In the case of ethanol production from lignocellulosic biomass, the main obstacle to be overcome is the fact that fermenting microorganisms are not able to assimi­late all the sugars released during pretreatment and hydrolysis of biomass in an effective way. Genetic engineering has been contributing to the development of microorganisms exhibiting this feature. To face this challenge, there exist two approaches (Aristodou and Penttila, 2000; Chotani et al., 2000; Zaldivar et al., 2001). The first one consists of modifying microorganisms in such a way that they can assimilate a wide spectrum of substrates, e. g., by introducing the metabolic pathways required for the utilization of xylose or arabinose to good ethanologenic microorganisms such as yeasts and Z. mobilis. The second approach is based on the modification of microorganisms that assimilate a great variety of substrates; in this case, genes encoding the conversion of pyruvate into ethanol are introduced to microorganisms, like Escherichia coli, capable of assimilating hexoses and pen­toses. Some examples illustrating these two approaches are shown in Table 6.3.

Through clonation of genes encoding xylose reductase and xylitol dehydro­genase in S. cerevisiae, the conversion of xylose into xylulose via xylitol can be achieved. The xylulose is a pentose, which can be assimilated by the yeasts. Unfortunately, the productivity is low and xylitol is formed as a by-product, which deviates part of the substrate that could be utilized for ethanol synthe­sis (Claassen et al., 1999). Ingram and Doran (1995) report the development of recombinant strains of gram-negative bacteria (E. coli, K. oxytoca, or Erwinia sp.) in whose chromosomes have been inserted into the genes of Z. mobilis encod­ing the metabolic pathway for ethanol production. In this way, these strains can efficiently convert all the sugars released during the hydrolysis of cellulose and hemicellulose. The saccharification of cellulose is more complicated, although one of the obtained recombinant strains of K. oxytoca has the natural ability for degrading cellobiose that employs a lower dosage of added cellulases for the effective conversion of purified cellulose into ethanol by a simultaneous hydro­lysis and fermentation process. In their documented review on the fermentation

of lignocellulosic hydrolyzates, Olsson and Hahn-Hagerdal (1996) discuss the performance of different pentose-utilizing microorganisms, among them several recombinant strains. Fermentation indexes for lignocellulosic hydrolyzates pre­treated and detoxified by different methods are also provided.

Lynd et al. (2002) point out that the feasibility of a CBP (see Chapter 9, Section 9.2.4) for lignocellulosic ethanol process will be established when a microorgan­ism or microbial consortium can be developed according to one of two strategies. The first of them, called native cellulolytic strategy (A in Figure 6.5), is oriented to engineer microorganisms having a high native cellulolytic activity in order to improve the ethanol production through the increase of their yield or tolerance, i. e., by the improvement of the fermentative properties of a good producer of cel — lulases. In this case, the modifications in the process microorganism should be aimed at reducing or eliminating the production of by-products, such as acetic acid or lactate, and at increasing the ethanol tolerance and titres (Cardona and Sanchez, 2007). Recent studies have demonstrated the possibility of obtaining ethanol-tolerant strains of C. thermocellum growing at ethanol concentrations exceeding 60 g/L, a titer not sufficient to put thermophiles at a disadvantage rela­tive to more conventional ethanol producers (Lynd et al., 2005).

The recombinant cellulolytic strategy (B in Figure 6.5) contemplates the genetic modification of microorganisms that present high ethanol yields and tol­erances in such a way that they are capable of utilizing cellulose within a CBP configuration, i. e., making a microorganism with good fermentative properties to produce cellulases (Cardona and Sanchez, 2007). The second strategy is con­sidered more difficult due to the complexity of cellulases system (Begum and Aubert, 1994). Currently, the production of cellulases by bacterial hosts produc­ing ethanol at high yield such as engineered E. coli, K. oxytoca, and Z. mobilis and by the yeast S. cerevisiae has been studied. For instance, the expression of cellulases in K. oxytoca has allowed an increased hydrolysis yield for microcrys­talline cellulose and an anaerobic growth on amorphous cellulose. Similarly, sev­eral cellobiohydrolases have been functionally expressed in S. cerevisiae (Lynd et al., 2005). Undoubtedly, ongoing research on genetic and metabolic engineering will make possible the development of effective and stable strains of microorgan­isms for converting cellulosic biomass into ethanol. This fact will surely lead to a qualitative improvement in the industrial production of fuel ethanol in the future (Cardona and Sanchez, 2007).

One of the bottlenecks of ethanol production from biomass is the high cost of enzymes, as noted above. The current cost of producing lignocellulolytic enzymes by submerged fermentation mainly using T. reesei, is about US$0.40 to 0.60/gal ethanol, but an increase in the specific activity of the enzymes or in the efficiency of their production through genetic engineering can be expected. This would allow an eventual cost reduction of the enzymes to US$0.07/gal ethanol, as suggested by (suggested by Tengerdy and Szakacs, 2003). However, these authors consider that the genetic improvement of fungi producing these enzymes by solid — state fermentation could have a greater potential than the genetic improvement of fungi synthesizing the same enzymes by submerged fermentation, considering the fact that fungi growing on the surface of biomass have enzymatic complexes with optimal characteristics and proportions for the hydrolysis of lignocellulosic materials. On the other hand, the U. S. Department of Energy has contracted with the world enzyme-producing leaders, Novozymes (Denmark) and Genencor (USA), with the aim of developing research for improving cellulase systems and reducing their costs. The research must be oriented not only to the enhancement of yield, stability, and specific activity of cellulases, but also to the development of an enzyme mixture that will remain active under hard conditions related to such steps as acid pretreatment (Mielenz, 2001).

Undoubtedly, if the worldwide use of fuel ethanol uses the development of the technology of lignocellulosic biomass utilization, genetic engineering will be called to supply the “tailored” microorganisms needed to meet the exigen­cies of this new technology. From this, the importance of the metabolic pathway engineering is inferred. Metabolic pathway engineering is aimed at establishing metabolic pathways and production hosts, which are capable of delivering opti­mal flow of carbon from substrate to final product at high yields and volumetric productivities. In particular, pathway engineering can achieve the integration of the process at the molecular level through the optimization of the primary meta­bolic pathways for the synthesis of the targeted product (Chotani et al., 2000).