Parallel to the use of agriculture residues instead of whole food crops as feedstock for biofuel generation, the use of whole non-food crops is also gaining popularity as feedstock for biomass conversion. Dedicated non-food crops which can be grown on unproductive lands are being considered as a more practical and eco­nomical option to even agricultural residues, as feedstock for biorefineries (whole crop biorefineries), as these would not use agricultural land for their generation as also, the entire plant could be used as feedstock. Such a whole crop biorefinery using the Jatropha plant for sustainable production of biodiesel has been described by Naik et al. [40]. Such a biorefinery can produce biodiesel as the main product, along with the production of other valuable chemicals as by-products from the solid residue obtained from the production of biodiesel. The oil cake remaining after removal of oil can be used to generate second-generation biofuelsand other important chemicals. The scheme for such a whole crop biorefinery using dedi­cated oil crops is shown in Fig. 1.23.

Takara et al. [41] investigated the suitability of banagrass for production of biofuel in a biorefinery concept. Banagrass (Pennisetum purpureum) is a perennial grass which is a good source of lignocellulosic biomass. Banagrass resembles sugarcane but shows a yield double to that of sugarcane and switchgrass in terms of biomass. A new concept of wet or green processing has been proposed. The biomass is taken as such without drying and subjected to dilute acid hydrolysis. It was found that the yield of fermentable sugars after the wet, juice processing was the maximum compared to dry, juice processing. This hydrolysate can be pro­cessed further for serving as a substrate for fermentation to ethanol. The process has a particular advantage in regions where banagrass can be grown throughout the year in that, it will reduce the time and cost of drying the biomass prior to pretreatment for extraction of cellulose. Co-products can also be obtained from the

nutrient-rich liquid substrates generated out of wet processing, which would be otherwise destroyed during downstream processing of the biomass. The biorefin­ery concept in this case offers a unique flexibility such that in times when the commodity value of ethanol is less, the process can be adapted such that the nitrogen-rich juice is utilized for the production of fungal biomass and aquaculture feed. The nitrogen-free clean fiber can be used for heat/steam electricity generation via gasification, whereas in times of high demand for ethanol, the process is used for the generation of ethanol.

Typically, there are three different operational parameters associated with the solids content of the feedstock to the digesters:

• High-solids (dry—stackable substrate)

• High-solids (wet—pumpable substrate)

• Low-solids (wet—pumpable substrate)

High-solids (dry) digesters are designed to process materials with high-solids content between *25 and 40%. Unlike wet digesters that process pumpable slurries, high solids (dry—stackable substrate) digesters are designed to process solid substrates deposited in tunnel-like chambers with a gas-tight door. They typically have few moving parts, require minimal or no pre-grinding or shredding, and do not use water addition. Solid state digestion of cattle dung is a suitable technology in which fresh cattle dung is anaerobically digested. Solid degradation of about 40-48% is observed in the effluent slurry that provides easy flowability to the oulet slurry [9].

Wet digesters can either be designed to operate in high solids content, with a total suspended solids (TSS) concentration greater than *20%, or a low solids concentration less than * 15%. High-solids (wet) digesters process a thick slurry that requires more energy input to move and process the feedstock. The thickness of the material may also lead to associated problems with abrasion. High-solids digesters will typically have a lower land requirement due to the lower volumes associated with the moisture.

Low-solids (wet) digesters can transport material through the system using standard pumps that require significantly lower energy input. Low-solids digesters require a larger amount of land than high-solids due to the increase volumes associated with the increased liquid-to-feedstock ratio of the digesters. There are benefits associated with operation in a liquid environment as it enables more thorough circulation of materials and contact between the bacteria and their food. This enables the bacteria to more readily access the substances they are feeding off and increases the speed of gas yields.

4.3.1 Analytical Techniques

Advances in a variety of analytical techniques have provided valuable insight into the mechanisms involved in delignification and cellulose dissolution. Optical and fluorescence microscopy enabled the study of wood expansion (Fig. 4.2) [70, 71] and switchgrass dissolution [42] in ionic liquids at the micron scale. Distinct autofluorescence from cellulose and lignin signatures distinguish the cellulose-rich cell walls and the lignin-rich cell corners and middle lamellae in poplar and switchgrass [42, 70, 71]. Optical and scanning electron microscopies have been particularly useful in visualizing IL interacting with heterogeneous native biomass. They revealed structural changes after dissolution, regeneration, and chemical functionalization [22, 30, 36, 38, 41, 42, 44, 72].

