For decades, D. salina has been cultivated for its natural ability to produce |3-carotene. This valuable bioproduct allows for large-scale cultivation and processing of the biomass to be very profitable, as D. salina is the predominant source of natural |3-carotene (Ye et al. 2008). This green alga is ideal for growth in outdoor ponds due to its ability to grow in high salinity waters — as much as eight times the salt concentration of seawater — greatly reducing the threat of contamination by local microbes and eliminating the need for large quantities of freshwater. Dunaliella spp. are similar to Chlamydomonas spp. in that they exist as single, flagellated, elongated cells in the size range of 10 microns; however, D. salina and D. tertiolecta, are capable of osmoregulation by a complex network of ion channels, a flexible cell membrane uninhibited by a cell wall, and glycerol biosynthesis to offset osmotic pressure (Goyal, 2007). As such, the lack of a rigid cell wall makes the algal biomass relatively simple to lyse for the purpose of downstream processing. Furthermore, the technique of "milking" microbial cells for certain metabolites has improved substantially in recent years. In this process, the cells are contacted with a biocompatible organic solvent in order to promote preferential transfer of desired compounds to the solvent phase, leaving the cells viable for continued bioproduction. This process has been successfully demonstrated with D. salina for the extraction of |3-carotene in a two-phase system (Hejazi et al, 2004b).

While the demand for natural |3-carotene dictates the high market price of this compound and continued use of D. salina, an increasing desire for biofuel production draws an inquisitive eye to the carotenogenesis pathways of D. salina (Lamers et al., 2004). Since all carotenoid compounds are composed of long-chain branched hydrocarbons, it is conceivable that the biosynthetic pathways of D. salina could be altered to produce hydrocarbons that are ideal for use as gasoline-like biofuel. With some molecular biology tools already developed for Dunaliella spp. (Polle et al., 2009), the sequencing and annotation of its 610 Mbp nuclear genome will now allow for more extensive genetic engineering endeavors with this organism. At the time of these experiments, only the chloroplast and mitochondrial genomes of D. salina CCAP 19/18 (GenBank GQ250046, GQ250045) were released. In light of its unique biotechnological application and long history of mass production, D. salina is an ideal organism for future development as a biofuel producing microalgae.

The first high-temperature, high-pressure ever used methanol synthesis catalysts were ZnO/Cr2O3 and were operated at 350°C and 250-350 bar. Catalyst compositions contained 20-75 atom% Zn and these catalysts demonstrated high activity and selectivity for methanol synthesis and proved robust enough resist sulphur poisoning which is inherent when converting syngas from coal gasification. Over the years, as gas purification technologies improved, interest in the easily poisoned Cu catalysts for methanol synthesis was renewed. In 1966, ICI introduced a new, more active Cu/ZnO/ Al2O3 catalyst was the first of a new generation of methanol production using lower temperature (220-275°C) and lower pressure (50-100 bar) than the established ZnO/ Cr2O3 catalysts. The last high temperature methanol synthesis plant was closed in the mid-1980s (Fiedler et al., 2003) and at the present, low temperature, low pressure processes based on Cu catalyst are used for all commercial production of methanol from syngas. The synthesis process has been optimised to the point that the modern methanol plants yield 1 kg of methanol/liter of catalyst/hr with >99.5% selectivity for methanol. Commercial methanol synthesis catalyst has lifetimes on the order of 3-5 years under normal operating conditions.

