The metabolic network encompasses all the primary reaction routes involved in the anaerobic growth of recombinant yeast such as glycolytic and pentose phosphate pathways, citric acid cycle, and reactions for pyruvate metabolism. In addition, two oxidoreductase reactions from xylose to xylulose catalyzed by the heterologous expression of XR and XDH enzymes are incorporated. Biochemical reactions participating in the metabolism of recombinant yeast are listed up in Table 2.1.

1.2 Introduction on phytopathogenicity, saprophytism

The primary choice of a microorganism (fungus) potentially providing cell wall degrading enzymes should be directed toward one naturally present in plant environment i. e. a phytopathogen or a saprophyte. Considering the ecology, fungi are qualified as "decomposers" in the opposite to plants, the producers and animals, the consumers. Some fungi, called saprotrophs, get nutrients from dead organisms, especially plants. Some other are pathogen, attacking living organisms. Invasive growth thanks to hyphae gets fungi very adapted to penetrate plants. Hyphae diameter (2-10 pm) permit cell penetration and their hyphal growth in several directions allow them to colonize quickly the plant material with very close contact. Many saprotrophs, phytopathogens and other fungi living in plant environment developed tools for gaining energy from plants during their evolution. Cell wall degrading enzymes (CWDE) are one of these tools which are also efficient for bioethanol production. This is the reason why this chapter focuses on CWDE produced and secreted by some fungi.

Presently, there are 17 factories operating with the total production capacity of 2.575 million liters/day but most of them have operated under their full production capacity due to oversupply of ethanol. The influencing factor for decreasing ethanol demand is other alternative energy for transportation, i. e. Liquid Petroleum Gas (LPG) and Compressed Natural Gas (CNG), being promoted by the government. LPG is a primary fuel for household use such as for cooking that is why it is important to control the price of LPG (being low at 18 Baht per kilogram or 0.59 USD per kilogram). This promotes an increase usage of LPG in automobile sector, as indicated by increasing automobile engine change from gasoline to LPG. For CNG, there are several policies being released to promote the use

of CNG in automobile sector in order to reduce the amount of gasoline consumption. Firstly, there is an exemption of tax for CNG fuel tank. Secondly, government agrees to cover the cost of changing car engine to CNG-using engine for taxi countrywide and tax reduction for CNG fuel cost. With the cost of production and fuel itself, the actual price is at 14.75 Baht per kilogram but the selling price is only at 8.50 Baht per kilogram. This difference requires a significant amount of subsidized oil fund to compensate the gap. At present, the government led by Ministry of Energy, has considered different mechanisms to intensify ethanol demands in transportation sector by promoting use of gasohol with a higher ethanol component (E85 and E100) for Flexible Fuel Vehicle (FFV), use of ethanol in motorbikes and use of ethanol as diesohol for trucks. These applications need technical support to acquire consumer’s confidence. In addition, supporting policy and effective mechanism for exporting bioethanol can help expand market demand.

The fiber chemical hydrolysis process can produce a large number of sugar degradation products which are known to inhibit bacteria and yeast and thus the conversion of fermentable sugars into bioethanol (Ban et al., 2008). The most important inhibitors are furfural, 5-hydroxymethylfurfural and acetic acid. After the acid hydrolysis, it is necessary to adjust the pH with alkalis in order to obtain the adequate conditions for the subsequent step of fermentation. Lime or calcium hydroxide is commonly added to increase the pH to 9­10. This alkali treatment precipitates inhibitors in the form of insoluble salts and therefore acts as detoxifying treatment (Kurian et al., 2010).

As mentioned before, during the conversion of LCB into monomeric sugars, other type of products are formed and some of them can be strong inhibitors in fermentation bioprocesses. When compared to the fermentation of pure sugars, LCB hydrolysates present slower kinetics with a lower ethanol yield and productivity and in some cases a complete inhibition of growth and ethanol production can be observed. The variety and concentration of toxic compounds in feedstocks depend on both, the raw material and the pretreatment conditions applied for polysaccharides hydrolysis. The maximum concentration allowed for each inhibitor, without losing fermentation efficiency, depends on several factors: the origin of toxic compound, the inhibition mechanism, the microbial strain used and its physiological state, and also the fermentative process technology, the dissolved oxygen concentration in the medium and the pH (Mussatto et al. 2004).

