Metal ions are introduced into the biodiesel fuel during the production process. Whereas alkali metals stem from catalyst residues, alkaline-earth metals may originate from hard washing water. Sodium and potassium are associated with the formation of ash within the engine, calcium soaps are responsible for injection pump sticking (Mittelbach 2000).These compounds are partially limited by the sulphated ash, however tighter controls are needed for vehicles with particulate traps. For this reason these substances are limited in the fuel specifications.

1.12 Phosphorus

Phosphorus in biodiesel stems from phospholipids (animal and vegetable material) and inorganic salts (used frying oil) contained in the feedstock. Phosphorus has a strongly negative impact on the long term activity of exhaust emission catalytic systems and for this reason its presence in biodiesel is limited by specification.

1.13 Distillation

This parameter is an important tool, like ester content, for determining the presence of other substances and in some cases meeting the legal definition of biodiesel (i. e. monoalkyl esters).

The bleomycin family of glycopeptide antibiotics is toxic to a wide range of organisms with as intercalator functionality able to cleave DNA. Bleomycin-resistance, attributed to the ble gene, is an ideal selectable marker as the BLE protein acts in stoichiometric equivalent. Occurring as a dimer, each protein has a strong affinity for binding and inactivating two molecules of bleomycin. Therefore, the level of expression of this exogenous gene can be directly correlated with antibiotic tolerance observed phenotypically. As such, it has been developed as a selectable marker system for nuclear transformation of C. reinhardtii and V. carteri and is likely to have similar applicability in other algae (Lumbreras et al., 1998; Hallmann et al., 1999). Since the protein must enter the nucleus, it requires high levels of expression to be active; thus, inherently selecting for clonal isolates with high-level expression. Commercial forms of bleomycin include bleocin™ and zeocinTM, which are known to affect mammalian, insect, yeast, bacterial, and plant cells.

Paromomycin has been used extensively as a selective agent for the genetic engineering of microorganisms as well as an antibiotic for the treatment of bacterial infections in humans. Paromomycin acts by binding to the 16S ribosomal RNA of microbes, effectively interfering with protein synthesis. The aminoglycoside 3′-phosphotransferase gene aph from Streptomyces rimosus already exists in a codon bias similar to that of Chlamydomonas and confers resistance to paromomycin, kanamycin, and neomycin. Paromomycin is highly toxic to green algae and has been used as an effective selective agent in both C. reinhardtii and V. carteri (Sizova et al., 2001; Jakobiak et al., 2004).

Phosphinothricin (PPT), also known as glufosinate-ammonium (GLA) and bialaphos, is a natural amino acid that competitively inhibits glutamine synthesis in plants and animals (Hoerlein, 1994). For this reason, it is used in a number of herbicides, including Basta® and RoundUp®. A number of agricultural crops have been engineered by to resist PPT through introduction of the bar (bialaphos resistance) gene from Streptomyces hygroscopicus, which encodes a phosphinothricin acetyl transferase (Thompson et al., 1987). In the academic community, PPT is used as a selective agent for work with Arabidopsis and the bar gene is carried on pGR117 for Agrobacterium-mediated transformation of A. thaliana (Akama et al., 1995).

Ethanol can be readily produced by fermentation of simple sugars that are hydrolyzed form starch crop. Feedstocks for such fermentation include corn, barley, potato, rice and wheat (Cybulski, 1994). Sugar ethanol can be called grain ethanol, whereas ethanol produced from cellulose biomass is called cellulosic or second generation ethanol. Both grain ethanol and cellulosic are produced via biochemical processes, whereas chemical ethanol is synthesised by chemical synthesis routes that do not involve fermentation. In fact, thermo-catalysis process route can be a good alternative to the bioprocess route. In a recently reported process (Chornet et al., 2009) the ethanol is produced by a two steps process: first methanol carbonylation to produce methyl acetate and second hydrogenolysis of the latter as illustrated in the figure 6.

Bulk Density: The goal of densification is to increase the bulk density of agricultural straw to facilitate economic storage, transportation and handling of the material. In addition, densification results in an increase in the net calorific content per unit volume. The bulk density of agricultural biomass depends on the type of biomass, moisture content, grind size, and pre-treatment (Mani et al., 2006). Lower bulk densities, and concerns with uneven and low flowability of straw grinds are critical issues to sustainable production of pellets using pellet mills (Adapa et al., 2010c; Larsson et al., 2008).

