Cellulose is a polysaccharide consisting of linear chains of several 100-10,000 b(1-4) linked D-glucose units [2]. The chains are assembled in both parallel and anti-parallel ways via hydrogen bonds, which adds more rigidity to the structure, and a subsequent packaging of bound-chains into microfibrils forms the ultimate building materials of nature. The formed rigid structure determines the insolubility characteristic in conventional solvents and thus limits its full exploitation of the potential of cellulose as feedstock. The story of dissolution of cellulose in ionic liquids may go back to a published patent by Charles Graenacher [15], in which they reported that molten benzylpyridinium chloride or N-ethylpyridinium chlo­ride in the presence of nitrogen-containing bases was able to dissolve cellulose. However, the potential of ILs for biomass processing was only recognized seri­ously till the discovery of imidazonium-based ionic liquids by Rogers et al. in 2002 [6]. It was found that the solubility of ionic liquids to cellulose is related to the anions in ILs, with the order of solubility of Cl- > Br — > SCN-, all of which have the same cation of 1-methyl-3-butyl imidazonium, and the BF-, PF — based ILs cannot dissolve cellulose. Furthermore, microwave irradiation could promote the dissolution both in dissolving rates and solubility of ILs [6]. Since then, with the aim to develop more efficient, economic and ‘greener’ ionic liquids for cel­lulose processing, a lot of ILs have been synthesized and screened for the disso­lution of cellulose and other biopolymers through tuning the structure of cations or anions, and using cheap and renewable resource as raw materials for the ILs synthesis [7]. Whereas, the imidazonium cation-based ILs companied with Cl-, acetate, formate, and dimethyl phosphate anions present better performance than that of quaternary ammonium, pyrrolidinium, phosphonium-based ILs [16]. For example, Zhang et al. reported that 1-allyl-3-methylimidazolium chloride ([Amim]Cl) was a high-efficient ILs for cellulose dissolution and derivation with advantages of low melting point and low viscosity [17]. Ohno et al. reported low viscosity, polar and halogen-free 1,3-dialkylimidazolium formats, and acetate ionic liquids which have superior solubility of various polysaccharides under mild conditions (10 wt% at even 60°C) [18]. Fukaya et al. found that alkylimidazolium salts containing dimethyl phosphate, methyl methylphosphonate, or methyl phos- phonate have the potential to dissolve cellulose under mild conditions. Especially, N-ethyl-N-methylimidazolium methylphosphonate enabled the preparation of cellulose solution (10 wt%) and rendered soluble cellulose (2-4 wt%) without pre­treatments and heating [19]. Due to the excellent solubility of phosphonate-derived ionic liquids to cellulose, it was found that 1-ethyl-3-methylimidazolium phosphinate could extract polysaccharides (or cellulose) from bran even without heating [20].

Lignocellulose, mainly composed of cellulose, lignin, hemicellulose, and extractives, represents an abundant carbon-neutral renewable resource. The three­dimensional cross-linked lignin network binds the whole wood architecture together, which determines their comparatively harder solubility in solvents than cellulose [2]. In 2007, Kilpelainen et al. first reported the details of the dissolution of woody lignocellulosic materials and defined the various variables that determine its solubilization efficiency in ILs [21, 22]. By stirring the mixtures mechanically, an up to 8 wt% wood solution was obtained by simple mixing of dried wood sawdust and thermo-mechanical fiber samples with the ILs ([Amim]Cl or [Bmim]Cl) at 80-120°C. The results showed that the solubility of wood-based lignocellulosic material is related to several key factors, such as ILs’ structure, size of lignocellulosic materials, water content of both the ILs and lignocellulosic materials, etc. Interestingly, an introduction of a phenyl group into ionic liquids ([Benzlymim]Cl) could result in a completely transparent, amber-colored but viscous solution. It was estimated that coulombic interactions, such as H bonding, p-p stacking, and van der Waals interactions in ILs can be up to 600 kJ/mol, whereas H-bonds (for water) or van der Waals forces are generally around 40 kJ/ mol [23]. It was proposed that a cationic moiety with an electro-rich aromatic p-system may create stronger interactions for polymers capable of undergoing p-p and n-p interactions according to the Abraham salvation equation [16, 21].