Selection of a detoxification methodology for a specific feedstock is mandatory for attaining good results in 2nd generation bioethanol production. The identification of the main and relevant inhibitors present in the feedstocks is crucial in order to choose a specific, efficient and low-cost detoxification methodology. Besides, this knowledge can helps to establish the best conditions in hydrolysis pretreatment in order to minimize the inhibitors formation.

Fermentation inhibitors are conventionally classified in four groups according to their origin in lignocellulosics and hydrolysis processing: sugar degradation products, lignin degradation products, compounds derived from extractives and heavy metal ions (Parajo et al. 1998; Mussatto et al. 2004). Sugar degradation products are formed during hydrolysis and the main compounds produced are furfural from pentoses and 5-hydroxymethylfurfural (HMF) from hexoses as mentioned above. Furfural can inhibit cell growth, affecting the specific growth rate and cell-mass yield (Palmqvist et al. 2000b). However, it was noticed that some bioethanol-producing microorganisms like Pichia stipitis are not affected by furfural in low concentrations up to 0.5 g. L-1 (Mussatto et al. 2004). Moreover it could have a positive effect on cell growth. Nigam (2001) referred that ethanol yield and productivity were not affected by 0.27 g. L-1 of furfural. However concentrations above 1.5 g. L-1 interfered in respiration and inhibited cell growth almost completely, decreasing ethanol yield in 90% and productivity in 85% (Nigam 2001b). HMF has an inhibitory effect similar to that of furfural, but at a lower extension. Usually HMF is present in lower concentrations than furfural, due to its high reactivity and also due to the experimental conditions in the hydrolysis process that degrades lower amounts of hexoses. It was reported that HMF increases the lag phase extension and decreases cell growth (Delgenes et al. 1996; Palmqvist et al. 2000b). Mussatto et al. (2004) reported that a synergistic effect occurs when these compounds are combined with several other compounds formed during lignin degradation. Different compounds, aromatic, polyaromatic, phenolic, and aldehydic can be released from lignin during hydrolysis of LCB materials, and they are considered more toxic to microorganisms than furfural and HMF, even in low concentrations. Phenolic compounds are the most toxic products for microorganisms present in lignocellulosic hydrolyzates. They promote a loss of integrity in biological membranes, thus, affecting their ability as selective barriers and as enzyme matrices and decreasing cell growth and sugar assimilation (Parajo et al. 1998; Palmqvist et al. 2000b). Syringaldehyde and vanillic acid affect cell growth (Mussatto et al. 2004; Cortez et al. 2010) and the ethanolic fermentative metabolism of several microorganisms, like P. stipitis (Delgenes et al. 1996). In SSL, these compounds are normally present in the sulphonated form, due to the cooking process (Marques et al. 2009).

Extractives (acidic resins, taninic, and terpene acids) and also acetic acid derived from acetyl groups present in the hemicellulose are released during the hydrolytic processes. In terms of toxicity, the extractives are considered less toxic to microbial growth, than lignin derivatives or acetic acid (Mussatto et al. 2004). Gallic acid and pyrogallol are low molecular weight phenolic compounds normally formed from hydrolysable tannins (Marques et al. 2009) and some authors have shown anti-fungal properties of these phenolics (Dix 1979; Panizzi et al. 2002; Upadhyay et al. 2010). Acetic acid is also known as antimicrobial compound and the mechanism of inhibition is well-understood. At low pH, in the undissociated form, it can diffuse across the cell membrane, promoting the decrease of the cytoplasmatic cell activity and even causing cell death (Lawford et al. 1998; Mussatto et al. 2004). It has been reported that acetic acid inhibition degree depends not only on its concentration, but also on oxygen concentration and on pH of fermentation medium (Vanzyl et al. 1991). Another type of inhibitors are heavy metal ions, namely iron, chromium, nickel and copper, which result from reactors corrosion during the acidic hydrolysis pretreatment. Their toxicity acts at metabolic pathways level, by inhibiting enzyme activity (Mussatto et al. 2004).

As previously mentioned, a detoxification step is required before the hydrolysates undergo fermentation. Therefore, after identification of the toxic compounds, the choice of the best hydrolysate detoxification method is crucial for an effective and economical feasible detoxification methodology, in order to improve the fermentative process (Mussatto et al. 2004; Sanchez et al. 2008). Three different approaches have been described to decrease the concentration of inhibitors: (1) prevention of formation of inhibitors during the pretreatment step as mentioned before; (2) detoxification of the raw-material before fermentation; (3) development of microorganisms able to resist to inhibition.

