High worldwide demand for energy, unstable and uncertain petroleum sources, and concern over global climate change has led to resurgence in the development of alternative energy that can displace fossil transportation fuel. Biomass is con­sidered to be an important renewable source for securing future energy supply, production of fine chemicals and sustainable development.

Having looked at a lot of integrated multi-disciplinary research on biomass conversion into energy and fine chemicals, I was delighted to find that this book does exactly what it says on the cover — it provides a guide to conversion of biomass into energy, biofuels and fine chemicals. This timely book covers many different topics: from biomass conversion to energy, the concept of green chem­istry (the applications of ionic liquids for biomass conversion), catalysts in ther­mochemical biomass conversion, production of biobutanol, bioethanol, bio-oil, biohydrogen and fine chemicals, the perceptive of biorefinery processing and bioextraction. The majority of chapters survey topics that will allow the reader to obtain a greater understanding about biomass conversion and the role of multi­disciplinary subjects which include biotechnology, microbiology, green chemistry, materials science and engineering.

I am pleased that the editors took on the challenge to give an excellent overview of the different techniques for biomass conversion applied in academia and industry. Their expertise and their valuable network of contributors have made this volume a highly respected work that has a central place in this series on renewable resources.

National University of Singapore Dr. Seeram Ramakrishna

Singapore, February 2012 Professor of Mechanical Engineering

and Bioengineering Vice-President (Research Strategy)

Most biorefineries have the capability to produce biofuels as well as high value chemicals. An important question that is obviously expected to arise in this context is, whether a biorefinery should be energy oriented or product oriented. A hybrid biorefinery or, as it is more commonly called, an integrated biorefinery, which can produce both category products efficiently, and has the capability of switching over to alternative feedstocks as and when required, is the answer to this question. An integrated biorefinery employs various combinations of feedstocks and con­version technologies to produce a variety of products with a main focus on pro­ducing biofuels. Thus an integrated biorefinery would involve the integration of all the above-mentioned biorefineries with respect to feedstock used and biomass conversion processes employed, in order to get maximum benefits from the bi­orefinery concept. The World’s first integrated biorefinery project was launched in October 2003 as a joint effort between the U. S. Department of Energy’s National Renewable Energy Laboratory (NREL) and Du Pont. Since then, a large number of such integrated biorefineries, at various stages of development, and operating with a variety of feedstock, yielding a range of products, have been successfully set up globally. Table 1.12 gives a list of integrated biorefineries operating in the US [54]. There are other such biorefineries being set up in the UK [55] and other parts of the world, which progressively incorporate state-of-the-art biomass conversion technologies and produce biofuels and other products in an effort to reduce GHG emissions and provide for a viable alternative to the fossil fuels.

For an integrated biorefinery to become a reality, it should be cost-effective. The logistics of feedstock availability, its generation and utilization will need to be very carefully planned for the purpose. Simultaneously, the bioconversion pro­cesses will also have to be integrated in a manner that is technically and eco­nomically feasible. The U. S. Department of Energy—Energy Efficiency and Renewable Energy has proposed a comprehensive outline for such a project (Fig. 1.30).

Anaerobic digestion in landfills is brought about by the microbial decomposition of the organic matter in refuse. The levels of organic matter produced per capita vary considerably from developed to developing countries. Worldwide, the urban population is growing at twice the rate of the total population growth, creating unprecedented demands for goods and services as well as increasing pressure on the environment and on safe waste disposal [10]. Landfill-generated gas is on average half methane and half carbon dioxide with energy content from 18 to 19 MJ/m3. Its production does not occur under pressure, and thus recovery pro­cesses must be active. Commercial production of land-gas can also aid with the leaching problems now increasingly associated with landfill sites. Local commu­nities neighboring landfill sites are becoming more aware of the potential for heavy metals and nutrients to leach into aquifers. Landfill processing reduces the volume of sludge to be disposed of, and the nutrient content, thus facilitating proper disposal. Methane is a powerful greenhouse gas, with substantial amounts being derived from unutilized methane production from landfill sites. Its recovery therefore, not only results in the stabilization of the landfill site, allowing faster reuse of the land, but also serves to lessen the impact of biosphere methane emissions on global warming.

