The extension of substrate utilisation is critical to determine the economic viability of ethanol production from LCB. This presumes a complete conversion of sugars presented in feedstocks to ethanol under industrial conditions. Under an industrial context, the microorganism chosen should meet some requirements, which are discussed in relation to four benchmarks: (1) Process water economy; (2) Inhibitor tolerance; (3) Ethanol yield; (4) Specific ethanol productivity. Several species of bacteria, yeast and filamentous fungi naturally ferment sugars to ethanol. Each microorganism has its advantages and disadvantages, some can use only hexoses for producing ethanol and others can use both, hexoses and pentoses, but many times with low ethanol yields (Hahn-Hagerdal et al. 2007). The mixture of sugars obtained after LCB hydrolysis, besides glucose, also contains other sugars e. g. xylose, mannose, galactose, arabinose and also some oligosaccharides. Therefore, in the fermentation process, microorganisms ferment these sugars into bioethanol according to reactions presented below. The calculation of the theoretical maximum yield should follow equation 1 for pentoses or equation 2 for hexoses:

3C5H10O5 ^ 5C2H5OH + 5CO2 (1)

C6H12O6 ^ 2C2H5OH + 2CO2 (2)

According to these equations, the theoretical maximum yield is 0.51 g bioethanol and 0.49 g carbon dioxide per g of xylose and glucose.

In order to obtain an economically feasible conversion process of any biomass, it is imperative that the microorganisms chosen should be able to convert efficiently all the sugars present into the desired end product, in this case bioethanol (Chu et al. 2007; Hahn — Hagerdal et al. 2007; Matsushika et al. 2009). The ideal yeast for bioethanol production from LCB should consume the sugars present and provide high production yields as well as specific productivities. Moreover it should not suffer any inhibition from the other components of the raw material (Hahn-Hagerdal et al. 2007).

One of the most effective and well-known ethanol producing microorganisms from hexose sugars is the yeast S. cerevisiae. This yeast is successfully employed at industrial scale, allowing for high ethanol productivity, since it bears high tolerance to ethanol and to inhibitors normally present in lignocellulosic residues. However, this yeast is unable to ferment xylose to ethanol efficiently, though it can only ferment its isomer, xylulose (Jeppsson et al. 2006; Chu et al. 2007; Hahn-Hagerdal et al. 2007; Matsushika et al. 2009). Some yeasts were reported to be efficient in xylose conversion to ethanol, such as, P. stipitis, Candida shehatae and Pachysolen tannophilus (Huang et al. 2009). Among them, P. stipitis exhibits the best potential for industrial application due to the high ethanol yield obtained (Huang et al. 2009). Nevertheless, this yeast is sensitive to organic acids, including acetic acid, which are present in lignocellulosic residues. These compounds inhibit both cell growth and the bioethanol production (Bajwa et al. 2009; Huang et al. 2009). Although, wild type S. cerevisiae cannot ferment xylose to ethanol, several genetic engineered strains have been already developed (Hahn-Hagerdal et al. 2007; Mussatto et al. 2010). Other yeasts, like P. stipitis, can naturally utilize both types of sugars with high yields and its use for producing 2nd generation bioethanol from HSSL is being developed (Xavier et al. 2010). Hence, it is important to improve the yeast strain with the most promising characteristics in order to optimize ethanol production from LCB hydrolysates through genetic engineering and/or strain adaptation (Chu et al. 2007; Hahn-Hagerdal et al. 2007; Matsushika et al. 2009). Table 7 summarizes the fermentation performance of several yeasts in different media. Among bacteria, the most promising for industrial implementation are Escherichia coli, Klebsiella oxytoca and Zymomonas mobilis. Z. mobilis is the bacteria which has the lowest energy efficiency resulting in a higher ethanol yield (up to 97% of theoretical maximum). However, this bacterium is only able to ferment glucose, fructose and sucrose to ethanol. Another problem appears when the medium has sucrose, due to the formation of the polysaccharide levan (made up of fructose), which increases the viscosity of fermentation broth, and of sorbitol, a product of fructose reduction that decreases the efficiency of the conversion of sucrose into ethanol (Lee et al. 2000). K. oxytoca, an enteric bacterium, found in paper, pulp streams and different sources of wood, is able to grow at low pH (minimum 5.0) and temperatures up to 35 °С. This bacterium is able to grow either on hexoses or pentoses, as well as on cellobiose and cellotriose (Lee et al. 2000; Cardona et al. 2007; Chen et al. 2010b).

