To make possible genetic manipulation in F. velutipes, we constructed the pFvT plasmid containing the hygromycin phosphotransferase gene (hph) under the control of the

tryptophan synthetase gene (trpl) promoter, and developed an easy transformation method for F. velutipes by the REMI method (Maehara et al., 2010a). Here, we focused on the promoter of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene because many tools such as promoters and selection markers are desirable for effective metabolic pathway engineering of F. velutipes Fv-1. The gpd promoters are the most frequently used constitutive promoters in basidiomycetes. GPD constitutes up to 5% of the soluble protein in Saccharomyces cerevisiae and other higher eukaryotic organisms (Piechaczyk et al., 1984; Punt et al., 1990), and gpd mRNA accounts for 2-5% of the poly (A)+ RNA in yeast (Holland & Holland, 1978).

In this section, we described that construction of new plasmids having the hph gene from Escherichia coli as a selection marker, which regulated by the gpd promoter and the potency of the gpd promoter from F. velutipes were evaluated.

First we constructed three vectors, pFvG, pFvTgh, and pFvGgh, by modification of pFvT. The pFvT vector possessed a trp1 promoter and terminator regulating the expression of the constructed genes, and the hph gene as selection marker (Fig. 8A, Maehara et al., 2010a). Vectors pFvG (Fig. 8B) and pFvGgh (Fig. 8D) contained the gpd promoter and the terminator of F. velutipes (Kuo et al., 2004) located upstream and downstream of a multiple cloning site (MCS), and both pFvTgh (Fig. 8C) and pFvGgh (Fig. 8D) contained the gpd promoter and the terminator located upstream and downstream of the hph gene (Maehara et al., 2010b).

To determine the potency of the gpd promoter, we compared transformation efficiency by the gpd promoter with that by the trp1 promoter. Gene integrations were performed by the REMI method. Protoplasts were prepared from mycelia of the F. velutipes Fv-1 strain, and then plasmids were transformed into the protoplasts with PstI (25 U). As shown in Table 2, about 10 transformants (10.7 to 12.3) were obtained by the transformation of pFvT and of pFvG, which contain the hph gene controlled by the trp1 promoter. In contrast, as for the results of the transformation of pFvTgh and pFvGgh, the numbers of transformants were significantly increased and about 24.7 to 33.3 transformants were obtained, suggesting that the activity of the gpd promoter was higher than that of the trp1 promoter in F. velutipes Fv-1. There is a difference of about 500-bp in the length of pFvT and pFvG, or pFvTgh and pFvGgh, but no significant difference in the number of transformants obtained by pFvT and by pFvG, and by pFvTgh and pFvGgh was not observed. It might suggest, that the difference of the sizes of these plasmids was not affected on transformation efficiency.

To compare the activity of the gpd and the trp1 promoter, the expression levels of the hph gene in each transformant were examined by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from each set of three transformants and equal amounts of RNAs from each set of three clones were mixed and used as template for RT — PCR. As shown in Fig. 10A, the intensities of the bands of the pFvTgh and pFvGgh transformants were stronger than that of the pFvT and pFvG transformants (upper panel), suggesting that the expression level of the hph gene in the pFvTgh and pFvGgh transformants was higher than that in the pFvT and pFvG transformants. The results were corresponded to the transformation efficiency presented in Table 2, and strongly suggest that the gpd promoter is functional in the heterologous gene expression system in F. velutipes Fv-1 to improve the expression level of the target gene.

Finally, in order to determine whether the plasmid vector was integrated into the genomic DNA by the REMI method, the genomic DNAs of 10 randomly selected pFvGgh transformants were analyzed by Southern blot using the digoxigenin-labeled hph gene as a probe (Fig. 10B). Hybridization signals were detected in all the transformants, and multiple

Ftrp-p-hph Fgpd-p-hph

Fig. 10. Analysis of the transformants obtained by REMI method hybridization signals were also detected in some transformants. There was no signal from the genomic DNA of wild-type Fv-1 as a negative control (data not shown). These results indicate that at least a single hph gene was introduced into all the transformants, and the hph gene is thought to exist as a multicopy in the genomic DNAs of many transformants (Fig. 10B, lanes 3, 4, 5, 6, 8, 9 and 11). The same size bands were detected between 2,027 and 3,530- bp in four transformants (Fig. 10B, lanes 3, 4, 8 and 9). These bands might represent about 2,700-bp of the full-length gpd promoter-hph-gpd terminator region. A 6.9-kb DNA fragment, corresponding to the size of the pFvGgh plasmid, was observed in the genome of only one clone (Fig. 10B, lane 6), indicating that the full length of the plasmid was successfully introduced into the transformant. Consequently, we estimate the probability of integration of full-length pFvGgh vector by the REMI method to be approximately 10%. In our previous study, the probability of integration of the full-length vector was 30% so that the frequency of REMI events of Fv-1 was 10-30% (Maehara et al., 2010a). This value seems to be the comparable level in the case of model mushroom, Coprinus cinereus (8-56%) (Granado et al., 1997).