The Cu crystallites in methanol synthesis catalysts have been identified as the active catalytic sites although the actual state (oxide, metallic…) of the active Cu site is still being debated. The most active catalysts all have high Cu content, optimum about 60 wt% Cu on the catalyst that is limited by the need to have enough refractory oxide to prevent sintering of the Cu crystallites. Hindering agglomeration is why ZnO creates a high Cu metal surface area. ZnO also interacts with Al2O3 to form a spinel that provides a robust catalyst support. Acidic materials like alumina, are known to catalyse methanol dehydration reactions to produce DME. By interacting with the Al2O3 support material, the ZnO effectively improves methanol selectivity by reducing the potential for DME formation. Catalysts are typically prepared by the co-precipitation of metal salts with a variety of precipitation agents. It is important to avoid contaminating methanol catalysts with metals that have FT (Fischer Tropsch) activity (Fe or Ni) during the synthesis. Incorporation of alkali metal in the catalyst formulation should also be avoided because they catalyse the increase of higher alcohols production. Table 4 shows catalyst formulation from several commercial manufacturers. Additional catalyst formulations have been presented in the literature with the purpose of improving per-pass methanol yields (Klier, 1982). The addition of Cs to Cu/ZnO mixtures has shown improved methanol synthesis yields. This only holds true for the heavier alkali metals, as the addition of K to methanol synthesis catalysts tends to enhance higher alcohols yields. The Cu/ThO2 intermetallic catalysts have also been investigated for methanol synthesis (Klier, 1982). These catalysts have demonstrated high activity for forming methanol from CO2-free syngas. Cu/ Zr catalysts have proven active for methanol synthesis in CO-free syngas at 5 atm and 160-300°C (Herman, 1991). Supported Pd catalysts have also demonstrated methanol synthesis activity in CO2-free syngas at 5-110 atm and 260-350°C (Spath and Dayton, 2003).

Baled agricultural biomass from the field does not have good flowing characteristics and may not flow easily into grinders such as hammer mills and disc refiners. Therefore, biomass needs to be chopped with a chopper (rotary shear shredder)/ knife mill/ tub grinder to accommodate bulk flow and uniformity of feed rate. A chopper, knife cutter, or knife mill is often used for coarse size reduction (>50 mm) of stalk, straw, and grass feed stocks (Bitra et al., 2009). Knife mills reportedly worked successfully for shredding forages under various crop and machine conditions (Cadoche and Lopez, 1989).

Bitra et al. (2009) reported that the total specific energy (including energy to operate the knife mill) for agricultural biomass chopping increases with knife mill speed. The total
specific energy for knife mill and tub grinder has been observed to have negative correlation with screen size and mass feed rate (Arthur et al., 1982; Bitra et al., 2009; Himmel et al., 1985). However, grinding rate (throughput) increases with an increase in screen size (Arthur et al., 1982).

For tub grinders, an increase in screen size results in an increase in geometric mean length of particles and throughput, but a decrease in bulk density of the particles and specific energy consumption (Kaliyan et al., 2010).

The oxidation of glucose on gold-platinum nanoparticles has been investigated in numerous studies (Habrioux et al., 2007; Sun et al., 2001). Jin and Chen (Jin & Chen, 2007) examined glucose oxidation catalyzed by Pt-Au prepared by a co-reduction of metallic salts. An oxidation peak of glucose was visible at much lower potentials than on gold electrode. Moreover, they showed that both metals favored the dehydrogenation of the glucose molecule. They concluded that the presence of gold prevents platinum from chemisorbed poisonous species. The efficiency of such catalysts towards glucose oxidation is thus not to be any more demonstrated, and greatly depends on the synthesis method used to elaborate the catalytic material.

1.1.4.1 Synthesis of gold-platinum nanoparticles

Various gold-platinum nanoparticles synthesis methods have been already studied: Polyol (Senthil Kumar & Phani, 2009), sol-gel (Devarajan et al., 2005), water-in-oil microemulsion
(Habrioux et al., 2007), electrodeposition (El Roustom et al., 2007) and Bonnemann (Atwan et al., 2006). Among all these methods, the water-in-oil microemulsion technique produces particles that exhibit high catalytic activity towards glucose electrooxidation (Habrioux et al., 2007). It consists in mixing two microemulsions, one containing the reducing agent in the aqueous phase and the other containing one or several metallic precursors in the aqueous phase. Collisions of water nanodroplets permit to obtain metallic nanoparticles which can be then cleaned and dispersed onto a carbon support. The choice of the different components of the microemulsions is not unique and influences the physical properties of the obtained nanoparticles. Actually, both surfactant molecules and oil-phase chemical nature have an effect on interfacial tension of the surfactant film that determines water solubility in micelles (Paul & Mitra, 2005). This greatly affects intermicellar exchanges. Moreover, the chemical nature of the reducing agent controls the rate of the nucleation step and subsequently the kinetic of particles formation. In the system described herein, n-heptan is used as oil phase, non-ionic polyethyleneglycol-dodecylether as emulsifier molecule and sodium borohydride as reducing agent. The synthesized particles have been dispersed onto Vulcan XC 72 R and then washed several times with acetone, ethanol and water, respectively to remove surfactant from their surface (Habrioux et al., 2009b). The removal of surfactant molecules from all the catalytic sites without modifying structural properties of the catalyst is currently a great challenge (Brimaud et al., 2007). Since electrocatalysis is a surface phenomenon depending on the chemical nature of the surface of the catalyst, on its crystalline structure and on the number of active sites, it is useful to precisely know the physico-chemical properties of the used nanoparticles to understand their electrochemical performances.