Further in-depth study of the influence of ionic liquids’ structure on their sol­ubility by Doherty et al., especially, on the effect of anions, demonstrated the relationship between the Kamlet-Taft a, b, and p* solvent polarity parameters of different ILs ([Emim][OAc], [Bmim][OAc], and [Bmim][MeSO4]) and effective pretreatments of lignocellulosic biomass. The b parameter provides an excellent predictor for fermentable sugar yields (b > 1.0, resulting in >65% glucose yields after 12 h cellulose hydrolysis following pretreatment) [24]. The hydrogen bond accepting ability of the anions of the ILs, as characterized by :H NMR and the b parameter of the ILs, are closely linked to the solubility of cellulose, which was also supported by other work [25, 26].

Traces of ILs can be significantly reduced with multiple washing with water. However, in order to reduce water use, the extraction or development of cellulases capable to hydrolyze biomass at high IL loading (>5 vol.%) is necessary [72]. An intense effort is underway to find compatible combinations of cellulases and ILs. A few cellulases were found to tolerate high concentrations of ILs. Endogluconases isolated from thermophilic organisms Thermatoga maritima and Pyrococcus hor — ikoshii retained 50 and 95% of their activities on carboxymethylcellulose after incubation in 15 vol.% [EMIM][OAc] for 30 min at 80°C, respectively. In contrast, commercial cellulases from Trichoderma viride were de-activated in 10 vol.% [EMIM][OAc] at 37°C. The activity of cellulases from Thermatoga maritima and Pyrococcus horikoshii only decreased by 34% and 11% on IL-pretreated corn stovers in 15 vol.% [EMIM][OAc] for 14 h at 80°C, compared to the hydrolysis without IL. The sugar yields were much higher than in the untreated corn stovers. The unfolding temperature of the cellulases, measured by differential scanning calorimetry, decreased with increasing [EMIM][OAc] concentration [131].

High-throughput techniques are necessary to screen the wide variety of IL/cel — lulase combinations. The activity of Celluclast 1.5L was assessed on numerous cellulose substrates with varying crystallinity in different buffers and at various pH using 96-well plates [127]. In a similar high-throughput approach, IL/biomass solutions were pipetted into wells in a 96-well plate filled with the anti-solvent to be regenerated before addition of the enzyme cocktail. This avoids the handling of solid regenerated biomass, which is difficult to pipette [72]. The amount of reducing sugars can then be assessed spectroscopically with the dinitrosalicylic acid reagent [80]. This high-throughput technique can not only identify quickly the cellulases capable to tolerate ILs, but also assess the effect of pH and substrate concentration [127].

Cellulases resistant to IL were identified using metagenomics, where microbial DNA was extracted from organisms found in a natural environment and then cloned in a host bacterium (for example, Escherichia coli) [126, 132-134]. Using metagenomics, 24 cellulase clones were identified and their activity was tested in six different ILs on carboxymethylcellulose at 37°C for 30 min. One of the isolated enzymes retained about 40% of its cellulase activity in 30 vol.% of 1-ethyl-3- methylimidazolium trifluoromethanesulfonate ([EMIM][CF3SO3]), [EMIM]

[TfAc], and [BMPy][CF3SO3]. However, no activity was observed on Avicel cel­lulose. Longer incubation of the cellulases in 60 vol.% any IL for 17 h deactivated the cellulases [126].

For lignocellulosic materials, the performance of batch and fed-batch fermentation processes has been examined using different bacteria. In one previous study, five different combinations of pretreatment of wheat straw and batch fermentation process have been performed. Results demonstrated that simultaneous hydrolysis and fermentation with agitation by gas stripping showed maximum productivity