In conclusion, we demonstrated that the gpd promoter from F. velutipes Fv-1 would be a useful in the transformation system of the strain. The transformation efficiency was about 3 times improved by the use of the gpd promoter. The vectors constructed in this study will be available to improve the genetic engineering of F. velutipes Fv-1 for ethanol fermentation from pentose.

2. Conclusion

In spite of CBP is gaining recognition of a low-cost biomass processing as it involves enzyme production, completely no enzyme process which does not add the saccharification enzymes have not been established. In this study, we demonstrated that F. velutipes can highly convert biomass to ethanol using only small amount of saccharification enzyme even in the quite high concentration of biomass such as 30% w/ v. These results suggest F. velutipes has favorable properties for CBP. Generally, artificial cultivation of mushrooms in polypropylene bottles is performed under the condition of water content 70 to 80%. The condition must be most suitable condition to cultivate the mushrooms. Therefore, F. velutipes will be especially effective in situations that CBP performed under the high concentration of biomass. We believe that this point would be advantage of F. velutipes compared with the other microorganisms engineered for CBP and even for fungus which is possible to ferment the both pentose and hexose. In the future, we would like to develop a novel bioethanol production process by using F. velutipes.

3. Acknowledgment

This work was financially supported by a grant-in-aid (Development of Biomass Utilization Technologies for Revitalizing Rural Areas) from the Ministry of Agriculture, Forestry, and Fisheries of Japan.

In this process, metabolically engineered microorganisms such as bacteria convert ethanol to hydrogen under the facilitation of hydrogenase enzymes which are metalloproteins, containing complicated metal active centres that catalyze the interconversion of protons and electrons with dihydrogen. According to literature reporting [3-5], two major classes of hydrogenases are recognized based on their metal active sites: [FeFe] and [NiFe]. Depending on whether light will be involved, this biological hydrogen production process can be simply classified as photo — and dark-fermentation processes [6].

During the photo-fermentation process, the hydrogenase enzyme synthesized and activated under dark anaerobic condition is used to convert ethanol to biohydrogen under light anaerobic condition. Since the light acts as the energy source, the consumption rate of substrate is less than that required for dark fermentation. However, the hydrogen efficiency will be dramatically reduced in the presence of oxygen concurrently produced through photosynthesis by bacteria, which has been evidenced by many researchers [7]. Furthermore, the ultra-violet wavelength radiation requirement and relatively slower production rate limit its industrial application at large scale.

Under the dark operation environment, there is no risk for hydrogenases exposed to oxygen, which makes the hydrogenase enzymes remain active throughout the whole process, leading to more efficient hydrogen production. Compared to photo-fermentation, the inherent continuous and fast production feature makes dark anaerobic digestion economically promising for industrial scale practice. In recent years, many publications have reported their efforts spent on optimization of operation parameters, development of genetically modified microorganism, metabolic engineering, improvement of reactor designs, use of different solid matrices for cell immobilization, etc. to maximize hydrogen yield. Among many considerations, the blockage of methanogenesis in the anaerobic pathway is crucial to improve hydrogen selectivity through the inhibition of methane formation.

Sweet sorghum juice can be used for syrup, molasses, sugar and ethanol production with average fermentation efficiencies from 85 to 90% (Almodares & Hadi, 2009; Prasad et al., 2007; Wang et al., 2009; Wu et al., 2010b). The sweet sorghum juice is not commonly used for crystallized sugar production because of the presence of significant amounts of inverted sugars (glucose and fructose) that makes difficult crystallization in large-scale processes. However, the sweet sorghum juice, rich in fermentable sugars, has an excellent potential for yeast fermentation (Turhollow et al., 2010; Woods, 2000).

The sweet sorghum juice is obtained through a mechanical operation with a roller mill composed by a set of cylinders, similar to the ones employed by the sugar cane mills. Water is added during the last stage of the crushing process with the aim to augment the solubilization of residual sugars associated to the bagasse. The sweet sorghum juice yields around 50% in relation to the initial weight of the stems (Wu et al., 2010b). However, these authors describe an extraction process by pressing, which results in lower yields compared to roller mills. Furthermore, pressing is a batch process which is difficult to optimize for industrial conditions.

