Page 69 - Advances in bioenergy the sustainability challenge

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equivalents (or 4H + 4e ) released, as the metabolic price for making two ATPs by substrate-
level phosphorylation, are disposed of by the further metabolism of two pyruvate molecules.
Glucose is converted to a mixture of metabolic products consisting primarily of acetate and
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formate, as well as smaller amounts of lactate, succinate, and ethanol. Furthermore, the
native ethanol pathway is not redox balanced when sugars, such as glucose or xylose, are
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fermented. Only one NADH is formed per acetyl-CoA synthesized, but two NADH are
needed to produce one ethanol molecule via this pathway. 74
Most scientific efforts dealing with the ethanol fermentation using E. coli as the fermenting
microorganism have focused on the improvement of ethanol production by introducing Z.
mobilis genes to E. coli strains. It was shown that the introduction of Z. mobilis homoethanol
pathway (PET operon) increased significantly ethanol production, reaching yields similar to
those achieved by some S. cerevisiae strains. Directed engineering and metabolic evolution of
E. coli strains have also been able to improve the tolerance toward inhibitory compounds
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present in lignocellulosic hydrolysates, giving more promises for the future. In 2007, Kim et
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al. showed that E. coli strains could be efficient ethanol producers without adding genes from
other microorganisms. In that study, the development of an ethanologenic E. coli mutant devoid
of foreign genes was described. An essential mutation in this mutant was mapped within the
pdh operon (pdhR aceEF lpd), which encodes components of the pyruvate dehydrogenase
complex. Anaerobic ethanol production by this mutant is apparently the result of a novel
pathway that combines the activities of pyruvate dehydrogenase (typically active during
aerobic, oxidative metabolism) with the fermentative alcohol dehydrogenase.

Ethanologenic Fungi

Monilia, Fusarium, Neurospora, Rhizopus, Aspergillus, Neocallimastix, Trametes, Phlebia,
and Trichoderma are some of the filamentous fungi genera that are known to produce ethanol
under anaerobic and/or microaerobic conditions. The fermentation characteristics by various
fungal species are shown in Table 2.2. The strategies to improve ethanol production by fungi
have focused on metabolic engineering techniques aiming at (1) the introduction of
heterologous genes, such as S. cerevisiae pyruvate decarboxylase (PDC) and alcohol
dehydrogenase (ADH) to enhance the classical ethanol synthetic pathway; (2) the knockout of
some fungal genes responsible for the production of by-products; or (3) the overexpression of
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existing genes playing a key role in sugars metabolism. The enhancement of the efficiency of
sugar transporters has also been proposed as a strategy to enhance the ethanol producing rate
by H. jecorina. 78

ETHANOL FERMENTATION OF CELLULOSE-DERIVED

SUGARS BY YEASTS

Many yeast species have been reported to convert simple sugars to ethanol under anaerobic
conditions. S. cerevisiae, Pichia stipitis, P. kudriavzevii (Candida krusei), Kluveromyces
marxianus, C. shehatae, C. tropicalis, C. guilliermondii, and Pachysolen tannophilus are