Xavier and co-workers (2010) reported HSSL containing nearly 25 g. L-1 of xylose to P. stipitis for bioethanol production. Four increasing concentrations of HSSL were accessed to evaluate its toxicity. The results showed that increasing HSSL content in the fermentation medium decreased dramatically the maximum cell growth rate (pmax), ethanol yield (Yp/s) and productivity (qpm) attained. It was reported that HSSL content higher than 40% (v/ v) was critical for bioethanol production (Table 3). Acetic acid has been appointed as the main inhibitor of P. stipitis and other microorganisms (Schneider 1996; Lawford et al. 1998; Nigam

2001a). After the removal of acetic acid, ethanol fermentations were still unsuccessful, meaning that other compounds present had a toxic effect (Xavier et al. 2010).

Several biological, physical and chemical detoxification methods were developed in order to reduce inhibitor concentrations. The efficiency of detoxification methodology depends on chemical composition of the hydrolysate, as well as on microorganism chosen for bioethanol production (Mussatto et al. 2004; Helle et al. 2008; Sanchez et al. 2008). For this reason, the detoxification methods cannot be directly compared since mechanisms of inhibition and degree of toxicity removal are completely different (Palmqvist et al. 2000a).

Evaporation with vapour and vacuum evaporation are physical detoxification methods, in order to reduce the concentration of volatile compounds present in the hydrolysates, such as acetic acid, furfural and formaldehyde, and at the same time, to increase sugars concentrations. However, these methods also increase the non-volatile toxic compounds content, such as extractives and lignin derivatives. A balance between these two effects should be achieved or, consequently, the degree of fermentation inhibition will increase. Furthermore, the energy required for these processes should be properly considered to attain a potential economical gain (Lawford et al. 1993; Mussatto et al. 2004). As mentioned above, in the particular case of HSSL, evaporation is already implemented in the pulp production process for liquor concentration, to prepare it to burn for energy and chemical recovery. This is an advantage for HSSL bioconversion, and it is possible to optimise the evaporation stage, in order to get a good balance between volatile and non-volatile toxic compounds and sugar concentration for the fermentation process. Additionally, the condensate obtained in this step is rich in furfural and acetic acid, that can be easily extracted and purified for selling purposes as added-value products (Evtuguin et al. 2010). Alkali treatment, in particular overliming, is the most common detoxification method and is considered one of the best technologies. This method consists on the addition of lime (Ca(OH)2), or other alkali compound such as sodium or potassium hydroxide, until pH 9-10 promoting the precipitation of toxic compounds. Acetic acid, furfural, HMF, soluble lignin and phenolic compounds are mostly removed with this methodology, increasing the fermentability of hydrolysates. Several authors obtained the best results with alkali treatment using calcium hydroxide (Lawford et al. 1993; Martinez et al. 2001; Helle et al. 2008; Sanchez et al. 2008). Martinez et al. (2001) reported for sugarcane bagasse hydrolysate at 60 °C that the addition of Ca(OH)2 to adjust the pH to 9.0, promoted the precipitation of furanic and phenolic compounds. The obtained results showed a removal of nearly 51% and 41% respectively, of furans and phenolics with only 8.7% of sugars loss. Lawford et al. (1993) also used Ca(OH)2 for HSSL treatment at pH 10, followed by neutralisation to pH 7 with 1N of H2SO4. This methodology resulted in the improvement of the volumetric productivity and conversion efficiency, 92%, of bioethanol production by a recombinant strain of Escherichia coli.

Toxic compounds can also be removed by adsorption. Several authors have studied the capacity of removal of toxic compounds using different materials as adsorbents such as, activated charcoal (Dominguez et al. 1996; Lee et al. 1999; Mussatto et al. 2001; Canilha et al. 2004) and ion-exchange resins (Vanzyl et al. 1991; Larsson et al. 1999; Lee et al. 1999; Nilvebrant et al. 2001; Xavier et al. 2010). In particular, a specific strategy of adsorption on ion-exchange resins was employed by Xavier et al. (2010) to toxic compounds removal from HSSL for subsequent sugar purification and then ethanol fermentation with P. stipitis (Fig. 8).

In order to remove the cations added during pulping processing, namely Mg2+, HSSL was initially treated with a cation-exchange resin column. Then free carboxylic acids and polyphenols, including lignosulphonates, were separated from sugars with an anion — exchange resin in the second column. This process provided a transparent solution (sugars faction) containing essentially neutral monomeric sugars with traces of neutral polyphenolics (Table 4). However, this separation process released the sugars with some dilution and a concentration step was required for fermentation. This procedure led to excellent results of ethanol production by P. stipitis: high fermentation efficiency, 96%, productivity, 1.22 g. L-Lh’1, and yield, 0.49 g of ethanol / g of sugar.