Most of the microfluidic devices employ patterned electrodes positioned in parallel on the bottom wall or on sides of the channel (Kjeang et al., 2008). Electrodes, with varying length and wide, are patterned by coating glass slides with conductive materials such as gold, graphite over an adhesive layer (often chromium or titanium) by standard sputtering techniques (Zebda et al., 2010) or by photolithography and sputtering (Moore et al., 2005; Lim et al., 2007; Togo et al., 2008). The inter-electrode gap varies between 0.2 mm and 1.4 mm (Lee et al., 2007; Togo et al., 2008; Zebda et al., 2010).

1.1.2 Fabrication of the microfluidic devices

The microfluidic device is finally obtained by physically clamping the PDMS slab with the glass substrate that accommodated the electrode pattern. This approach works well with elastomer polymer like PDMS. Alternatively, an irreversible seal may be achieved between both parts by oxygen-plasma treating prior to improve the adhesion (Lim et al., 2007; Lee et al., 2007). Alignment of the flow channel over the microelectrodes is often aided by a microscope. As an example, a device, consisted of a Y-shaped channel with two inlets and two outlets, is presented in Fig. 4. The pressure-driven laminar flow required for injection of fuel and oxidant is typically driven by a syringe pump via polyethylene tubing.

1.2 Performances of microfluidic biofuel cells

This paragraph mainly describes microfluidic BFCs involving mediated monoenzymatic systems, which are capable of only partial oxidation of the fuel. Devices have been
developed either with diffusional enzymes flowing through the microchannel, or with immobilized enzymes on electrode surface. This paragraph also includes preliminary works on devices allowing improvement of fuel utilization by complete oxidation, which have been designed with multienzymatic systems.

The performances of microfluidic BFCs are evaluated from cell voltage and current density. The cell voltage of the biofuel cell reflects both the open circuit voltage (OCV), partially controlled by the formal potential of the two redox mediators and the overpotential losses. The delivered current density reflects the rate of catalytic turnover and transport processes as a function of the surface area of the electrode. The power density is the product of cell voltage and cell current density. As already mentioned, the performances of the microfluidic BFCs are limited (i) by cross-diffusional mixing (SmiX) of fuel and oxidant at the interface between the two streams, (ii) by formation of depletion boundary layers at the surface of the electrodes as the result of the reaction of fuel and oxidant, and (iii) by low concentration and low diffusion coefficient of oxygen. These factors depends on geometric and process parameters such as the microchannel dimensions, the electrode parameters (number of electrodes, electrode surface area, electrode spacing), and operating conditions (electrolyte, flow rate, pH, concentration of species). The influence of these parameters on open circuit voltage, current density and power density, have been evaluated both experimentally and theoretically in literature.

This cereal relies heavily on the availability of nitrogen in the soil; it has high contents of both glucan and xylan (40.8% and 22.3% respectively) (Petersson et al., 2007).

1.8.3 Corn stover

This is what remains on the ground after maize has been harvested. This raw material is abundantly available and demands no further investment in biomass, although not all of the corn stover can be removed — 30% of it must be left on the ground to prevent erosion (by facilitating water infiltration and reducing evaporation), and as the main source of soil organic carbon (SOC) in order to preserve the soil’s productivity. Corn stover contains polymeric hemicellulose and cellulose, but their biodegradability by glycosidase is strongly inhibited by a small quantity (12-15%) of lignin (Gressel, 2008; Varvel et al., 2007).