Typically, the bulk density of ground straw increases with a decrease in hammer mill screen size. Also, pre-treatments usually results in a decrease in bulk density since the organized lignocellulosic structure of biomass is disturbed/ disintegrated. In addition, the bulk density and geometric mean particle size of material is correlated by either power or exponential relations (Adapa et al., 2010b; Mani et al., 2004). Table 5 shows a summary of average bulk density of various agricultural biomasses ground using a hammer mill.

Particle Density: Particle size of the grinds will have direct effect on the final pellet density. Theoretically, the density of pellet can be as high as the particle density of the ground biomass. Similar to bulk density, particle density also depends on the type of biomass, moisture content, grind size, and pre-treatment (Adapa et al., 2010b). The particle density is observed to have negative correlation with hammer mill screen size. Application of pre­treatment increases the particle density since disturbance/ disintegration of lignocellulosic structure results in finer components (Adapa et al., 2010b). Table 5 shows a summary of average particle density of various agricultural biomasses ground using a hammer mill. Geometric Mean Particle Size and Distribution: It has been reported that wider particle size distribution is suitable for compaction (pelleting/briquetting) process (Mani et al., 2004a). During compaction, smaller (fine) particles rearrange and fill in the void space of larger (coarse) particles producing denser and durable compacts (Tabil, 1996). Therefore, ideally the grinds should be normally distributed, should have near zero skewness and lower peak than expected for the normal and wider distribution of data (negative Kurtosis values). In addition, a decrease in the biomass grind size has been observed to have a positive effect on pellet mill throughput (Adapa et al., 2004).

Frictional Properties: Prior to densification, biomass grinds need to be efficiently stored, handled and transported. Physical and frictional properties of biomass have significant effect on design of new and modification of existing bins, hoppers and feeders (Fasina et al., 2006). The frictional behavior of biomass grinds in all engineering applications is described by two independent parameters: the coefficient of internal friction, and the coefficient of wall friction. The former determines the stress distribution within particles undergoing strain, and the latter describes the magnitude of the stresses between the particle and the walls of its container (Seville et al., 1997). The classic law of friction states that frictional force is directly proportional to the total force that acts normal to the shear surfaces (Chancellor, 1994; Chung and Verma, 1989; Larsson, 2010). Frictional force depends on the nature of the materials in contact but is independent of the area of contact or sliding velocity (Mohsenin, 1970). Material properties such as moisture content and particle size affect the frictional properties and densification performance of an individual feedstock (Larsson 2010; Shaw and Tabil, 2006). In addition, the determination of coefficient of friction is essential for the design of production and handling equipment and in storage structures (Adapa et al., 2010a; Puchalski and Brusewitz, 1996). A comprehensive summary of literature review on coefficient of internal friction and cohesion of agricultural biomass is provided in Table 6.

Predominantly, a linear correlation exists between normal and shear stress for agricultural straw grinds (Adapa et al., 2010a; Chevanan et al., 2008; Richter, 1954) at any specific hammer mill screen size. An increase in hammer mill screen size significantly decreases the shear stress for ground straw at any specific normal stress (Adapa et al., 2010a).

The correlation for coefficient of internal friction and cohesion with average geometric mean particle sizes for agricultural straw grinds is provided in Adapa et al. (2010a). These correlations can be used to predict the coefficient of internal friction (slope of the linear plot) and the cohesion (intercept of the linear plot) for various geometric mean particle sizes. In general, the coefficient of internal friction for ground agricultural straw decreases with an increase in average geometric mean particle diameter. The coefficient of cohesion for straw grinds increases with an increase in average geometric mean particle size (Adapa et al., 2010a).