Biological methods for detoxification of hydrolysates involve the use of specific enzymes or microorganisms that can degrade or consume the toxic compounds present in the hydrolysates. Jonsson et al. (1998) reported an increasing glucose consumption and ethanol productivity when wood hydrolysates were detoxified with laccase and peroxidase enzymes from Trametes versicolor, a white-rot fungus. These oxidative enzymes have the capability to degrade acid and phenolic compounds (Jonsson et al. 1998). The use of

anot detected

microorganisms was also proposed to remove inhibitors from HSSL. Xavier and co-workers

(2010) presented the first approach for HSSL biological detoxification, specifically for acetic acid removal. Four yeasts commonly used for acetic acid removal from wine were chosen, Candida tropicalis, Candida utilis, S. cerevisiae and Pichia anomala, and results are presented in Table 5.

According to these results, S. cerevisiae was selected for biological deacidification of HSSL. Sequential strategy of deacidification by S. cerevisiae and fermentation by P. stipitis on 60% of HSSL was carried out. Despite the acetic acid consumption by S. cerevisiae, xylose fermentation by P. stipitis produced only cell biomass, and no ethanol was detected in the medium. These results clearly showed the presence of other toxic compounds from HSSL, eventually phenolic compounds, probably inhibiting the sugars conversion to ethanol by P. stipitis (Xavier et al. 2010).

A different approach for performing biological detoxification of HSSL, with better results, was made in the same research group, using the Paecilomyces variotti filamentous fungus. This fungus can be found in air and soils of tropical countries, and has been studied for single cell protein (SCP) production, another important added-value product, normally used in animal feeding (Nigam 1999). Besides, P. variotti presents a good performance to grow in residues like HSSL and consumes substrates, including phenolic compounds, as carbon source. Pereira et al. (2011) showed for the first time the possibility of using this fungus to detoxify HSSL hydrolysates for subsequent ethanol fermentation. The biological treatment with P. variotti yielded HSSL with very low levels of acetic acid. Moreover, toxic compounds like gallic acid, pyrogalol and other low molecular phenolics were completely consumed and metabolized by P. variotti, indicating that this detoxification method can be suitable for treating HSSL into a proper feedstock for further bioprocessing. A successful fermentation of this detoxified HSSL by P. stipitis was performed, attaining an ethanol yield of 0.24 gethanol. gsugars-1. However, more research is required in order to improve the ethanol fermentation yields and productivities (Pereira et al. 2011).

Comparing the four different detoxification methodologies described, ion-exchange resins provided the best results on subsequent bioethanol fermentation (Table 6). High percentages of different toxic compounds from the hydrolysate were removed and provided the highest ethanol yield (0.49 g. g-1) and volumetric productivity (1.22 g. L-1.h-1). However, ion-exchange resins are expensive and difficult to implement and operate in large scale industries. P. variotti treatment, despite the fact of having promoted low ethanol fermentation yields in preliminary results (Table 6), appeared to be a very promising detoxification method. Furthermore the biomass of P. variotti can be used as SCP for animal feeding, increasing the economic potential of the process. More research work is being developed to combine this coupled strategy of biological detoxification of HSSL with simultaneous SCP production (Pereira et al. 2011). Other approaches for detoxification of hydrolysates were proposed and different methods can be used sequentially to improve their own capacity (Mussatto et al. 2004).

