Page 68 - Advances in bioenergy (2016)

BIODIVERSITY IN ETHANOL FERMENTATION

Significant differences are observed in the biochemical paths used by different fermenting
microorganisms (Figure 2.3). The differences may refer to very important characteristics for
ethanol production in industrial scale, such as the enzymes used, the metabolites produced, the
energy gain for the cell, and the final fermentation products. In recent trends of biofuels
production processes (Biorefinery concept), the successful fermentation of undiluted, not
fractionated lignocellulosic hydrolysates is substantial not only to exploit all biomass
components but also to make the process simpler. Thus, apart from ethanol yields and

productivities, the ‘robustness’ of the fermenting microorganisms (mainly reflected to toxicity
and ethanol tolerances, and to a lesser extent to thermotolerance and to pH tolerance) is also
important for their success in industrial applications. The formation of by-products is
considered a major disadvantage not only because of their impact on the overall ethanol yield,
but also because of the difficulties in separation and down processing of end products.
Although the natural fermenting microorganisms, S. cerevisiae and Z. mobilis, are the more
suitable for large-scale applications, using genetic engineering and system biology tools, the
fermentative performances of bacteria such as E. coli, Klebsiella oxytoca, and C.
thermocellum have also been significantly improved.

Fermentation of Biomass-Derived Sugars by Zymomonas mobilis

The anaerobic bacterium Z. mobilis ferments glucose through the Entner–Doudoroff (ED)
pathway, in which one mole of glucose is converted to one mole of pyruvate, one mole of
NADPH, one mole of NADH and one mole of ATP (Figure 2.3). Although many bacterial

species are using the ED pathway, Z. mobilis is the only known natural species that uses this
path under anaerobic conditions. The ED pathway yields only half as much ATP per mole of
glucose as the Embden–Meyerhof (EM) pathway. As a consequence, Zymomonas produces
less biomass than yeast, and more carbon is channelized to fermentation products. Also, as a
consequence of the low ATP yield, Zymomonas maintains a high glucose flux through the ED
pathway. All the enzymes involved in fermentation are expressed constitutively, and
fermentation enzymes comprise as much as 50% of the cells’ total protein. 71

72
The complete genome sequence of Z. mobilis ZM4 has been reported at 2005, revealing that
2056416 base pairs form a circular chromosome with 1998 open reading frames (ORFs) and
three ribosomal RNA transcription units. Recognizable genes for 6-phosphofructokinase, and
for two enzymes in the tricarboxylic acid (TCA) pathway were absent and, therefore, it was

confirmed that glucose could be metabolized only by the ED pathway.

Escherichia coli

Although E. coli is naturally able to ferment a wide spectrum of sugars (much wider than
natural S. cerevisiae and Z. mobilis strains), the ethanol yields are very low because of the
formation of other fermentation products in higher rates and final concentrations. In E. coli, the
fermentation of glucose may be regarded as a process in which the four extra reducing