of 0.31 g l-1 h-1 over the other combinations (fermentation of pretreated wheat straw, separate hydrolysis and fermentation of wheat straw without removing sediments, simultaneous hydrolysis and fermentation of wheat straw without agitation, simultaneous hydrolysis and fermentation of wheat straw with addition of sugar supplements). Also, the successful consumption of hydrolysate sugars (glucose, xylose, arabinose, galactose, and mannose) of wheat straw has been seen during the study [33]. For the same feedstock, fed-batch process enabled to pro­duce butanol with more than twofold productivity of batch fermentation (0.77 g l-1 h-1) [60]. Further, pH-stat fed-batch fermentation synthesized 16 g l-1 of butanol with 72% higher productivity than conventional batch pro­cesses. The pH was maintained constant by adding butyric acid. The other advantage of butyric acid was observed in enhancing the solventogenesis phase during the metabolic pathways of clostridium bacteria [43]. Solventogenesis phase

is the second phase of metabolic pathway, which includes the production of sol­vents (acetone, butanol, and ethanol) on the consumption of acids (acetic and butyric acid), produced in first phase or acidogenesis.

The productivity and performance of fermentation processes also depend on pretreatment or hydrolysis methods, which convert the complex biomass compo­sition into simple sugars. Two traditional pretreatment methods such as acidic and enzymatic methods were compared on the basis of yield of batch fermentation. It was observed that enzymatic method eliminates some inhibitors from the sugar solution and generates better yield (0.35 g g-1) over the acidic method [25]. Other studies also suggested that the inhibitors of cellulosic hydrosylates, produced in hydrolysis processes, can be reduced by treating with Ca(OH)2 [49, 63].

The integrated fermentation process with recovery has provided significant ele­vation to total solvent concentration in the broth. It was recorded 51.5 g l-1 with the comparison 24.2 g l-1 in non-integrated batch process using C. beijerinckii BA 101. In integrated process, sugar consumption was also increased to as high as 150 g l-1 over 60 g l-1 in the case of non-integrated process [64]. Due to the toxicity of high sugar concentration to the bacterial culture, the fed-batch process also has great advantages [67]. In fed-batch reactor, a total concentration of 165.1 g l-1of the solvent was achieved compared to 25.3 g l-1 in batch reactor [68].

In a heterologous host such as E. coli, a non-native pathway introduces non­native metabolites and potential toxicity, difficult to express in heterologous enzymes. Consequently, metabolic imbalance and cytotoxicity that poses as a barrier for large quantity production. It is therefore, necessary to seek for path­ways that are compatible with the host. Biosynthesis of amino acid generates many keto acid intermediates. By using decarboxylation and reduction catalyzed by keto acid decarboxylase and alcohol dehydogenase, these keto acids can be converted into alcohols. For example, the isoleucine biosynthesis pathway gen­erates n-propanol and 2-methyl-1-butanol, valine biosynthesis pathway produces 2-keto-isovalerate which is the precursor for isobutanol, the leucine biosynthesis generates 2-keto-4-methyl-pentanoate, which is the substrate for 3-methyl-1- butanol, the phenylalanine biosynthesis pathway leads to 2-phenylethanol and nor-valine biosynthesis pathway produces a substrate for n-butanol [9].These pathways have been recently used for the production of alcohols in E. coli with good results (Fig. 9.11).

An ideal gasification process converts biomass completely into carbon monoxide and hydrogen, i. e., syngas. This syngas can be used directly for the generation of electricity via internal or external combustion engines or for synthesis of liquid biofuel and other chemicals. However, the hot syngas from the gasifier may be contaminated to a varying degree, depending on the design of the gasifier used, process controls exerted, medium of gasification (oxygen, air, steam, etc.), and nature of biomass feedstock used. The product, instead of pure syngas may consist of a mixture of syn-gas and carbon dioxide, methane, water, and smaller hydro — carbons—condensable and non-condensable. This gaseous product may be further contaminated by contaminants comprising particulate matter, alkali compounds, nitrogen compounds, sulfur compounds, and condensable tars. Hence, syngas cleaning is an important component of downstream processing in the gasification process. Cold cleaning (at temperatures <30°C), warm cleaning (at temperatures between 30 and 300°C) and hot cleaning (at temperatures >300°C) may be done depending on the final application of the syngas. The particulate matter in the syngas is removed through physical means by using ceramic or metal based filters. Particle agglomeration techniques may precede the physical filtration process in order to facilitate the filtration process and prevent the clogging of the filter surface by fine ash deposits. High porosity nanoparticle membrane filters are also being developed for removal of sticky materials from the gaseous product. The alkali and elemental contaminants may be removed by passing the gas through a bed of suitable high capacity sorbents. Tar formation is one of the major issues in the gasification process. The tar present in the syngas can condense into a thick viscous liquid which can stick on the various surfaces of the process equipment such as turbines and engines. Primary tar formation, i. e., the tar formation in the gasifier can be controlled by using optimum operating conditions as well as by use of appropriate catalysts which cause cracking of the tar. Secondary tar removal,