Satoshi Kaneko, Ryoji Mizuno, Tomoko Maehara and Hitomi Ichinose

National Food Research Institute

Japan

1. Introduction

Plant cell walls are the most abundant biomass source in nature and are of increasing importance because worldwide attention has now focused on bioethanol production to combat global warming and to safeguard global energy. Because of competition between food and fuel production, lignocelluloses are expected to be utilized for future fuel ethanol production. One of the major problems in producing ethanol from lignocellulosic biomass is the expensive production cost. Consolidated bioprocessing (CBP) is gaining recognition as a potential breakthrough for low-cost biomass processing (Lynd, 1996; Lynd et al., 2002; Lynd et al., 2005; Van Zyl et al., 2007; Xu et al. 2009). CBP of lignocellulose to bioethanol refers to the combination of the 4 biological events required for this conversion process (production of lignocellulose-degrading enzymes, hydrolysis of polysaccharides present in pre-treated biomass and fermentation of hexose and pentose sugars) in one reactor. However, no natural microorganism exhibits all the features desired for CBP. Bacteria and yeast have been the primary candidates for CBP research and some progress has been made in this regard. Traditionally, proponents of CBP processes have identified two primary developmental pathways capable of producing industrially viable CBP microbial strains. These are category I, engineering a cellulase producer, such as Clostridium thermocellum, to be ethanologenic; and category II, engineering an ethanologen, such as Saccharomyces cerevisiae or Zymomonas mobilis, to be cellulolytic (Lynd, 1996; Lynd et al., 2002; Lynd et al., 2005; Van Zyl et al., 2007; Xu et al., 2009). However, the both categories have advantages and disadvantages. Cellulase producer lacks ethanol tolerance, and it is very difficult to coexpress of multiple saccharification enzyme genes in ethanol producer. Especially, heterologous expression of Trichoderma reesei cellobiohydrolases (cellobiohydrolase I and cellobiohydrolase II), which play the crucial role in cellulose degradation, are generally poor.

Basidiomycetes, also known as wood-rotting fungi, can achieve the complete breakdown of lignins (Cooke & Rayner, 1984; Cullen, 1997), and are considered primary agents of plant litter decomposition in terrestrial ecosystems (Thorn et al., 1996). Furthermore, some basidiomycetes produce alcohol dehydrogenases, thus allowing the production of wine using a mushroom (Okamura et al., 2000; Okamura et al., 2001). These properties of basidiomycetes appear suitable for use in CBP. In a preliminary study, we screened some edible mushrooms for their ability to produce ethanol and found that Flammulina velutipes is a good producer of ethanol. F. velutipes is a white-rot fungus that grows from spring through

late autumn on a variety of hardwood tree stubs and dead stems and is widely distributed in temperate to subarctic regions. Currently, F. velutipes is the most produced mushroom in bed cultivation in Japan, the annual production being 130,000 tons/year. Artificial cultivation of mushrooms in polypropylene bottles is popular in Japan. F. velutipes has been characterized as wide adapted strain for various kinds of substance of artificial cultivation media, thus suggesting that the strain may be useful in the conversion of a wide variety of biomass types.

In this study, we investigated the properties of ethanol fermentation by F. velutipes to determine its suitability for CBP, because the use of basidiomycetes for bioethanol production is not common and the ethanol fermentation abilities of basidiomycetes are not well characterized. Furthermore, several biomass such as sorghums and rice straw were used as raw material to evaluate the detail conversion from biomass to ethanol by F. velutipes.

Yeast is an efficient industrial host with a high productivity of ethanol and with well- developed genetic tools. However, yeast does not possess endogenous cellulolytic ability. Several heterologous cellulases have, therefore, been expressed in yeast for direct conversion of cellulose into ethanol. Endoglucanase genes from Bacillus spp. were successfully integrated (randomly, at approximately 44 sites) into the chromosome of yeast, resulting in the direct conversion of cellodextrin into ethanol (Cho et al. 1999).

With the advent of cell-surface display technologies, it has become possible to express artificial cellulosomes (rather than free cellulases) in yeast. Cellulosomes facilitate the assembly of different cellulolytic enzymes in close proximity, and thereby favor a proper synergy between the enzymes (Tsai et al. 2010). Surface display of endoglucanase from T. reesei and ^-glucosidase from A. aculeatus in yeast helped in the successful conversion of barley ^-glucan into ethanol with 93% of the theoretical yield and without any pretreatment (Fujita et al. 2002). Co-displaying the exoglucanase from Aspergillus spp. along with the endoglucanase and ^-glucosidase in yeast has resulted in the direct conversion of amorphous cellulose into ethanol (Fujita et al. 2004; McBride et al. 2005). Very recently, recombinant yeast has been further modified to express ^-glucosidase within the cell. A high-affinity transporter for cellobiose and cellodextrin has also been cloned into the recombinant yeast. This strain co-metabolizes xylose and cellobiose more efficiently (Ha et al. 2011).