Approximately 90% of fermentable sugars from sorghum stalks can be obtained after conventional roller-mining, yielding an extraction ratio of 0.7 in relation to the initial plant weight (Almodares & Hadi, 2009). Gnansounou et al. (2005) reported extraction ratios ranging from 0.59 to 0.65 for the sweet sorghum cultivars Kelley, Wray, Rio and Tianza. On the other hand, Kundiyana (1996) observed that extraction percentages varied between 47 to 58%, close to values observed by our research group in central Mexico (unpublished data). After extraction, the sweet sorghum juice is fermented, distilled and the ethanol finally dehydrated (Fig. 1). This is the simplest way to produce fuel ethanol because the grain and fiber processes require the hydrolysis of starch and fiber components into fermentable sugars. These steps are considered expensive, take time and expend energy and other additional resources (i. e. enzymes, chemical reagents, etc.) (Fig. 2 and 3). Despite these benefits, some challenges must be solved in order to efficiently convert the sweet sorghum crop into fuel ethanol. The main setbacks are the relatively higher rate of sugar degradation at ambient temperature and the low nitrogen content for yeast growth (Mei et al., 2009; Wu et al., 2010b). Thus, the logistics of just in time harvesting and the storage of the feedstock in facilities that retard decomposition and degradation of fermentable carbohydrates should be considered and stressed. In relation to nitrogen availability, this disadvantage can be overcome with the supplementation of urea, ammonia or yeast extract in order to avoid sluggish fermentation.

Besides sugar and nitrogen content, fermentation performance of sweet sorghum juice can also be affected with processing parameters and bioreactor configuration. Nuanpeng et al. (2011) observed in a repeated-batch study that very high gravity (VHG) fermentation is a good alternative to produce high ethanol concentrations from sweet sorghum juice when an adequate level of yeast cell concentration, nitrogen, and agitation are used. On the other hand, Laopaiboon et al. (2007) reported better results in fed-batch fermentation compared to batch configuration, in terms of ethanol concentration and product yield but not in productivity (measured as grams of ethanol generated/L/hr). These findings indicate the need to optimize parameters as feeding and withdrawn rate in order to optimize yields.

The microorganism used, as indicated in the next sections, is also a factor that is worthwhile exploring. In the case of sweet sorghum juice, fermentation with different yeast strains has been evaluated and productivity varies significantly, but most of the strains showed an efficiency of more than 90% (Wu et al., 2010b). Liu et al. (2008) reported the use of immobilized yeast in a fluidized bed reactor that shortened process time and increased conversion efficiency. These results can be optimized when parameters as temperature, agitation rate, particles stuffing rate and pH are modified. Liu & Shen (2008) found that fermentation with immobilized yeast at 37°C, 200 rpm, 25% particles stuffing rate and pH of 5.0 in shaking flasks and 5 L bioreactor corresponds to the optimal conditions derived from an orthogonal experimental design.

Lignocellulosic biomass is typically nonedible plant material, including dedicated crops of wood and grass, and agro-forest residues. Lignocellulosics are mainly composed of cellulose, hemicellulose, and lignin.

Cellulose is a homopolysaccharide composed of P-D-pyranose units, linked by P-1, 4- glycosidic bonds. Cellobiose is the smallest repetitive unit and it is formed by two glucose monomers. The long-chain cellulose polymers are packed together into microfibrils by hydrogen and van der Waals bonds. Hemicellulose and lignin cover the microfibils (Fig.1). Hemicellulose is a mixture of polysaccharides, including pentoses, hexoses and uronic acids. Lignin is the most complex natural polymer consisting of a predominant building block of phenylpropane units. More specifically, p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol are the most commonly encountered alcohols (Harmesen et al., 2010). Lignocellulosic materials also contain small amounts of pectin, proteins, extractives (i. e. no — structural sugars, nitrogenous material, chlorophyll and waxes) and ash (Kumar et al., 2009).

The composition of the biomass constituents can vary greatly among various sources (Table 1). Accurate measurements of the biomass constituents, mainly lignin and carbohydrates, are of prime importance because they assist tailored process designs for the maximum recovery of energy and products from the raw materials.

Since 1900, researchers have developed several methods to measure the lignin and carbohydrates content of lignocellulosic biomass. Globally recognized Organizations, such as American Society for Testing and Materials (ASTM), Technical Association of the Pulp and Paper Industry (TAPPI) and National Renewable energy and Laboratory (NREL) have developed methods to determine the chemical composition of biomass, based on modifications of the two main procedures developed by Ritter (Ritter et al., 1932) and by Seaman (Saeman et al., 1954), (Table 2).

