Fatty acid biosynthesis pathway uses acetyl-CoA as a starting molecule [114]. Acetyl-CoA is converted into malonyl-CoA by the addition of a carboxyl group using acetyl-CoA carboxylase as a catalyst. The acetyl and malonyl groups on acetyl-CoA and malonyl-CoA are transferred to a small protein called acyl carrier protein (ACP), which has 77 amino acid residues with a phosphopantothene group specifically attached to a serine residue. Acetyl-ACP and malonyl-ACP are con­densed to generate acetoacyl-ACP. This molecule, then goes through reduction, dehydration, and another reduction step to form a 2,3,4-saturated fatty acyl-ACP. The fatty acids synthesized have a long carbon chain backbone, which stores a large amount of energy. To transform fatty acids into combustible fuels, pathways leading to biodiesels and long-chain alkanes/alkenes have been proposed. The fatty acyl-CoA can be reduced to the corresponding fatty aldehydes, which are in turn decarboxylated to long-chain alkanes or further reduced to fatty alcohols that can also be esterified to biodiesel with acetyl-CoA by an alcohol acyltransferase or ester synthase [1, 148]. Biodiesel as a possible substitute for petroleum-based diesel fuel is made from plant oils through transesterification of triacylglycerols with methanol or ethanol. Large-scale application of biodiesel seems difficult because of the seasonal restrictions and the costliness of the transesterification procedure [89]. To overcome these drawbacks, E. coli was engineered to produce fatty acid ethyl esters, where the traditional pathway of ethanol consisting of pyruvate decarboxylase (PDC) and alcohol dehydrogenase was introduced to supply ethanol as building units. The metabolically engineered E. coli was reported to have capability to produce fatty acid ethyl esters at a titer of 1.28 g/l, by using glucose and oleic acid as substrates.

9.4.3.1 Isoprenoid Pathway

Isoprenoids are natural hydrocarbons biosynthesized for a wide variety of func­tions. The isoprenoid pathway has been engineered in heterologous hosts to pro­duce nutraceuticals or pharmaceuticals [155]. Despite this, isoprenoids synthesized from isoprenyl diphosphate and dimethylallyl pyrophosphate which are either synthesized from glyceraldehydes-3-phosphate or pyruvate. Recently, two genes in Bacillus subtilis 6051 whose products can convert the prenyl diphosphate pre­cursors into corresponding isoprenoids have been reported [195].

The production of landfill gas from landfill sites is carried out by a complex process involving a succession of microbial population. The initial fermentation is aerobic fermentation which is carried out by bacteria already present in waste. This stage is followed by an anaerobic digestion stage. During the aerobic fermentation stage, the carbon in the biomass is converted into carbon dioxide and water. These reactions are strongly exothermic and increase the temperature of the landfill waste, consequently increasing the activity of the other microorganisms. If this stage continues for long, the amount of methane, which is the product of the
anaerobic digestion stage, decreases. In order to shorten the aerobic fermentation stage, and consequently prevent the reduction in methane yield, the landfill waste is made to pass through compactors, which serves a dual purpose of increasing the density of the landfill waste and simultaneously removing a substantial amount of oxygen from the waste. After an initial phase of aerobic fermentation, the waste gradually becomes anaerobic as oxygen is depleted from it. The subsequent fer­mentation causes the breakdown of complex polymeric components in the biomass into simpler compounds, followed by volatile fatty acids, followed by carbon dioxide, hydrogen, and acetic acid.

High compaction technology or binderless technology consists of the piston press and the screw press. Most of the units currently installed, are the reciprocating type, where the biomass is pressed in a die by a reciprocating ram at a very high pressure. In a screw extruder press, the biomass is extruded continuously by a screw through a heated taper die. In a piston press the wear of the contact parts e. g., the ram and die is less compared to the wear of the screw and die in a screw extruder press. The power consumption in the former is less than that of the latter. But in terms of briquette quality and production procedure screw press is definitely superior to the piston press technology. The central hole incorporated into the briquettes produced by a screw extruder helps to achieve uniform and efficient combustion and, also, these briquettes can be carbonized. Table 2.2 shows a comparison between a screw extruder and a piston press.