Normal stress from 10 to 400 kPa • Coefficient of wall friction were 0.68 Shaw and Tabil,

Geometric mean particles sizes of 0.74 (peat (peat moss), 0.45 (wheat straw), 0.39 2006

moss), 0.65 (wheat straw), 0.47 (oat hulls) (oat hulls), and 0.41 (flax shives)

and 0.64 (flax shives) mm • Adhesion coefficients were 0.2635 kPa

Moisture content 9-10% (wb) (peat moss), 10.687 kPa (wheat straw),

Mild steel surface 4.719 kPa (oat hulls), and 16.203 kPa

(flax shives)

Coefficient if friction on steel surface Afzalinia and for alfalfa and barley straw increased Roberge, 2007 with moisture content and was from 0.15 to 0.26, and 0.14 and 0.27, respectively

Coefficient of friction for wheat straw and whole green barley were 0.13 and 0.21, respectively

No effect of chop size on coefficient of internal friction on barley straw

2. Summary

The current chapter has explored the effects of pre-treatment (chemical, physico-chemical, and biological) and pre-processing (size reduction) techniques on densification of agricultural straw resulting in high quality (density and durability) pellets. It has been determined that an increase in bulk density of biomass also increases the net calorific content per unit volume of pellets, and facilitates easy and economical storage, transport and handling of the biomass. Pre-treatment and pre-processing methods disintegrate the basic lignocellulosic structure of biomass, and change the relative composition of lignin, cellulose and hemicelluloses in the material. In addition, physical and frictional properties of agricultural straw are altered. The application of pre-treatments breaks the long-chain hydrogen bond in cellulose, making hemicelluloses amorphous, and loosening the lignin out of the lignocellulosic matrix, resulting in better quality (physically) pellets. During this process, the high molecular amorphous polysaccharides are reduced to low molecular components to become more cohesive in the presence of moisture during densification process. Particle size reduction increases the total surface area, pore size of the material and the number of contact points for inter-particle bonding in the compaction process.

It has been shown that the Fourier Transform Infrared Spectroscopy (FTIR) can be used to rapidly characterize and quantify cellulose-hemicellulose-lignin composition prior to and after application of various pre-processing and pre-treatment methods. Regression equations were developed to predict the lignocellulosic content of agricultural biomass using pure cellulose, hemicelluloses and lignin as reference samples.

It is difficult to tailor non-enzymatic catalyst, capable of exhibiting electrochemical performances similar to those shown by laccase or BOD in physiological type media. The major problem with enzymes lies in the natural lack of stability of the proteins. One of the possibilities to tailor new efficient and stable cathode catalysts for glucose/O2 biofuel cells is to artificially reproduce active centers of enzymes and to stabilize their environment by mimicking the structure of enzymatic proteins and by removing all organic parts responsible for instability of enzymes. The possibility of designing this kind of catalyst has already been discussed (Ma & Balbuena, 2007).

2. Design of glucose/O2 biofuel cells

The global reaction associated to the glucose/O2 biofuel cell can be described according to Eq. 13 :

C6H12O6 + ±O2 ^ C6H10O6 + H2O (13)

Gibbs free energy associated to this reaction is ArG0 = -251 kJ mol-1. This implies that the theoretical cell voltage is E0 = 1.3 V (Kerzenmacher et al., 2008). Furthermore, when the cell delivers a current j, the cell voltage E(j) can be expressed as follows:

E(j) = Eeq — rj, — ъ — Rj (14)

where is the anodic overvoltage, the cathodic one, R the cell resistance and Eeq the equilibrium cell voltage. In Eq.14, it clearly appears that both values of and R must be very low in order to increase the cell performances.

Since the development of the first biofuel cell realized by Yahiro et al. (Yahiro et al., 1964) that consisted in a two-compartment anionic membrane cell in which two platinum foils were used as conducting supports, numerous progress have been realized in designing devices. Nowadays, four main designs are developed. The first one has been developed by Heller’s group. It simply consists in using two carbon fibers of 7 pm diameter as electrode materials. On these fibers, enzymes are immobilized in a redox osmium based hydrogel capable of immobilizing enzymes. These two electrodes are directly dipped into the electrolyte. In a physiological medium containing 15 mM glucose, the device was primarily able to deliver a power density of 431 pW cm-2 at a cell voltage of 0.52 V (Mano et al., 2002c). The device exhibited a high stability, since after one week of continuous working, it was still capable of delivering 227 pW cm-2. Based on this study, and by replacing carbon fibers by newly engineered porous microwires comprised of assembled and oriented carbon nanotubes, Mano’s group (Gao et al., 2010) recently made the most efficient glucose/O2 biofuel cell ever designed. It indeed achieved a remarkably high power density of 740 pW cm-2 at a cell voltage of 0.57 V. The success of the experiment probably lies in the increase of the mass transfer of substrates. Other promising but presently less performing designs of glucose/O2 biofuel cells have been developed in the recent past years. The first one consists in using a microfluidic channel to build a glucose/O2 biofuel cell. The laminar flow obtained in the channel at low Reynold’s number prevents the electrodes from depolarization phenomena and/or from degradation. The mixing of the reactants indeed occurs only on a very small distance in the middle of the channel. The development of such glucose/O2 biofuel cells seems of great interest for various applications. It is very simple to use abiotic and non-specific materials as catalysts. Moreover, it offers the possibility of working with two different pH values for the catholyte and the anolyte what can be interesting to improve electrochemical performances of each electrode (Zebda et al., 2009a). Nowadays, these devices are capable of delivering 110 pW cm-2 for a cell voltage of 0.3 V (Zebda et al., 2009b) by using GOD and laccase as catalysts. Glucose/O2 biofuel cells realized with classical fuel cell stacks have also been carried out (Habrioux et al., 2010). Both the used system and the obtained performances are described in Fig. 13.

