A wide variety of biomass feedstock/IL combinations has been studied for their potential in biomass pretreatment. Multiple wood species have been studied: poplar [29], spruce [7, 30-34], eucalyptus [31, 32], pine [4, 6, 7, 31, 32, 35-37], maple [25, 38], Metasequoia glyptostroboides [16], red oak [36], common beech [34], cork [39], and Japanese fir [40]. Other biomass feedstocks currently under investigation include grasses, such as switchgrass [41, 42], Miscanthus grasses [26, 43], and agricultural wastes, such as corn stovers [6, 33, 35, 43-45], wheat straw [27] and rice straw [6, 35, 46].

Among the most successful and widely used ILs in native wood pretreatment are the imidazolium-based ILs with the chloride or acetate anion. The ILs 1-allyl-

3- methylimidazolium chloride ([AMIM][Cl]) and [BMIM][Cl] could dissolve
maple wood flour at solubilities above 30 g/IL kg at 80°C under nitrogen atmo­sphere after 24 h [25]. Ball-milled pine powder and spruce sawdust (size 0.1-2 mm) were completely dissolved in [BMIM][Cl] and [AMIM][Cl] at a weight ratio of up to 8% at 80-110°C in 8 h with mechanical stirring [7]. [AMIM][Cl] was able to dissolve completely 5 wt% of spruce, silver fir, beech, chestnut wood chips (particle size 1-2 mm) at 90°C in 12 h, whereas the same wood samples were only partially dissolved in 1-ethyl-3-methylimidazolium chloride ([EMIM][Cl]), [BMIM][Cl], and 1,3-dimethylimidazolium dimethyl — phosphate ([MMIM][Me2PO4]) in the same conditions [34]. The IL 1-ethyl-3- methylimidazolium chloride ([EMIM][Cl]) can partially dissolve wheat straw and pine wood particles (<1 mm, 5 wt%) at 100°C in 24 h, [BMIM][Cl] can only partially dissolve wheat straw, and 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]) could dissolve neither [47]. Ground pine, poplar, eucalyptus, and oak were dissolved in [BMIM][Cl] with a 5 wt% solubility at 100°C. After 24 h, about 45 wt% of the cellulosic material was extracted from the native biomass. The extraction rates were higher for softwoods, such as pine and poplar. 13C Nuclear Magnetic Resonance (NMR) confirmed the presence of dissolved polysaccharides in the wood/IL mixture [4].

[EMIM][OAc] dissolved spruce, beech, chestnut completely (5 wt%), but not silver fir [34]. In another study, [EMIM][OAc] could dissolve 5 wt% of red oak (particle size 0.125-0.250 mm) completely in 25 h at 110°C, while it took 46 h to dissolve 5 wt% of southern yellow pine in the same conditions [36]. Pretreatment of maple wood flour with [EMIM][OAc] or 1-butyl-3-methylimi — dazolium acetate ([BMIM][OAc]) at 90°C increased significantly the sugar yield and the amount of extracted lignin [38]. Pretreatment with 1-butyl-3-methyl — imidazolium methyl sulfate ([BMIM][MeSO4]) at the same temperature for the same duration resulted in sugar yields comparable to the untreated wood flour and an amount of extracted lignin lower than with [EMIM][OAc] or [BMIM][OAc]. This was explained by the fact that [BMIM][MeSO4] only delignified the middle lamella and not the primary cell wall and cellulose-rich secondary cell wall. Also, the [EMIM][OAc] or [BMIM][OAc] pretreatment for 12 h reduced the wood fiber diameter from an average of 250 pm in the untreated flour to about 17 pm. Pretreatment with [BMIM][MeSO4] had no effect on the wood fiber diameter [38].

Other combinations of IL/native wood were studied. Dry wood (Metasequoia glyptostroboides, 60 mesh sawdust) was partially dissolved in 1-butyl-3-allylim — idazolium chloride ([BAIM][Cl]) or 1-methyl-3-allylimidazolium chloride ([MAIM][Cl]) at a weight ratio from 4.5:1 to 10.5:1 (60-90°C for 10-40 min) [16]. Phenyl-containing ionic liquids were synthesized to see if the aromatic n-systems would be better at disrupting the strong n-n interactions between aro­matic groups in lignin. Indeed, after the wood was dissolved in 1-benzyl-3- methylimidazolium chloride ([BzMIM][Cl]), the solution was clear, free of any residual lignin [7]. Ball-milled poplar was soluble in 1-allylpyridinium chloride, cyanomethylpyridinium chloride, and pyridinium chloride within 1 h at 60°C with solubilities ranging from 35 to 80 mg/g [29].