LIGNIN

Fig. 1. Lignocellulosic materials: composition of major compounds (Kumar, 2009)

Research on bioethanol production from lignocellulose-containing substrates has made great progress over the last decades. As shown by other authors and our own results, the theoretical yield of bioethanol from cellulose (690 l/t cellulose, 283 l/t straw) is almost achievable. The yield of bioethanol from hemicelluloses still has to be increased. Compared to bioethanol production of the first generation, cost-effectiveness also has to be improved. No commercial bioethanol plant using lignocellulose-containing residues as feedstock is in operation in 2011. However, diverse pilot plants are in operation and the first demonstration plants have been completed and running succesfully.

The production of biofuels such as bioethanol is often criticized because of the negative impact of the feedstock on biodiversity. The competition of the raw materials for use either as biofuel or for food production is also a major obstacle to increasing bioethanol production capacity. Therefore, lignocellulose-containing residues offer a possibility to satisfy part of the increasing demand for fuel by means of biofuel.

Diverse scenarios are possible — only using first generation fuel, resulting in dramatic increases in world prices for feedstock crops. The stimulation of the second generation results in reduced pressure on world prices for feedstock crop. It is the authors’ opinion that the higher demand for biofuels will necessarily lead to the use of lignocelluloses as feedstock to produce biofuels. In order to replace fossil fuels to a larger extent, not only agricultural residue must be used as feedstock. Agriculture has to be geared towards food as well as towards energy production. This will only be possible in the context of a coordinated international effort.

2. Acknowledgment

This work was supported by the following projects: FH Plus in Coin SteamExplo 818383; Bioethanolproduktion aus Lignocellulosen mit Steamexplosion (Fabrik der Zukunft, Projekt 814953); REGIO 13/ EFRE regional production of energy and by the country of Upper Austria and FH OOE basic financing — bioenergy.

In this study the main focus was on production of bioethanol from rape straw, but there are a lot of other possible feedstocks suitable for bioethanol production in general, all cellulosic material can be converted by physical/chemical pretreatment followed by enzymatic hydrolysis into glucose. It is also possible to produce bioethanol from sugar and starch from crops such as corn, wheat, sugarcane, and sugar beet, but since sugar is a food source, using it could decrease food availability for future generations. Using food sources or available agricultural land for pure energy production is generally classified as a 1st generation technology, and is not normally regarded as a sustainable energy source. To compare the different feedstocks Table 4 is produced. However, there are numerous ways to co-produce food and feedstocks for bioenergy when utilizing the lignocellulosic residues from agricultural production as shown in table 4.

As table 4 shows, bast fibers have a very high cellulose content (60-63%) and a low lignin content (3-4 %) which should make them ideal for producing bioethanol, but bast fibers as a feedstock would fall under the category of 1st generation bioethanol, because the production of bast fibers requires land that could otherwise be used for food production. Rape straw has a low cellulose content compared to other straw fibers (32%) but in return the hemicellulose (14%) and lignin (18%) content is also low compared to wheat straw (20%) and corn stover (19 — 21%). Low lignin content is good for the enzymatic hydrolysis, since lignin can denaturize cellulase enzymes (Thygesen et. al 2003). Low hemicellulose content will result in a slightly lower concentration of microbial growth inhibitors derived from oxidation of the hemicellulose. Sugarcane bagasse seems to be the ideal 2nd generation feedstock with its high cellulose content (43%) and low lignin content (11%) but sugarcanes require high temperatures and a lot of rainfall to grow and are therefore only energy efficient when grown in tropical regions, which limits the amount of ethanol produced from sugarcane bagasse worldwide. It is possible to produce bioethanol from wood fibers, like waste wood from carpentry or

willow, which can grow on land not suitable for agriculture, using pretreatment methods such as steam explosion (Soderstrom et. al. 2002). The high lignin content in wood fibers increases the amount of enzymes needed and the time period of the fermentation. Furthermore wood fibers have other uses and can easily be burned to produce electricity and heat in a cogeneration plant.

Marine biomass has the advantage that it does not use the same space as agriculture and even though it is not a waste product from food production it is still a viable feedstock because it does not reduce food availability. Chaetomorpha linum has very low lignin content (6 — 8 %) and cellulose content similar to straw fibers (34 — 40 %). C. linum and other types of useable macroalgae are easy to grow in most of the world, and is therefore a suitable candidate for expanding the bioethanol production to more than what can be obtained from waste products (Schultz-Jensen et. al. 2011).

