Sensitivity analysis reveals the dependence of the ethanol productivity on the overexpression of enzymes catalyzing EM fluxes. The sensitivity of the ethanol productivity is calculated as follows:

Sensitivity of Peth — M, j dPETH, j є {1,2,…,12} (8)

Peth daM, j

The sensitivity plot (Fig. 3.1(a)) shows that all xylose-consuming EMs (EM4 to EM7) are effective in increasing the ethanol productivity but the highest sensitivity is found among glucose-consuming modes (i. e., EM2). Both can contribute to increasing the productivity but in different ways. The former (i. e., amplifying fluxes of EM4 to EM7) promotes the simultaneous consumption of mixed sugars as illustrated in Fig. 1.1(b). On the other hand, the latter (i. e., amplifying EM2 flux) effectively increases the biomass formation as the growth rate of EM2 is the highest among others.

It should be noted that information provided from the sensitivity analysis is local because it shows only the change of productivity with respect to the "infinitesimal" change of enzyme
expression level. It is more important to know how the productivity will change with respect to the "appreciable" change of enzyme levels. This information on nonlinear cellular behaviors can be acquired from dynamic simulations. The results are shown in Fig. 3.1(b) where mixed-sugar-consuming modes (EM8 to EM12) are excluded due to their negligible level of activation (Song et al., 2009). From this investigation, EM6 (red line) is chosen as the "best" mode, while EM2 is the second.

Non-monotonic profiles are observed in Fig. 3.1(b). For example, as the overexpression level of mode 5 increases, the productivity goes up initially but comes down afterwards. This may be seen as the outcome of competition between amplification of throughput flux of EM4 (i. e., benefit) and metabolic burden (i. e., cost).

Genome, transcriptome and proteome studies lead to interesting but perturbing questions. Several studies indicate that fungi could secrete up to 50 different CWDE in order to degrade cellulose, hemicellulose, pectin and, for some of them, lignin. Among these enzymes, some display the same EC number and/or belong to the same glycosyl hydrolase family (GH). Why fungi use up so much energy to secrete enzymes with quite similar activities? Is it true or apparent redundancy?

Several clues indicate that this is apparent redundancy. Most of the CWDE, still putative, wait for potent substrate specificities characterization. By analogy with the enzymes already characterized, it means that slightly different specificities are likely to be discovered and could be essential to complete plant cell wall degradation. Furthermore, quantitative studies performed on Fusarium hemicellulases demonstrate that on hop cell wall, the expression level of the 30 putative enzymes varies greatly from 1 (the less abundant) to 1500 (Hatsch et al., 2006). When another biomass is used for growth, the pattern of secreted enzymes is different, clearly indicating that there is no "general response" to the presence of plant material but specific responses to a given biomass. Taken together these studies mean that
the fungus exhibits a large flexibility in its response. Then it could be thought that the observed redundancy actually reflects the multitude of different structures of plant cell wall. Consequently, it is of primary importance that in silico studies should be carefully validated by enzymatic measurements. For example, an exponential increase of putative CAZY (Carbohydrate-Active enZYmes) described is observed, but unfortunately only a small proportion of them are biochemically characterized yet (Cantarel et al., 2009). Whereas entries in CAZY database increased exponentially from 1999 to 2007 (a 14-fold increase), less than 10% of them have been enzymatically characterized and less than 1% of the enzyme structures have been solved. This means that there is a real lack in enzyme knowledge regarding to the huge potential of new activities undiscovered yet. It should be noticed that the increasing difference between the number of putative enzymes and well characterized ones also lead to the possibility of mis-annotation and/or false identification. This phenomenon has been known for a long time by molecular biologists and correction of errors and inconsistencies in data bases became an authentic research area (Ghisalberti et al., 2010). In order to thoroughly characterize the enzyme activities and their capacity to degrade the complex structures found in plant cell wall, CWDE substrate specificity should be determined with both artificial and natural substrates. This absolute necessity drives us to perform the characterization of the enzyme cocktail produced by F. graminearum on hop cell wall. We used 29 different substrates, poly-or oligosaccharides, natural or artificial (Phalip et al., 2009). Enzyme activities were evaluated by assays of the products (monomers) or by their visualization by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). The conclusion of this study is that the enzymes constituting this cocktail are no more putative but active on each layer of the plant cell wall. On the opposite, the enzyme cocktail produced on glucose displays very tiny activities, furthermore on a small number of substrates. The proof is then provided that to get a large diversity of cell wall degrading enzymes, it is very important to choose the right substrate for a given fungus to grow.

