Biofuels in Perspective

206 Biofuels
Clostridium thermocellum and Tt. maritima suggesting that also these thermophiles contain
a NADH-dependent hydrogenase (Table 11.3). Alternatively, NADH may transfer its
electrons first to ferredoxin by an NADH:ferredoxin reductase, and the reduced ferredoxin
is subsequently used for proton reduction. An NADH:ferredoxin reductase (EC 1.18.1.3)
was described by Jungermann et al. 43 and Gottschalk et al.; 44 and this activity has since
been demonstrated in many anaerobic fermentative bacteria including Tt. maritima 26 and
Clostridium cellulolyticum. 45 Cl. butyricum appears to have one bidirectional enzyme,
whereas Clostridium acetobutylicum probably has two enzymes (forward and reverse). 46
However, despite the extensive research on these enzymes in the Sixties and Seventies,
these enzymes have never been purified and gene sequences are also not known (according
to BRENDA database). More recently, NADH:ferredoxin oxidoreductases have been
purified from Cl. tetanomorphum, Acidaminococcus fermentans and Fusobacterium nucleatu
and they appeared to be membrane-bound enzymes. 36 They either use the energy
difference between ferredoxin and NAD to generate a Na + gradient, or they catalyze the
reverse reaction and use a Na + gradient to reduce ferredoxin by NADH. Obviously, not all
NADH:ferredoxin oxidoreductase interconversions occur at the membrane, because e.g.
P. furiosus harbors two cytoplasmic ferredoxin:NADPH oxidoreductases (sulfhydrogenase
and sulfide dehydrogenase) 37,47 (Table 11.3).
Hydrogen can also be produced in (facultative) anaerobes without ferredoxin or separate
hydrogenases. For instance, the facultative anaerobic enterobacteria differ in their hydrogen
enzymology by the sequential use of a pyruvate formate lyase and a formate hydrogen
lyase.
11.5 Enterobacteria
Species of the genus Enterobacteriaceae are facultative anaerobic. In the absence of oxygen
and inorganic electron acceptors they perform a mixed acid fermentation, resulting in the
formation of hydrogen and other products like formate, acetate, ethanol, lactate, succinate
and butane-diol. The ratio at which these products are formed is dependent on the species
and the culture conditions. Escherichia coli produces little butane-diol, while this is a
main end product of Enterobacter aerogenes (Table 11.4). Interestingly, E. coli forms
formate as main product when grown at high pH, while hydrogen is only formed at a low
pH. In the aerobic metabolism enterobacteria convert pyruvate to acetyl-CoA and CO2
via an NAD-dependent pyruvate dehydrogenase complex. NADH is oxidized to NAD in
the electron transport chain. However, under fermentative conditions a pyruvate formate
lyase is involved in pyruvate conversion which results in the formation of acetyl-CoA
and formate. Formate is converted to hydrogen and carbon dioxide by means of a formate
hydrogen lyase. 49 The role of formate in hydrogen production in E. coli has been discussed
by Sawers. 50 Formate is excreted by this bacterium, but at an acid pH it is taken up and
split into hydrogen and carbon dioxide. Cleavage of formate results in energy conservation
by means of a proton gradient. 51 A proton pumping hydrogenase (Ech) as described by
Hedderich and Forzi 41 may be involved. Thermodynamically hydrogen formation from
formate (formate − + H2O → H2 + HCO3 − ; �G 0′ =+1.3kJ/mol formate) is intriguing.
The �G ◦′ of this conversion is + 1.3 kJ, which implies that this conversion is affected by
the P(H2) aswell.