Although several studies have demonstrated efficient ethanol production from amorphous cellulose, attempts to engineer yeast to hydrolyze crystalline cellulose have been unsuccessful because of low exoglucanase activity (la Grange et al. 2010). The exoglucanase and ^-glucosidase activities in recombinant cellulolytic yeast strains are insufficient to support growth with cellulose as a sole carbon source. Hence, a synthetic yeast consortium has been developed with four engineered yeast strains, each expressing either the scaffoldin from Clostridium spp. and Ruminococcus spp. or the three enzymes, namely, exo — and endoglucanases from Clostridium spp. and ^-glucosidase from Ruminococcus spp. (Fig 4) (Tsai et al. 2010). However, investigators have been unable to completely decipher the efficiency of the synthetic consortium, because the ratio of the different cellulases needed for a proper synergy has not been established. A cocktail 8-integration tool has been developed in yeast to predict the optimum ratio of different cellulases, but with little success (Yamada et al. 2010).

Another major problem with recombinant cellulase expression is that heterologous cellulases are made to function at a suboptimal temperature. The optimal temperature for the growth of recombinant hosts is 37°C, but cellulases are more active at temperatures

above 50°C. Therefore, the thermotolerant yeast Kluyveromyces marxianus has been engineered to display thermostable endoglucanase and ^-glucosidase on its surface. This engineered, thermostable yeast ferments ^-glucan directly to ethanol at 48°C (Yanase et al. 2010).

4.2 E. coli

The broad substrate range of E. coli, together with its ample genetic tools and its substantial fermentation capacity, renders the species to be a potential candidate for bioethanol production. E. coli, with chromosomally integrated genes encoding pyruvate decarboxylase and alcohol dehydrogenase, is an efficient ethanol producer (Ohta et al. 1991). Several attempts have been made to engineer cellulolytic ability in ethanologenic E. coli. The species also has endogenous cryptic genes for cellobiose metabolism and an endoglucanase for the hydrolysis of soluble cellulose (Park and Yun 1999; Kachroo et al. 2007; Vinuselvi and Lee 2011). Achieving a higher extracellular titer of cellulases is a bottleneck in the development of a recombinant cellulolytic E. coli for ethanol production. E. coli does not have a proper protein secretion system (Shin and Chen 2008). Because E. coli is a gram-negative bacterium, it has an outer membrane rich in peptidoglycan, which acts as a barrier for protein secretion. The extracellular protein concentration observed with E. coli is 0.0088 g/L, one hundred-fold less than that observed with native cellulolytic organisms (Qian et al. 2008; Xu et al. 2009;

Vinuselvi et al. 2011). Gram-negative bacteria possess five different protein-export pathways (Types I-V), two of which are found in E. coli (Type I and Type II).

Several attempts have been made to increase the extracellular titer of recombinant proteins in E. coli: by exploiting the Sec/TAT signal sequence (Zhou et al. 1999; Angelini et al. 2001), by fusion of recombinant proteins with extracellular proteins such as OsmY (Qian et al. 2008), or by increasing membrane permeability (Shin and Chen 2008). Cellulase secretion in E. coli has been achieved through the expression of endoglucanase, along with the out genes of Erwinia chrysanthemi, under the control of a surrogate promoter (Zhou et al. 1999). Deletion of Ipp weakens the outer membrane, allowing any proteins targeted to the periplasmic space to be secreted into the medium. Approximately 70% of the cellulases produced were secreted into the medium in an lpp knockout E. coli strain (Shin and Chen 2008). Several studies have used OsmY as a fusion partner for recombinant protein secretion in E. coli. However, this technique has not been exploited for cellulase secretion because of the large size of cellulases (Aristidou and Penttila 2000; Qian et al. 2008) (Fig 5).

The cellobiose metabolic operon from Klebsiella oxytoca has been introduced into E. coli, but the expression level of the cellobiose transporter and metabolic genes was poor, and hence could not support the growth of E. coli on cellobiose (Moniruzzaman et al. 1997). Cellulases from several species of Clostridium, Bacillus, Cellulomonas, and Ruminococcus have been expressed and characterized in E. coli (Hinchliffe 1984; Zappe et al. 1986; Fierobe et al. 1991; ReverbelLeroy et al. 1996; Lam et al. 1997; ReverbelLeroy et al. 1997; Lee et al. 2008; Li et al. 2009). Co-expression of endoglucanase from B. pumilus and ^-glucosidase from

Fervidobacterium spp. in E. coli favored growth of the recombinant strain, with soluble carboxymethyl cellulose (CMC) as the sole carbon source (Rodrigues et al 2010).

Sergio O. Serna-Saldivar* Cristina Chuck-Hernandez, Esther Perez-Carrillo and Erick Heredia-Olea

Departamento de Biotecnologia e Ingenieria de Alimentos, Centro de Biotecnologia. Tecnologico de Monterrey, Monterrey, N. L.