2.6.1.1 Piston Press

The piston presses which are currently in operation, are also known as ram and die technology. In this case the biomass is punched into a die by a reciprocating ram with a very high pressure thereby compressing the mass to obtain a briquette.

The briquette produced is 60 mm in external diameter. This machine has a 700 kg/ h capacity and the power requirement is 25 kW. The ram moves approximately 270 times/min in this process.

Merits and Demerits of Piston Press Technology

1. There is less relative motion between the ram and the biomass hence, the wear of the ram is considerably reduced.

2. It is the most cost-effective technology currently offered.

3. Some operational experience has now been gained using different types of biomass.

4. The moisture content of the raw material should be less than 12% for the best results.

5. The quality of the briquettes goes down with an increase in production for the same power.

6. Carbonization of the outer layer is not possible. Briquettes are somewhat brittle.

[AMIM][Cl] dissolved Eucalyptus grandis, southern pine sawdust (particle size 0.1-2 mm) and Norway spruce thermomechanical pulp almost completely in 5 h at 120°C. IL pretreatment of southern pine improved the glucose yield after enzymatic hydrolysis from 7 to 17 wt%. But the improvement of the glucose yield after IL pretreatment was found to decrease with increasing wood density. Higher density wood (Eucalyptus grandis) requires an IL pretreatment at a higher temperature than low-density wood (southern pine) to achieve the same pretreatment efficiency [31]. Hardwoods such as red oak usually have a higher density than softwoods such as pine, but softwoods also tend to have higher lignin content. Lignin in softwoods is also rich in guaiacyl units, while lignin in hardwoods is a mixture of guaiacyl and syringyl units [36]. Similarly, wheat straw (low lignin content) could be dissolved in [EMIM][OAc] with acetic acid at a lower temperature (100°C) than pine wood (higher lignin content) (120°C) for the same particle size (<1 mm) [47].

Nickel-based catalysts have been widely used for syngas production in the petrochemical industry. These types of catalysts are very effective for the catalytic hot gas cleanup during biomass gasification. Elimination of tar is also accomplished by Ni-based catalysts with a high rate. The mechanism of tar elimination can be summarized as follows [15]. Adsorption of hydrocarbons (C1-C7) and water onto the nickel surface is the first step in tar removal. Then, the OH radicals migrate to the metal sites at suitable temperatures and this leads to the oxidation of the intermediate hydrocarbon fragments and surface carbon to CO + H2.

High tar levels on the generated gases lead to coke deposition on the nickel surface and deactivation occurs restricting the routine use of the catalyst. Regeneration of the catalysts might have a positive effect on removal of coke.

Simell et al. investigated the effect of different process parameters on sulfur poisoning of nickel catalysts in tar (toluene), ammonia and methane decomposi­tion [16]. Removing sulfur from the gas mixture leads to the recovered catalyst activity for tar removal. Not only sulfur, but also chlorine and alkali metals might show a poisoning effect.

Ni-based catalysts have also been used for the production of hydrogen-rich product gas as proposed by Wang et al. [17]. They produced significant amounts of hydrogen from acetic acid and hydroxyacetaldehyde in the presence of a Ni-based catalyst. In addition, noble metal catalysts such as Ru, Pt and Rh are considered to be the most important catalysts in hot gas cleaning processes. They are highly effective to remove tar and to help improve the content of syngas. However, they are more expensive than nickel-based catalysts.