i. e., removal of tar present in the syn-gas already collected from the gasifier can be done by filtering, gas scrubbing, passing through cyclone separators, catalytic cracking, steam reforming, or by thermal cracking methods. Kumar et al. [22] have reviewed the various aspects of downstream processing in gasification. The main methods for tar removal are steam reforming and catalyst cracking. In steam reforming, the tar is reacted with at temperatures of around 650-700°C where the tar gets converted to syngas:

CxHy + XH2O xCO + (x + 2^ H2

For obtaining hydrogen as the major product, the above reaction is followed by water-gas shift reaction:

CO + H2O $ CO2 + H2

Steam reforming may be a part of the gasification process or may be performed separately after the gasification. Tar cracking involves thermal cracking of the long

chain hydrocarbons in the tar into smaller molecules, finally forming CO and H2. This can also be performed in situ, in the gasifier or separately. Bridgwater [13] describes the use of catalysts such as dolomite, fluid catalytic cracking catalysts, and metal catalysts for removal of tar from the gasification product.

Biomass gasification is the latest generation of thermochemical biomass-to — energy conversion processes. A number of commercial gasifiers, based on the various types described previously in this section, are operational, and some of them have successfully completed several years of operation. The Atlanta-based Future Energy Resources Corporation (FERCO) has commercialized the Silva — Gas TM gasification technology in partnership with the US Department of Energy and the Burlington Electric Department at Vermont, USA. This facility, which was operational in 2001, is a low inlet gas velocity, high throughput biomass gasification process, which can convert more than 285 tons of biomass such as forest residue, MSW, agricultural waste, and energy crops into SilvaGas, a medium Btu gas. This gas is piped directly to Burlington Electric Department’s McNeil generating plant, where it produces more than 140 MWh of electric power. Another circulating fluidized-bed gasifier developed by Foster Wheeler at Lahiti Kymijarvi, Finland, has completed several years of operation and can produce 60 MWth energy. It uses paper, textiles, wood, and peat fuels with an average moisture content of about 50%. It provides hot but low calorific value gas around 2 MJ/Nm3 which can be used for heating purposes only. A demon­stration plant with a bubbling fluidized-bed gasifier design has been developed as a part of the Carbona Project, at Skive, Denmark, and commissioned in late 2007. It uses wood-based biomass and has an efficiency of 90%. The product generated is a mixture of carbon monoxide, hydrogen, and methane, having a heating value of 5 MJ/kg, which is used for CHP application and generates

6,0 kW of power. Plasma gasification plants, mainly for the processing of MSW have also been developed, one of them being the Hitachi demonstration plant set up at Yoshii, Japan, commissioned in 1999. This plant was developed as a solution to dioxin, ash, and energy recovery problems from incineration-to — waste energy plants in Japan. It processes 20 tons/day of MSW and produces steam for industrial use. The plant emissions are much reduced, and the slag produced is a glassy product which can be used as a construction material. Another plasma gasification plant—the Hitachi combined MSW and Sewage Sludge gasification plant, is a commercial scale plant, Mihama and Mikdata, Japan, and commissioned in 2002. This plant treats 24 tons/day MSW and 4 tons/day sewage sludge to generate energy which is used in the municipal wastewater treatment facility. The same company has a 200 tons/day plasma gasification plant which has been fully operational since 2003, at Utashinai, Japan which uses automobile shredder residue as feed. The syngas produced is used to generate power and process steam. The hydrothermal gasification process is still at the development stage, but considering the numerous advantages it offers over other conventional gasification processes, it will not be long before commercial-scale plants see the light of the day.

