Biofuels in Perspective

Biological Hydrogen Production by Anaerobic Microorganisms 213
hydrogen formation from ferredoxin is thermodynamically more favorable (vide infra),
Pyrococcus should be able to produce hydrogen more easily. However, batch culturing has
shown that in addition to acetate, CO2 and alanine, P. furiosus produces ∼2.6–3.0 H2 per
glucose equivalent, 29 which is not superior to the mesophiles, and even worse compared
to Tt. maritima. Nevertheless, the amount of hydrogen that is produced is strongly influenced
by the P(H2). 29 At low P(H2) more hydrogen (3.7 H2/glucose) is produced as was
shown by coculturing of P. furiosus with a hyperthermophilic methanogen. Likewise, in
substrate-limited chemostat cultures, during N2-flushing, hydrogen levels amounted to 3.9
H2 per glucose (Tuininga and Kengen unpublished results).
The enzyme involved in transfer of the electrons (ferredoxin) to hydrogen has been investigated
in detail for P. furiosus. 30,83 P. furiosus contains two cytoplasmic [NiFe]-containing
hydrogenases (Sulfhydrogenase I and II) that are capable of reducing protons to hydrogen
but also to reduce polysulfide to H2S (37, 91) (Table 11.3). However, NADPH is the preferred
electron donor for these bifunctional enzymes and not ferredoxin. A third cytopasmic
enzyme has been described, which contains Fe-S centers and a flavin and which is also
capable of polysulfide reduction. In addition, this sulfide dehydrogenase was shown to have
ferredoxin:NADP oxidoreductase activity, and it was therefore initially hypothesized that
reductant is transferred via ferredoxin to NADP and subsequently to hydrogen. 47 However,
Silva et al. showed that the turnover of the sulfhydrogenase is too low to account for the flux
of reductant from the glycolysis. Moreover, they could demonstrate that P. furiosus contains
another membrane-bound [Ni-Fe] hydrogenase complex which can accept electrons from
ferredoxin directly, without the involvement of NADP(H). This hydrogenase complex is
encoded by the mbh operon, consisting of 14 ORFs. This hydrogenase was found to be
unique in that it is rather resistant to inhibition by CO and that it exhibits an extremely
high H2 evolution to H2 uptake activity ratio compared to other hydrogenases. 83 Moreover,
it was shown that the enzyme could act as a proton pump, generating a proton-motive
force, and coupled to the synthesis of ATP. 30 The genome contains a second gene cluster
(mbx operon) coding for another [NiFe]-containing hydrogenase complex, and highly
homologous to the mbh cluster (Table 11.3). Its physiological role is as yet unknown.
The ferredoxin:NADP oxidoreductase (or sulfide dehydrogenase) can become active
under conditions of high P(H2), by transferring electrons from ferredoxin to
polysulfide or to NADP. Subsequently, NADPH can be reoxidized by producing alanine
from pyruvate using glutamate dehydrogenase (NADP-dependent) and alanine
aminotransferase. 92
11.11 Approaches for Improving Hydrogen Production
Ways to improve fermentative hydrogen production have been reviewed by Nath and
Das. 93 Improvements can be made by the proper choice of the fermentative bacterium, by
applying genetic modification to redirect biochemical pathways in such a way that less side
products are formed and by a proper process design and process operation (gas sparging,
creating a large biofilm surface, etc.). All these measures will optimize hydrogen formation
only to maximally 4 molecules of hydrogen per molecule of glucose and may result in an
increase of the hydrogen production rate. However, the challenge is to extend the yield to
more than 4 hydrogen per glucose. In principle this might be possible by blocking certain