The dry matter content (DM) of the solid straw fraction after wet oxidation (filtercake) is measured by drying at 105°C overnight. The procedure for SSF is as follows: 250 mL flasks are loaded with 100 ml substrate with either 17 g DM or with 8 g DM. The substrate is adjusted to pH 4.8 with 6 mol/L of NaOH. After pH adjustment, 15 FPU/ g DM of Celluclast (Novozymes mixture of endo — and exo-glucanase) is added together with 0.20 ml of Novozym 188 (|3- glycosidase) per ml Celluclast using sterile conditions. The flasks are shaken at 50 °C with 120 rpm for 24 hours during the pre-hydrolysis. After the pre-hydrolysis, 10FPU/ g DM of Celluclast and 0.20 ml of Novozym 188 (|3-glycosidase) per ml Celluclast are added together with 1-4 g/L of Turbo yeast and 0.8 g/L of urea and the pH value re-adjusted to 4.8 with addition of NaOH. The flasks are sealed with glycerol yeast locks and incubated at 37°C with shaking at 120 rpm. During SSF, the flasks are weighed to measure ethanol production with respect to time, and after the SSF termination a sample of the supernatant is analyzed by

HPLC (High Performance Liquid Chromatography, Shimadzu) with a Bio-Rad Aminex HPX87H column and 0.6 mL/min flow of the eluent (4 mmol/L of H2SO4) at 63°C for ethanol and monosaccharides (Thomsen et al., 2009).

The energy required for ethanol reforming can also be provided by the electrical discharge powered by high voltage transformer. The ethanol solution fed can thereafter be ionized to plasma state under such discharge, leading to the creation of a variety of chemically active species and energetic electrons which will quickly react with each other to form product gases. Depending on their energy level, temperature, and electronic density, plasma state can be generally classified as thermal and non-thermal plasma. Compared to thermal plasma, the hydrogen production under non-thermal plasma condition has much lower energy consumption. The features of low temperature operation, rapid reaction start-up, no involvement of catalyst handling, and non-equilibrium properties make non-thermal plasma technique very promising for energy conversion and fuel gas treatment [27]. Comparable performance has been reported through non-thermal plasma process toward hydrogen production, which is very close to the ones obtained from catalytic reactors [28]. However, its relatively high energy requirement, complicated reaction network, and low selectivity remain the main obstacles preventing it from industrial application at current stage.

Since 2004, the world production of cassava roots has been greater than 200 million tons and reaches 240 million tons in 2009 (Food and Agriculture Organization [FAO], 2011; Table 1). The major cassava producers are located in three continental regions which are Nigeria, Brazil and Thailand, accounting approximately for 20, 11 and 12% of total world production, respectively. In the last two decades, the world production of cassava continuously increases (Table 1), as primarily driven by the market demand, in particular an expansion of global starch market. The growth rate of root production in the last decade (2000-2009) is even greater than the previous one (1990-1999) due to markedly rising demand of cassava for bioethanol production in Asia especially in China and Thailand. Interestingly, the root productivity of cassava has been dramatically increased in some countries including Vietnam, India, Indonesia and Thailand by 8.46, 7.46, 6.22 and 5.85 tons/hectare in the past 10 years. The root productivity of India is the greatest (34.37 tons/hectare), followed by Thailand (22.68 tons/hectare) and Vietnam (16.82 tons/hectare) while the world average is

1 The numbers in parenthesis represent the percentage of total world production. n. a. = not available

Source: Food and Agriculture Organization of the United Nations [FAO], 2011

Table 1. Annual production of cassava roots by major producers.

12.64 tons/hectare (Table 2). The world leading cassava producers, i. e. Nigeria and Brazil, however, do not have much improvement in root productivities in the past 10 years; only by 2.10 tons/hectare (from 9.70 to 11.80 tons/hectare since 2000 to 2008) and by 0.35 tons/hectare (from 13.55 to 13.90 tons/hectare since 2000 to 2009), respectively.