1.2.1.2

Biochemical Conversion Processes

The biochemical methods of conversion of biomass are more environment friendly than the thermochemical processes described above. These processes are mainly used for conversion of organic wastes, both agricultural and municipal solid waste (Fig. 1.16), which are relatively difficult to process due to their very low energy density and, difficulty in handling. The schemes for processing the two types of wastes are outlined in Figs. 1.17 and 1.18.

In principle, these methods can be considered to be the reverse of photosyn­thesis. The products of biochemical conversion methods comprise biogas and

landfill gas; liquid biofuels such as biodiesels, bioethanol, biomethanol, and pyrolysis oils; and hydrogen. Biogas is a mixture of methane and carbon dioxide, generated as a result of the decay of sewage or animal waste. A similar product generated at landfill sites is called landfill gas. The landfill gas generated from landfill sites, if it is not collected, escapes into the atmosphere. Pipelines laid at the landfill sites before the construction of the site makes it possible to collect the landfill gas which can be used to generate electricity with the help of large internal combustion engines. The output of the landfill gas can be as high as 1,000 m3/h [16]. Processing lignocellulosic feedstock, including lignocellulosic waste gives liquid biofuels such as ethanol which can be used as a transport fuel by mixing with gasoline. A host of other products such as methane, producer gas, esters, and other chemicals having a cellulosic origin can also be obtained. Hydrogen is going to be an important energy carrier of the future. The biochemical processes can be classified into three categories:

• Aerobic fermentation (which can also be considered as biogasification of bio­mass), which produces compost, carbon dioxide, and water.

• Anaerobic digestion which produces fertilizer gas and biogas.

• Alcoholic fermentation which produces ethanol, carbon dioxide, and waste.

The main disadvantage of biochemical processes is that they are slow, involving very long time periods.

The operation of both up — and downdraught gasifiers is influenced by the mor­phological, physical and chemical properties of the fuel. Problems commonly encountered are: lack of bunker flow, slagging and extreme pressure drop over the gasifier. Air is blown through a bed of solid particles at a sufficient velocity to keep these in a state of suspension. The bed is originally externally heated and the feedstock is introduced as soon as a sufficiently high temperature is reached.

The fuel particles are introduced at the bottom of the reactor, very quickly mixed with the bed material and almost instantaneously heated up to the bed temperature. As a result of this treatment the fuel is pyrolyzed very fast, resulting in a com­ponent mix with a relatively large amount of gaseous materials. Further gasifi­cation and tar-conversion reactions occur in the gas phase. Most systems are equipped with an internal cyclone in order to minimize char blow-out as much as possible. Ash particles are also carried over the top of the reactor and have to be removed from the gas stream if the gas is used in engine applications [5].

2.5.2 Other Types of Gasifiers

A number of other biomass gasifier systems (double fired, entrained bed, molten bath), which are partly spin-offs from the coal gasification technology, are cur­rently under development. In some cases these systems incorporate unnecessary refinements and complications, in others both the size and sophistication of the equipment make near-term application in developing countries unlikely.

Most IL pretreatments were conducted at high temperatures ranging from 70 to 190°C [7, 22, 25, 27, 36, 37, 43, 48-50]. Higher sugar yields are usually reported for pretreatments at higher temperatures for longer times [7, 22, 25, 27, 50]. Wood dissolution at temperatures above 100°C was faster in [BMIM][Cl] and [AMIM][Cl] [7]. Short pretreatments at higher temperatures resulted in higher glucose yields from oil palm fronds [22]. Increasing temperatures from 50 to 130°C during the dissolution of maple wood flour in [EMIM][OAc] increased the amount of extracted lignin and reduced the recovered wood flour [25]. In another study, increasing the temperatures from 70 to 150°C increased the sol­ubility of lignin in triticale straw residues. The cellulose and hemicellulose content in the residues decreased with higher temperatures. The IL treatment at higher temperatures also resulted in higher glucose yields after enzymatic hydrolysis. More than 95% of the initial cellulose extracted above 130°C after 11 h was hydrolyzed [27]. This can be partially explained by the fact that, at higher temperatures, the self-diffusion coefficients of the IL anions and cations increase dramatically [49].