X-ray diffraction provided an insight into structural changes occurring at the atomic scale in cellulose during its dissolution and regeneration [7, 36, 38, 71, 73]. It was used to monitor in situ the loss of cellulose crystallinity in poplar, ramie fibers [71], switchgrass, eucalyptus, and pine wood [73]. After the regeneration of dissolved pine and spruce sawdust, X-ray diffraction revealed a change of crystal structure from the native cellulose I to the cellulose II structure [7, 36]. Neutron scattering was used to estimate the surface roughness of switchgrass, eucalyptus, and pine after their IL pretreatment [73].

These structural changes were supplemented by analyses of the biomass chemical composition. The distinct Raman signatures of cellulose and lignin have made hyperspectral Raman imaging a powerful tool to map the chemical com­position of native biomass [74] and its evolution during pretreatments [70, 71]. IR spectroscopy was commonly used to assess purity [4, 39, 42], loss of hemicel — luloses/lignin after the dissolution [36,42], chemical functionalization [6, 7, 30,48], cleavage of b-O-4 bonds in lignin models [75,76]. It can also probe the interactions between the anion and cation in the IL and the hydrogen bonding network [77]. FTIR spectroscopy combined with principal component analysis was used to distinguish lignins from bagasse, softwoods, and hardwoods [78]. Efforts were made to use IR spectroscopy as a method to quantify glucose and cellobiose in [EMIM] [OAc]. The IR absorption of multiple bands in glucose and cellobiose was found to vary with concentration and empirical nonlinear relations between the absorbance and the concentration were derived [79].

Optical absorption spectroscopy offers a quick way to quantify the saccharification of purified substrates, such as Avicel, and native biomass.

The 2,4-dinitrosalicyclic reagent acid assay has been widely used to quantify reducing sugars, including glucose [26,41,44,45,80]. However, on native substrates (municipal solid waste, paper mill wastes, or agricultural wastes), the method suffers from the interference from other chemicals and impurities [78,81]. Due to the variety and heterogeneity of native biomass, it has also been difficult to find adequate standards to establish Beer-Lambert relations between the absorbance and the sugar concentration, particularly for lignin which has different ratio of syringyl and gua — iacyl units [78]. ILs, such as [BMIM][Cl], also absorb strongly in the UV range. Optical absorption analyses are also complicated by chemical alterations of the biomass during pretreatments [78].

Analytical techniques, such as mass spectrometry [44, 69], HPLC [26, 43, 44, 46], high-performance anion-exchange chromatography (HPAEC) [41, 45, 50], have been used to identify hydrolysis products. HPLC and HPAEC can quantitate the amount of reducing sugars produced during cellulose hydrolysis. Size exclu­sion chromatography was used to determine the molecular weight distribution of milled woods and their dissolution products in ILs [33].

Another widely used analytical technique is NMR. The variety of isotopes available ( H, C, P, Cl) has made NMR spectroscopy a versatile method to characterize chemical functionalization [7, 30, 82], assess purity of products [32], identify/quantify hydrolysis/dissolution products [3, 4, 31, 32, 36], study the structure of milled native biomass (poplar, switchgrass) [29] and lignin [83], and study hydrogen bonding in cellulose dissolution [84, 85].

The combination of all these techniques has provided a wealth of information at multiple length scales about the chemical composition and structure of the pre­treated biomass. Quantitation of reaction products allowed for the optimization of reaction conditions, such as temperature and IL composition, and revealed the critical factors affecting the delignification and hydrolysis of cellulose.

The selective decarboxylation/decarboxylation of fatty acids is a relatively new technique which emerged very recently [7]. The idea of the process is to selec­tively decarboxylate/decarbonylate fatty acids to n-1-carbon alkanes with the selectivity above 95%. It was shown that fatty acid conversion can be achieved at around 300oC without using hydrogen [8]. Moreover, the pressure of the process is around 2 MPa using typically argon or 5% vol. H2 in argon atmosphere, compared to HDO over Ni-Mo and Co-Mo catalysts, where pressure is around 3-8 MPa of hydrogen.