4. Conclusion

The amount of yeast needed for SSF of pretreated rape straw is dependent on the DM content, despite the fact that enzymes continue to be the primary rate-determining factor. The positive feedback effect from the yeast lowering the sugar concentration can have high relevance when running SSF with high DM content. After prolonged testing of Turbo yeast, the optimal temperature of the SSF is found to be 37°C. Furfuryl alcohol and possibly other growth inhibitors as well, show a positive effect on the rate of fermentation when added in small dosages, since yeast will increase its metabolism under stress. The positive effect of growth inhibitors is so strong that the fermentation rate in sugar media is lower than the fermentation rates in a medium produced from wet oxidized rape straw (filter cake), given the DM concentration does not exceed critical levels.

5. Acknowledgement

The Danish Research Council, DSF is gratefully acknowledged for supporting the research project: Biorefinery for sustainable reliable economical fuel production from energy crops (2104-06-0004). The European Union is acknowledged for supporting the EU-project: Integration of biology and engineering into an economical and energy-efficient 2G bioethanol biorefinery (Proethanol nr. 251151). Efthalia Arvaniti is acknowledged for academic advice. Tomas Fernqvist, Ingelis Larsen and Annette Eva Jensen are thanked for technical assistance and HTX Roskilde for providence of microscope cameras.

Compared to significant amount of experimental efforts spent on catalytic BESR for surface reaction mechansim investigation, computational approach at molecular level still remains barely untouched in the past several decades probably due to its extreme complicacy and limited computation resources. However, recent years have witnessed the rapid development of computational technology, making the reaction simulation at catalyst surface technically feasible. For simplifying simulation work, many publications have purely focused on the ethanol or water alone adsorption and associated decomposition on single metal clusters [135-139].

Various methodologies have been developed to reasonably represent catalyst surface for obtaining more accurate simulation results. The slab geometry in contrast to cluster model is widely adopted to model the catalyst surface with certain thickness. In addition to the top atomic layer, several successive layers below are also included to simulate the bulk effect on the surface layer. The surface layer is thereafter allowed to be reconstructed in response to the constraint from bulk layers. Usually, a vacuum region with certain length is created right above the top layer of the slab model to prevent the interaction of adsorbed molecules with its periodic images [140]. The choice of supercell size comes from the compromise between computation accuracy and computation time span. "Nudged Elastic Band (NEB)" method [141, 142] is proven by many papers to be effective in transition state and associated energy barrier estimation and very useful in minimum energy pathway determination especially for complex chemical reactions. Most of recently published computational results are based on the self-consistent periodic density functional theory (DFT) calculation, which is more accurate than other commonly used computational methods such as ab initio, semi­empirical, and empirical methods.

According to the published papers, although there are some disagreements on the ethanol decomposition on model catalyst surface, the proposed pathways can still be generally classified into two routes. One is CH3CH2OH ^ CH3CH2O(a) ^ CH2CH2O(a) ^ CH2CHO(a) ^ CH2CO(a) ^ CH2(a)+CO(a)+4H(a) ^ CH4(g)+CO(g)+H2(g). In this route, ethanol molecule first prefers to adsorb at atop sites and binds to the surface through the oxygen atom after O-H bond cleavage, followed by a six-membered ring of an oxametallacyclic compound formation through the elimination of the hydrogen atom attached to the P-carbon. This six — membered ring is usually located at the interface of active metal and support, creating a bridge between them. The ethanol decomposition process then continues with two consecutive eliminations of hydrogen atom attached to a-carbon. Scission of C-C bond then occurs under the facilitation of active metal, resulting in the formations of a series of adsorbates which subsequently desorb from substrate at elevated temperature to yield final gas products such as CH4, CO, and H2 [142-144]. The other suggested route follows the track of CH3CH2OH ^ CHsCHOH(a) ^ CHsCHO(a) ^ CHsCO(a) ^ CH2CO(a) ^ CHCO(a) ^

CH(a)+CO(a) ^ CH4(g)+CO(g)+H2(g)+C(s) [145].

Unlike ethanol decomposition, water dissociation completes only in two steps (i. e., H2O ^ H(a)+OH(a) ^ 2H(a)+O(a)), which is obviously due to its rather simple formulation. Compared to the second O-H bond breakage, the first one can take place with much lower activation energy [146]. Therefore, it can be easily predicted that hydroxyl group will have much higher chance to participate in BESR for ethanol oxidation than O* after water complete dissociation.