For example, nickel-based catalysts were reported as very effective for tar conversion in the secondary reactor at around 700-800°C, resulting in about 98% tar removal from product gas [18]. Asadullah et al. [19] used Rh/CeO2/M (M5 SiO2, Al2O3, and ZrO2) type catalysts with various compositions for the gasification of cellulose in a fluidized bed reactor at 500-700°C. Compared with the conventional nickel and dolomite catalysts and other compositions of Rh/CeO2 catalyst, Rh/CeO2/ SiO2 with 35% CeO2 was found to be the best catalyst with respect to the carbon conversion to gas and product distribution. Addition of steam contributed to the complete conversion of cellulose to gas even at 600°C. Moreover, although they directly used the catalyst in the primary reactor, tar formation was not observed. This is an encouraging result because even if the use of catalyst in the primary reactor offers the benefit of simplification of the overall process, there are very few studies focusing on the direct use of catalysts in the primary bed due to severe catalyst deactivation. Ni-based catalysts are regarded as popular and also very effective for hot gas cleaning [20]. The recent advancement of nanocatalysts has made it possi — bleto upgrade the produced syngas and to reduce the tar formation in gasification of biomass. In a direct gasification of sawdust, Li et al. [21] used nano-Ni catalyst (NiO/ g-Al2O3), and demonstrated that their catalyst can significantly improve the quality of the produced gas and meanwhile efficiently eliminate the tar generation.

Bioethanol appears to have been firmly established as an important form of alternate fuel. With the second and later generation of bioethanol production focusing on the use of cellulosic biomass, the need for improvement of biomass plants is evident from the above discussion. Despite the occasional controversies raised, bioethanol is an environmentally friendly renewable energy source, and its large-scale use will lead to significant reduction in net emission of GHG. Alternate forms of biofuels such as oils to be used as biodiesel either from plants or from algae are also being explored. The emerging field of synthetic biology strives to convert microalgae into an efficient fuel oil production system. Although it is in its infancy, based on the underlying biological facts, synthetic biology for biofuel production by microalgae is expected to be successful in the coming decades.

It is important to phase out the use of food grains for fuel production in the coming decades. Because of the significant increase in demand for food grain expected, the conflicting demands on agricultural land will lead to serious social conflicts. Therefore, improving the efficiency and scaling up production of cellulosic ethanol is imperative. In order to achieve this, it is important to generate sufficient amounts of cellulosic biomass. Well over a trillion liters of ethanol (theoretical yield per year) can be obtained if all the available corn stover, rice straw and wheat straw (estimated 3 billion tons per year, [6]) are utilized for biofuel production. This represents one year’s oil demand of USA or approxi­mately 25% of the annual world usage of petroleum. Currently, a significant amount of straw is either burnt and disposed off or used for animal feed. Therefore, use of non-food crop biomass plants becomes essential to broaden the availability of raw material for bioethanol production. Unlike with food crops, objections will be minimal if genetic modification strategies are applied to the biofuel plants to enhance yield, be tolerant to stresses and adverse growth conditions.

We have identified manipulation of the intermediates of phytohormone sig­naling pathways as an important strategy for enhancing plant biomass. The key developmental processes affecting biomass, which include reduced apical domi­nance and increased branching, plant height, leaf area and root to shoot ratio etc., are strongly influenced by phytohormones. The fact that phytohormones have pleiotropic effects on growth and development combined with the recent findings of the multiple signaling intermediates presents tremendous untapped opportuni­ties for modifying specific traits listed above for improvement of the biofuel plants. The various signaling intermediates and downstream target genes can serve as candidates for biotechnological improvement or future marker-assisted breeding efforts.

The foregoing discussion has highlighted the need for and feasibility of using genetic and biotechnological approaches to enhance biomass production from a unit land area. Knowledge gained from model plants can be adapted to the biofuel crops in order to achieve this and to ensure sustainable biofuel production as a valuable alternative fuel in the decades to come.

Acknowledgments We thank Ms. Petra Stamm for helping to prepare Fig. 8.1. Research in the author’s laboratory is funded by the Science and Engineering Research Council (SERC Grant No.: 0921390036) of the Agency for Science Technology and Research, Singapore; and the National University of Singapore.

Mash must be enriched with secondary nutrients in addition to the sugar source for ethanol production. Secondary nutrients are necessary for cell maintenance and growth [82]. Yeast extract NH4C1, MgSO4, CaCl2 are a few of the ingredients which promote very rapid cell growth and ethanol production at laboratory scale [30, 31]. Ammonium ions provide nitrogen for protein and nucleic acid synthesis. Yeast extract contains all the necessary yeast growth factors viz., amino acid, purines, pyrimidines, vitamins, and minerals. Phosphorous, potassium (from yeast extract), magnesium, and calcium are incorporated into cell mass and are also cofactors activating several enzymes. The wide variation in media compositions used for different yeasts for alcohol production resulted in different yields.

