For enzymatic hydrolysis and fermentation, different strategies have been explored including separate hydrolysis and fermentation (SHF), SSF nonisothermal simultaneous saccharification and fermentation (NSSF), simultaneous saccharification and cofermentation (SSCF), or consolidated bioprocessing (CBP) (Lynd et al., 2002; Taherzadeh and Karimi, 2007). Each process has advantages and disadvantages.

For SHF, the main advantage is the possibility to separately optimize hydrolysis and fermentation steps and the main drawback is the inhibition of cellulase activ­ity by the released sugars, mainly cellobiose and glucose (Taherzadeh and Karimi, 2007). SSF, different from SHF, combines the enzymatic hydrolysis and fermentation in one step, thus minimizing the product inhibition of cellulase enzymes as the released sugars are immediately consumed by the microorganism. In addition, cellulase production and fermentation of hemicellulose hydrolysis products occur in two additional, discrete process steps. This process has many advantages over SHF such as increased ethanol yield, decreased enzyme loading, decreased contamination, and lower capital cost. The dis­advantages are differences between optimum tempera­tures for enzyme hydrolysis and fermentation and inhibition of cellulase by the produced ethanol (Lynd et al., 2002; Olofsson et al., 2008).

To solve the issue of temperature difference, the NSSF process was proposed (Wu and Lee, 1998) in which saccharification and fermentation occur simultaneously but in two separate reactors, each operated at its own optimum temperature. Compared to SSF, NSSF increased ethanol yield and productivity with a reduced overall enzyme loading of 30—40%. The disadvantage is increased capital cost for extra equipment.

In SSCF, enzymatic biomass hydrolysis and fermen­tation of both cellulose and hemicellulose hydrolysis products all occur in a single bioreactor with a single microorganism (Teixeira et al., 2000). It is considered an improved process compared to SSF, which requires two bioreactors with two different microorganisms and two different biomass production setups (Hame — linck et al., 2005; McMillan, 1997; McMillan et al.,

1999) . However, SSCF usually requires a metabolically engineered microorganism that can robustly coferment both glucose and xylose (Teixeira et al., 2000) without synthesis of side products. For example, when a natu­rally occurring strain, Lactobacillus pentosus (American Type Culture Collection, ATCC 8041), was used in an SSCF process using pretreated corn stover as substrate and the commercial cellulase Spezyme-CP for hydroly­sis, the maximum yield of lactic acid was >90% of the theoretical maximum on the basis of all available fermentable sugars. However, acetic acid was also pro­duced through a different metabolic pathway that as­similates pentoses (xylose and arabinose). Another drawback of the process is the difficulty in improving lactic acid concentration due to end-product inhibition of the nonengineered strain (Zhu et al., 2007).

All the above-mentioned processes require a separate enzyme production step or an external supply of en­zymes for biomass hydrolysis. In CBP, enzyme produc­tion, biomass hydrolysis, and fermentation of pentoses and hexoses are accomplished in a single reactor by mono — or cocultures of microorganisms (Lynd et al.,

2002) . The obvious advantages of CBP are decreased cap­ital costs and no extra cost for enzyme production or pur­chasing (Hamelinck et al., 2005; Lynd et al., 2005). However, since naturally occurring microorganisms cannot simultaneously synthesize enough of the neces­sary saccharolytic enzymes and convert released sugars into the desired end products, the CBP configuration requires the development of engineered microorganisms (Hasunuma and Kondo, 2012a; Xu et al., 2009). Such "superbugs" need to not only secrete high titer, robust en­zymes, but also efficiently produce ethanol and other bio­products at high yields under harsh environments containing toxic compounds. CBP is gaining increasing recognition as a potential breakthrough for low-cost biomass processing (Hasunuma and Kondo, 2012a; van Zyl et al., 2007). The company Mascoma Corporation claims to have successfully engineered microorganisms for industrial CBP application (http://www. mascoma. com/).

Commonly Used Microorganisms in Biomass Conversion and Some Application Examples

A large number of microorganisms are capable of degrading plant cell walls including bacteria and fungi. With few exceptions, two distinct cellulolytic strategies have been adapted by the aerobic and anaerobic groups. While aerobic bacteria and fungi produce numerous individual, extracellular enzymes with many of them acting in synergy for effective hydrolysis, anaerobic bacteria and fungi possess a unique extracellular multi­enzyme complex, termed the cellulosome, that can efficiently hydrolyze crystalline cellulose (Bayer et al., 2004, 1998; Doi and Kosugi, 2004; Fontes and Gilbert, 2010; Lamed et al., 1983; Lynd et al., 2002; Schwarz, 2001; Shoham et al., 1999; Steenbakkers et al., 2003). Metabolic utilization of the monomeric sugars from hydrolyzed biomass leads to the natural production of biofuels and bioproducts, mostly as side products by different microorganisms. For ethanol fermentation of lignocellulosic biomass, most frequently considered microorganisms include the bacteria E. coli, Z. mobilis and Clostridium phytofermentans; themophilic bacteria such as Clostridium thermocellum; yeasts such as S. cerevi — siaeand Pichia stipitis; and filamentous fungi (Amore and Faraco, 2012; Hahn-Hagerdal et al., 2007; Weber et al., 2010; Xu et al., 2009).

Like ethanol, the majority of other potential biofuels and bioproducts are naturally produced by various mi­croorganisms as side products. The viability of a fermentation process for industrial application depends on its cost-competitiveness. As listed in Table 5.1, most microorganisms cannot use polymeric carbohydrates directly as fermentation substrates; therefore, biomass has to be broken down into monomeric sugars to be used as fermentation substrates. For an economically viable manufacturing process of biofuels from ligno — cellulosic biomass, pentose utilization is essential. Therefore, an optimal microorganism should be able to simultaneously ferment both hexose and pentose sugars and give rise to high productivities and yields. In addition, it should have high tolerance to fermenta­tion inhibitors and end products and resist microbial

contamination, e. g. bacteriophage infections (Weber et al., 2010).