After a careful literature review, it is worth noting that the role of catalyst support and co­adsorption of ethanol and water are barely considered, which is probably attributed to its awful computational complicacy. In order to give a clear picture of what is really happening on catalyst surface during BESR and provide a theoretical support to our experimental observations and proposed reaction mechanism, we launched a computational task in collaboration with the Chemistry Department at Ohio State University. We employed plane — wave periodic DFT method implemented in the Vienna ab initio simulation program (VASP) to investigate the ethanol steam reforming reactions [147-149]. The projector augmented wave (PAW) method [150, 151], combined with a plane-wave basis set, was utilized to describe the core and valence electrons. The generalized gradient approximation (GGA) [152] of Perdew and Wang (PW91) [153] was applied for the exchange-correlation functional. The convergence of the plane-wave expansion was obtained with moderate truncation energy of 500 eV, while the electronic relaxation was converged to a tolerance of 1 x 10-4 eV. The Monkhorst-Pack grid [154] served in the generation of the k-points, and a (4 x 4 x 1) k-point grid was used for Brillouin zone sampling for surface calculations. Spin polarization was applied in all calculations.

The relaxed bulk structure of CeO2 with a lattice parameter of 5.46 A was used to construct the slab model. The CeO2 (111) and Co/CeO2 (111) surfaces were modeled as 2 x 1 super cells. A three molecular CeO2 thick slab model was constructed, thus nine atomic layers in total. The super cell has dimensions: a = 7.72 A, b = 6.69 A, and c = 23.88 A, and a 16 A thick vacuum region is included to ensure that there is no interaction between the surface adsorbates of one layer and the next slab. To optimize the surface structure, the top three atomic layers of the slab with the adsorbates were allowed to relax. The bottom six atomic layers were fixed at the bulk positions of ceria. The NEB method [155-157] was employed to locate the transition states of various reactions over the catalyst surface. After numerical differentiation, each transition state was confirmed to have a single imaginary vibrational frequency.

Ethanol decomposition via steam reforming reaction was computationally studied on the CeO2(111) and Co/ CeO2(111) surfaces. From our results, the most likely reaction pathway is described below. The decomposition of ethanol starts with the breaking of the O-H bond on the catalyst surface. The produced ethoxide unit prefers to be adsorbed on the catalyst surface by the Oe-Co interaction. With the assistance of a surface-bound hydroxyl moiety, derived from water dissociation, the Ca-H bond breaking of the ethoxide unit could proceed to yield the thermodynamically stable product (adsorbed acetaldehyde and hydrogen atom). The surface-bound hydroxyl group could act as a better hydrogen acceptor to assist the Ca — H bond-breaking reaction as compared to the surface oxygen atom of ceria. In the subsequent step, the surface-bound hydroxyl addition to acetaldehyde produces the hydroxyl adduct, СНзСН(0)(0Н), as an intermediate. This СНзСН(0)(0Н) intermediate further undergoes the loss of H from the Ca position to generate acetic acid. Acetic acid can then lose the acidic hydrogen from the hydroxyl unit, yielding an adsorbed acetate and hydrogen. The acetate could be further converted to the CH2(OH)COO intermediate via H — atom abstraction and subsequent surface-bound hydroxyl addition reactions. As suggested by the calculations, the Ca-Cp bond rupture from the chemisorbed CH2(O)COO intermediate generates formaldehyde and CO2. Similar to acetaldehyde, the generated formaldehyde could react with a surface-bound hydroxyl group to produce the HCH(O)(OH) adduct that subsequently undergoes a H-atom abstraction reaction to yield formic acid. Then, formic acid loses the acidic hydrogen of the hydroxyl unit to generate surface-bound formate. Finally, formate could be converted to CO2. Throughout the favorable reaction pathway from ethanol to CO2, one of the most energetically costly steps on the potential energy surface is the Cp-H bond-breaking step of acetate for ethanol decomposition with the participation of surface-bound hydroxyl groups on the Co/CeO2(111) surface.

Our modeling indicates that surface-bound hydroxyl groups, which is formed from water dissociation, plays two critical roles in the ethanol steam reforming reaction. The first is to assist the hydrogen-abstraction reactions from carbon atoms. The second is their involvement in addition reactions to form the C=O or C=C double bond intermediates. Thus, a catalyst on which water could more effectively dissociate to form surface-bound hydroxyl and hydrogen might be a potentially better catalyst for steam reforming reactions. On the Co/CeO2(111) surface, our computational work elucidates the formation of acetaldehyde and acetate intermediates and is consistent with extant experimental observations [133]. The present computational studies do not account for the generation of acetone, carbon monoxide, and methane, which are byproducts observed in experimental studies. A model that includes larger Co particles with some surface-bound hydroxyl groups would be more realistic and may account for the formation of other byproducts.