Mexico

1. Introduction

Nowadays, there is a growing interest for alternative energy sources because of the reduction of fossil fuel production. Ethanol used as automotive fuel has increased at least six times in the current century. According to the Renewable Fuels Association, in 2010 the USA bio-refineries generated 13 billion gallons of fuel ethanol and the year before worldwide production reached 19 billion. This noteworthy increment is in its majority based on maize and sugar cane as raw materials (Berg, 2004; Renewable Fuels Association, 2010). The use of these feedstocks has triggered concerns related to food security especially today when the world population has reached 7 billion people.

The relatively sudden rise in food prices during 2008, 2010 and 2011 has been attributed mainly to the use of maize for bioethanol even when other factors like droughts or changes in global consumption patterns have also played a major role (World Food Program, 2008). Food price projections indicate that this situation will worsen, breaking the downward trend registered in food prices in the last thirty years (The Economist, 2007).

Even if there was not a food-fuel controversy especially due to the current conversion of millions of tons of maize for bioethanol, the use of only this crop cannot support the ambitious objectives of renewable fuel legislation in countries like the United States of America, where a target of 36 billion gallons of liquid biofuels have been established for 2022. In order to meet this requirement all the 333 million tons of maize yearly produced by USA should be channelled to biorefineries. This production represents 2 and 16 times the maize harvested in countries like China and Mexico respectively, which in turn are two of the five top world producers.

Environmental factors have been also pushing for the quest of new crops dedicated exclusively for liquid automotive fuel in order to reduce the use of prime farming land, irrigation water and other resources. A dedicated energy crop ideally must meet several requirements such as: high biomass yield and growth rate, perennial, with reduced input necessities, fully adapted to the geographic regions where will be planted, easy to manipulate via genetic improvement, non-invasive, tolerant to stress and with a good carbon sequestration rate among others (Jessup, 2009). At the present time, energy crops are

mainly represented by perennial grasses as switchgrass (Panicum virgatum L.), energy cane (Saccharum spp), sweet and forage sorghum (Sorghum bicolor), miscanthus (Miscanthus spp.) as well as other short-rotation forest resources (willow — Salix spp- and poplar — Populus spp) (Jessup, 2009; McCutchen et al., 2008).

The development of new and improved enzymes, bioprocesses and feedstocks could lead to cost reduction from an estimated of 0.69 cents to below 0.51 cents/L that nowadays is the benchmark established for starchy raw materials (Kim & Day, 2011). Besides the development of dedicated crops for energy, one of the best approaches for cost reduction and optimal use of resources is the use of flexible facilities allowing the integration of different streams of same or different feedstocks. Flexibility, balance, diversification and regionalization are indeed keywords in the development of solutions to meet future world energy demands.

In tropical, subtropical, and arid regions from the United States, Mexico, China, India, Southern Africa, and other developing countries, where agronomic harsh conditions prevail, one of the most promising crops for fuel is sorghum (Sorghum bicolor (L.) Moench) (Reddy et al., 2005; Zhang et al., 2010). This is a high efficient photosynthetic crop that reached a worldwide production of 56 million tons of grain in 2009 (FAOSTAT, 2011), just behind maize, wheat, rice and barley. Almost 30% of this production is harvested in North America where sorghum is mainly used for feed. Sorghum is a C4 plant, highly resistant to biotic and abiotic factors as insects, drought, salinity, and soil alkalinity. Furthermore, this crop has one of the best rates of carbon assimilation (50 g/m2/day) which in turn allows a fast growth and a better rate of net CO2 use (Prasad et al., 2007). Sorghum requires one third of the water with respect to sugar cane and 80 to 90% compared to maize (Almodares & Hadi, 2009; Wu et al., 2010b). Thus, sorghum is considered as one of the most drought resistant crops. Furthermore, sorghum requires approximately one third of the fertilizer required by sugar cane (Kim & Day, 2011) and its growth cycle is between 3 to 5 months allowing two or three crops per year instead of one commonly obtained with sugarcane. Besides environmental advantages, sorghum is one of the more acquiescent plants to genetic modification because is highly variable in terms of genetic resources and germplasm. This facilitates plant breeding and development of new cultivars adapted to different regions around the globe (Zhang et al., 2010).