In this chapter, starch as carbon source will be primarily discussed in the application for single-step or direct bioconversion. Starch is a polysaccharide and the most abundant class of organic material found in nature. Sources of starch that are normally used in the production of ethanol are derived from seeds or cereals such as corn, wheat, sorghum, barley, soy and oat. Other sources of starch can be from tuber or roots such as potato, yam or cassava. By using starch as substrate for bioethanol production has distinct advantages in terms of its economical pretreatment and transportation compared to other types of biomass. For example cassava or tapioca tuber that has received an enormous attention in the production of biofuel in particular bioethanol in East Asia region such as China, Thailand, Malaysia and Indonesia (Dai et al., 2005; Hu et al., 2004; Nguyen et al., 2006). Cassava is a perennial woody shrub, ranks second to sugarcane and is better than both maize and sorghum as an efficient carbohydrate producer under optimal growing conditions. It is also the most efficient producer under suboptimal conditions of uncertain rainfall, infertile soil and limited input encountered in the tropic (Fregene and Puonti-Kaerlas, 2002).

Before undergo conventional or traditional fermentation, starch regardless of its sources required to be hydrolyzed. Two types of hydrolysis usually applied are mineral acid hydrolysis and enzymatic hydrolysis. The mineral acid or acid-base involved in the hydrolysis can be of diluted or concentrated form. The dilute acid process at 1-5% concentration is conducted under high temperature and pressure and has fast reaction time in the range of seconds or minutes. The concentrated acid process on the other hand uses relatively mild temperatures and the reaction times are typically much longer as compared to those in the dilute acid hydrolysis. The biggest advantage of dilute acid processes is their fast reaction rate, which facilitates continuous processing for hydrolysis of both starch and cellulosic materials. Their prime disadvantage is the low sugar yield and this has opened up a new challenge to increase glucose yields higher than 70% (especially in cellulosic material) in an economically viable industrial process while maintaining high hydrolysis rate and minimizing glucose decomposition (Xiang et al., 2004; McConnell, 2008). The concentrated acid hydrolysis offers high sugar recovery efficiency, up to 90% of both hemicelluloses and cellulose sugars. Its drawback such as highly corrosive and volatility can be compensated by low temperatures and pressures employed allowed the use of relatively low cost materials such as fiberglass tanks and piping. Without acid recovery, large quantities of lime must be used to neutralize the acid in the sugar solution. This neutralization forms large quantities of calcium sulfate, which requires disposal and creates additional expense. In addition to that, this type of hydrolysis has resulted in the production of unnatural compounds that have adverse effect on yeast fermentation (Tamalampudi et al., 2009).