The dissolution of cellulose was usually attributed to the ability of the IL to disrupt the hydrogen-bond network in cellulose by forming hydrogen bonds with cellulose. For example, in [AMIM][Cl], the chloride anion is a hydrogen-bond acceptor, while the proton at the 2-position of the imidazolium ring is a hydrogen — bond donor [32]. NMR studies of [BMIM][Cl] showed that the chloride anion has an active role in the solubility of cellulose through hydrogen bonding with the hydroxyl groups of cellulose [84]. Density-functional theory calculations showed that the anions in imidazolium-based ILs tended to form hydrogen bonds with the O2 and O3 hydroxyl groups of cellulose. The strength of the hydrogen bonds increased for the following anions in the order: hexafluorophosphate < tetrafluo — roborate < alkyl phosphate < acetate. The trend matched the one observed in the dissolution of cellulose in the corresponding imidazolium-based ILs, where cellulose solubility was highest with the acetate anion [25, 92]. The strong hydrogen bonding ability of ILs means that they can disrupt the hydrogen bonding network in lignocellulosic biomass by displacing the lignocellulose components to form stronger hydrogen bonds [34].

Fig. 4.3 A time series of X-ray diffraction images recorded from a radial section of Poplar sp. As [EMIM][OAc] is applied and then expelled with water; a Untreated sample, b-e application of [EMIM][OAc], f-i application of water.

The fiber diffraction direction is approximately vertical. Note the presence of two superimposed equators in a, h, and i with a relative orientation of approximately 25°. Reprinted from [71], copyright (2011), with permission from Elsevier

Molecular dynamics simulations were conducted to study the interaction between [EMIM][OAc] with glucose oligomers (5-20 units). The total interaction energy between [EMIM][OAc] with cellulose (around -75 kcal/mol) was larger than the one between water and cellulose (around -50 kcal/mol) and the one between methanol and cellulose (around -45 kcal/mol). The difference between [EMIM][OAc] and water/methanol became larger with the cellulose chain length [49]. The acetate anion is also a hydrogen-bond acceptor, with the potential to form hydrogen bonds with the three hydroxyl groups of each unit of cellulose. The strength of these hydrogen bonds (14 kcal/mol) was estimated to be three times higher than the hydrogen bonds in water (5 kcal/mol) and methanol (4 kcal/mol). The simulations showed that the imidazolium cation interacts strongly with the glucose ring structure via van der Waals forces. Also, the interactions between [EMIM][OAc] and cellulose led to conformation changes in the cellulose chains, which can explain the loss in crystallinity and structural changes in regenerated cellulose [49].

The hydrogen bonding ability of ILs was probed by IR spectroscopy. ILs were prepared with the same anion [Tf2N]- and different cations with increasing hydrogen bonding ability: 1,2,3-trimethylimidazolium, 1,3-dimethylimidazolium, 1,2-dimethylimidazolium, and 1-methylimidazolium. The increasing strength of hydrogen bonds was indicated by a shift of the IR absorption band below 150 cm-1 toward higher wave numbers. This band shifted from 62 cm-1 for the 1,2,3-trimethylimidazolium cation to 101 cm-1 for the 1-methylimidazolium cation. There was a linear relationship between the measured peak position and the average interaction energies in IL clusters from ab initio calculations. Ab initio calculations also showed that the interaction energy is minimal for the 1,2,3,4,5- pentamethylimidazolium cation where all protons were substituted by methyl groups and the hydrogen bonding ability was reduced [77].

Formation of hydrogen bonding between [EMIM][OAc] and cellobiose was also studied by :H NMR. A broadening of the OH resonances was observed as the molar ratio between [EMIM] [OAc] and cellobiose was increased, which was explained by the interaction between the O atoms in the hydroxyl groups and the protons of the imidazolium ring. The accompanying downshift of the OH resonances was attributed to the hydrogen bonding between the acetate anion and the hydrogen atoms in the cellulose hydroxyl groups. NMR spectra of [EMIM][OAc] with increasing cellobiose concentration indicated that the strongest hydrogen bonding between the imidazolium cation and cellobiose involves the proton at the 2-position of the imidazolium ring. The next stron­gest hydrogen bonds involve the protons at the 4- and 5-position, which are much weaker hydrogen-bond donors [84]. When all the hydroxyl groups in cellobiose were acetylated, the NMR spectra of the [EMIM][OAc]/cellobiose octaacetate remained unchanged as the IL concentration increased. This showed that hydrogen bonding between cellobiose and the IL cation/anion is the main reason cellobiose dissolves in [EMIM][OAc]. In order to dissolve cellulose effectively, it was proposed that the IL must have an anion that is a good hydrogen acceptor, and a cation that is a moderate hydrogen-bond donor and not too large [84].