Fig. 13 shows that the maximum power density obtained is 170 pW cm-2 for a cell voltage of 600 mV. However, let’s notice that performances of the biofuel cell rapidly decrease for current densities higher than 300 pA cm-2. This is clearly due to a very low ionic exchange rate between the two compartments of the cell since this value is too weak to correspond to mass transfer limitation of glucose molecule. The last design of glucose/O2 biofuel cell developed in the last past years is the concentric device (Habrioux et al., 2008; Habrioux et al., 2009a). It is based on concentric carbon tubes as electrodes and operates at physiological pH. An oxygen saturated solution circulates inside the internal tube composed of porous carbon, which is capable of providing oxygen diffusion. The whole system is immersed in a phosphate buffered solution (pH 7.4, 0.1 M) containing various glucose concentrations. Oxygen consumption occurs at the cathode such that no oxygen diffuses towards the anode. This allows to use in this device both abiotic and enzymatic materials as anode and cathode catalysts, respectively. BOD/ ABTS/Vulcan XC 72 R system is immobilized on the internal surface of the inner tube whereas Au-Pt nanocatalysts are immobilized on the internal surface of the outer tube. The surfaces of the cathode and anode were 3.14 and 4.4 cm2, respectively. The system is fully described in Fig. 14.

Different fuel cell tests realized by using various nominal compositions of Au-Pt nanomaterials have been realized. The best performances are obtained with Au70Pt30 as anodic catalyst. Actually, the maximum power density achieved is approximately of 90 pW

When Au8oPt20 is used as anode catalyst, the open circuit voltage is lower (i. e. 0.64 V). This is clearly explained by the surface composition of the catalyst which only contains 29 at. % of platinum. In the case of pure platinum, the open circuit voltage is very low due to strong competitions between phosphate species and glucose for adsorption. Such competition also occurs on other Au-Pt catalysts but the presence of gold allows a weaker interaction between phosphate species and the metallic surface. Consequently, higher glucose concentrations were used so as to improve biofuel cell performances. The obtained results are given in Fig. 16.

The data show a strong increase in cell voltage with glucose concentration. The raise observed in cell voltage between 0.1 M and 0.3 M can be attributed to the slow adsorption of phosphate species due to the presence of a higher glucose concentration. The maximum power density was also increased from 90 pW cm-2 (for a glucose concentration of 10 mM) up to 190 pW cm-2 (for a glucose concentration of 0.7 M). Nevertheless, in all cases, the fuel cell performances are greatly limited by resistance of the cell.

3. Conclusion

In this chapter we clearly show the importance of both electrodes assembly and global design of the cell on power output of the glucose/O2 biofuel cell. Moreover, it seems that a suitable choice of well-characterized nanocatalysts materials can lead both to an increase of the cell performances and to an improvement of their lifetime resulting in the abiotic nature of these materials. The approach, which consists of the utilization of an abiotic anode catalyst and an enzyme for a four electrons reduction, can undoubtedly open new outlooks for biofuel cells applications. This hybrid biofuel cell combines the optimized fuel electrooxidation, as developed in classical fuel cells, with the complete reduction of dioxygen to H2O without H2O2 production. Moreover, a concentric membrane-less design associated with an appropriate immobilization of the catalysts can avoid a costly separator of the cell events. Nevertheless, progresses to develop an efficient cell design are still necessary.