Sorghum can be classified in four broad groups: grain, sweet, forage and high biomass. All belong basically to the same species and virtually there are no biological or taxonomic differences (Wang et al., 2009). Grain sorghum is used mainly as food, feed and for starch production. In the United States only a small percentage of fuel ethanol (around 2-3%) is obtained from grain sorghum (Renewable Fuels Association, 2010; Turhollow et al., 2010; Zhao et al., 2008), but in 2009 about 30% of the U. S. grain sorghum crop was used for ethanol production (Blake, 2010).

On the other hand, forage sorghum is characterized as a high biomass crop. This capacity has been boosted by intensive research programs worldwide, focused in the design of new varieties tailored for ethanol production (Rooney et al., 2007). The main product obtained from sweet sorghums is the fermentable sugar rich juice that is produced and accumulated in the stalks in a similar fashion as sugar cane. The extracted sweet juice is mainly composed of sucrose, glucose, and fructose, and thus can be directly fermented into ethanol with efficiencies of more than 90% (Wu et al., 2010b). According to Almodares & Hadi (2009) sorghum yields a better energy output/input ratio compared to other feedstocks such as sugar cane, sugar beet, maize and wheat. Altogether with the juice, the residue or bagasse can be also converted to ethanol or used for other traditional applications.

In summary, sorghum is a crop well adapted to adverse climatic conditions which at this time is one of the growing concerns in agronomic projections. This is mainly due to the change of rain patterns and climate, greenhouse effect and the steadily rise of world temperature. Given all these advantages of sorghum as a potential source of biofuels, the objective of this chapter is to explore its potential, as an integrated crop for fuel production in terms of yield and technologies available for processing. The chapter especially focuses on optimum technologies to produce bioethanol from sweet sorghums, starchy grains and biomass from dedicated crops.

Traditionally, ethanol has been produced batch wise. However, high labor costs and the low productivity offered by the batch process have led many commercial operators to consider the continuous fermentation. Continuous fermentation can be performed in different kind of bioreactors — stirred tank reactors or plug flow reactors. Continuous fermentation often gives a higher productivity, offers ease of control and is less labor intensive than batch fermentation (Cheng et al., 2007). However contamination is more serious in this operation (Skinner & Leathers, 2004). In the fuel ethanol industry, control of bacterial contamination is achieved by acidification and using antibiotics such as penicillin G, streptomycin, tetracycline (Aquarone E,1960; Day et al., 1954), virginiamycin(Hamdy et al., 1996; Hynes et

al., 1997; Islam et al., 1999), monensin(Stroppa et al., 2000), or mixtures thereof. Fig 9 shows the process of continuous fermentation of molasses and sugarcane juice to produce ethanol. A high cell density of microbes in the continuous fermenter is locked in the exponential phase, which allows high productivity and overall short processing of 6 — 12 h as compared to the conventional batch fermentation (30 — 60 h). This results in substantial savings in labor and minimizes investment costs by achieving a given production level with a much smaller plant.

Fig. 7. Approach for evolutionary engineering
Temperature
• л	8 °С
Ethanol 15%(V/V)

The pretreatment of the straw was carried out in a steam explosion pilot unit using a reactor with a reaction volume of 0,015 m3. Explosion was carried out into a cyclonic separator to separate the treated straw from the vapour phase. The vapour was then condensed in a regenerative cooler. The maximum steam temperature of the steam generator was 200°C

(equivalent to a steam vapour pressure of 1.55 MPa). The operation temperature in the reactor is reached via a temperature ramp. In most experiments the mass of added water was 1.0 kg per kg of straw. The operation temperature was generally 200°C and the duration of the treatment was usually 10 minutes. This results in a severity factor of 9500 (log (R0) = 3.98).

The bulk density of the straw in the reactor was 60 kg m-3 for chopped straw. When straw pellets (mixture of Triticale (Triticosecale Wittmack) and wheat straw) were pretreated, the bulk density increased to 520 kg m-3. However, in these cases the volumetric use of reactor volume had to be reduced. Also the ratio of added water was lower.

The steam consumption in the pilot tests was more than two times the calculated value due to only partial thermal insulation of the reactor. In the case of a cold start of the system, steam consumption was even higher.

Figs. 5a and 5b show an example of wheat straw before and after pretreatment. The scanning electron microscope (SEM) images show wheat straw with intact bundles of fibres before preatrement (Fig. 5a) and the same material after pretreatment (Fig. 5b), where the morphological structure has been broken down. This material is now accessible to the cellulytic enzyme complex.

Fig. 5a. Wheat straw untreated (SEM)

Fig. 5b. Wheat straw treated (SEM)