The most employed microorganisms for fuel ethanol production are the yeasts of the Saccharomyces cerevisiae species that convert hexoses, such as glucose and fructose, into pyruvate through glycolysis, which is finally reduced to etha­nol generating two moles of ATP for each molecule of consumed hexose under anaerobic conditions (Claassen et al., 1999) as shown in Figure 6.1. This micro­organism also has the ability to convert hexoses into CO2 by aerobic respiration. One of the two processes may be favored depending on the oxygen concentration in the culture medium and the carbon source. In the latter case, mainly biomass is formed and it is the base for large-scale production of baker’s yeast. In addition to their ability to be grown under anaerobic conditions, yeasts have the advantage of tolerating relatively high concentrations of ethanol (up to 150 g/L). Below a concentration of 30 g/L the inhibition is negligible (Kargupta et al., 1998). The yeast Schizosaccharomyces pombe has the additional advantage of tolerating high osmotic pressures (high amounts of salts) and high solids content (Bullock, 2002; Goyes and Bolanos, 2005). In fact, a fermentation process using a wild strain of this yeast has been patented (Carrascosa, 2006).

Other yeasts having the capability of growing under thermophilic conditions have been evaluated from an industrial viewpoint. Increased fermentation temper­ature accelerates metabolic processes and lowers the refrigeration requirements. For this reason, one of the yeasts that is most studied for ethanol production is Kluyveromyces marxianus, which can be cultivated at temperatures higher than 40°C (Ballesteros et al., 2001). This condition makes this yeast very promising in the case of cellulose conversion schemes for ethanol production by the simul­taneous accomplishment of hydrolysis and fermentation (Ballesteros et al., 2004) because the cellulases have greater activity at temperatures much higher (50 to 60°C) than those of conventional fermentations.

One of the main problems during ethanol production from lignocellulosic mate­rials is that S. cerevisiae can ferment only certain mono — and disaccharides, such as glucose, fructose, maltose, and sucrose. Nevertheless, this microorganism cannot
assimilate either cellulose or hemicellulose directly. Furthermore, this yeast does not assimilate the pentoses obtained during the pretreatment of lignocellulosic biomass when hemicellulose is hydrolyzed at a higher degree. This hemicellulose hydrolyzate contains pentoses (mostly xylose, though also arabinose) as well as other hexoses (glucose, mannose, and galactose). For this reason, the utilization of pentose-utilizing microorganisms has been proposed similarly to some species of yeasts. Yeasts, such as Pichia stipitis, Candida shehatae, and Pachysolen tan — nophilus can assimilate both pentoses and hexoses (Olsson and Hahn-Hagerdal, 1996) as shown in Table 6.2. One key aspect in the metabolism of xylose is its conversion into xylulose that is integrated to the metabolic pathways for pyruvate synthesis (final product of glycolysis) from which ethanol is derived. Pyruvate is also the starting point for the cycle of tricarboxylic acids (Krebs cycle; Figure 6.2). The cultivation of these yeasts requires a thorough control to ensure low levels of oxygen in the medium needed for the oxidative respiratory metabolism.

For pentose-assimilating yeasts, the hexoses are, however, the most readily and rapidly assimilable substrate during ethanol production. This implies a diauxic growth, which means that the hexoses are consumed firstly before the pentoses if the fermentation is extended enough. After a relatively short lag-phase in which the enzymes necessary for pentose metabolism are synthesized, the pentoses are consumed until the end of fermentation. This means that the microorganisms do not utilize the two types of sugar at the same time, which causes a decrease in the

biomass utilization rate. As a rule, the microorganisms prefer the glucose over the galactose, followed by the xylose and arabinose (Gong et al., 1999), which is explained by the catabolic repression the glucose exerts on the consumption rate of xylose and other pentoses as in the case of C. shehatae. In addition, ethanol productivity achieved using xylose-assimilating yeasts is lower than that of micro­organisms fermenting only hexoses. Thus, their ethanol production rate from glu­cose is at least five times lower than that observed in S. cerevisiae. Moreover, their culture requires oxygen and ethanol tolerance that is two to four times lower (Claassen et al., 1999). Most xylose-utilizing yeasts are mesophiles, i. e., they are cultivated at temperatures near 30°C; likewise S. cerevisiae, though there exist reports about the methylotrophic yeast Hansenula polymorpha cultivated at 37°C in a xylose-containing medium (Ryabova et al., 2003).