Rapid transport of reactants to the electrodes is essential to provide high power densities. When a heterogeneous reaction occurs at electrode surface, depletion of the reactant results in formation of a depletion zone near the electrodes surface where lower conversion rates occur as the reactant concentration is lower than in the bulk region. The thickness of the depletion layer increases usually in the direction of the convective flow (Fig. 5), thus resulting in the decrease of the current density along the electrode length.

Concentration profiles of reactive species are usually described computationally by resolving the convection-diffusion Eq. 9 and by setting appropriate boundary conditions. Modelling results in targeting optimal electrodes configuration in microchannel, fuel utilization and flow rate. Fig. 6 describes the 2D profile concentration during the operation of a glucose/O2 biofuel cell based on Y-shaped microfluidic channel of height, h, and with electrodes length L. As observed, the concentration of the active species decreases near the gold electrode surface that generates a depletion zone gradually increasing. The thickness of the depletion zone is a function of the distance from the inlet edge of the microchannel, and decreases with high flow rates. According to the Fick’s law (Eq. 11), it results that the simulated current density decreases sharply because of the concomitant increase of the depletion zone.

Reduction of the thickness of the depletion boundary layer is necessary to increase mass transport since the diffusion distance will be shorter and the diffusional flux will be higher. Several approaches have been developed to control the transport rate of reactants towards the electrode surface and thus the current density. It was demonstrated experimentally that the adjustment of flow rates controls the electrochemical processes that take place at the electrodes and regulate the depletion layer thickness. As observed in Fig. 7, the delivered current densities are influenced by flow rate of streams containing GOx for the anolyte and laccase for the catholyte during the operation of a glucose/O2 biofuel cell based on Y-shaped microfluidic channel.

The shape of the voltage-current density curves indicates that the current density is maximal when the voltage is almost zero, due to the consumption of all the fuel instantaneously at the electrode. Maximal current densities increase with flow rate from ~ 0.4 to 0.7 mA cm-2 as the impact of mass transport limitations is reduced from the bulk solution to the electrode surface. The maximum current density is thus limited by the diffusion of fuel and oxygen to their respective electrodes. The performance of the biofuel cell was evaluated at the operating flow rate of 1000 pL min-1. With an oxidant stream under oxygen at pH 3 and a fuel stream under nitrogen gas at pH 7, the maximum power density delivered by the biofuel cell is 110 pW cm-2 at 0.3 V (Fig. 7). Moreover, the pumping power to sustain the necessary flow in the microchannel was evaluated, according to Eq. 13, and compared with the delivered power by the cell. By varying the flow rate, it was found that the ratio of the input power to the output power increased from 1.5 % at 100 pL min-1 to 76 % at 1000 pL min-1. This experiment pointed out the importance of the flow rate on the power output delivered by the microfluidic BFC.

Another way to reduce depletion layer limitation and to enhance the transport rate of reactants towards the electrode surface lies in optimization of electrode geometry. Detailed experiments and simulations have revealed that current density decreases with increasing length of electrodes in the direction of the convective flow (Lim et al., 2007). To promote uniforme current density across the entire electrode assembly, Palmore and co-workers have demonstrated that splitting electrodes into smaller units separated by a gap in a microfluidic cell decreased the diffusion layer and improved the delivered power density by about 25% (Lee et al., 2007). In this work, the microfluidic fuel cell was built from a biocathode operating with laccase and ABTS as mediator to perform oxygen reduction, and from an anode operating with ABTS under N2. When operated at pH 4, this microfluidic cell exhibited a maximum power density of 26 pW cm-2 at the open-circuit voltage 0.4 V with a flow rate of 100 pL min-1. However, since the geometrical surface area required for the gaps did not contribute to any net current, the overall current density was not improved.

The depletion layer can be also manipulated to overcome mass transfer limitations by the development of original microfluidic configurations. This strategy was pointed out by simulation and experiments in the case of a microfluidic fuel cell working from formic acid (Yoon et al., 2006). These configurations featured, along the electrodes, either multiple periodically located outlets to remove consumed species or multiple periodically located inlets to add fresh reactants in the microfluidic channel. Such devices require controlling the volumetric flow rate trough each segments of the fluidic network. For both configurations, mass transfer was enhanced and reactant conversion at the electrodes was increased from 10 to 100 %.