During cassava bioethanol production, wastes are generated; the quantity and quality are depending significantly on feedstock quality and processing types. Since dry milling process is more widely used for bioethanol production from cassava feedstock, the information provided here is based on dry milling process of cassava chips. Similar to dry milling process for bioethanol production of corn grains, both of solid and liquid wastes are obtained at the end of distillation. The waste can be generated as a whole stillage containing both solid and liquid waste if the whole beer is subjected to the mash column without fiber separation. This process is applied in order to minimize ethanol loss in the solid pulp if fiber separation is accomplished prior to distillation. Recently, the process is adjusted by separating the fiber first and the fiber is washed to collect ethanol in pulp. At the production capacity of 150,000 liters of anhydrous ethanol/day, the total whole stillage is produced approximately 1,400-1,600 m3/day, being wet cake 100-200 ton/day and the stillages 1,200­1,400 m3/day (Sriroth et al., 2006).

Table 8. Parameters and results of ethanol production from cassava chips by SSF and SLSF process.

One of the most significant problems in ethanol production from lignocellulose is production cost (Mizuno et al., 2009) because the fiber conversion requires of high energy investments in order to obtain high concentrations of fermentable sugars from the insoluble polymers (Kurian et al., 2010; Mamma et al., 1996). A pre-hydrolysis step releases both the hemicellulosic and cellulosic fractions of the fiber (Herrera et al., 2003). The main processes related to the pretreatment of sorghum biomass for ethanol production are the acid and/or enzyme-catalyzed hydrolyses (Mamma et al., 1996; Sipos et al., 2009). Generally, the acid hydrolysis precedes the enzymatic in order to optimize production of C6 and C5 fermentable sugars (Sipos et al., 2009).

4.3 Pretreatments used for sorghum bagasse

The extraction of structural carbohydrates from bagasse cell walls is highly related to the effectiveness of pretreatments. Nowadays there are many proposed treatments for cellulose and hemicellulose extraction, but only few have been commercially implemented. In the following sections some of the proposed technologies for sorghum biomass are discussed.

Daniel L. A. Fernandes, Susana R. Pereira, Luisa S. Serafim, Dmitry V. Evtuguin and Ana M. R. B. Xavier CICECO, Department of Chemistry, University of Aveiro

Portugal

1. Introduction

The world is facing a reduction of global fossil fuels resources, like petroleum, natural gas, or charcoal, while energy requirements are progressively growing up. Fossil fuels should be replaced, at least partially, by biofuels once the current fuel supply is suspected to be unsustainable in the foreseen future. In fact, the search for sustainable alternatives to produce fuel and chemicals from non-fossil feedstocks has attracted considerable interest around the world, to face the needs of energy supply and to response to climate change issues. Alternative resources of energy are being explored in order to reduce oil dependence and increase energy production by exploring of solar, wind, hydraulic and other natural phenomena. Besides these sources of energy, also biomass possesses a potential target for fuel and power production as well as for chemicals or materials feedstocks. Thus biomass can efficiently replace petroleum-based fuels for a long term. (Sanchez et al. 2008; Alvarado- Morales et al. 2009; Brehmer et al. 2009; Gonzalez-Garcia et al. 2009; Singhania et al. 2009; Mussatto et al. 2010; Sannigrahi et al. 2010).

Many countries in Europe, North and South America and Asia are replacing fossil fuels by biomass-based fuels according to international regulations. One of the directives of European Union (2009/28/CE) imposes a quota of 10% for biofuels on all traffic fuel until 2020 (Rutz et al. 2008; Xavier et al. 2010). Also economic incentives for research on biofuels are being implemented all over the world. Bioethanol can be produced from different raw materials containing simple sugars, starch or more complex substrates as lignocellulosics. New methodologies for biofuels (e. g. ethanol and biodiesel) production have been developed in the last years, to achieve new and non cost-intensive technologies for bioconversion of lignocellulosic renewable resources. The most common renewable fuel is ethanol, which is produced from direct fermentation of sugars (e. g. from sucrose of sugarcane or sugar beet) or polysaccharides (e. g. starch from corn and wheat grains) (Gonzalez-Garcia et al. 2009; Mussatto et al. 2010). The selection of the best raw material is strongly dependent on the local conditions where feedstock is obtained. Evidently, ethanol in Brazil is produced from sugarcane, whereas, in North America or Europe the ethanol industry is based on starchy materials. Besides, energy considerations should be attained: not only the energy input required for ethanol production and the content in fermentable

sugars of the feedstock must be considered, but also the annual ethanol yield per cultivated hectare. As suggested, for beet molasses, the yield of ethanol per ton of feedstock is lower than that for corn, but on the other hand, when compared to starchy materials the beet productivity per cultivated hectare, expressed in L/(ha year), is considerably higher, (Sanchez et al. 2008).