Another possible explanation of the benefits of high temperatures on biomass pretreatment is the improved access of enzymes to cellulose. The surface area, pore size distribution, and pore volume of switchgrass (3 wt%, 40 mesh) pretreated with [EMIM][OAc] at 110-160°C for 3 h, were measured by nitrogen porosi — metry. The switchgrass pretreated at 160°C adsorbed significantly more gas than the untreated sample or the one treated at 120°C, with a specific surface area 30 times higher (15.8 m2/g at 160°C, 0.7 m2/g at 120°C, and 0.5 m2/g for the untreated sample). The increased surface area and pore volume were correlated with an increase in the initial rates of enzymatic hydrolysis. After 30 min of hydrolysis with cellulases from Trichoderma reesei, the concentration of reducing sugars in the broth was 2.84 g/L for a 3-h IL treatment at 110°C and 7.44 g/l for the sample treated at 160°C. The lignin removal efficiency also increased from 25% at 110°C to 74% at 160°C. The improved delignification and sugar yields observed for pretreatments above 150°C was attributed mostly to the softening or melting of lignin. The average glass transition temperature of lignin is around 165°C, but varies considerably depending on its chemical composition and the ratio of monolignol units [50].

Cellulose degradation was reported when IL pretreatment is conducted at higher incubation time and higher temperature, leading to lower sugar yields after enzymatic hydrolysis [22]. There was evidence of degradation of the IL and cel­lulose, when pine wood chips were pretreated in [EMIM][OAc] at 110°C for 16 h. The appearance of additional peaks on 13C NMR spectra of the pine/IL solution was attributed to the generation of glucose oligomers and the degradation of [EMIM] [OAc] [36].

Alkali metals such as lithium, sodium, potassium, rubidium, cesium can be used directly as catalysts in the form of alkali metal carbonates or supported on other materials such as alumina and silica. Alkali salts are mixed directly with the biomass as it is fed into the gasifier. Addition of alkali metals to biomass can also be achieved by impregnation. These metals are highly reactive. Alkali metals as catalysts lead to an enhancement for the biomass gasification reactions, especially for the char formation reactions.

Alkali metals could act as promoters present in commercial steam-reforming catalysts by enhancing the gasification reaction of carbon intermediates deposited on the catalyst surface [8]. But, the major disadvantages of these catalysts are their loss of activity due to particle agglomeration. In addition, the recovery of the alkali metals appears to be difficult. Ashes often contain high concentrations of alkali metals and these can also be added to biomass. Alkali metal catalysts are also active as secondary catalysts. Potassium carbonate supported on alumina is more resistant to carbon deposition although not as active as nickel [8].

According to previous studies, the addition of Na2CO3 enhances the catalytic gasification of rice straw compared with nickel catalyst and significantly increases the formation of gas, and the catalytic activity of single salts in steam gasification depends on the gasification temperature, with the following order of activity: K2CO3 > Ni(NO3)2 > K2SO4 > Ba(NO3)2 > FeSO4 [12].

Better interaction between feedstock and catalyst should be provided to get the enhanced performance of the catalyst. Impregnation has many advantages over mixing directly. Mudge et al. studied the catalytic steam gasification of wood using alkali carbonates and naturally occurring minerals, which were either impregnated or mixed with the biomass. They reported that the impregnation decreased particle agglomeration [13].

According to Hallen et al. the presence of Na2CO3, K2CO3 or CsCO3 as catalyst in biomass steam gasification decreased the carbon conversion degree to gas. However, an increase in the rate and total amount of gas produced was observed. The presence of a catalyst increased the char yield during the volatilization stage but then decreased the char yield during the second stage of the gasification process. [14].

