceptible against oxygen. Currently, 3 enzymes, capable of catalyzing reaction for hydrogen production, are known (Hallenbeck et al., 2002):
The enzyme nitrogenase is a two-component-protein system, which uses MgATP. The electrons are obtained from reduced ferrodoxin or flavodoxin. A Fe-protein-MgATP-complex associate with a MoFe-protein. Two molecules of ATP are hydrolysed and one electron is transferred to the MoFe-protein, resulting in dissociation of the complex. The turn-over account for 6.4 s^-1^ and is very low. Enormous enzyme amounts are necessary and therefore it is not efficient for hydrogen production.
Fe-Hydrogenase is a periplasmatic enzyme with a complex Fe-S centre. One of the iron atoms is connected with CO and CN. This enzyme removes excess reduction equivalents during fermentation of stringent anaerobic microorganisms. The conversion rate of this enzyme is much higher than that of nitrogenases – in the range of 6,000-9,000 s^-1^.
The NiFe-hydrogenase is a so-called "uptake"-hydrogenase, where electrons of hydrogen are used directly or indirectly for NAD(P) reduction by a quinon-pool. This enzyme is a heterodimeric protein, divided into a small subunit, consisting of 3 iron-sulfur clusters and a large subunit, consisting of a unique complex Ni-Fe centre. Conversion of this enzyme accounts for 98 s^-1^.
During fermentation of organic substrate, for energy production and cell structure of heterotrophic bacteria, electrons are produced, which bacteria have to dispose of, in order to sustain their electric neutrality. Therefore, in case of aerobic organisms, oxygen is reduced and water produced. For anaerobic fermentations, other electron acceptors are necessary. Therefore, protons are reduced to molecular hydrogen. Reduction equivalents, such as formate, reduced ferredoxin and NADH function as electron donors (compare reaction equations of electron transfer) (Nath el al., 2004).
The sugar, used as carbon source, is oxygenated to pyruvate by bacteria during various metabolic pathways, such as pentose phosphate pathway, Embden-Meyerhof pathway (part of the glycolysis) or Entner-Doudoroff pathway. Thereby, 2 mol pyruvate and 2 mol NADH are produced from one mol hexose. In a second step, pyruvate is further converted during oxidative decarboxylation. Conversion of pyruvate can be catalyzed by 2 enzyme systems (Hallenbeck et al., 2002) (compare reaction equations with PFL and PFOR):
- Pyruvat-formate lyase (PFL) and
- Pyruvate-ferredoxin oxidoreductase (PFOR)
Stringent anaerobes prefer that metabolic pathway, where the reduction equivalent ferredoxine is produced. For fermentative hydrogen production, this is the most commonly reaction. The product acetyl-CoA is used for ATP production and as by-product, acetate is produced.
All these reactions result in the total reaction equation of fermentative hydrogen production, describing the most optimal conversion process of sugar (Thauer et al., 1977). Compare reaction equation of dark-fermentation.
Some of the fermentation products, e.g. ethanol or lactate, are alternative pyruvate metabolic pathways, which compete against the enzyme complex PFOR and thus constrain hydrogen production. Presence of uptake-hydrogenases results in further physiological limitations, as they consume a part of the produced hydrogen (Hallenbeck et al., 2002).
Additionally, hydrogen production depends on type of intracellular electron carrier. With - 400 mV, reduced ferredoxin and formate have similar redox potentials, compared to hydrogen and therefore being more suitable. If the redox potentials of carriers and hydrogen significantly differ, only very low hydrogen partial pressures act self-limiting (Chou et al., 2008).
Granted that reducing and oxidized forms of electron carriers are equally available, the maximal hydrogen partial pressure can be calculated:
Fd: pH2,max ≤ 3∙104 Pa
NADH: pH2,max ≤ 60 Pa
If this maximum level of hydrogen partial pressure is exceeded, other metabolic pathways are activated and hydrogen production is constrained. Higher maximal hydrogen partial pressures can be achieved, if the ratio of reduced to oxidized form is greater than 1.
Figure 1: Effects of hydrogen partial pressures on biological hydrogen production (source: Angenent et al., 2004).
Figure 1 shows the effects of partial pressure on the reaction. Figure 1 (a) shows the fermentation at a hydrogen partial pressure of less than 60 Pa. The oxidation of NADH is thermally advantageous. At higher partial pressures, (compare figure 1 (b)), other fermentation products have to be produced for NADH oxygenation. Conversion of glucose during glycolysis or Entner-Doudoroff pathway is indicated with 1. Oxidative decarboxylation of pyruvate with PFOR is marked with 2. Labeled with 3, the reactions for hydrogen production with aid of hydrogenases are shown. Reaction 4 is fermentation of butyric acid.