1.9 Jerusalem artichoke (helianthus tuberosus)

This plant grows in summer, reaching its maximum height in July and dying in October. The tubers are rich in inulin (a fructose polymer), which can be used to obtain a syrup for use both in the foodstuffs industry and in the production of ethanol. It was demonstrated (Curt et al., 2006) that, towards the end of the season, the potential for bioethanol production of the stems of clones is 38% of that of the tubers.

2.3 Finite element approach for optimization of electrodes design

For our simulation approach, we used commercially available COMSOL 3.5 software multiphysics software, which solves partial differential equations (PDEs) by finite element technique. In the model we assume that 3D carbon microelectrode arrays were uniformly immobilized with glucose oxidase and laccase on anode and cathode respectively with out the use of any mediators. The proposed implantable membraneless EBFC is assumed to be placed inside a blood artery of the human body thus utilizes the glucose extracted from blood as a fuel. In principle, glucose oxidase reacts with glucose and produces gluconolactone and hydrogen peroxide. This hydrogen peroxide oxidizes on the anode to generate electron and hydrogen ions. The hydrogen ions travel from electrolyte to cathode, while electrons flow through an external load and generate electricity. On cathode, dissolved oxygen is reduced via laccase enzyme and by combining with electrons and hydrogen ions forms water.

We applied Michaelis-Menten theory in our 2D model to analyze phenomenon between enzyme kinetics on the electrode surface and glucose diffusion and thus optimize the electrode microarray design rule according to the enzyme reaction rate In order to determine the output potential in developing biofuel cell, we also incorporated Nernst equation. The numerical simulations have been performed with various electrodes heights and well widths (distance between any two electrodes) to obtain the relation between design rule and EBFCs performance. Various 2D models are investigated for same foot print length (600 pm) of SiO2, with fixed electrode diameter of 30 pm and fixed enzyme layer thickness of 10 pm. The height of electrodes is chosen as 60 pm, 120 pm and 240 pm for different well widths (WW-distance between any two electrodes) of 10pm, 20 pm, 40 pm, 60 pm, 80 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm and 200 pm.

The quantification of reaction rates of enzymes on anode and cathode is showed in Fig. 6.

From the results, we observe that the reaction rate decreased from the top to the bottom along the surface of both electrodes due to the lack of diffusion of the substrate as we go towards the bottom; also the outer surfaces of the electrodes have the larger reaction rate in the enzyme layer. The reaction rate along the surface of both electrodes is plotted in Fig. 6. The reaction rate is increased from the bottom to the top along the electrode surface and reached the maximum at edge of the top due to the edge effect. The maximum reaction rates of GOx enzymes vs. different well widths is shown in Fig. 7. for three different heights of electrodes: 60 pm, 120 pm and 240 pm, with 10 pm, 20 pm, 40 pm, 60 pm, 80 pm, 100 pm and 120 pm well widths, respectively. In the case of 60 pm height of electrodes, the maximum reaction rate is obtained when the well width is about 30 pm. For the height of 120 pm and 240 pm, reaction rate reached the highest at the well width of 60 pm and 120 pm respectively. From all these three sets of models both in anode and cathode, we can conclude that the reaction rates of one pair of electrodes reach the maximum when the well width is half as the height of electrodes.

The open circuit output potential also has been simulated for the same heights and well widths of electrodes by applying the Nernst equation. The current collectors are assumed at the bottom of the electrodes and hence these potentials are calculated from the bottom. Fig. 8. shows the open circuit output potential vs. well width of electrodes at different height of electrodes. From the results of simulation, we could find out an empirical relationship between electrodes height and well width to achieve optimized output potential is when height of electrodes is twice than that of well width which is in agreement to the results we obtain for the diffusion of the substrate.