Several organic and inorganic nitrogen sources in media for ethanol production by Z. mobilis were tested [176]. Urea and yeast extract were found to be better sources and calcium pantothenate was found to be an essential vitamin for ethanol production.

The conversion of oil crops to biofuels—Fatty Acid Methyl Esters (FAME), more commonly known as biodiesel involves processes like transesterification, where the vegetable oil is chemically reacted with an alcohol, in presence of a homog­enous or a heterogenous catalyst. In the catalytic conversion process, in turn the catalyst plays an important role in an oil seed biorefinery. Catalysts that selectively convert a particular substrate to the desired product and the catalytic reactor

parameters related to the design, operation, and control of the catalytic reactor are the key factors responsible for the development of an economically viable process. Chew et al. [42] have proposed a biorefinery based on palm oil and palm biomass for the production of biofuels. The conceptual biorefinery would consist of two plants. One plant would treat the solid portions of the Palm tree biomass whereas the other plant would treat the expressed oil portion. The solids processing plant will carry out liquefaction using supercritical water, pyrolysis, and gasification of the biomass, and the liquid/oil processing plant will carry out transesterification and catalytic cracking of the expressed oil portion of the palm biomass. The products of the biorefinery would comprise of different biofuels, gaseous hydro­carbons, hydrogen, glycerine, olefinic, and aromatic compounds. Figure 1.24 shows the scheme for such a biorefinery along with the various products obtained therein.

The catalytic processes proposed by them for the purpose include (a) catalytic cracking of palm oil for production of biodiesel, (b) production of hydrogen and

syngas from biomass gasification, (c) Fischer-Tropsh synthesis for conversion of syngas into liquid fuels, and (d) upgrading of liquid fuels obtained from lique — faction/pyrolysis of biomass. Catalyst plays a key role in all the above processes in terms of economy and product distribution. They have reviewed a number of catalysts which can be used for the palm oil-based biorefinery.

The biorefinery approach has been used for the valorization of coconut oil by Abderrahim Bouaid et al. [43]. Coconut oil, the main substrate for obtaining biodiesel by a process of transesterification, is a very costly raw material. The high cost of this substrate makes the process of obtaining biodiesel from coconut oil economically unviable. Hence, a process outlined in Fig. 1.25 has been proposed to get multiple products from coconut oil which would then make the process of conversion of coconut oil to biodiesel economically effective. Coconut oil contains 42-49% lauric acid. This lauric acid can be converted to methyl laurate, by a transesterification process. Methyl laurate forms the basis for the production of a number of products like lauryl sulfate (biodegradable surfactants), coconut estolide esters (LMWME’s) which serve as a base for biodegradable lubricants, and recently, biodiesel (methyl ester) a HMWME, which is emerging as a promising substitute for conventional fuels. The proposed biorefinery approach uses an integrated process for generation of HMWME (biodiesel) and LMWME (laurate and myristate methyl esters which can used as biolubricants and biosolvents). The lauric fraction has importance in the detergent industry as it is the preferred material for the manufacture of soaps and detergents due to its exceptional

cleansing properties. Under optimum conditions, a yield of 77.54% for HMWME’s and 20.63% for LMWME’s was obtained.

Gary Luo et al. [44] proposed a novel biorefinery concept for the production of biofuels (biodiesel and bioethanol) from rapeseed and straw. The process effluents from this process were further used for additional production of biofuels (biohy­drogen and biomethane). The overall bioenergy recovery was found to increase to 60% compared to an energy recovery of 20% in case of a conventional biodiesel conversion process.

