Biofuels in Perspective

214 Biofuels
genes of the glycolysis and expressing genes that encode for enzymes of the pentose
phosphate pathway. While under fermentative conditions the EM pathway can produce up
to4H2/glucose (concomitant with 2 acetate), the PP cycle can theoretically produce 12
H2/glucose because glucose can be entirely oxidized to CO2. This was demonstrated in an in
vitro system using the enzymes of the oxidative pentose phosphate cycle. 94,95 By combining
the enzymes of the PP cycle (from Yeast) with the NADP-dependent sulfhydrogenase of
P. furiosus, in an in vitro system,11.6 mol of hydrogen could be formed per mol of
glucose-6-P. However, the instability of some of the enzymes and of NADP(H) hampers
the practical application of the method. To overcome this problem some oxidative PP cycle
enzymes from Tt. maritima were cloned into E. coli, 96 but an efficient hydrogen producing
system has not been obtained yet. The genome sequences of Tt. maritima, P. furiosus
and Caldicellusiruptor saccharolyticus are available now, enabling genetic engineering
approaches within these promising hydrogen-producing microorganisms, provided that
genetic systems can be developed.
A main breakthrough would be achieved when acetate would be efficiently converted
to carbon dioxide and hydrogen. Thermophilic bacteria, like Thermoacetogenium phaeum
and Clostridium ultenense 97,98 have the biochemical potential to do so. At low P(H2), these
bacteria employ the acetyl-CoA cleavage pathway to convert acetate to carbon dioxide and
hydrogen. They even can grow by acetate conversion. In principle also homoacetogenic
bacteria and enterobacteria have the biochemical potential to oxidize acetate and form
hydrogen, the former via the acetyl-CoA cleavage pathway and the latter via the citric
acid cycle. However, energy input is needed to enable hydrogen production to high levels.
One way to introduce extra energy is light. Light energy enables phototrophic bacteria of
the genus Rhodopseudomonas to produce high levels of hydrogen from acetate. Such an
integrated dark-light fermentation is under investigation 99 and its perspectives have been
discussed by Nath and Das. 93
Another way to introduce energy in a dark fermentation is via electricity. 100 The concept
of electricity-mediated electrolysis of organic compounds was introduced by Liu et al. 101
and Rozendal et al. 102 They showed that in a biofuel cell acetate can yield hydrogen
and carbon dioxide and that this type of hydrogen formation is much more cost-effective
than electricity mediated electrolysis of water to form hydrogen and oxygen. A rough
calculation showed that the equivalent of one hydrogen is needed to produce the electricity
for the electrolysis of acetate. This implies that (a) a net yield of 10 molecules of hydrogen
per molecule of glucose can be obtained, and (b) hydrogen formation is not restricted
to (poly)saccharides as substrates, but that all kinds of organic (waste) components are
feasible substrates.
11.12 Concluding Remarks
Dark hydrogen formation is performed by many anaerobic and facultative anaerobic microorganisms,
which differ, however, in the amount of hydrogen that is produced per
glucose. Thermophiles and extreme thermophiles appear to be superior in this respect,
as the amount of hydrogen approaches the apparent maximum of 4 H2/glucose and less
side-products are produced. These observations are supported by the thermodynamics of