Enzymatic hydrolysis of starch required at least two types of enzymes. This is due to that the starch or amylum comprises of two major components, namely amylose, a mainly linear polysaccharide consisting of a-1,4-linked n-glucopyranose units and the highly branched amylopectin fraction that consists of a-1,4 and a-1,6-linked n-glucopyranose units (Knox et al., 2004). Depending on type of plants, starch typically contains 20 to 25% amylose (van der Maarel et al., 2002) and 75 to 80% amylopectin (Knox et al., 2004). These two type linkage, a-1,4 and a-1,6-linked required an efficient starch hydrolysis agent or enzyme that can fraction a-1,4 and promote a-1,6 debranching activity. Since starch contains amylose and amylopectin, single or mono-culture cells are usually added during fermentation stage where starch has already been hydrolyzed to reducing sugar by hydrolysis agent such as acid-base or microbial enzymes in pretreatment and saccharification steps. The microbial enzyme of a — amylase cleaves a-1,4 bonds in amylose and amylopectin which leads to a reduction in viscosity of gelatinized starch in the liquefaction process. The process is the hydration of starch by heating the starch in aqueous suspension to give a-amylase an access to hydrolyze the starch. Dextrin and small amount of glucose and maltose are the end products. Exoamylases such as glucoamylase is then added during saccharafication which hydrolyses 1,4 and 1,6-alpha linkages in liquefied starch (van der Maarel et al., 2002). Enzyme has an advantage over acid — based hydrolysis. Amylolytic enzymes hydrolysis work at milder condition with the temperature lower 110°C (Cardona et al., 2010). However, enzyme is expensive especially cellulosic enzyme where it was reported the most expensive route accounted for approximately 22%-40% of total total cost (Wooley et al., 1999; Yang and Wyman, 200; Rakshit, 2006). Furthermore, fermentation of high concentration of starch to obtain high yield of ethanol is unfeasible due to reducing sugar inhibition on enzyme. This was shown in the work of Kolusheva and Marinova (2007) where the high reducing sugar produced from hydrolysis of high concentration not only inhibited the enzyme activity but also the fermenting yeast.

Lei Liang, Riyi Xu, Qiwei Li, Xiangyang Huang, Yuxing An, Yuanping Zhang and Yishan Guo

Bio-engineering Institute, Guangdong Academy of Industrial Technology Guangdong Key Laboratory of Sugarcane Improvement and Biorefinery, Guangzhou Sugarcane Industry Research Institute

P. R. China

1. Introduction

With the ever growing concern on the speed at which fossil fuel reserves are being used up and the damage that burning them does to the environment, the development of sustainable fuels has become an increasingly attractive topic (Wyman & Hinman, 1990; Lynd & Wang, 2004; Herrera, 2004; Tanaka, 2006; Chandel et al., 2007; Dien et al., 2006; Marelne Cot, et al., 2007). The interest partially caused by environment concern, especially global warming due to emission of Greenhouse Gas (GHG). Other factors include the rise of oil prices due to its unrenewability, interest in diversifying the energy matrix, security of energy supply and, in some cases, rural development (Walter et al., 2008). The bioethanol such as sugarcane ethanol is an important part of energy substitutes (Wheals et al., 1999). This chapter was focused on the development and trends of the sugarcane ethanol in China. Based on the analysis of the challenge and the chance during the development of the sugarcane ethanol in China, it introduced a novel process which is suitable for China, and mainly talked about simultaneous production of sugar and ethanol from sugarcane, the development of sugarcane varieties, ethanol production technology, and prospect aspects. We hope it will provide references for evaluation the feasibility of sugarcane ethanol in China, and will be helpful to the fuel ethanol development in China.

Bioethanol production of the first generation from sugar cane and from wheat or corn is well established in Brazil as well as in the US and Europe. The world’s ethanol production in more than 75 countries amounted in 2008 to more than 77 billion litres of ethanol (Sucrogen bioethanol, 2011).

Bioethanol production of the second generation can use lignocelluloses from non-food crops (not counted in the animal or the human food chain), including waste and remnant biomass e. g. wheat straw, corn stover, wood, and grass. These feedstocks are composed mainly of lignocellulose (cellulose, hemicelluloses and lignin).

The process of bioethanol production of the first generation is well established and shown in Fig. 1.