The production of cassava can be simply increased by expanding planting areas. Nevertheless, in most regions, no new marginal land is accessible as well as forestry areas are not allowed for area expansion. Moreover, in some countries, there is a competition for land uses among other economic crops such as sugar cane and maize in Thailand. The sustainable and effective means of increasing root production should be achieved by an increase in root productivity. Yields or root productivities of cassava roots vary significantly with varieties, growing conditions such as soil, climate, rainfall as well as agronomic practices. Better root yields can be obtained by well-managed farm practices including time of planting (early of a wet season), land preparation (plowing by hand or mechanically and ridging), preparation of planting materials (ages of mother plants, storage of stems, length & angle of cuttings, chemical treatment), planting method (position, depth of planting and spacing), fertilization (type of fertilizers — chemical vs. organic, dose, time and method of fertilizer application), erosion control, weed control, irrigation and intercropping (Howeler, 2001; 2007). The agronomic practices implemented by farmers vary markedly from regions to regions, depending greatly on farm size, availability of labor, soil and climatic conditions as well as socio-economic circumstances of each region (Table 3). It is very interesting to note that the highest root productivity was reported in India (i. e. 40 tons/hectare) which was irrigated cassava rather than rainfed one, with a highest amount of fertilizer application. In some planting areas such as in Thailand, irrigation is now introduced instead of relying only on rainfall. Yet, the investment cost is high and farmer’s decision is upto market demand, price of cassava roots as well as other competitive crops. By effective farm management, it is expected that the root productivity can be increased twice, from 25 to 50 tons/hectare. By combining that with varietal improvement, the root productivity can be potentially improved upto 80 tons/hectare (Tanticharoen, 2009).

The production cost of cassava is classified into fixed costs and variable costs. The fixed costs include land rent, machinery, depreciation cost and taxes. The variable costs are consisted of labor costs (for land preparation, planting material preparation, planting, fertilizer & chemical application, weeding, harvesting and irrigation) and others including planting materials, chemicals (herbicides, sacks), fuels and tools. Except China, all countries demonstrate that the labor cost is greater than 40% of total production cost. In particular, the labor cost as well as the fixed costs of cassava plantation in India is quite high comparatively to other countries, making their production cost quite high. A semi-mechanized practice for cassava plantation is therefore developed in some countries such as Brazil and Thailand in order to minimize the labor cost, and hence total production cost.

Germinated or sprouted regular and high-tannin sorghums have improved ethanol yields compared to the unmalted kernels. Yan et al. (2009, 2010) reported a reduction in fermentation time and reported higher yields when sprouted sorghum was processed. The improved yield and efficiency is attributed to the action of intrinsic enzymes in starch, proteins and cell walls. Thus, the use of purposely malted or field sprouted sorghums can be advantageous for fuel ethanol biorefineries. Nevertheless, the industries should consider that malting requires important inputs in terms of water, labor, energy for drying and logistics.

A pretreatment step is necessary for the enzymatic hydrolysis process. It is able to remove the lignin layer and to decristallize cellullose so that the hydrolytic enzymes can easily access the biopolymers. The pretreatment is a critical step in the cellulosic bioethanol technology because it affects the quality and the cost of the carbohydrates containing streams (Balat et al., 2008). Pretreatments methods can be classified into different categories: physical, physiochemical, chemical, biological, electrical, or a combination of these (kumar et al., 2009), (Table 3).

On the whole, the final yield of the enzymatic process depends on the combination of several factors: biomass composition, type of pretreatment, dosage and efficiency of the hydrolytic enzymes (Alvira et al., 2010).

The use of enzymes in the hydrolysis of cellulose is more advantageous than use of chemicals, because enzymes are highly specific and can work at mild process conditions. Despite these advantages, the use of enzymes in industrial applications is still limited by several factors: the costs of enzymes isolation and purification are high; the specific activity of enzyme is low compared to the corresponding starch degrading enzymes. As consequence, the process yields increase at raising the enzymatic proteins dosage and the hydrolysis time ( up to 4 days) while, on the contrary, decrease at raising the solids loadings. One typical index used to evaluate the performances of the cellulase preparations during the enzymatic hydrolysis is the conversion rate to say the obtained glucose concentration per time required to achieve it (g glucose/L/h/). Some authors reported conversion rates of softwoods substrates (5%w/ v solids loading) in the range 0.3-1.2 g/L/h (Berlin et al., 2007). In general, compromise conditions are necessary between enzymes dosages and process time to contain the process costs.

In 2001, the cost to produce cellulase enzymes was 3-5$ per gallon of ethanol (0.8-1.32$/liter ethanol), (Novozymes and NREL)[1]. In order to reduce the cost of cellulases for bioethanol production, in 2000 the National Renewable Laboratory (NREL) of USA has started collaborations with Genencor Corporation and Novozymes. In particular, in 2004, Genencor has achieved an estimated cellulase cost in the range $0.10-0.20 per gallon of ethanol (0.03-

0. 05$/liter ethanol) in NREL’s cost model (Genencor, 2004)[2]. Similarly, collaboration between Novozymes and NREL has yielded a cost reduction in the range $0.10-0.18 per gallon of ethanol (0.03-0.047$/liter ethanol), a 30-fold reduction since 2001 (Mathew et al., 2008).