In addition to woods, ILs could at least partially dissolve or delignify other feedstocks, including leaves and agricultural wastes. Triticale straw, flax shives, and wheat straw were soluble in [EMIM][OAc] and [BMIM][Cl] [27]. The dis­solution of shredded oil palm fronds in [BMIM][Cl] was studied for temperatures ranging from 60 to 100°C [22]. Lignin was extracted from bagasse using the IL 1-ethyl-3-methylimidazolium alkylbenzenesulfonate at high temperatures (170-190°C) [48]. Rice straw powder (<2 mm) could be dissolved in [BMIM] [Cl], [EMIM][Cl], and [EMIM][OAc] completely in 24 h at 130°C. The amount of regenerated cellulose and glucose after enzymatic hydrolysis was highest for [EMIM][OAc] [46]. Milled corn cob had solubilities above 30 g/kg at 130°C in 1-methyl-3-methylimidazolium dimethylphosphite ([MMIM][DMP]), 1-ethyl-3- methylimidazolium diethylphosphate ([EMIM][DEP]), 2-ethyl-3-methylimidazo — lium dimethylphosphite ([EMIM][DEP]), [BMIM][Cl], and 1-butyl-1-meth- ylpyrrolidinium chloride ([BMPy][Cl]). Pretreatment with chloride ILs resulted in the doubling of reducing sugar yield after enzymatic hydrolysis [44].

5.2.1 Known Catalyst Types for Biomass Gasification

5.2.1.1 The Synthesis Gas

The main product of biomass gasification is the synthesis gas. The synthesis gas is produced in the presence of steam. The following reactions are observed during biomass gasification.

C + H2O! CO + H2(syngas) AH°298 = 323.1kJ/mol (5.1)

AH°298 = —394kJ/mol (5.2)

AH°298 = 282.1 kJ/mol (5.3)

The first reaction, between carbon and steam, is strongly endothermic, pro­ducing carbon monoxide (CO) and hydrogen (H2). When the coke bed has cooled to a temperature at which the endothermic reaction can no longer proceed, the steam is then replaced by a blast of air.

The reactions (5.2) and (5.3) take place, producing an exothermic reaction— forming initially carbon dioxide—raising the temperature of the coke bed— followed by the second endothermic reaction, in which the latter is converted to CO. The overall reaction is exothermic, forming ‘‘producer gas’’. Steam can then be re-injected, then air etc., to give an endless series of cycles until the coke is finally consumed. Producer gas has a much lower energy value, relative to syngas, primarily due to dilution with atmospheric nitrogen. Pure oxygen can be substituted for air to avoid the dilution effect, producing gas of much higher calorific value.

The synthesis gas can be used for power/heat generation or further transformed into diesel range hydrocarbons by Fischer-Tropsch synthesis. Since products of synthesis gas conversion by the Fischer-Tropsch reaction contain olefins and oxygenates, there is considerable interest in combining a Fischer-Tropsch metal, such as Fe, Co or Ru, with ZSM-5 to form a bifunctional catalyst. These catalysts exhibit improved selectivity for a gasoline-range product, and synthesis gas can be converted to gasoline-range hydrocarbons in one step.

A widely used and accepted method of functional genomics is to disrupt the genes through mutations and study the effects in the following generations. There are several methods employed for this, e. g., the insertional mutagenesis such as T-DNA insertions in model plants such as Arabidopsis [38] and rice [39]. Also, transposon tagging is another method of choice for functional genomics in the model plants. The transposons or jumping genes were identified and isolated by Barbara McClintock from maize and it was cloned by [40]. The Ac/Ds system based on the transposons is being used as a tool for functional genomics in several plant systems [41,42]. Using these methods one can generate and study a pool of insertion mutants in the biofuel crops and look for desirable phenotypes and genes associated with them. Such experiments will increase our understating of the genetics of biofuel crops and will open the doors for genetic modifications of such crops to enhance biomass and biofuel production. However, generating large number of mutants is not feasible in all cases, and to address that there are several alternate tools. For example, if the crop has synteny with model crops such as rice that can be tested in the biofuel species. Thus, an aluminum tolerance (Alt3) locus was mapped in rye using rice/rye synteny [43]. Another valuable reverse genetics technique called ‘Targeting Induced Local Lesions IN Genomes’ (TILLING), involves high throughput PCR screens of genomic DNA from M2 mutant populations induced by chemical mutagens [44, 45].