The process of bioethanol production from wheat normally consists of five major process steps:

1. Milling of the grain

2. Liquefication at high temperatures

3. Saccharification (enzymatic degradation of starch)

4. Fermentation with yeast

5. Distillation (rectification) of ethanol

The production of bioethanol from lignocelluloses follows more or less the same principle and is composed of the following sub-steps: milling, thermophysical pretreatment hydrolysis, fermentation, distillation and product separation/processing (Fig. 2).

The cellulose in the lignocellulose is not accessible to enzymes. Therefore, lignin and/or hemicelluloses have to be removed in order to make the enzymatic degradation of the cellulose possible. Ideal pretreatment should lead to better performance during bioethanol production from lignocelluloses.

The pretreament should cause the hydrolysis of hemicelluloses, high recovery of all carbohydrates, and high digestibility of the cellulose in enzymatic hydrolysis. No sugars should either be degraded or converted into inhibitory compounds. A high solid matter content and high concentration of sugars should be possible. The process should have low energy demands and require low capital and operational cost.

The pretreament methods can be classified roughly into three types: thermophysical methods, acid-based methods and alkaline methods. Thermophysical methods like steam pretreament, steam explosion or hydrothermolysis solubilise most of the cellulose and hemicelluloses. There is only a low level of sugar conversion. Cellulose and hemicelluloses have to be converted enzymatically into C6 sugars (mainly glucose) and to C5 sugars (mainly xylose). Acid-based methods use mineral acids like sulphuric acid and phosphoric acid. Hemicelluloses are degraded to sugar monomers, cellulose has to be converted to glucose enzymatically. Alkaline methods like ammonia fibre explosion leave some of the hydrocarbons in the solid fraction. Hemicellulases acting both on solid and dissolved hemicelluloses are required as well as the celluloytic enzymes.

Lignocellulose containing substrates are mainly composed of cellulose (40-50%), hemicellulose (25-35%) and lignin (15-20%). Cellulose is a glucose polymer, hemicellulose is a heteropolymer of mainly xylose and arabinose, and lignin is a complex poly-aromatic compound. The different pretreatment methods are necessary to loosen the close bonding between cellulose, hemicellulose and lignin. Wheat (Triticum aestivum L.) straw is composed of 45% cellulose, 26% hemicellulose and 19% lignin. Maize (Zea mays) straw is composed of 39% cellulose, 30% hemicellulose and 17% lignin.

The high percentage of hemicelluloses and the resulting pentoses, e. g. xylose from the hydrolysis of the polymer, are a further challenge to a cost-competitive bioethanol process with lignocelluloses as carbon source.

Yeasts used for the conversion of sugars into ethanol (mostly Saccharomyces spec.) usually only convert glucose into ethanol. C5 sugars like xylose are only converted into ethanol at low rates by very few yeast (Pichia spec.) strains. Research programs are underway either to adapt yeasts for the use of both C5 and C6 sugars or to modify Saccharomyces genetically to obtain yeast that produces ethanol simultaneously from C5 and C6 sugars.

Nevertheless, because of its ready availability and low costs, lignocellulosic biomass is the most promising feedstock for the production of fuel bioethanol. Large-scale commercial production of bioethanol from lignocellulose containing materials has still not been implemented.

1.2 Development of a gene transfer system for the mycelia of F. velutipes

As shown in above, we found the edible mushroom F. velutipes Fv-1 strain to be an efficient ethanol producer, and, we demonstrated its preferable properties of ethanol fermentation from various sugars (Mizuno et al., 2009b), whole crop sorghums and rice straw (Mizuno et al., 2009a). However, the strain can only slightly convert pentoses, which account for approximately 20-30% of plant cell walls, into ethanol (Mizuno et al., 2009a). Therefore, genetic engineering of the pentose metabolism is necessary to make possible the ethanol fermentation from pentose. Furthermore, more efficient (low cost) conversion of biomass to ethanol could be expected if saccharification ability was strengthened by expressing cellulases. A transformation method of F. velutipes by the electroporation protocol for basidiospores has been reported (Kuo et al., 2004), but it requires a long period to produce basidiospores because it must go through fruiting body formation, and cannot eliminate the risk of contamination in the process of spore harvest. Since screening of many transformants is needed for improvement of the metabolic pathway by genetic engineering, the development of a simpler transformation method is desired to obtain high numbers of transformants.