Unlike the acid hydrolysis, the enzymatic hydrolysis, still has not reached the industrial scale. Only few plants are available worldwide to investigate the process (pretreatment and bioconversion) at demo scale. More recently, the steam explosion pretreatment, investigated for several years in Italy at the ENEA research Center of Trisaia (De Bari et al., 2002, 2007), is now going to be developed at industrial scale thanks to investments from the Italian Mossi & Ghisolfi Group.

Among many recombinant yeast strains currently available, we specifically choose S. cerevisiae 1400 (pLNH33) developed by Ho and coworkers (Krishnan et al., 1997). The strain was constructed by transforming the recombinant plasmids with two exogenous genes XYL1 and XYL2 (introduced from xylose-metabolizing Pichia stipitis), and one endogenous gene XKS1 (introduced from S. cerevisiae) into the host strain Saccharomyces yeast 1400 with high ethanol tolerance (Krishnan et al., 1997). The first two genes encode xylose reductase (XR) and xylitol dehydrogenase (XDH), which convert xylose to xylitol, and xylitol to xylulose, respectively, and the last one encodes xylulokinase (XK), which converts xylulose to xylulose-5-phophaste.

The HCM for the recombinant yeast 1400 (pLNH33) is presented below. The model has been previously developed by the authors (Song et al., 2009). The formulation of HCM is

composed of (i) construction of metabolic network, (ii) computation and selection of EMs, and (iii) parameter identification by model fitting.

The plant cell wall structures are highly diverse. Various lignocellulosic species have been used for biofuels production, woods, crop by-products, herbaceous plants, beet pulp, municipal and paper industry wastes. Although all these different biomasses contain typically four major components (i. e. cellulose, hemicelluloses, pectin and lignin), the architecture of the cell wall, the fine biochemical structures of these components and their interactions into the cell wall could be quite different. Nevertheless, cellulose and hemicelluloses leading, with lignins, to the formation of an insoluble, tridimensional network is a constant behavior. A schematic drawing of the plant cell wall polysaccharides is shown in Fig. 1.

As another example of variable composition of plant cell wall, lignins and sugars in cell walls of different origins were quantified (Table 1). Although this study is not exhaustive, it is clear that every plant has its own characteristic. These biomasses have been used as growth substrates for Fusarium graminearum (see paragraph 4).

Regarding cell wall composition variation, it could be postulated that cell wall degradation recalcitrance could be related to cell wall structure and ultra structure variability.

In Thailand, cassava is considered as one of the most important economic crops with the annual production around 25-30 million tons. The role of cassava in Thailand is not only as a subsistent cash crop of farmers, but it also serves as an industrial crop for the production of chips and starch, being supplied for food, feed and other products. This can be indicated by a continuous increase in root production since 2000 and be greater than 20 million tons since 2006. With the national policy on bioethanol use as liquid fuel, it significantly drives a rise in root demand. Various scenarios have been proposed to balance root supply and demand, in order to reduce the conflict on food vs. fuel security. Under the normal circumstance, root surplus should be used for bioethanol production, which initiates another industrial demand of roots and helps stabilize root price for farmers. Figure 11 is an example of projecting plan for root consumption by various industries, which corresponds to the targeted root production, proposed by Ministry of Agriculture and current root demand for chip and starch production. Another scenario is to reduce the amount of exporting chips and allocate those locally to existing industries. Meanwhile, the campaign for increasing root productivity (ton per unit area) by transferring good farming and agricultural practices has been distributed throughout the countrywide. In spite of that root shortage occurs in the last few years, caused by unexpected climatic change and widespread disease, i. e. mealy bugs. This, in fact, critically affects starch industries at a much greater extent than ethanol industry. Nevertheless, the starch industry is more competitive for higher root prices than ethanol industry. This situation of an unusual reduction of root supply emphasizes the need of increasing root production. A short-term policy on increasing root productivity from 25 tons/hectare by good farm management and cultivation practice has continuously pursued and expected to be 50 tons/hectare. Furthermore, long-term plan on R&D for varietal improvement is also greatly significant in order to develop varieties with higher root productivity (potentially be upto 80 tons/hectare), good disease resistance and good adaptation to climatic change such as higher growing temperatures or very dry condition.