The process of the catalytic cracking over highly acidic catalysts was proposed for transformation of triglycerides to obtain chemicals or fuels. The catalytic cracking of fatty acids is a highly unselective process involving cleavage of C-C bond of the fatty acids. In the case of HDO and decarboxylation/decarbonylation process oxygen removal is based on highly selective reactions over a-carbon, whereas in the case of catalytic cracking the reaction can occur independently on the position of carbon-carbon bond in the fatty acid.

Microporous, highly acidic zeolites, such as ZSM-5, have also been used as catalyst in the transformation of fatty acids esters [40] and triglycerides [41]. Transformation of the methyl octanoate gave 99% conversion of the ester at 500°C with a broad distribution of the products from C1 to C7 hydrocarbons from which the highest selectivity was achieved for ethane (34%). Aromatic products such as benzene, toluene, C8, and C9+ aromatics were found in the reaction mixture and their yield exceeds 20% [40]. Transformation of triglycerides over ZSM-5 at temperature of 400°C gave similar product distribution, but with different selec — tivities [41]. After 90% of triolein conversion, propylene was the main product with the selectivity of 44%. Selectivity toward benzene and toluene reached 40 and 20%, respectively.

The example of ZSM-5 suggests that zeolites are not good catalysts for pro­duction of the green diesel because of highly unselective transformation of fatty acids and their derivatives, leading to the formation of undesired aromatic com­pounds. However, it was recently proposed that catalytic cracking of fatty acid esters over MgO/Al2O3 catalysts resulted in minor formation of aromatic com­pounds, maintaining good deoxygenation activity [42].

The effect of MgO loading in Al2O3 was studied in oleic acid deoxygenation. The experiments were carried out at the temperature of 300, 350 and 400°C in presence of MgO-Al2O3 catalysts with magnesium oxide loading of 30, 63, and 70 wt% of the total catalyst weight [42]. With the MG-63 (MgO 63 wt%) and MG-70 (MgO 70 wt%) the oleic acid was converted in 98%. The oxygen content in the reaction mixture was below 1 wt%, for reaction at 400°C. The reaction mixture was composed from hydrocarbons in the range of C7-C17, and minor aromatics compounds. The reaction should be performed above 350°C to avoid saponification of fatty acids.

Recently, the use of Cs-containing zeolites (CsNaX) as a deoxygenation cata­lyst was proposed [43]. The CsNaX catalyst has an advantage of high selectivity toward n to n—2 carbon length hydrocarbons (n—length of fatty acid chain) compared to non-Cs-containing zeolites over which poor selectivities have been achieved. The transformation over CsNaX catalyst of 10% methyl octanoate was performed in methanol which sustains catalyst activity, at 425°C and inert atmospheric pressure. The CsNaX catalyst is superior to MgO, in the activity, which indicates that not only basic sites are needed for the conversion of fatty acids esters, but also synergy of basic and acidic zeolite sites is required.

Lactose is a milk sugar. In dairy products, the fermentation of this sugar plays a vital role. Lactose is a disaccharide of D-galactose and D-glucose bonded to each other by b-1,4 glycosidic linkage. Lactose cannot be taken up freely by the microbial cells. A specific transport system is required for the translocation of this sugar to the site of metabolism. Lactose transported through PTS gets phosphorylated as lactose-6-P, while the other system translocates it unphos — phorylated. Once lactose is translocated, it is fermented first undergoing hydrolysis into monosaccharides with the help of b-galactosidase, also called lactase. The former enzyme is present in the lactic acid bacteria. Approxi­mately, 80% of the galactose originated from lactose is metabolized via taga- tose pathway. Figure 9.5 shows the structure of lactose.

