Biofuels in Perspective

Biological Hydrogen Production by Anaerobic Microorganisms 211
ethanolicus. It has recently been argued that the ech operon is an example of horizontal
gene transfer, coming from the Methanosarcina genus or related species. 79
11.9 The Genus Thermotoga
Members of the order Thermotogales are Gram − , anaerobic, thermophilic, and heterotrophic
bacteria characterized by the presence of a toga, an outer sheath-like structure
surrounding the cells. All members of this order (Fervidobacterium, Thermotoga, Geotoga,
Petrotoga, Marinitoga and Thermosipho) are able to utilize complex carbohydrates and
proteins, and all produce hydrogen to some extent. Sulfur compounds (thiosulfate, S ◦ )
can be used as electron acceptors. The genus Thermotoga has been investigated in more
detail and for some species high levels of hydrogen production have been observed. Both
Tt. maritima and Tt. neapolitana have been reported to produce up to 4 hydrogen per
glucose. 26,80 Apparently these species are capable of directing all reducing equivalents
towards proton reduction. Tt. maritima harbors a cytoplasmic Fe-only hydrogenase which
contains a flavin binding site, suggesting that it may use NAD(P)H as electron donor. 81,82
Moreover, cell extracts of Tt. maritima catalyzed H2-dependent NAD reduction. However,
although the purified enzyme showed hydrogenase activity using the artificial electron
carriers methyl viologen- and AQDS, it did not catalyze NAD(P)H-dependent H2 formation
or the reverse reaction. 82 Cl. pasteurianum contains a similar Fe-only hydrogenase
which is ferredoxin-dependent, but which does not contain the flavin binding domain. 42
However, ferredoxin could not act as electron donor for the Tt. maritima hydrogenase and
it is therefore assumed that the physiological role is the oxidation of NADH. Moreover,
a homologous Fe-only hydrogenase was purified from Ta. tengcongensis which did show
NADH-dependent hydrogenase activity (see above).
Tt. maritima also produces reduced ferredoxin in the pyruvate: ferredoxin oxidoreductase
reaction, which is commonly recycled by proton reduction in a [NiFe]-hydrogenase.
Such a ferredoxin-dependent hydrogenase complex (ech operon) has been purified from
Ta. tengcongensis and the corresponding gene cluster has been identified. 28 Although, a
similar hydrogenase cluster can be found in Cl. thermocellum, Cl. phytofermentans, and
Ca. saccharolyticus, it is absent in Tt. maritima. Tt. maritima, on the other hand, contains
another gene cluster (Mbx operon; 13 genes), which is highly homologous to gene clusters
in the hyperthermophilic archaea P. furiosus, P. abyssi, P. horikoschii and Thermococcus
kodakaraensis. These gene clusters are suggested to code for an energy transducing hydrogenase
complex. 30,83 Whether the mbx operon in Tt. maritima also codes for such an
energy transducing and ferredoxin-dependent hydrogenase, has not been experimentally
confirmed.
Biochemical analyses and genomics of Tt. maritima have revealed that it uses the classical
EM-pathway for glucose catabolism and that reducing equivalents are produced as NADH
(glyceraldehyde-3-P dehydrogenase) and ferredoxin (pyruvate:ferredoxin oxidoreductase).
Tt. maritima also contains the classical Entner-Doudoroff (ED) pathway which is apparently
used alongside the EM pathway in a ratio of 15 %/85 %, respectively. 84 Whether the ED
pathway may become dominant under certain conditions is not known. In the upper part
of the ED pathway reducing equivalents are produced as NADPH, 85 which may only be
used for anabolism, and thus are not available for hydrogen production.