The growth of the biofuels industry raised questions regarding the sustainability of these "first generation" biofuels. The feedstocks described play an essential role in human and animal food chains, therefore the rise of prices of food all over the world resulted in social disturbance (Gonzalez-Garcia et al. 2009; Mussatto et al. 2010; Xavier et al. 2010). These raw materials were also expected to be limited due to the reassign of arable lands from food to fuel production leading to competition for feedstocks (Gray et al. 2006; Bacovsky et al. 2010). Moreover, first generation biofuels were accused of not contributing to reduce gas emissions, therefore the use of this technology was highly criticized. For all these reasons additional research in this area is mandatory, in order to search for non-food crops, like wastes from agriculture and/or industry as sources of raw-material. European Union strongly incentives research focusing biotechnological solutions for energy and chemical demands from renewable resources, such as, forestry wastes, agricultural biomass residues and food industrial wastes for "second generation" biofuels production.

The great advantage for the choice of lignocellulosic biomass as feedstock is the non­interference with food chain, which allows the production of bioethanol without using arable lands (Sanchez et al. 2008; Zhang 2008). Lignocellulosic biomass is a complex raw material which can be processed in different ways to obtain other value-added compounds contributing to the possibility of establishing a biorefinery. Different value-added products such as lactic acid, acetic acid, furfural, methanol, hydrogen and many other products can be obtained from its sugars. Lignin, the non-carbohydrate component, can be used for the production of advanced materials, polymers and aromatic aldehydes (Sanchez et al. 2008; Zhang 2008; Sannigrahi et al. 2010; Santos et al. 2001). In this way, lignocellulosic biomass can be used as substrate for the production of second generation biofuels, contributing to the diversification of energy supply and gas mitigation, offering less competition for the food and feed industry (Rutz et al. 2008; Bacovsky et al. 2010). The use of these raw materials to produce fuel, power and value-added chemicals, fits well into the biorefinery concept invoked to decrease the dependence from fossil resources and to improve the economic sustainability (Alvarado-Morales et al. 2009; Xavier et al. 2010). However, for a world massive utilization of fuel ethanol, a cost-effective technology for ethanol production is also required. In other words, ethanol production costs should be lowered (Sanchez et al. 2008). In a biorefinery, different technologies, including fermentation, biocatalytic, thermal and chemical technologies, must be used simultaneously for biomass conversion for the production and the purification of different value-added products (Alvarado-Morales et al. 2009).

Bioethanol is one of the products that can be obtained via biorefinery using bio-based resources. It is one of the most attractive biofuels, since it can be easily produced in large amounts and blended with gasoline or used pure as a "green" fuel. Furthermore, due to the higher oxygen content, ethanol allows a better oxidation of the gasoline and reduces CO and particulate emissions. Other advantages of ethanol versus gasoline are the higher octane number, broader flammability limits, higher flame speeds, heat of vaporization and compression ratio and a shorter burn time (Balat et al. 2008; Mussatto et al. 2010). The use of bioethanol can also contribute for the reduction of CO2 build-up, while the CO2 content of fossil fuels will remain in storage (Sanchez et al. 2008; Gonzalez-Garcia et al. 2009; Chen et al. 2010a; Balat 2011). Moreover, combustion of ethanol results also in lower NOx emissions, being free of sulphur dioxide. However, as disadvantages, ethanol has an energy density lower than gasoline, it is fully miscible in water and its lower vapour pressure makes motor cold start more difficult (Balat et al. 2008; Gonzalez-Garcia et al. 2009; Chen et al. 2010a; Mussatto et al. 2010; Balat 2011). Simultaneously, bioethanol is a building block for the production of several other chemicals, usually petrochemical-based, like acetaldehyde, ethane, ethylene, propylene, butadiene, carbon monoxide or hydrogen (Idriss et al. 2000; Wang et al. 2008; Yu et al. 2009; Lippits et al. 2010; Oakley et al. 2010; Song et al. 2010). Today nearly 95% of hydrogen is produced from fossil-based materials such as methane and naphtha. Bioethanol as chemical reagent for hydrogen production could be a way to support hydrogen economy from a renewable and clean energy source (Yu et al. 2009; Lippits et al. 2010). Besides, the production of olefins from ethanol has attracted much attention since it valorises bioethanol production under a biorefinery context (Thygesen et al. 2010).

In this context Hardwood Sulphite Spent Liquor (HSSL) is a subproduct of pulp and paper industry that results from the acidic sulphite pulping process in high amounts per day. The main objective of acidic sulphite pulping process is to remove lignin and hemicelluloses from wood and to maintain cellulose integrity as much as possible. In this process, lignin and hemicelluloses are hydrolysed and released in the aqueous phase. HSSL can be a suitable substrate for 2nd generation bioethanol production as well as other biobased products since it is rich in monosaccharides obtained during the acidic sulphite pulping process.