Fermentation of the sugars generated from enzymatic hydrolysis of biomass is another important step where a lot of technical advances are needed to make lignocellulosic ethanol technology feasible. What is desired in an ideal organism for biomass-ethanol technology would be a high yield of ethanol, broad substrate utilization range, resistance to inhibitory compounds generated during the course of lignocellulose hydrolysis and ethanol fermentation, ability to withstand high sugar and alcohol concentrations, higher temperatures and lower pH, and minimal by-product formation [143]. Unfortunately, all these features seldom exist together in any wild organism and the need of the industry would be to develop an organism which will at least partially satisfy these requirements [208].

The ability to use the hemicellulose component in biomass feedstock is critical for any bioethanol project. S. cerevisiae and Z. mobilis, the commonly employed organisms used in alcohol fermentation, lack the ability to ferment hemicellulose and derived pentose (C5) sugars. While there are organisms that can ferment C5 sugars (e. g., Pichia stipitis, Pachysolen tannophilus, Candida shehatae), the effi­ciencies are low. These organisms also need microaerophilic conditions and are sensitive to inhibitors, higher concentrations of ethanol, and lower pH [26]. Worldwide, a lot of R&D efforts are being directed to engineer organisms for fermenting both hexose (C6) and pentose (C5 sugars) with considerable amount of success [4]. There are a large number of microorganisms including bacteria and fungi that are capable of breaking down cellulose into monosaccharides either aerobically or anaerobically. The anaerobic bacteria include Bacteroids cellulo — solvents, Bacillus spp. Clostridium cellulolyticum, Clostridium cellulovorans, Cellvibrio gilvus, Candida lusitance, etc. The fermentation of cellulose yields a variety of products, e. g., ethanol, lactate, acetate, butyrate, H2, CO2, etc.

Introduction of bacteria has been the greatest microbiological innovation because they produce less biomass, low concentration of by-products, and high productivity. The bacterium Z. mobilis ferments glucose to ethanol by with a typical yield of 5-10% higher than that of most of the yeasts though it is lesser ethanol tolerant than industrial yeast strains [151]. However, the small bacterium is difficult to centrifuge. Zymomonas being a simple prokaryote, an important possibility for the future is development of genetically modified organisms especially tuned to more ethanol tolerance and improved centrifugability [109].

Clostridium thermosaccharolyticum, Thermoanaerobacter ethanolicus, and other thermophillic bacteria as well as Pachysolen tannophilus yeast [177] are employed in fermenting pentose sugars which are nonfermentable by other organisms usually employed in ethanol production. These bacteria also convert hexose sugars. They have minimum end-product inhibition because very high temperature reactions would allow simple continuous stripping of ethanol from the active fermenting mixture. The yield of alcohol was further improved by cocul­turing C. thermocellum with C. thermosaccharolyticum or C. thermomophydro- sulphuricum [156]. However, the organisms so far studied produce excessive quantities of undesirable by-products and require strict anaerobic conditions which would be difficult to maintain on an industrial scale [53, 154].

Several microorganisms, including bacteria, yeasts, and filamentous fungi, have capacity to ferment lignocellulosic hydrolysates generating ethanol. Among them, Escherichia coli, Z. mobilis, S. cerevisiae, and P. stipitis are the most relevant in the context of lignocellulosic ethanol bioprocesses. These microorganisms have different natural characteristics that can be regarded as either advantageous or disadvantageous in processes of ethanol production from hemicelluloses (Table 9.8).

Pure and mixed cultures of Z. mobilis and Saccharomyces sp. were tested for the production of ethanol by fermentation of medium containing sucrose (200 g/l) at 30°C. The best results were obtained using fermentation for 63 h by a mixed culture and the average hourly ethanol productivity was 1.5 g/l [2, 161]. Ethanol fermentation from culled apple juice was compared by using Sacharomyces and Zymomonas spp. Ethanol production from culled apple juice showed that fer — mentability of the juice could be enhanced by addition of Di-ammonium hydrogen phosphate (DAPH) or ammonium sulfate in Saccharomyces and DAHP in Zymomonas. Trace elements however, inhibited the fermentation in both the cases. Physicochemical characteristics of the fermented apple juices were also analyzed.

Overall, S. cerevisiae proved better than Zymomonas for fermentation of apple juice [161].