Therefore, an adequate condition for protoplast preparation from mycelia of F. velutipes Fv-1 strain was investigated, and simpler a transformation protocol for this fungus was developed by the calcium-PEG method and the restriction enzyme-mediated-integration (REMI) method.

First, we constructed a pFvT vector for transformation of the F. velutipes Fv-1 strain (Fig. 8A). The vector possessed a F. velutipes tryptophan synthetase gene promoter and terminator (GenBank no. AB028647) to regulate expression of the constructed genes, and the hygromycin phosphotransferase gene (hph) from Escherichia coli as selection marker. The hph gene was obtained from pCAMBIA1201 vector (CAMBIA; http://www. cambia. org/).

Next, conditions to prepare protoplast from the mycelia of F. velutipes were optimized by modifying a method for Phanerochaete sordida (Yamagishi et al., 2007). The F. velutipes Fv-1

MCS, multiple cloning sites; Ftrp-p, trpl promoter from F. velutipes; Ftrp-t, trpl terminator from F. velutipes; Fgpd-p, gpd promoter from F. velutipes; Fgpd-t, gpd terminator from F. velutipes; hph, hygromycin B phosphotransferase gene; ampr, ampicilin resistance gene; Eori, pUC19 ori. (Reproduced from Maehara et al., 2010b)

Fig. 8. Structures of the plasmids used in this study

strain was grown in PCMY (1% polypeptone, 0.2% casamino acid, 1% malt extract, and 0.4% yeast extract) medium at 25°C for 3 d. Then the mycelia were collected and incubated in enzyme solution [1.5% cellulase Onozuka-RS (Yakult Pharmaceutical, Tokyo) and 1.5% lysis enzyme (Sigma, St. Louis, MO) in 0.75 M MgOsm (0.75 M MgSO4, 20 mM MES, pH 6.3)] at 30°C for 5 h. The protoplasts were filtered through Miracloth (Cosmo Bio, Tokyo), washed at twice with 1 M SorbOsm (1.0 M sorbitol, 10 mM MES, pH 6.3), and suspended in SorbOsm plus 40 mM CaCl2 solution to a final concentration of approximately 108 protoplasts ml-1. Genetic transformation was investigated using the pFvT vector and the protoplasts prepared as described above. The transformation procedures for Lentinus edodes (Sato et al., 1998) and Schizophyllum commune (Van Peer et al., 2009) were modified for the transformation of F. velutipes Fv-1. In the course of the transformation process, the effect of the structure of the plasmid DNA on transformation was evaluated using circular and linear pFvT plasmids. Approximately 6-fold transformants were obtained when the plasmid DNA was linearized (Table 2).

Because the REMI method is a popular transformation tool for fungi (Hirano et al., 2000; Maier & Schafer, 1999; Riggle & Kumamoto, 1998; Sato et al., 1998), we evaluated the effect of REMI on the transformation for F. velutipes Fv-1. The F. velutipes Fv-1 strain was transformed by pFvT with a restriction enzyme, BglI, Kpnl, or Pstl. The addition of the restriction enzymes increased the number of transformants by about 1.6- to 5.8-fold (Table 2). The suggests that the addition of restriction enzymes enhanced the transformation efficiency of F. velutipes. Therefore, to find the optimum enzyme concentration for REMI, we

performed transformation using circular pFvT plasmid with the presence of various concentrations of PstI (Fig. 9). As for the results, the number of transformants obtained was affected by the amount of restriction enzyme. The efficiency was significantly increased by the addition of PstI at 25 units, by it gradually decreased when the PstI amount was over 25 units, suggesting that the optimal value for transformation mediated by the PstI is 25 units. In conclusion, we found a simple transformation procedure for the mycelia of F. velutipes Fv — 1 strain by the calcium-PEG method combined with REMI. The transformation method of F. velutipes Fv-1 strain does not require a process of spore formation, because the mycelia could be used as starting material. Moreover, a high efficiency of transformation was obtained by the adoption of REMI.