Fig. 9.2 Pathway of glucose degradation. a hexokinase, b phosphoglucose isomerise, c phosphofructokinase, d aldolase, e triosephosphate, f glyceraldehydes-3-P- defydrogenase, g phosphoglycerate kinase, h phosphoglycerate mutase, i enolase, j pyruvate kinase

— Fructose-6-P

9.4.1.4 Starch

Starch is a homopolysaccharide of D-glucose units that are joined to each other through a 1,4-glycosidic bond. Starch has two components, amylose and amylo- pectin (Fig. 9.6). Amylose is an unbranched molecule with molecular weight ranging from a few thousands to 5,000,00. One end of each chain with free hemiacetal group is reducing while the other is nonreducing in nature. The typical blue color with starch is due to its ability to form a helical structure. It is soluble in water. Amylopectin is a branched polysaccharide with b 1-6 linkage at every

25-30 glucose units. The molecular weight and branching per chain differ for different sources of starch.

Starch is widely distributed from lower microalgae such as Chlamydomonas to higher plants. In plants, it is the major storage material. A great diversity of microorganisms is able to utilize this polysaccharide. The hydrolysis of starch into glucose in biological systems is carried out with multiple enzymes. For the commercial application of amylolytic enzymes, the reader is referred to an earlier review [62].

Pyrolysis processes can be classified on the basis of the rate of heating as: Slow pyrolysis, fast pyrolysis, and intermediate pyrolysis. All these types of pyrolysis processes are carried out in the absence of oxygen. Depending on the medium in which it is carried out, it is classified as either hydrous pyrolysis (carried out in presence of water) or hydropyrolysis (carried out in presence of hydrogen).

Slow pyrolysis

The process of slow pyrolysis is used mainly for production of char and involves slow heating of the biomass over long periods of time (ranging from minutes to days).

Torrefaction is a slow pyrolysis process carried out at low temperatures (230-300°C) in the absence of oxygen. It is a form of pretreatment of biomass to improve its energy density, reduce the oxygen/carbon and hydrogen/carbon ratio, and reduce its hygroscopicity. This pretreatment makes the biomass more suitable for other biomass conversion processes. For example, the high oxygen content of biomass increases thermodynamic losses during the gasification process. Reduc­tion of the oxygen/carbon ratio as a result of torrefaction reduces the thermody­namic losses during the gasification process. The microfibrils in biomass, comprising cellulose, are supported or bound together by hemicellulose. The process of torrefaction depolymerizes this hemicellulose, causing a consequent reduction in binding of cellulose fibrils. This causes the structure to become friable and brittle, reducing the energy requirement for size reduction process which precedes most bioconversion processes. Torrefaction is also accompanied with a color change in most cases. The process of roasting of coffee beans by heating the green beans to 200-300°C over a long period of time is the most popular example of the torrefaction process. During the process of torrefaction, there is some reduction in the energy content of the biomass due to the partial devolatilization occurring during the process. However, this reduction in energy content is com­pensated by the increase in energy density of the biomass during the process. Basu [11] gives details of the changes in terms of energy density, heating value, etc. in bagasse after torrefaction.

2.4.2.1 Pyrolysis

Pyrolysis is a thermochemical decomposition of organic material at elevated temperatures in the absence of oxygen. Pyrolysis typically occurs under pressure and at operating temperatures above 430°C (800°F). In general, pyrolysis of organic substances produces gas and liquid products and leaves a solid residue richer in carbon content. Extreme pyrolysis, which leaves mostly carbon as the residue, is called carbonization.