Bioethanol production processes vary considerably depending on the raw material involved, but some of the main stages in the process remain the same, even though they take place in different conditions of temperature and pressure, and they sometimes involve different microorganisms. These stages include hydrolysis (achieved chemically and enzymatically), fermentation and distillation.

Hydrolysis is a preliminary treatment that enables sugars to be obtained from the raw materials that are then fermented. In the case of enzymatic hydrolysis, effective pretreatments are needed, however, to increase the susceptibility of lignocellulose materials to the action of the enzymes. The following paragraphs describe the various production methods, distinguishing them according to the type of raw material involved.

1.11 Lignocellulose biomass

The biofuels obtained from wood cellulose and from organic materials in general offer considerable advantages over conventional biofuels. Burning ethanol obtained from cellulose produces 87% lower emissions than burning petrol, while for the ethanol from cereals the figure is no more than 28%. Ethanol obtained from cellulose contains 16 times the energy needed to produce it (Martinez et al., 2008), petrol only 5 times and ethanol from maize only 1.3 times. The problem is a matter of how to disrupt the bonds of this molecule in order to convert it into fermentable sugars.

In fact, this is unquestionably the type of raw material that is the most complicated to process. The starting material may be farming and forest waste, scrap woods, grassy crops grown for energy purposes or even municipal solid waste. Lignocellulose occurs in the walls of vegetable cells and consists of cellulose microfibers contained in the lignin, hemicellulose and pectin. The procedure to obtain ethanol consists first in depolymerizing the carbohydrates into their monomeric sugars, then fermenting the sugars with the aid of appropriate microorganisms. The lignocellulose biomass consists mainly of three basic polymers: cellulose, hemicellulose (such as xylane), lignin and other minor components (essential oils, acids, salts and minerals).

Jean-Michel Lavoie12, Romain Beauchet1, Veronique Berberi12 and Michel Chornet12

1Industrial Research Chair on Cellulosic Ethanol Department of chemical and biotechnological engineering, Universite de Sherbrooke Quebec 2CRB Innovations Sherbooke, Quebec

Canada

1. Introduction

The first generation of biofuels, made out of starch (ethanol) or triacyglycerol (biodiesel) uses expensive homogeneous feedstocks (sugar cane, corn, wheat and edible oils) coupled with relatively inexpensive technologies known and practiced for years at an industrial level. First generation biofuels have had a bad press: high water and energy consumption (very significant is the energy used in the production of the fertilizers needed by agriculture) and the fuel versus food controversy. Increased use of biofuels requires alternative sources of biomass that lower water and energy consumption and do not compete with food supplies. Lignocellulosic biomass, either from forestry or agriculture offers such potential. Cellulose, the most abundant carbohydrate on the planet, is a fraction of the complex lignocellulosic matrix along with other macromolecules, lignin, hemicelluloses, and extractives. The cellulose macromolecule is composed of glucose units linked together via |3-1,4-glycosidic bonds (or acetal bonds) creating long chains that combine together to form fibrils and eventually fibres. The polar hydroxyl groups are oriented one toward the other so that interaction with a polar medium (as a solvent) is fairly difficult making cellulose water resistant. The natural macromolecule is usually present in nature in two forms: crystalline and amorphous. A typical fibril will have zones that are crystalline separated by zones that are amorphous. Whilst the crystalline form is difficult to disassemble with hydrolyzing agents, the amorphous phase has a certain level of disorder that makes relatively easy the penetration and action of hydrolysing agents, either enzymes or ionic species. The other macromolecules found in the lignocellulosic matrix are also of interest. Lignin is a macromolecule composed of phenylpropane units bond together via, predominantly, ether bonds although C-C between moieties are also significant. Lignin, has low oxygen content and thus a high energetic value. Hemicelluloses are, as cellulose, macromolecules composed of carbohydrates. Upon hydrolysis, the C6 fraction of these carbohydrates can effectively be converted to ethanol via fermentation using classical yeast strands (Girio, 2009). Studies have shown that fermentation of all the glucidic part of the hemicelluloses, both C6 and C5 sugars, was feasible using nontraditional microorganisms (Agbogbo & Coward-Kelly, 2008; Casey et al., 2009; Chu & Lee, 2007). It is also well known

that hydrolysis of hemicelluloses produces / liberates organic acids that could inhibit fermentation of the carbohydrate.