Genomic and functional genomics projects are being applied to one of the model grass species, Brachypodium, and its whole genome sequence has been released [46] similar to what has been achieved with the rice genome project. Also, functional genomics tools are being employed to the biomass crops such as switchgrass, Miscanthus and sorghum. These studies include genome-wide analysis of miRNA targets, developing low input switchgrass biomass using its bacterial endophytes and studying root physiology using root hair response to abiotic stresses. To study the gene functions there are other functional genomics approaches such as microarray studies which are useful to understand the global changes in gene expression. All these approaches will yield valuable information on the genetic nature of the biofuel crops, which have been ignored for a long time primarily due to the lack of investment in this area of research. Using these powerful genetic tools, one can generate and study a pool of insertion mutants in the biofuel crops and look for desirable phenotypes and genes associated with it. A better understating of genetic nature of biofuel crops will open the doors for genetic modifications to enhance biomass and biofuel production.

9.6.1.1 Yeast Metabolic Pathways

Glucose is converted into ethanol and CO2 via glycolysis, in the anaerobic pathway:

С6Н12Об ! 2C2H5OH + 2CO2 + Energy (Stored as ATP)

The overall reaction produces two moles of ethanol and CO2 for every mole of glucose consumed, with the reaction energy stored in 2 mol of ATP. Every gram of glucose converted will yield 0.511 g of ethanol, via this pathway. Secondary reactions consume a small portion of the glucose feed, however, to produce bio­mass and secondary products, Pasteur found that the actual yield of ethanol from fermentation by yeast is reduced to 95% of the theoretical maximum (Table 9.10). For maximum ethanol productivity, aerobic reaction should be avoided as in this

reaction, sugar is completely converted into CO2, cell mass and by-product with no ethanol formed.

9.6.1.2 Effect of Sugar Concentration

The primary reactant in the yeast metabolism is hexose sugar (glucose, fructose). The rate of ethanol production is related to the available sugar concentration by a Monod-type equation under fermentative conditions:

V = VmaxC/(Ks + Cs),

where

V = specific ethanol productivity (g ethanol/g cells/h)

Cs = Sugar substrate concentration (/g)

Ks = Saturation constant having a very low value (typically 0.2-9.4 g/l).

The yeast is starved at very low substrate concentrations (below 3 g/l) conse­quently, the productivity decreases [105]. At higher concentrations, a saturation limit is reached so that the rate of ethanol production per cell is essentially at its maximum up to 150 g/l sugar concentration. The catabolic (sugar) inhibition of enzymes in the fermentative pathway becomes important above 150 g/l, and the conversion rate is slowed down [72, 192].

An important secondary effect of sugar is catabolic repression of the oxidative pathways—Crabtree Effect. At above 3-30 g/l sugar concentration (depending on the yeast strain), the production of oxidative enzymes is inhibited [34, 127] thus, fermentative pathway is adopted. The Crabtree effect is not found in all the yeasts and is a desirable character in the industrial strains of yeast selected.

Biorefineries can be classified into a number of categories depending on the feedstock used.

1. Biorefinery based on agriculture sector feedstock (dedicated crops and residue) including oilseed biorefinery

2. Forest biorefinery (forest residue mainly lignocellulosic feedstock)

3. Biorefinery based on industry (process residues and leftovers), and municipal solid waste and waste water (domestic waste)

4. Aquaculture-based biorefinery (algae — and seaweed-based biorefinery).

The salient features of all these types of biorefineries are given in Table 1.9.

Biorefinery Based on Agriculture Sector Feedstock (Including Dedicated Crops and Residue, and Oilseed Feedstock)

These biorefineries use as their feedstock, dedicated crops (food or non-food crops), such as cereal crops, oilseed crops, grasses, and other non-food green plants, or residues generated from the agricultural crops. The first — and second- generation biofuels can be produced from this type of biorefinery. Presently, biodiesel, bioethanol, and biogas are the main types of biofuels which are pro­duced by commercially viable technologies. An overview of the conversion pro­cesses for these biorefineries is shown in Fig. 1.22.