Biotechnological approaches are well known rapid ways of enhancing the plant traits. Genetic transformation of useful genes into the biofuel crops is demon­strated to be feasible. Thus, there are several successful reports on genetic transformation of switchgrass [49-52], Miscanthus [53] and sugarcane [54, 55]. Selected genes (or their homologs) that cause biomass enhancement in a given (crop) plant species can be the candidate genes for genetic transformation of biofuel crops. For example root-specific expression of cell wall invertase gene CIN1 from Chenopodium rubrum displayed enhanced shoot and root biomass in Arabidopsis [56]. Hence, similar genetic modification using either this gene or its homolog from the biofuel species may increase the shoot and root biomass. In another study, the overexpression of sugar metabolism enzymes such as UDP — glucose pyrophosphorylase, sucrose synthase and sucrose phosphate synthase was shown to result in increased plant biomass [57]. Suppression of Arabidopsis GA2ox homolog in tobacco enhanced fiber, wood formations and overall biomass yields [29]. Mutation in one of the WRKY transcription factors induces sec­ondary wall formation in pith cells and leads to increased stem biomass in Medicago [58]. Also, delayed flowering will increase the biomass due to the availability of more time for vegetative growth. Thus, overexpression of floral repressor FLC in tobacco caused delayed flowering and as a result, the plants had accumulated significantly more biomass [59]. Similarly, another flowering time regulator mutant in maize called indeterminate1 (id1) showed delayed flowering and increase in biomass [60], suggesting that various candidate genes are already available for genetic modification of the plants used for cellulosic bioethanol production.

Although biofuel crops can grow in marginal land and yield significant amounts of biomass there are several potential problems with them. These include sus­ceptibility to abiotic, biotic stresses and difficulties associated with conversion of cellulose into simple sugars during downstream processing. Prolonged cold and drought stresses may lead to significant yield loss, and to overcome this biofuel crops can be genetically engineered using proven cold — and drought-tolerant genes. Another major problem identified is biotic stresses such as insect and pest attack (e. g., plant-parasitic root nematodes) associated with decline in biomass produc­tion [61]. Generation of plants resistant to nematode and insect attacks might be the solution for this problem, which is possible to be achieved by genetic modi­fication and biotechnological approaches.

Other approaches for genetic improvement of plants used for cellulosic ethanol production are to modify the chemical composition of the cell wall, specifically, to alter the lignocellulosic content or to incorporate genes for stable/inducible forms of enzymes such as cellulase into the plants so that downstream processing will be facilitated. Recently, genetic modification involving RNAi suppression of caffeic acid 3-O-methyltransferase (COMT) gene in switchgrass has been demonstrated to reduce lignin content and increase ethanol yield by up to 30% [51]. To convert the cellulose (which is a polymer of glucose units) to simple sugars either acid hydrolysis at high temperatures (with high energy input) or treatment with fungal cellulase enzyme is used. This is a rate limiting and costly step and to avoid this, temporal expression of cellulase gene in biofuel crops using specific promoters has been suggested. Efforts are underway in various laboratories to achieve this.

Aerobic metabolism which leads to utilization of sugar substrate but produces no alcohol must be avoided to a great extent. However, the trace amounts of oxygen may greatly stimulate yeast fermentation. Oxygen is required for yeast growth as a building block for the biosynthesis of polyunsaturated fats and lipids required in mitochondria and plasma membrane [69]. High sugar concentration is adequate to repress aerobic sugar consumption in yeasts which shows the Crabtree effect. For other yeasts or at low sugar concentrations, the oxygen supply should be limited. Trace amounts of oxygen (0.7 mm Hg Oxygen tension) are adequate and do not promote aerobic metabolism [30].

9.6.1.3 Effect of pH

Fermentation rate is sensitive to pH, but most distiller’s yeasts show a broad pH optima from 4 to 6 [29]. Most yeast strains are capable of tolerating high acidic pH (2) in the solutions without any permanent damage [71].

9.6.1.4 Effect of Temperature

High temperature tolerance is a desirable quality selected for distillery yeasts and most distillery yeasts have a temperature growth optima of 30-35°C [56]. For low alcohol concentrations, the optimum fermentation temperature is slightly higher (up to 38 °C) but alcohol tolerance is improved at reduced temperatures [70]. Exposure to temperatures above the optimum results in excessive enzyme deg­radation and loss of yeast viability. Yeast metabolism liberates 11.7 KCal of heat per kg of substrate consumed [103]. Yeast is inactive at low temperature (0°C) and can be stored at that temperature and readily revived [178].