Cellulosics being an alternative for ethanol production, there is still an important aspect that has to be considered: the cost of the feedstock. Lignocellulosics for sugar production and subsequent fermentation can be considered to belong to three categories which interlink biomass cost, quality and transformability: homogeneous, quasi-homogeneous and non­homogeneous. The first category comprises structural and furniture wood and chips for pulp production which requires a single species (or a mixtures of comparable species). Such homogeneous biomass has also a rather homogenous chemical composition and it is used for high end products with well established markets. Homogeneous biomass is expensive, above $US 100 / tonne (anhydrous basis equivalent) in the NorthEast of America (2011 basis, prices range is a courtesy of CRB Innovations). Besides cost, such biomass also has a large market, in structural wood and in the pulp and paper industry. Quasi homogeneous biomass is usually composed of a mixture of species and to a certain extent, of tissues. This category embraces the residual lignocellulosic biomass produced during forest or agricultural operations. Cost range for this biomass will vary, FOB conversion plant in 2011, NorthEast of America, from $US 60 to 80 per tonne, dry basis, mostly related to transportation costs in a radius of a maximum of 100 km from harvesting operations (prices are a courtesy of CRB Innovations). Contrary to the homogeneous feedstock which has a wide and diversified market (yet very competitive), the quasi-homogeneous feedstock, since such biomass is of lesser homogeneity and often includes a higher quantity of ashes, is less coveted. Therefore, this biomass could be the main feedstock of the upcoming cellulosic ethanol market since the competition is actually low, the feedstock does not compete with food supply and the biomass is readily available close to major cities in the world. The last category of biomass is non-homogeneous. It is of lesser quality than the previous categories and usually costs close to 0 USD ( it may even come with a tipping fee in some cases). The low cost is of course attractive but the conversion process will have to use such biomass as a whole complex mixture converting it to a more homogeneous intermediary. Although we acknowledge the availability and the potential of each type of biomass, this chapter will be focused on the residual lignocellulosic material generated from established forest and agricultural industries (quasi-homogeneous biomass) as well as on plantation biomass. Plantation biomass can be identified as "energy crops" which are grown, ideally on marginal lands, with two objectives; sequestering carbon and bioconverting it into carbon — based structured macromolecules that could be used for the production of bioenergy. In North America, some cultures that have gained attention during the last 10 years, amongst many: willows, poplars, miscanthus, switchgrass, panic, reed canary grass, etc. Depending on the targeted market, these energy crops could be oriented towards high yields of cellulose (if the ethanol market is the main target) or high yield of lignin and less ashes (if the combustion market is targeted).

Biomass cost and composition are the main concerns of a cellulosic ethanol plant. A technological issue is how to convert the carbonhydrates to low cost monomeric sugars in high yields. Cellulose has been separated from plants for decades by the pulp and paper industry, the latter having developed industrial scale facilities that converted large quantity of lignocellulosic biomass to pulp and paper. However, the established processes to isolate the cellulose use large quantities of water putting a stress on water supplies. Furthermore, the pulp and paper does not actually use the hemicellulose and lignin other than for CHP production.. Research around the world have been focusing in the past decades toward processes that recover and use most of the carbon present in biomass to create true biomass refineries from which multiproducts would be obtained. This requires a careful consideration of which biomass to use to achieve valuable multiproducts and which biomass to use to provide heat and power.

The key technological challenge for the production of cellulosic ethanol is depolymerizing the cellulose to obtain high yields of glucose. As mentioned earlier, cellulose is a compact macromolecule, particularly its crystalline fractions, and it requires specific enzymes or chemicals to allow hydrolysis of the |3-1,4-glycosidic bonds. Accessibility of enzymes and chemical hydrolytic agents is a function of the three-dimensional ultrastructure of cellulose. Therefore, before going forward with production of cellulosic ethanol, the composition of the original feedstock and the ultrastructure of its isolated cellulosic fraction has to be known in order to adapt the hydrolytic processes to such ultrastructure.

This chapter focuses on three aspects that should be closely related to the production of second generation ethanol. In a first section, the composition of different substrate will be review as for their cellulosic, hemicellulosic and lignin contents. These data are essential for adaptation of the downstream process of a biorefinery. The second section of this chapter will be aimed at reviewing the steam treatments from our experience with the Feedstock Impregnation Rapid and Sequential Steam Treatment (FIRSST) process developed through two and a half decades of effort within our extended team (fundamentals at the academic level; engineering and technology via the spin-off company, CRB). Finally, the third section of this chapter will be an overview of the chemical treatments for cellulose hydrolysis compared with the CRB decrystallyzation and depolymerization process whose fundamental basis was developed by our team at the Universite de Sherbrooke.