These biofuels are presently produced from agriculture sector feedstock. The agriculture sector feedstock mainly contains sugar, starch, and cellulosic biomass which can be converted to biofuel (bioethanol) mainly by fermentation processes.

For this purpose, the macromolecular starch and cellulose is first hydrolyzed by enzymatic hydrolysis into smaller molecules like glucose. Fermentation of these sugars by either aerobic or anaerobic fermentation processes converts them to ethanol. The process details are already discussed in Sect. 1.2.1.2. The alcohol produced from food crops is called grain alcohol and that produced from ligno — cellulosic feedstock such as agricultural residues (wheat straw, rice straw, etc.), grasses (switch grass) is called biomass ethanol or bioethanol or lignocellulosic ethanol. The agriculture-based feedstock, which was, until recent times used as the major source for biofuel generation, is gradually being replaced by agriculture sector residue such as wheat straw.

A batch system is the simplest form of digestion. Biomass is added to the reactor at the start of the process in a batch and is sealed for the duration of the process. Batch reactors suffer from odor issues that can be a severe problem when they are emptied. Typically, biogas production will be formed with a normal distribution pattern over time. The operator can use this fact to determine when they believe the process of digestion of the organic matter has completed. As the batch digestion is simple and requires less equipment and lower levels of design work it is typically a cheaper form of digestion.

In continuous digestion processes organic matter is constantly added (contin­uous complete mixed) or added in stages to the reactor (continuous plug flow; first in-first out). Here the end products are constantly or periodically removed, resulting in constant production of biogas. Single or multiple digesters in sequence may be used. Examples of this form of anaerobic digestion include continuous stirred-tank reactors (CSTRs), upflow anaerobic sludge blanket (UASB), expanded granular sludge bed (EGSB) and internal circulation reactors (IC).

The wide range of biomass solubility in ILs reported in the literature could be partially explained by the contamination with water, which can significantly affect their physicochemical properties [58]. Even hydrophobic ILs, such as 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([EMIM][Tf2N]), are hygroscopic and can contain up to 25% of water (molar ratio) when exposed to an environment with a relative humidity of 81% [59]. Water can also be produced during the reaction of biomass with IL by the hydrolysis of acetate groups [47]. Traces of water can be detected by :H NMR or IR spectroscopy [60]. They can be quantified by Karl-Fischer titration or gravimetrically [59]. Water can be removed in a vacuum oven or in a freeze dryer [7, 61]. Its presence complicates the IL recycling and removal is energy-intensive.

Addition of water above 4 wt% to loblolly pine wood before its pretreatment in [BMIM][Cl] led to a notable decrease in soluble products, monosaccharides, and

5- hydroxymethylfurfural. This was attributed to the competition with the cellulose hydroxyl groups to form hydrogen bonds with Cl — ions [53]. The presence of water reduced the solubility of wood in ILs and the yield of sugars released in the dissolution of maple wood flour in [BMIM][OAc] and [EMIM][OAc] [7]. Water also prevented the IL from effectively reducing the cellulose crystallinity [38]. Water can also prevent the formation of by-products such as 5-hydroxymethyl­furfural during the dissolution of cellulose in [EMIM][Cl] catalyzed by HCl or H2SO4 [62].

The IL hygroscopicity is the result of the adsorption of water on the IL surface, diffusion from the surface and/or the formation of complexes through hydrogen bonding [59, 63, 64]. The hygroscopicity depends on the IL composition and structure [65]. Adsorption would depend on the charge distribution and structure of the cation and anion, while diffusion would be affected by the IL viscosity [59]. The length of alkyl chains and substitution on the cation ring (e. g., pyridinium, imidazolium) affected the mutual solubility of the IL with water [65, 66]. For ILs with the [EMIM] cation, water uptake increased with different anions in the fol­lowing order: dicyanamide < diethyl phosphate < chloride < acetate [61].