Multiple techniques have been developed during the past decades to convert bioethanol to hydrogen by following the reaction (1).

C2HsOH(l) + 3 H2O(l) 2 CO2 + 6 H2 (AHr,298K = 348 kJ/mol)

It is clearly observed that 6 moles of hydrogen can be produced per mole of ethanol fed. However, the highly endothermic feature of this reaction requires external energy supply. Depending on the type of energy input, the current hydrogen production technologies can be categorized into two areas: non-thermal including bio, photo, plasma, and thermal — chemical processes. Besides, several hybrid systems have also been recently developed to produce hydrogen relying on the energy supply of more than one source (e. g., photo­fermentation and thermal plasma). Compared to thermochemical conversion, non-thermal hydrogen production can take place at much mild conditions with minimal thermo-energy input requirement from surroundings. However, the biological or photo hydrogen production efficiency is much lower than acceptable scale for industrial application. Unlike biological or photo process, thermochemical conversion can happen at much higher reaction rate, but under relatively severe conditions (e. g., high temperature and pressure) with notable amount of thermo-energy input. In addition to water, CO2 (dry reforming) and O2 (partial oxidation or oxidative reforming) can also act as oxidant to oxidize ethanol for hydrogen production. Among all the available techniques described in details in this section, steam reforming might possess the highest potential to be commercialized in the near term.

As stated in section 3.1, besides water-soluble sugars (sucrose, glucose and fructose), sorghum is composed by structural cell wall carbohydrates primarily cellulose and hemicellulose, which in turn can be hydrolyzed and used as substrate for ethanol production (Sipos et al., 2009).

Sorghum bagasse is the residual fraction obtained after juice extraction from sweet sorghum whereas sorghum straw is the remaining material usually left on the field after threshing. The composition and proportion of fibrous-structural fractions in sorghum is widely reported and varies according to intrinsic and extrinsic factors such as cultivar type, maturity and climatic conditions. An average of 15% of the total weight corresponds to the fibrous portion within a range from 12 to 17% (Woods, 2000).

In sweet sorghum bagasse, average content of cellulose, hemicelluloses and lignin is 34-44%, 27-25%, and 18-20% respectively (Ballesteros et al., 2003; Kim & Day, 2011; Sipos et al., 2009).

Table 1. Fiber composition of different ethanol feedstock 1

Alessandra Verardi1, Isabella De Bari2*, Emanuele Ricca1 and Vincenza Calabro1

1Department of Engineering Modeling, University of Calabria, Rende (CS) 2ENEA Italian National Agency for New Technologies, Energy and the Sustainable Economical Development, Rotondella (MT)

Italy

1. Introduction

Bioethanol can be produced from several different biomass feedstocks: sucrose rich feedstocks (e. g. sugar-cane), starchy materials (e. g. corn grain), and lignocellulosic biomass. This last category, including biomass such as corn stover and wheat straw, woody residues from forest thinning and paper, is promising especially in those countries with limited lands availability. In fact, residues are often widely available and do not compete with food production in terms of land destination. The process converting the biomass biopolymers to fermentable sugars is called hydrolysis. There are two major categories of methods employed. The first and older method uses acids as catalysts, while the second uses enzymes called cellulases. Feedstock pretreatment has been recognized as a necessary upstream process to remove lignin and enhance the porosity of the lignocellulosic materials prior to the enzymatic process (Zhu & Pan, 2010; Kumar et al., 2009).