Iodine number is a measure of the total unsaturation within a mixture of fatty acids, and is expressed in grams of iodine which react with 100 grams of biodiesel. Engine manufacturers have argued that fuels with higher iodine number tend to polymerize and form deposits on injector nozzles, piston rings and piston ring grooves when heated (Kosmehl and Heinrich 1997). Moreover, unsaturated esters introduced into the engine oil are suspected of forming high-molecular compounds which negatively affect the lubricating quality, resulting in engine damage (Schaefer et al 1997). However, the results of various engine tests indicate that polymerization reactions appear to a significant extent only in fatty acid esters containing three or more double bonds (Worgetter et al. 1998, Prankl and Worgetter 1996, Prankl et al 1999).Three or more-fold unsaturated esters only constitute a minor share in the fatty acid pattern of various promising seed oils, which are excluded as feedstock according to some regional standards due to their high iodine value. Some biodiesel experts have suggested limiting the content of linolenic acid methyl esters and polyunsaturated biodiesel rather than the total degree of unsaturation as it is expressed by the iodine value.

1.9 Methanol or ethanol

Methanol (MeOH) or ethanol (EtOH) can cause fuel system corrosion, low lubricity, adverse affects on injectors due to its high volatility, and is harmful to some materials in fuel distribution and vehicle fuel systems. Both methanol and ethanol affect the flash point of esters. For these reasons, methanol and ethanol are controlled in the specification.

The low density, strength, and stiffness of aspen make it unsuitable for many structural applications (Mackes & Lynch, 2001). However, aspen has an alternative use as bedding material or feedstock for biofuel or bio-oil. The amount of biomass produced per unit area by canola depends on irrigation and varies from 5 to 10 tons/ha (Enayati et al., 2009). According to the US Canola Association, canola was cultivated on 3.2 million ha during 2009, resulting in 16-32 million tons of canola straw. Early in the US history corn cobs were an important feedstock for heating houses, farm buildings, and small businesses. Now, corn cobs are reemerging as a potential feedstock for direct combustion, gasification, and cellulosic ethanol due to numerous advantages (dense and relatively uniform size, high heat value, and low N and S contents) over many competing feedstocks. The average cob yield is about 14 % of the grain yield and represents about 16 % of the corn stover biomass in a field on a dry matter basis (Roth & Gustafson, 2010). According to Blaschek and Ezeji (2010), 15% of corn stover is corn cob. Various sources have independently estimated that anywhere from 200 to 250 million dry tons of corn stover are produced per year (Sokhansanj et al., 2002; Kadam & McMillan, 2003). Based on the 15-16% of stover as cobs, the US annual production of corn cob is estimated at 30-40 million metric tons.

1.1 Introduction

In the past, it was assumed that the catalytic hydrotreatment of pyrolysis oils shows strong resemblances with conventional hydrotreatment processes of fossil feeds (hydrodesulphurisation, hydrodenitrogenation) and typical catalysts, process conditions and reactor configuration were taken from these conventional processes (Elliott, 2007). For all, the objective is a reduction of certain elements (oxygen, sulphur, nitrogen) in the feed by the action of hydrogen and a catalyst. However, common reaction conditions for the hydroprocessing of crude oil derivatives cannot be adopted directly for pyrolysis oils. For instance, pyrolysis oil cannot be treated as such at temperatures exceeding 300 oC because of its high charring tendency.

Numerous papers on hydrotreating pyrolysis oil in packed beds and autoclaves were published in the eighties and nineties, and now very recently as well (Elliott, 2007). Specifically the older literature seems rather phenomenological in nature, and merely focuses on ‘fact-finding’. Large differences in operating conditions like in pressure, temperature, residence times and hydrogen consumption are reported. In any case, without active catalyst or (high pressure) hydrogen, significant charring of pyrolysis oil occurs. Reduction of charring is possible by applying a (relatively) low temperature catalytic hydrotreatment at 175 to 250 oC, in which reactive components in the oil are ‘stabilized’. Subsequently, the stable product is further processed at higher temperatures (> 300 oC) and pressures (> 150 bar). High pressures are believed to be essential to keep the water in the pyrolysis oil feed in a liquid state (and as such probably reduce the charring reactions), to promote the solubility of hydrogen in the initially polar bio-oil, and to increase the rate of the actual hydrogenation reactions.