Deactivation of the Ni-Mo and Co-Mo catalysts in the HDO is caused by:

• desulfurization of the catalyst

• coke formation

• water inhibition

• catalyst poisons

Desulfurization of the catalyst is one of the main reasons of deactivation. Sulfur compounds could be removed from the surface of the catalyst by reactions with compounds from the reaction mixture [11, 12, 14] or hydrogen [19]. Commonly accepted way to inhibit deactivation is addition of the sulfur-containing compounds to the feedstock. When H2S and CS2 were added to the feedstock no significant decrease of deactivation of the catalyst was observed. Sulfur content in the Ni-Mo catalyst was not affected when H2S or CS2 were added. In the case of Co-Mo catalyst the addition of different amounts of sulfur agents did not stop desulfur­ization of the catalyst. Moreover, addition of CS2 caused lower yield of hydro­carbons, compared to H2S, but increase of fatty acid production from esters [12].

Coke formation was observed over Ni-Mo and Co-Mo catalyst in the deoxy­genation of the aliphatic esters (Table 6.1). It can be partially responsible for deactivation of the catalyst by blocking the catalyst active sites. The Ni-Mo and Co-Mo catalysts on y-Al2O3 can be regenerated by burning away the coke deposits with hot air, but there is no published data available for the influence of regen­eration on the sulfided catalysts properties.

The water inhibition was studied with aliphatic esters, showing deactivation of Ni-Mo and Co-Mo catalysts. Water inhibition is an important issue because it can be created in different stages during the reaction (Fig. 6.2a). Increase of water in the reaction mixture causes not only deactivation but it also affects product dis­tribution by decreasing strongly decarboxylation/decarbonylation reaction rate, compared to hydrogenation rate, increasing the ratio between n-carbon and n-1- carbon hydrocarbons [20]. A negative effect of water can be diminished by addition of H2S but that will promote decarboxylation/decarbonylation reaction, as mentioned before (see Sect. 6.2.1.2).

The negative effect of phosphorus and alkali, impurities from vegetable oils feedstock, was studied over sulphided Co-Mo/y-Al2O3 catalyst at 310°C and

3.5 MPa of hydrogen pressure [21]. Alkali metals block/poison active sites leading to deactivation of the catalyst. When alkalis and phosphorus were present, deac­tivation of the catalyst was even higher than in the absence of them because of the formation of phosphates locating above charge-compensating alkalis. In the absence of alkalis, phospholipids produced phosphoric acid which catalyzed oligomerization reactions and lead to deactivation of the catalyst by carbonaceous deposits.

9.3.1 Microorganisms and Their Characteristics

Microorganisms are a key component of the technology used in different fermen­tation regimes, including ethanol. Diverse groups of microorganisms are capable of producing ethanol. These include yeasts, Saccharomyces cerevisiae, Schizosac — charomyces pombe, bacteria Zymomonas mobilis, fungus Fusarium oxysporum, yeast-like fungus Pachysolen tannophylus, and thermophilic bacteria [28]. Saccharomyces cerevisae and Schizosaccharomyces pombe represent the organism of choice for the industrial production of ethanol due to the following features:

• Capable of fermenting a diverse range of sugars and sole production of ethanol, CO2 under anaerobic conditions

• Due to their comparatively bigger size, they flocculate well to supply clean wash to the still and the wash and distillate lack offensive odor

• Contamination problem is under control as the fermentation process operates at low pH and high sugar concentration

• Are genetically stable and ferment 20-25% (w/v) sugar in molasses solution completely

Wheat straw like any other biomass of lignocellulosic composition is a complex mixture of cellulose, hemicellulose, and lignin, as three main components, and a small amount of soluble substrates (also known as extractives) and ash. The cel­lulose strains are bundled together and tightly packed in such a way that neither water nor enzyme can penetrate through the structure [104]. Hemicellulose serves as a connection between lignin and cellulose fibers, and it is readily hydrolyzed by dilute acid or base, as well as hemicellulase enzyme. Lignin is covalently linked to cellulose and xylan (predominant hemicellulose carbohydrate polymer in wheat straw) such that lignin-cellulose-xylan interactions exert a great influence on the digestibility of lignocellulosic materials [104]. Due to this, the structural com­plexity of the lignocellulosic matrix, ethanol production from wheat straw requires at least four major unit operations including pretreatment, hydrolysis, fermenta­tion, and distillation. Unlike sucrose or starch, lignocellulosic biomass such as wheat straw need to be pretreated to make cellulose accessible for efficient enzymatic depolymerization.