Direct combustion of solid biomass occurs through evaporation combustion, decomposition combustion, surface combustion, and smoldering combustion. Components in the biomass which have a relatively simple structure and a low fusion temperature, fuse and evaporate when heated, and burn by reacting with oxygen in the gas phase. This is called evaporation combustion. The heavy oils present in the biomass first decompose due to the high temperatures encountered during combustion. The gas produced from thermal decomposition by heating reacts with oxygen in gas phase, flames, and then burns. This is called decom­position combustion. The char which remains after these forms of combustion, burns by surface combustion. Smoldering combustion is the thermal combustion reaction at temperature lower than the ignition temperature of the volatile com­ponents of the reactive fuels such as wood. If the ignition is forced to smoke, or temperature exceeds ignition point, flammable combustion occurs. In industrial direct combustion of biomass, decomposition combustion and surface combustion are the main forms of combustion [8].

This term refers to an area that is used to grow biomass for energy purposes. The idea behind energy plantation programme is to grow selected strains of tree and plant species on a short rotation system on waste or arable land. The sources of energy plantation depend on the availability of land and water and careful management of the plants. Energy crops, also called ‘‘bioenergy crops’’, are fast­growing crops that are grown for the specific purpose of producing energy (electricity or liquid fuels) from all or part of the resulting plant. They are selected for their advantageous environmental qualities such as erosion control, soil organic matter build-up and reduced fertilizer and pesticide requirements. As far as suitability of land for energy plantation is concerned the following criterion is used:

(1) It should have a minimum of 60-cm annual precipitation and (2) arable land having slope equal to or less than 30% is suitable for energy plantation.

The economics of energy plantation depends on the cost of planting and availability of market for fuel. Whereas these two factors are location specific, they vary from place to place. Further productivity of this programme depends on the microclimate of the locality, the choice of the species, the planting spacing, the inputs available and the age of harvest. There are many suitable species for energy plantation, for example, Acacia nilotica. There are many other perennial plant species which could be used for energy crops. In addition, some parts of traditional agricultural crops such as the stems or stalks of alfalfa, corn or sorghum may be used for energy production.

Although ILs have been demonstrated to be highly effective solvents for the dissolution of cellulose and lignocellulosic biomass, to date, the mechanism of this dissolution process remains not well understood. There is no definitive rationale for selecting ionic liquids that are capable of dissolving these biopolymers. Most current work is based on the hypothesis that cellulose insolubility is due to the strong intermolecular hydrogen bonds between cellulose chains. The dissolution of cellulose by a solvent is dependent on the destruction of these hydrogen bonds.

The outstanding solubility of ionic liquids to cellulose is due to the hydrogen basicity of anions, which can disrupt the hydrogen-bonding network among cellulose and lead to the dissolution. So far, there have been a few theoretical and experimental studies, including molecular dynamic studies and NMR analyses.

In 2007, Remsing et al. reported that 35/37Cl NMR relaxation measurements could be employed to study Cl-H hydrogen bonds in [Bmim] Cl [27]. It was found that the solvation of cellulose by the ionic liquid 1-n-butyl-3-methylimidazolium chloride ([Bmim]Cl) involves hydrogen bonding between the carbohydrate hydroxyl proton and the chloride ion in a 1:1 stoichiometry. Their further study demonstrated that the anions in these ILs are involved in specific interactions with the solutes, and govern the solvation process by analysis of Cl and C relaxation data for sugar solutions in both imidazolium chlorides and [Emim] [OAc] [28]. Variable-temperature NMR spectroscopy was also applied in the investigation on the dissolution mechanism of cellulose in 1-ethyl-3-methylimi — dazolium acetate ([Emim] [OAc]) in DMSO-d6. The results confirmed that the hydrogen bonding of hydroxyls with the acetate anion and imidazolium cation of EmimAc is the major force for cellulose dissolution in the ILs. The relatively small acetate anion favors the formation of a hydrogen bond with the hydrogen atoms of the hydroxyls, while the aromatic protons in the bulky cation imidazolium, especially H2, prefer to associate with the oxygen atoms of hydroxyls with less steric hindrance [29].

Since glucose is one of the main repeating units of polysaccharides, a better understanding of the interaction mechanism of glucose with ILs will provide in-depth understanding of the interaction of ILs and polysaccharides. In 2006, Youngs et al. investigated the molecular dynamics simulations of the solvation environment of isolated glucose monomers in a chloride-based IL (1, 3-dime — thylimidazolium chloride); the results revealed that the sugar prefers to bind to four chloride anions. Coordination shells involve only three anions, two of which are bridging chlorides. The low value of chloride: glucose ratio explains the unexpected high solvation degree of glucose in ILs [30]. Few glucose-glucose hydrogen bonds, but chloride anions hydrogen bonding to different glucose molecules simultaneously were found, partially explain the high solubility of glucose/cellulose in ILs [31].