The biomass feedstock is subjected to high temperatures at low oxygen levels, thus inhibiting complete combustion, and may be carried out under pressure. Biomass is degraded to single carbon molecules (CH4 and CO) and H2 producing a gaseous mixture called ‘‘producer gas’’. Carbon dioxide may be produced as well, but under the pyrolytic conditions of the reactor it is reduced back to CO and H2O; this water further aids the reaction. Liquid-phase products result from temperatures which are too low to crack all the long chain carbon molecules thus resulting in the production of tars, oils, methanol, acetone, etc. Once all the volatiles have been driven off, the residual biomass is in the form of char which is virtually pure carbon. Pyrolysis has received attention recently for the production of liquid fuels from cellulosic feedstocks by ‘‘fast’’ and ‘‘flash’’ pyrolysis in which the biomass has a short residence time in the reactor. A more detailed understanding of the physical and chemical properties governing the pyrolytic reactions has allowed the optimization of reactor conditions necessary for these types of pyrolysis. Further work is now concentrating on the use of high-pressure reactor conditions to pro­duce hydrogen and on low-pressure catalytic techniques (requiring zeolites) for alcohol production from the pyrolytic oil [3].

The pyrolysis process is used heavily in the chemical industry, for example, to produce charcoal, activated carbon, methanol and other chemicals from wood, to convert ethylene dichloride into vinyl chloride to make PVC, to produce coke from coal, to convert biomass into syngas, to turn waste into safely disposable sub­stances, and for transforming medium-weight hydrocarbons from oil into lighter ones like gasoline. These specialized uses of pyrolysis are called by various names, such as dry distillation, destructive distillation or cracking.

Pyrolysis differs from other high-temperature processes like combustion and hydrolysis in that it does not involve reactions with oxygen, water or any other reagents. In practice, it is not possible to achieve a completely oxygen-free atmosphere. Because some oxygen is present in any pyrolysis system, a small amount of oxidation occurs. The term has also been applied to the decomposition of organic material in the presence of superheated water or steam (hydrous pyrolysis), for example, in the steam cracking of oil. Pyrolysis is the basis of several methods that are being developed for producing fuel from biomass, which may include either crops grown for the purpose or biological waste products from other industries. Fuel bio-oil resembling light crude oil can also be produced by hydrous pyrolysis from many kinds of feedstock by a process called thermal depolymerization (which may however include other reactions besides pyrolysis).

Although ILs have proven to be ideal solvents for biomass pretreatment and homogeneous chemical catalytic conversion of biomass into monosugars, the process still suffered a shortage of high cost cellulose regeneration. Considering the fact that ILs are also regarded as ideal solvents for biocatalysis due to their unique advantages compared to conventional solvents, researchers are devoting to develop an integrated process of pretreatment and enzymatic hydrolysis in one batch, which will eliminate the need to recover the regenerated lignocellulosic materials, and will lead to a more economic and environmentally friendly conversion process for bio-energy production [5]. It is rational to postulate that ILs are potentially ideal media for the enzymatic conversion of cellulose and ligno — cellulosic materials into sugar. However, carbohydrate-dissolving ILs are typically composed of Cl-, dca-, HCOO-, — OAc, i. e., anions which form strong hydrogen bonds with the carbohydrate. These interactions facilitate the dissolution of bio­mass, but denaturation of enzymes can be a problem which hinders the enzymatic conversion of dissolved cellulose in ILs. To overcome this obstacle, the design and synthesis of enzyme-compatible ionic liquids which are capable of dissolving cellulose, and do not considerably deactivate enzymes is essentially necessary. In addition, factors such as IL polarity, IL network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, surfactant effect, etc., may also influence the catalytic performance of enzymes [76]. To improve the enzyme solubility and activity in ILs, various attempts have been made, including immobilized enzymes, microemulsions, whole cells catalysis, multi-phase partitioning (TPP) reaction, the use of additives (NaHCO3, Na2CO3, or triethylamine), enzyme-coated micro­crystals, and lipase lyophilization with cyclodextrins [77].

In 2008, Kamiya et al. first reported a one-batch enzymatic process for the saccharification of cellulose in aqueous-IL [1-methyl-3-methyl-imidazolium] [Diethyl phosphate] system, which showed initial information on the potential of [1-methyl-3-methyl-imidazolium] [Diethyl phosphate] as the solvent for in situ pretreatment and enzymatic hydrolysis of lignocellulosic materials in ILs media [78]. Further study by Yang et al. with the diethyl phosphate-based ionic liquids showed that ultrasonic pretreatment could enhance the in situ enzymatic sac­charification of cellulose in aqueous-ionic liquid media, as a result 95.5% con­version of cellulose could be obtained [79]. Furthermore, they also found that the pretreatment of corn cob in 1-methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) in view of its biocompatibility with both lignocellulose solubility and cellulase activity (more than 70% saccharification rate), did not bring negative effects on saccharification, cell growth, and accumulation of lipid of R. opacus ACCC41043 [80].

