The co-fermentation of lignocellulosic hydrolyzates represents another techno­logical option for utilizing all the sugars released during biomass pretreatment and cellulose hydrolysis. This kind of cultivation process is aimed at the com­plete assimilation of all the sugars resulting from lignocellulosic degradation by the microbial cells and consists of the employment of a mixture of two or more compatible microorganisms that assimilate both the hexoses and pentoses present in the medium. This means that the fermentation is carried out by a mixed culture. Some examples of mixed culture are summarized in Table 7.3. However, the use of mixed cultures presents the problem that microorganisms

utilizing only hexoses grow faster than pentose-utilizing microorganisms lead­ing to a more elevated conversion of hexoses into ethanol (Cardona and Sanchez, 2007). To solve this problem, the utilization of respiratory-deficient mutants of the hexose-fermenting microorganisms has been proposed. In this way, the fer­mentation and growth activities of the pentose-fermenting microorganisms are increased as they grow very slowly when cultivated along with rapid hexose-fer — menting yeasts. In addition, the presence of hexose-assimilating microorganisms allows the reduction of the catabolic repression exerted by glucose on the pen­tose consumption in pentose-assimilating microorganisms (Laplace et al., 1993). Considering the indicators for the process using only the glucose-assimilating bacterium Z. mobilis grown on the biomass hydrolyzate, the productivities of the mixed culture are less than those of the bacterium, but the yields are com­parable, which offers a space for further research (Delgenes et al., 1996). One of the additional problems in this kind of configuration is that pentose-fermenting yeasts present a greater inhibition by ethanol, which limits the use of concen­trated substrates in the system.

Another variant of co-fermentation consists of the utilization of a single micro­organism capable of assimilating both hexoses and pentoses in an optimal way allowing high conversion and ethanol yield. Although these microorganisms exist in nature (see previous section), their efficiency and ethanol conversion rates are reduced for the implementation of an industrial process. Hence, the addition to the culture medium of an enzyme transforming the xylose into xylulose (xylose- isomerase) has been proposed (see Table 7.3.). In this way, microorganisms exhib­iting high rates of conversion to ethanol and elevated yields (like S. cerevisiae) can assimilate the xylulose, involving it in the metabolic pathways leading to the ethanol biosynthesis (see Chapter 6, Figure 6.2). On the other hand, a high effi­ciency in the conversion to ethanol can be reached through the genetic modifica­tion of yeasts or bacteria already adapted to the ethanolic fermentation—a topic that was discussed in Chapter 6. The microorganisms most commonly modified for this purpose are S. cerevisiae and Z. mobilis to which genes encoding the assimilation of pentoses have been introduced (see Chapter 6, Table 6.3). The other approach for genetic modification is the introduction of genes encoding the metabolic pathways for ethanol production to microorganisms that are capable of fermenting both hexoses and pentoses in their native form. The “design” of etha — nologenic bacteria like E. coli or Klebsiella oxytoca is an example of such type of modification (see Chapter 6, Table 6.3). Using these recombinant microorganisms allows implementing the co-fermentation process intended to the more complete utilization of the sugars contained in the hydrolyzates of lignocellulosic biomass (Cardona and Sanchez, 2007).