Digestion systems can be configured with different levels of complexity:

• One-stage or single-stage

• Two-stage or multistage

A single-stage digestion system is one in which all of the biological reactions occur within a single sealed reactor or holding tank. Utilizing a single stage reduces construction costs; however, it facilitates less control of the reactions occurring within the system. Acidogenic bacteria, through the production of acids, reduce the pH of the tank. Methanogenic bacteria, as outlined earlier, operate in a strictly defined pH range. Therefore, the biological reactions of the different species in a single stage reactor can be in direct competition with each other. Another one-stage reaction system is an anaerobic lagoon. These lagoons are pond-like earthen basins used for the treatment and long-term storage of manures. Here, the anaerobic reactions are contained within the natural anaerobic sludge contained in the pool.

In a two-stage or multistage digestion system different digestion vessels are optimized to bring maximum control over the bacterial communities living within the digesters. Acidogenic bacteria produce organic acids and grow and reproduce more quickly than methanogenic bacteria. Methanogenic bacteria require stable pH and temperature in order to optimize their performance.

Typically hydrolysis, acetogenesis and acidogenesis occur within the first reaction vessel. The organic material is then heated to the required operational temperature (either mesophilic or thermophilic) prior to being pumped into a methanogenic reactor. The initial hydrolysis or acidogenesis tanks prior to the methanogenic reactor can provide a buffer to the rate at which feedstock is added. It should be noted that it is not possible to completely isolate the different reaction phases and often there is some biogas that is produced in the hydrolysis or acidogenesis tanks.

Due to the complexity and variability of native biomass, early studies on possible mechanisms have focused on purified cellulose/lignin substrates [3, 14, 28, 67, 86, 87], oligomers of glucose and lignin models [26, 28, 75, 76]. Indeed, biomass is a complex heterogeneous substrate constituted of cellulose, hemicellulose, and lig­nin at varying ratios depending on the biomass feedstock. Cellulose can have several different crystalline structures [71]. Lignin is a branched polymer com­posed of different types of aryl-ether units and bonds that ionic liquids can cleave (aryl-ethers and aryl-alkyl linkages) [75]. The composition of lignin can affect its structure. Hardwood lignins have usually a higher ratio of syringyl/guaicyl units, giving them a more linear structure. In contrast, softwoods contain mostly guaiacyl phenolic units, giving them a branched structure [32].

The dissolution of Avicel cellulose was studied in different 1-alkyl-3-methyl — imidazolium chloride ILs prepared with alkyl chains of various lengths (2-10 atoms). It was found that Avicel cellulose was more soluble with alkyl chains with an even number of carbon atoms [61]. The depolymerization of cellulose was studied also in [BMIM][Cl] using an acid resin as a catalyst [88, 89]. It was proposed that the hydrolysis of cellulose is initiated with the protonation of the oxygen atom in the glycosidic bond. The glycosidic bond then breaks to form a cyclic carbocation, followed by a nucleophilic attack of water to add a hydroxyl group [89].

The cleavage of a particular type of linkage was studied on specifically designed lignin models with the desired linkage. For example, the IL 1-H-3- methylimidazolium chloride was effective in the cleavage of the b-O-4 bond in guaiacylglycerol-b-guaiacyl ether and veratrylglycerol-b-guaiacyl ether [75]. The cleavage of the same lignin models in [BMIM][Cl] required the presence of metal chloride catalysts, such as FeCl3, CuCl2, and AlCl3 [76]. The reactivity of 2-methoxy-4-(2-propenyl)phenol (similar to guaiacyl unit), 4-ethyl-2-methoxy — phenol (alkyl substitution), and 2-phenylethyl phenyl ether (with b-aryl ethers linkage) was studied in 1-ethyl-3-methylimidazolium triflate and [EMIM][Cl] with metal chlorides and acid catalysts [28].

Another study focused on the dissolution of pine kraft lignin. It was found soluble at temperatures above 50°C in 1,3-dimethylimidazolium methylsulfate ([MMIM][MeSO4]), 1 — hexyl-3-methylimidazolium trifluoromethanesulfonate

([HMIM][CF3SO3]), [BMIM][MeSO4]. However, it was insoluble in 1-butyl-3- methylimidazolium hexafluorophosphate ([BMIM][PF6]) even at 120°C. The anion in imidazolium-based ILs affected the solubility dramatically: the methyl — sulfate anion was more effective than the chloride and bromide anions at dis­solving lignin [3].