Yeast Strain Description Type of medium Detoxification method Fermentative process Xylinitial (gL-1) Ye (gff1) Reference Pichia stipitis BCRC21777 Wild type Rice straw hydrolysate Overliming Batch 30°C;100rpm 21 0.37 (Huang et al. 2009) BCRC21777 Adapted Rice straw hydrolysate Overliming Batch 30°C;100rpm 21 0.45 (Huang et al. 2009) NRRL Y -7124 Adapted HSSL Overliming Batch 30°C 40 0.30 (Nigam 2001a) NRRLY-7124 Wild type HSSL Ion-exchange resins Batch 29°C;180rpm 21 0.48 (Xavier et al. 2010) Saccharomyces cerevisiae ADAP8 XYLAi, XKSl/ SUT1 Complex None Batch 30°C;200rpm 20 0.35 (Madhavan et al. 2009) MA-N5 XYL1/XYL2/X KS1 Complex None Batch 45 0.36 (Matsushika et al. 2009) MA-R4 XYL1/XYL2/X KS1 Complex None Batch 45 0.35 (Matsushika et al. 2009) MA-R5 XYL1/XYL2/X KS1 Complex None Batch 45 0.37 (Matsushika et al. 2009) TMB3400 n. a.2 Spruce hydrolysate n. a2. Fed-batch 6 0.43 (Hahn — Hagerdal et al. 2004) TMB 3006 n. a2. Spruce hydrolysate n. a2. Fed-batch 6 0.37 (Hahn — Hagerdal et al. 2004) MT8-1 XYL1/XYL2/X KS1 Lignocellulosic Hydrolysate Biological with enzymes Batch 30°C;100rpm 9 0.41 (Katahira et al. 2006) F12 XYL1/XYL2/X KS1 Vinasse residue Biological with enzymes Batch 30°C;300rpm 6 0.27 (Olsson et al. 2006)

Cf) T3 (D

Several metabolic engineering and genetic modification strategies to enhance an efficient fermentation of xylose to ethanol were studied for S. cerevisiae (Chu et al. 2007; Hahn — Hagerdal et al. 2007; Matsushika et al. 2009). Although the genes that allow for xylose utilization are present in S. cerevisiae, they are expressed in low levels resulting in production rates of ethanol from xylose ten times lower than the verified for glucose as substrate (Chu et al. 2007; Hahn-Hagerdal et al. 2007). In pentose-fermenting yeasts, xylose catabolism begins with its reduction to xylitol by a NADH — or NADPH-dependent xylose reductase (XR), as seen in Fig. 9. Then, xylitol is oxidized to xylulose by NAD-dependent xylitol dehydrogenase (XDH) (Chu et al. 2007; Hahn-Hagerdal et al. 2007; Bengtsson et al. 2009). Xylulose is phosphorylated by the enzyme xylulokinase (XK) to produce xylulose-5- phosphate (X5P). This enters in glycolytic pathway and then in the pentose phosphate pathway (PPP). The formed intermediates are converted to pyruvate in the Embden — Meyerhof-Parnas pathway. Under anaerobic conditions, fermentation of pyruvate occurs by decarboxylation promoted by pyruvate decarboxylase to acetaldehyde which is then reduced to ethanol by alcohol dehydrogenase (Chu et al. 2007; Hahn-Hagerdal et al. 2007).

The most straightforward metabolic engineering strategy was the expression of a bacterial xylose isomerase (XI) gene, so that xylose can directly be converted to xylulose (Jeppsson et al. 2006). The XI gene from the thermophilic bacterium Thermus thermophilus was successfully expressed in S. cerevisiae, generating xylose-fermenting recombinant strains (Karhumaa et al. 2005). Also, the genes of Piromyces sp. XI were also successfully expressed in S. cereviasiae (Kuyper et al. 2003). Another possible metabolic engineering strategy consisted in expressing fungal XR and XDH genes. Stable xylose-fermenting S. cerevisiae strains were obtained by integrating the P. stipitis XYL1 and XYL2 genes encoding XR and XDH, respectively, and over expressing the endogenous XKS1 gene encoding xylulokinase (XK) (Bengtsson et al. 2009; Matsushika et al. 2009). However, ethanol yield attained with these strains was far from the theoretical maximum of 0.51 g. g-1, as can be seen in Table 7 because the metabolic pathway stopped in xylitol. This situation was attributed to the fact that since XR is NAD(P)H-dependent and XDH is strictly NAD+-dependent the relation between the two cofactors sometimes becomes unbalanced (Jeppsson et al. 2006; Chu et al. 2007; Bengtsson et al. 2009).