Transesterification, also known as alcoholysis is the reaction of oil or fat with an alcohol to form esters and glycerol. To complete a transesterification reaction, stoichiometrically, a 3:1 molar ratio of alcohol to triglyceride is needed. In practice, to have a maximum ester yield, this ratio needs to be higher than the stoichiometric ratio. A catalyst is usually used to improve the reaction rate and yield. Because the reaction is reversible, excess alcohol is used to shift the equilibrium to the products side (Ma & Hanna, 1999). The reaction is shown in Fig. 1.

Fig. 1. Transesterification of triglycerides with alcohols.

The conventional technology of biodiesel production employs a basic homogeneous catalyst such as sodium or potassium hydroxides but when oil rich in free fatty acids (FFA) is used the basic catalyst and the FFA will interact to produce soap. This makes the amount of available catalyst for the transesterification reaction to be reduced and also complicates the down streaming separation and the biodiesel purification. Alternative processes for fatty acid ethyl ester (FAEE) production have been under development in order to employ different catalysts as heterogeneous ones (Marchetti & Errazu, 2011) such as metal oxides, metal complexes, active metals loaded on supports, zeolite, resins, membranes, and lipases (Kansedo et al., 2009).

Transesterification consists of a sequence of three consecutive reversible reactions, as can be seen on Figure 2. The first step is the conversion of triglycerides to diglycerides, followed by the conversion of diglycerides to monoglycerides, and finally monoglycerides to glycerol, yielding one ester molecule for each glyceride at each step.

Fig. 2. The transesterification reactions of vegetable oil with alcohol to esters and glycerol (Freedman et al., 1986).

The main parameters affecting the transesterification reaction are molar ratio of vegetable oil to alcohol, catalyst type and amount, reaction time and temperature, the contents of free fatty acids (FFAs) and water in substrate oil (Freedman et al., 1984) and also the intensity of mixing during the chemical reaction.

An overview of batch, fed-batch and two variants of continuous bioreactor fermentation experiments using glucose cultivation medium and the strain C. pasteurianum NRRL B-598 is presented in Table 2. Both batch and fed-batch cultivations were operated about 50h and a ratio of produced solvents (B:A:E) was about 2:1:0.1 in all cases. Batch cultivations were performed in media with initial glucose concentration 40 g. L-1 and if usual total solvents yields referred in literature are about 30% (Ezeji et al., 2005; Shaheen et al., 2000) then similar solvents concentrations like those shown in Table 2 were usually obtained. Therefore, higher initial glucose concentrations (60 and 80 g. L-1) were tested in flasks cultivations, however solvents concentrations remained either at the same level (for 60 g. L-1 glucose) or they were lower (for 80 g. L-1 glucose) in comparison with use of glucose concentration 40 g. L-1 and significant portion of glucose stayed in media unconsumed what might indicate a phenomenon of substrate inhibition.

Consequently, fed-batch cultivations were employed (see Table 2) in which butanol and total ABE concentrations were moderately increased (about 10%) and lag growth phase was reduced to 50% i. e. 3 h (data not shown). Nevertheless, yield and productivity for both 1- butanol and total solvents remained almost the same as in case of batch cultivations. The reached maximal butanol concentration (8.3 g. L-1) is probably near the highest value tolerated by the used strain and a substantial improvement in an overall amount of produced butanol could be attained only by an integration of the cultivation with some on­line separation step.

Table 2. Parameters of batch, fed-batch and continuous fermentations using C. pasteurianum

Surprisingly, glucose-limited fermentation experiment showed superior results in comparison with glucose non-limited fermentation (see Table 2). The only exception was solvents productivity that was higher at the expense of unused substrate. In glucose non­limited continuous experiment, mutually adverse oscillations of butanol and glucose concentrations occurred unlike butanol concentration near constant value (pseudo steady state) achieved in glucose-limited fermentation. The glucose limitation is also believed to support long-term stability and to reduce strain degeneration (Fick et al., 1985). Fermentation courses in both cases were presented in Patakova et al., 2011a.