There are several enzymes generally used to convert cellulose and hemicellulose into soluble sugars. They are a mixture of pectinases, cellulases and hemicellulases (Lin et al., 2011; Reddy & Yang, 2005). Cellulose can be hydrolyzed by the synergistic action of endo — acting enzymes knows as endoglucanases, and exo-acting enzymes, known as exoglucanases (Lin et al., 2011). Today it is common to employ enzyme complexes consisting of seven or more degrading enzymes that act synergistically. The enzyme mixture is added before or after chemical or mechanical treatments (Reddy & Yang, 2005). Enzymes appear to be the best prospects for continued improvements because can reduce production costs (Gnansounou et al., 2005).

Sipos et al. (2009) observed that the separation of the solid and the liquid phases after chemical pretreatment is beneficial to the whole process because the xylose-rich liquid fraction can be fermented into ethanol through the pentose pathway or as substrate for microbial cellulase production or transformed into other various valuable products. On the other hand, the solid fraction can be further hydrolyzed and fermented into ethanol. The use of alkali treatment before enzyme hydrolysis generated 540 g glucose/kg raw material, equivalent to a 90% conversion of available cellulose to monomeric sugars. On the other hand, 235 g xylose/kg was released after pretreatment of sorghum straw (McIntosh & Vancov, 2010). These hydrolysates were obtained with an enzyme complex containing endoglucanase, exoglucanase, xylanase, beta-glucosidase and cellulase.

1.2 Acidic sulphite wood pulping process and (H)SSL composition

In pulp-and-paper industry the removal of lignin (fibre consolidating material) from wood is carried out during the pulping process to obtain a fibre material (cellulose pulp) suitable for papermaking or as a chemical feedstock. About 10% of chemical pulps are produced worldwide employing sulphite methods. The acidic sulphite chemical pulping is carried out under acidic conditions (pH 1-2) at 135-145 °C for 6-12h in batch digesters using SO2/MeHSO3 (Me — pulping base) aqueous solution (Sjostrom 1993). During sulphite pulping process, lignin and part of hemicelluloses (about 50% based on wood) are dissolved in sulphite spent liquors (SSLs) composed by monomeric sugars already in the fermentable form. Roughly 1 ton of solid waste is dissolved in the spent liquor (SSL 11-14% solids) per ton of pulp produced. SSLs are produced in large amounts, about 90 billion litres annually worldwide (Lawford 1993). SSL is usually burned, for chemical and energy recovery after its concentration by evaporation (Fig. 7). The utilization of SSL is considered for a long time to produce value-added products fitting well to the biorefinery concept (Lawford et al. 1993; Marques et al. 2009).

Fig. 7. Representation of acidic sulphite wood pulping process with Spent Sulphite Liquor release

For this reason, the use of raw materials like SSL is advantageous over other agro-forestry wastes, since the more complex lignocellulosic components were previously hydrolysed, releasing most of the sugars as monosaccharides. Consequently this process is already cost — effective for pulp production, improving the 2nd generation bioethanol process economy from SSL (Lawford et al. 1993; Helle et al. 2008; Marques et al. 2009; Xavier et al. 2010). However, besides monosacharides, SSL contains several fermentation inhibitors that require a preliminary detoxification step (Lawford et al. 1993; Xavier et al. 2010).

The major organic components of SSLs are, lignosulphonates, and sugars, and their composition varies notably among softwoods and hardwoods (Table 2). Softwood sulphite spent liquor (SSSL) from coniferous, yields a high proportion of hexose sugars content (about 76%), mainly mannose and glucose, while HSSL, from hardwood Eucalyptus globulus, produces a liquor with high content of pentose sugars (xylose about 70%). Hexoses bioprocessing is well studied and already implemented in different processes, while pentoses are difficult to use as feedstock for industrial bioprocesses, because pentoses are not fermented by the yeasts currently used on ethanol production, namely Saccharomyces cerevisiae. Therefore, while the use of SSSL has been studied since 1907, when SSSL was used in Sweden for bioethanol production and also during the World War II, for yeast production as a source of protein and vitamins, the HSSL bioprocessing only recently become investigated (Lawford et al. 1993; Helle et al. 2008; Marques et al. 2009; Xavier et al. 2010). Pichia stipitis, recently reclassified as Scheffersomyces stipitis (Kurtzman and Suzuki, 2010), is the most studied yeast capable to convert pentoses to ethanol. However, this yeast is highly sensitive to HSSL inhibitors, namely formic and acetic acids, furfural, levulinic acid and phenolics. For this reason, HSSL needs a special pretreatment for inhibitors removal, which is another technical issue to consider (Helle et al. 2008; Xavier et al. 2010).