Biofuels in Perspective

210 Biofuels
ferredoxin-dependent membrane-bound NiFe hydrogenase. The corresponding genes could
be identified in the draft genome sequence of Ca. saccharolyticus, based on homology to
the Ta. tengcongensis genes. 28 An NADH: ferredoxin reductase could not be found.
11.8 The Genus Thermoanaerobacter
The genus Thermoanaerobacter comprises a group of extreme thermophilic obligate anaerobes
that ferment sugars to acetate, lactate, ethanol, CO2 and hydrogen in different amounts.
About 16 valid species have been recognized and they differ from Caldicellulosiruptor
species in that they are able to respire with thiosulfate or sulfur, do not utilize cellulose, and
may form endospores. 74 Some species are able to oxidize H2 using thiosulfate or Fe(III) as
electron- acceptor. 75,76 Thermoanaerobacter ethanolicus has been studied for its capacity
to produce substantial amounts of ethanol, especially from pentoses. 77,78 In addition to
ethanol also lactate is produced as reduced end product by several Thermoanaerobacter
species. 27 A similar fermentation pattern was described for Ta. tengcongensis. 27 In correspondence
with the formation of ethanol and lactate, hydrogen levels are usually low
(Table 11.2). Despite the low hydrogen production by Thermoanaerobacter species under
normal batch conditions, high levels (up to 4 H2/glucose) can be attained in N2-flushed
fermentor systems, as was described for Ta. tengcongensis. 28
Two different types of hydrogenases are responsible for H2 formation in this organism: a
ferredoxin dependent [NiFe]-hydrogenase and an NADH-dependent Fe-only hydrogenase.
The former is membrane-bound and related to the energy-converting hydrogenase (Ech)
from Methanosarcina barkeri, whereas the latter is located in the cytoplasm and contains
FMN. The presence of an NADH-dependent hydrogenase in addition to a ferredoxindependent
hydrogenase is unusual. This suggests that NADH can be directly used for
hydrogen formation without the intermediate involvement of a NADH- ferredoxin reductase
(oxidoreductase) as described for certain clostridia. 43,44 Culturing under a high P(H2)
resulted in the formation of ethanol, and was accompanied by lower levels of the NADHdependent
hydrogenase and higher levels of NADPH-dependent alcohol- and acetaldehyde
dehydrogenases. 28 These observations suggest that the activity of the NADH-dependent
hydrogenase is regulated by the P(H2) and that under conditions of high P(H2) NADH is not
used for H2 formation but recycled via the formation of ethanol. However, the preference
of the ethanol-forming enzymes for NADPH instead of NADH implies the involvement of
an NADH:NADP transferase. Homologs of the NADH-dependent hydrogenase encoding
genes (hydA, hydB, hydC, hydD) can be found in the genome of Th ethanolicus, but also in
Ca. saccharolyticus, Cl. thermocellum, Cl. phytofermentans and Tt. maritima, suggesting
that these microorganisms all are able to produce hydrogen directly from NADH.
The membrane-bound [NiFe]-hydrogenase of Ta. tengcongsis has been purified and appeared
to consist of six polypeptides. The encoding genes were identified (echABCDEF)
and shown to be clustered and followed by a gene cluster required for the biosynthesis
of the [NiFe] center. 28 It was proposed that the membrane-bound Ech hydrogenase of Ta.
tengcongensis may be able to generate a pmf and thus conserve energy by ‘proton respiration’
as was described for the Mbh hydrogenase of P. furiosus. 30 Comparable genomics
revealed that the Ech gene cluster is present also in the genomes of Cl. thermocellum, Cl.
phytofermentans, Ca. saccharolyticus, but not in Tt. maritima or the closely related Ta.