Cellulases are proteins that have been conventionally divided into three major groups: endoglucanase, which attacks low cristallinity regions in the cellulose fibers by endoaction, creating free chain-ends; exoglucanases or cellobiohydrolases which hydrolyze the 1, 4- glycocidyl linkages to form cellobiose; and p-glucosidase which converts cello — oligosaccharides and disaccharide cellobiose into glucose residues. In addition to the three major groups of cellulose enzymes, there are also a number of other enzymes that attack hemicelluloses, such as glucoronide, acetylesterase, xylanase, p-xylosidase,

galactomannase and glucomannase. These enzymes work together synergistically to attack cellulose and hemicellulose. Cellulases are produced by various bacteria and fungi that can have cellulolytic mechanisms significantly different.

The use of enzymes in the hydrolysis of cellulose is more effective than the use of inorganic catalysts, because enzymes are highly specific and can work at mild process conditions. In spite of these advantages, the use of enzymes in industrial processes is still limited by

Corresponding Author

several factors: most enzymes are relatively unstable at high temperatures, the costs of enzyme isolation and purification are high and it is quite difficult to recover them from the reaction mixtures. Currently, extensive research is being carried out on cellulases with improved thermostability. These enzymes have high specific activity and increased flexibility. For these reasons they could work at low dosages and the higher working temperatures could speed up the hydrolysis reaction time. As consequence, the overall process costs could be reduced. Thermostable enzymes could play an important role in assisting the liquefaction of concentrated biomass suspensions necessary to achieve ethanol concentrations in the range 4-5 wt%.

The immobilization of enzymes has also been proposed to remove some limitations in the enzymatic process (Hong et al., 2008). The main advantage is an easier recovery and reuse of the catalysts for more reaction loops. Also, enzyme immobilization frequently results in improved thermostability or resistance to shear inactivation and so, in general, it can help to extend the enzymes lifetime.

This chapter contains an overview of the lignocellulosic hydrolysis process. Several process issues will be deepened: cellulase enzyme systems and hydrolysis mechanisms of cellulose; commercial mixtures; currents limits in the cellulose hydrolysis; innovative bioprocesses and improved biocatalysts.

Diverse concepts for the use of lignocellulose-containing plants for bioethanol production are available. In the simplest concept, only the glucose is fermented to bioethanol, with the by-products xylose solution and lignin pellets. The xylose sugars can be used as barrier films, hydrogels, paper additives (Soderqvist et al., 2001; Lima et al., 2003; Gronholm et al., 2004) or in xylitol production (reviewed in Chen et al., 2010). At the moment, the utilization of lignin is unsatisfactory; therefore, the lignin pellets are used as solid biofuel.

The economy of bioethanol production from lignocellulose-containing materials can be improved in a cost-effective concept by simultaneous fermentation of both sugars (glucose and xylose) to bioethanol by diverse microorganisms. In the last twenty years, diverse microorganisms were genetically modified to ferment both glucose and xylose, with good results (reviewed in Hahn-Hagerdal et al., 2007; Matsushika et al., 2009; Jojima et al., Kim et al., Mussatto et al., Weber et al, Young et al., 2010). Furthermore, diverse adaptation programs, mutagenesis and breeding were performed to produce yeasts and other microorganisms with improved xylose fermentation (reviewed in Hahn-Hagerdal et al., 2007; Matsushika et al., 2009; Mussatto et al., 2010). However, in several countries production with GMO is only possible under strict standards and acceptance of GMO in these countries is poor.

In a biorefinery concept, co-production of biofuels, bioenergy and marketable chemicals from renewable biomass sources take place simultaneously. Diverse biorefinery concepts for wheat straw were developed such as: bioethanol from glucose, biohydrogen from xylose and the residual effluents from bioethanol and biohydrogen processes being used for biogas production (Kaparaju et al., 2009). The biorefinery concept including higher-value chemical by-products and autonomous power supplies will enhance economic competitiveness of second generation plants and, therefore, will make this type of plant economical in the near future.