Hydrogen production by dark fermentation is achieved by strictly anaerobic or facultative anaerobic bacteria under anaerobic conditions. Hydrogen is an important compound for the metabolism of many anaerobic and a few aerobic microorganisms. Oxidation of hydrogen can be used by many organisms to drive energy generation. When external electron acceptors are absent, some organisms dispose of excess electrons generated during metabolism by reducing protons to hydrogen. Hydrogenase is the key enzyme for both situations. Ni-Fe hydrogenases and [FeFe] hydrogenases are the two main types of hydrogenases. They are phylogenetically distinct and contain different active sites. With some exceptions NiFe hydrogenases are known to catalyze hydrogen oxidation and [FeFe] hydrogenases are active in proton reduction catalysis of hydrogen evolution depends upon the organism. For example for Clostridia types hydrogen evolution

is catalyzed by soluble [FeFe] hydrogenase and for Escherichia coli is catalyzed by membrane bound NiFe hydrogenase [6].

Although in principle a variety of organic compounds; carbohydrates, proteins and lipids can be used for hydrogen production by dark fermentation, in reality hydrogen can only be obtained in practical yields by fermentation of carbohy­drates. Amino acids, obtained by protein hydrolysis, are fermented by Strickland reactions where one amino acid serves as the electron acceptor for the oxidation of the second amino acid. These reactions generate energy for the microorganisms carrying them out, but they do not yield hydrogen. Lipids can be hydrolyzed to glycerol and long chain fatty acids which in turn can be further degraded to acetate and hydrogen by synthropic bacteria. However, these reactions only occur at extremely low partial pressures of hydrogen maintained by associated methano — genic or sulfate-reducing bacteria [6].

Thus, a practical process for producing hydrogen must rely on fermentation of sugars derived from carbohydrates. This process can be modeled by considering hydrogen production from glucose, a typical hexose derived from various residues and wastes. The degradation of glucose to acetate (Fig. 10.1) is generally taken into account to estimate the theoretical yields, or to describe the reaction steps. By using the conversion of glucose to produce hydrogen the reaction is (Eq. 10.1) [7]:

C6H12O6 + 4H2O! 2 CH3COO— + 2HCO— + 4H+ + 4H2 AG0 = -206.3kJ/mol ( ‘ )

While in principle 4 mol of hydrogen can also be produced from glucose in two steps (Eqs. 10.2, 10.3) [7]:

Acetate reaction:

C6H12O6 + 2H2O! 2 CH3COO— + 2HCOO — + 4H+ + 2H2 AG0 = —209.1kJ/mol

2HCOOH! 2CO2 + 2H2 AG0 =-6kJ/mol

most organisms converting formate to hydrogen, giving 2 H2, are not capable of making hydrogen from NADH and thus are restricted to 2 H2/glucose.

Butyrate can also be an end product in anaerobic fermentation (Eq. 10.4) [7]:

C6H12O6 + 2H2O! CH3CH2CH2COO— + 2HCO— + 3H+ + 2H2 AG0 = —254.8kJ/mol ( ‘ )

To metabolize glucose to pyruvate the Embden-Meyerhoff-Parnas (i. e. Gly­colysis) or the Entner-Doudoroff pathways can be used [8]:

C6H12O6 + 2NAD+ ! 2 CH3COCOO— + 4H+ + 2NADH AG0 = -112.1 kJ/mol

As seen in reaction (10.4) 1 mol glucose can produce 2 mol pyruvate and 2 mol NADH. The NADH produced during glucose metabolism can in principle be used to provide electrons to reduce H+ to H2, but this reaction is thermodynamically unfavorable and hence cannot go to completion at high hydrogen partial pressures. A low NADH concentration, brought about by its oxidation during the production of other products; ethanol, lactate, butyrate, etc. is assumed by many researchers to result in low hydrogen yields.

The anaerobic metabolism of pyruvate formed during the catabolism of various substrates is the main reaction of hydrogen production. Two enzyme systems can catalyze the breakdown of pyruvate reactions (Eqs. 10.6, 10.7) [3]:

Pyruvate formate lyase:

Pyruvate + CoA $ acetylCoA + formate AG0 = — 16.3kJ/mol (10.6)

This reaction is a typical example of an enteric type fermentation, the metab­olism of Enterobacter species and Escherchia coli. Hydrogen production can become an advantage for the bacterium when the pH drops due to active metab­olism which causes induction of the FHL and the conversion of formic acid to hydrogen to prevent further acidification [6]. Thus, 2 mol of hydrogen can be produced by one mole of glucose by facultative anaerobic microorganisms [9].

Pyruvate: ferredoxin oxido reductase (PFOR)

Pyruvate + CoA + Fdox $ acetylCoA + CO2 + Fdred AG0 = — 19.2kJ/mol

(10.7)

This reaction is an example of H2 production by strict anaerobes. Clostridia can convert pyruvate to acetyl-CoA and CO2 producing reduced ferredoxin in a reaction catalyzed by the enzyme pyruvate:ferrodoxin oxidoreductase with ferre — doxin as electron acceptor. This enzyme can be found in many strictly anaerobic bacteria as well as facultative bacteria and some cyanobacteria. The acetyl-CoA which is produced can be metabolized to produce acetate and butyrate. Reoxi­dation of ferredoxin results in the formation of hydrogen by hydrogenase (One H2 per pyruvate). If acetate is the final product, one additional mole of hydrogen can be produced from the oxidation of each mole of NADH (NADH+H+?NAD++H2) that was produced during glycolysis, making the total hydrogen yield 4 mol H2/ mol glucose. If the final product is butyrate, NADH produced from glycolysis will be used for oxidation of acetyl-CoA to butyrate, giving a hydrogen yield of 2 mol H2/mol glucose. These reactions are typical examples for Clostridia species [6, 7]. As a result of glucose fermentation, the generation of other products; propionate, succinate and lactate, can also occur besides acetate, butyrate and formate. Since their production is at the expense of hydrogen production, these metabolites are undesired by-products of dark fermentation.