As a natural polymer, cellulose is significantly amphiphilic and hydrophobic interactions are important for explaining the solubility pattern of cellulose. Lindman et al. presented strong evidence that cellulose is amphiphilic and that the low aqueous solubility must have a marked contribution from hydrophobic interactions [32]. Thus, we should reconsider the molecule interaction between lignocellulosic biomass and ILs. Liu et al. developed an all-atom force field for 1-ethyl-3-methylimidazolium acetate [Emim][OAc] and the behavior of cellulose in this IL was examined using molecular dynamics simulations of a series of (1-4) linked b-D-glucose oligomers (degree of polymerization n = 5, 6, 10, and 20). They found that there is strong interaction energy between the polysaccharide chain and the IL, and the conformation (b-(1,4)-glycosidic linkage) of the cellulose was altered. The anion acetate formed strong hydrogen bonds with hydroxyl groups of the cellulose, and some of the cations were found to be in close contact with the polysaccharides through hydrophobic interactions. These results supported the fact that the cations play a significant role in the dissolution of cellulose in anion acetate ILs [33]. Guo et al. calculated geometries, energies, IR characteristics, and electronic properties of the cellulose-anion (acetate, alkyl phosphate, tetrafluoro — borate and hexafluorophosphate) complexes using density functional theory calcu­lations (DFT). They found that the strength of interactions of anions with cellulose follows the order: acetate anion > alkyl phosphate anion > tetrafluoroborate anion > hexafluorophosphate anion, and the favorable sites of cellulose for the chloride anion attack are around the O2 and O3 hydroxyls [34].

Singh et al. reported that autofluorescent mapping of plant cell walls was used to visualize cellulose and lignin in pristine switchgrass (Panicum virgatum) stems to determine the mechanisms of biomass dissolution during ionic liquid pretreat­ment. Swelling of the plant cell wall, attributed to the disruption of inter — and intra­molecular hydrogen bonding between cellulose fibrils and lignin, followed by complete dissolution of biomass, was observed without using imaging techniques that require staining, embedding, and processing of biomass [35]. This could be applied to the elucidation of structural information of wood and wood components.

The origin of enzyme deactivation in ILs was studied by comparing enzyme activity in different ILs. The effect of IL chemical composition, structure, and

functionalization were studied to design new ionic liquids suitable for cellulose dissolution and cellulase activity. It was found that ionic liquids with a higher molecular weight would maintain enzyme (lipase) activity at a high level. Adding a longer alkyl chain on the imidazolium cation would accomplish that, but this would lead to a higher viscosity, which has a negative effect on cellulose disso­lution. To reduce the viscosity, the long alkyl chain was substituted by oxygenated chains, such as poly(ethylene glycol) and poly(propylene glycol). A long oxy­genated chain reduced the cellulose solubility, so an optimum chain length was determined to maintain both the cellulase activity and cellulose solubility high. The imidazolium cation with the best overall performance was derived from tri­ethylene glycol monomethyl ether. The oxygen atoms introduced are believed to form hydrogen bonds with cellulose, facilitating its dissolution. Adding a longer alkyl or oxygenated chain on the other side of the imidazolium cation led to a significant decrease in cellulose solubility, which was attributed to the reduction of hydrogen bonding with cellulose due to steric hindrance. The acetate anion led to higher cellulase activity and cellulose solubility than the chloride anion [135].

From the solvent productivity point of view, batch process produced solvents with such a low productivity of 0.35-0.40 g l-1 h-1 [64]. For scaling up of such a process, high volume of broth, high capital cost, and operational cost became severe problems. This leads to uneconomical production of solvents. Continuous fermen­tation reactors show several advantages over batch reactors such as one inoculum culture is sufficient for the long time, drastically reduced sterilization and inocu­lation time. Various continuous processes, such as free cells, immobilized cells, and cell recycling and bleeding, have been investigated. However, immobilized cell process has shown significant potential with as high as 15.8 g l-1 h-1 of solvent productivity (about 40 times than batch process) [69]. Also, cell immobilization allowed long survival time to cells of C. acetobutylicum in solventogenesis phase, which resulted that 20% higher yield than conventional fermentation [30].