It is well recognized that ILs can be designed with different cation and anion combinations, which allows the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ILs provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. In order to avoid denaturing enzyme, Zhao et al. designed a series of glycol-substituted cation and acetate anion ILs that are able to dissolve carbohydrates but do not considerably inactivate the enzyme (immobi­lized lipase B from Candida Antarctica). The ILs could dissolve more than 10% (wt) cellulose and up to 80% (wt) D-glucose. The transesterification activities of the lipase in these ILs are comparable with those in hydrophobic ILs [81]. Garcia et al. reported a class of biocompatible and biodegradable cholinium-based ILs, the cholinium alkanoates, which showed a highly efficient and specific dissolution of the suberin domains from cork biopolymers. These results are almost more efficient than any system reported so far [82]. However, they did not perform the in situ conversion experiments in these ILs. Bose et al. employed tryptophyl fluo­rescence and DSC to investigate the reactivity and stability of a commercial mixture of cellulases in eight ILs. Only 1-methylimidazolium chloride (mim Cl) and tris-(2-hydroxyethyl) methylammonium methylsulfate (HEMA) provided a medium hydrolysis [83]. Although we can conclude that high concentrated ILs can make the enzyme lose its activity, there are still many new ILs or enzymes that show good biocompatability or IL-tolerance. These results provide us a green approach to the production of biofuels. At present, it is evident that the pretreat­ment of lignocellulose in ILs is a good choice for the fast enzymatic hydrolysis of cellulose.

With the aim to search for cellulose hydrolyzing enzymes that are stable in ILs, in 2009, Pottkamper et al. applied metagenomics for the identification of bacterial cellulases that are stable in ILs. By screening metagenomic libraries, 24 novel cellulase clones were identified and tested for their performance in the presence of ILs. Most enzyme clones showed only very poor or no activities. Three enzyme clones,(i. e.,. pCosJP10, pCosJP20, and pCosJP24) were moderately active and stable in the presence of 1-butyl-1-methyl-pyrrolidinium trifluoromethanesulfo — nate. The corresponding genes of these environment-derived cosmids were similar to known cellulases from Cellvibrio japonicus and a salt-tolerant cellulase from an uncultured microorganism. It was found that the most active protein (CelA10) belonged to GH5 family cellulases and was active at IL concentrations of up to 30% (v/v). Recombinant CelA10 was extremely tolerant to 4 M NaCl and KCl. In addition, improved cellulase variants of CelA10 were isolated in a directed evolution experiment employing SeSaM-technology. The analysis of these vari­ants revealed that the N-terminal cellulose binding domain played a pivotal role for IL resistance [84]. Meanwhile, Datta et al. found that both hyperthermophilic enzymes were active on [Emim] [OAc] pretreated Avicel and corn stover. Furthermore, these enzymes could be recovered with little loss in activity after exposure to 15% [Emim] [OAc] for 15 h. These results demonstrated the potential of using IL-tolerant extremophilic cellulases for hydrolysis of IL-pretreated lig — nocellulosic biomass and for biofuel production [85].

The potential persistence, accumulation in soils and water and biodegradability of ILs was assessed using standardized methods, such as the closed bottle test or the CO2 headspace test [60, 111, 114]. Studies on IL biodegradability included the major types of cations: ammonium, imidazolium, phosphonium, and pyridinium ions [111]. The widely used 1-butyl-3-methylimidazolium IL remains stable after 28 days in an aqueous suspension with waste-water microorganisms [60]. It was found that ILs with halogens, branched alkyl chains, pyridine rings, aliphatic ethers are usually more resistant to biodegradation [111]. With these observations, efforts are underway to develop biodegradable ILs. Current strategies include replacing branched alkyl chains with linear alkyl chains, functionalization to enable enzymatic hydrolysis, and the incorporation of phenyl rings [60].

4.5 Applications