Wahlbom and Hahn-Hagerdal (2002) found that the addition of electron acceptors such as acetoin, furfural and acetaldehyde re-oxidized NAD+ needed by XDH and decreased the amount of xylitol formed. Shifting the cofactor utilization in the XR step from NADPH to NADH was also a successful strategy for decreasing xylitol (Jeppsson et al. 2006). Since S. cerevisiae lacks the xylose-specific transporter, another common approach is to express in this microorganism the gene that encodes the transport of monosaccharides from P. stipitis (Van Vleet et al. 2009). Hence, xylose uptake occurs by facilitated diffusion mainly through non-specific hexose transporters, which have lower affinity for xylose (Matsushika et al. 2009). This approach enhanced xylose fermentation to ethanol by S. cerevisiae (Van Vleet et al. 2009).

In addition to metabolic engineering, natural selection of strains and random mutation are also alternatives to obtain improved xylose-fermentative yeasts. These evolutionary engineering approaches were successfully applied to several S. cerevisiae strains for effective xylose fermentation. These methods are particularly useful since they are non-invasive and can identify bottlenecks in the xylose metabolic pathway that can then be targeted to be overcome by genetic engineering (Chu et al. 2007; Matsushika et al. 2009). Chu and Lee (2007) suggested that an intense selection pressure will favour the presence of S. cerevisiae mutants able to grow slowly on xylose.

Recent studies have redirected their attention to the xylose-fermenting yeast, P. stipitis. In this case, the major issue is the inhibitors tolerance which can be critical when real raw materials are tested. Hence, an evolutionary strategy has been adopted. The strains adaptation was normally accomplished by sequential transfer of culture samples to different media composed by increasing concentrations of the residue in study (Mohandas et al. 1995; Bajwa et al. 2009; Huang et al. 2009). To accelerate the mutations, ultra violet radiation (UV) was also tested by Bajwa and co-workers (Bajwa et al. 2009).

Many challenges in ethanol production from xylose using metabolically engineered strains were being overcome. Several approaches were successfully employed to engineer xylose metabolism. Nevertheless, these approaches are insufficient for industrial bio-processes mainly due to the low fermentation rate of xylose when compared with glucose. Another bottleneck is the lack of tolerance to the major inhibitors present in lignocellulosic feedstocks. A successful fermentation of LCB hydrolysates requires not only a producing strain that consumes all the sugars present but with tolerance towards lignocellulose degradation products. Moreover, most of the methodologies tested were applied to defined synthetic media containing pure substrates and their applicability to real complex substrates should be validated. However, the composition of the inhibitors in raw materials as lignocellulosic wastes changes frequently and, consequently, the metabolic engineering method probably need some modifications to be applied (Hahn-Hagerdal et al. 2007; Matsushika et al. 2009). Metabolic engineering approaches to improve inhibitor tolerance were so far limited to the over expression of specific enzymes including laccase, phenylacrylic acid decarboxylase, glucose 6-phosphate dehydrogenase and alcohol dehydrogenase (Hahn-Hagerdal et al. 2007). These enzymes can transform some of the inhibitors (mainly the aromatic compounds) into products that microorganisms can assimilate.

In brief, the technical and economic issues related to the choice of fermenting microorganism are the conversion efficiency uniformity, the tolerance to inhibitors, the process requirements (aeration, temperature, pH, sterilization) and the bioprocess licensing (Lawford et al. 1993). Further intensive studies that combine functional genomics analysis with metabolic engineering are required for developing robust yeast strains, tolerant to several inhibitors and to the variability of the substrate and with the ability to ferment xylose from lignocellulosic feedstocks, in order to produce ethanol, at similar rates as those attained with glucose, to be applied at industrial level (Chu et al. 2007; Hahn-Hagerdal et al. 2007; Matsushika et al. 2009).