No naturally occurring microorganism has all the required features. Promising means to develop a microorganism for sustainable bioethanol/bioproduct production include breeding technologies, genetic en­gineering and the search for undiscovered species (Weber et al., 2010). For production of a particular product from a specific biomass, native organisms can be selected from a group of different species of mi­crobes based on their fermentation performance, such as substrate utilization efficiency, inhibitor resistance, and productivity (Rumbold et al., 2010, 2009). The yeast S. cerevisiae is by far the most widely used organ­ism in the existing fermentation industry. To improve its application in bioethanol fermentation from biomass, targeted evolution strategy has been applied to obtain inhibitor-tolerant S. cerevisiae that can resist
individual or multiple inhibitors (Ding et al., 2012; Heer and Sauer, 2008; Liu, 2006). When adaptation and selection processes were applied to the parental fungus Rhizopus oryzae, a new strain was obtained that exhibited significantly improved efficiency of sub­strate utilization and enhanced production of l-(+)- lactic acid from corncob hydrolysate. The final product concentration, yield, and volumetric productivity more than doubled compared with its parental strain (Bai et al., 2008).

Applications of thermotolerant mesophilic microor­ganisms in the fermentation process have considerable potential for cost-effective ethanol and other bioproduct production. The thermotolerant yeast Kluyveromyces marxianus grows well at temperatures as high as 45—52 °C and can efficiently ferment ethanol at temper­atures of between 38 and 45 °C. A 5 °C increase in the fermentation temperature can greatly decrease fuel

ethanol production costs (Babiker et al., 2010). Results from solid state fermentation of sweet sorghum stalk to ethanol with the thermotolerant yeast strain Issatchen — kia orientalis IPE 100A showed great potential for its practical application in large-scale, deep-bed solid state fermentation (Kwon et al., 2011).

The thermotolerant Bacillus coagulans strain 36D1 can ferment both hexoses and pentoses from enzymatically hydrolyzed biomass at 50—55 °C and pH 5.0 producing l (+)-lactic acid as the primary fermentation product. Since such conditions are closer to the optimum fungal enzyme functioning requirements, the amount of enzyme required for cellulose conversion is signifi­cantly reduced in comparison with yeast or lactic acid bacteria currently used by the industry as microbial biocatalysts. In addition, both biomass conversion efficiency and product yield are greatly increased with a dramatically decreased fermentation time, thus reducing the cost of both the process and final product (Ou et al., 2009).

The anaerobic mesophilic bacterium C. phytofermen- tans (ATCC 700,394) is a promising native microor­ganism for biomass conversion since its genome encodes the highest number of enzymes for degradation of lignocellulosic material among sequenced Clostridial genomes (Warnick et al., 2002; Weber et al., 2010). It se­cretes noncomplex, individual enzymes to hydrolyze both cellulose and hemicelluloses to both hexose and pentose sugars, which are mostly directly consumed, producing ethanol and acetate as the major products (Warnick et al., 2002; Weber et al., 2010). When used in the CBP process with pretreated corn stover as sub­strate, at optimal conditions with low solid loading (0.5% w/w), C. phytofermentans hydrolyzed 76% of glucan and 88.6% of xylan in 10 days. These values reach 87% and 102% of those obtained by SSCF process using commercial enzymes and S. cerevisiae 424A with an ethanol titer of 2.8 g/l corresponding to 71.8% of that yielded by SSCF (3.9 g/l) (Jin et al., 2011a). Howev­er, using a similar process with high solid loading (4% w/w), the side product acetate became a major product (Jin et al., 2012).

Even though C. thermocellum seems a good candidate for ethanol fermentation from cellulosic biomass, there are a few disadvantages as listed in Table 5.1. Despite its ability to degrade lignocellulosic waste to both hex — ose and pentose sugars, it can only utilize hexose sugars from cellulose and not the pentose sugars derived from hemicellulose (Lynd et al., 2002; Taylor et al., 2009). This drawback could be solved by the use of mixed cultures for the degradation and fermentation of all sugars derived from lignocellulosic materials. For example, the anaerobic thermophile Thermoanaerobacterium saccha- rolyticum, which can ferment xylan and almost all soluble biomass sugars, would be a good candidate for coculture with C. thermocellum. A twofold reduction of the bioethanol production cost from lignocellulose could be achieved when using thermophilic anaerobic mixed cultures (Demain et al., 2005; Lynd et al., 2002). Since there is currently no perfect CBP microbe that can degrade lignocellulosic biomass efficiently and at the same time utilize all the sugars released from biomass to produce mostly ethanol, coculture or community/ mixed fermentation may be a suitable option (Barnard et al., 2010; Demain, 2009; Jin et al., 2011a). Chen reviewed 35 coculture systems for ethanol production by cofermentation of glucose and xylose and concluded that even though still in its infancy, this strategy is prom­ising as it can increase ethanol yield and productivity, shorten fermentation time, and reduce process costs (Chen, 2011).

FUTURE PERSPECTIVES

For a particular product made from lignocellulosic biomass fermentation, it will be difficult to predict which particular microorganism should be finally used in commercial production. For different processes, it is possible that different species may be required. For bioethanol production, S. cerevisiae has some advan­tages since it is already widely used in large-scale, first-generation bioethanol production with well- established processes and technology. An ideal biomass sugar fermentation process needs to reach high product yield by fermenting all biomass sugars including glucose, xylose, arabinose, mannose, and galactose with an optimal microorganism that is resistant to toxic materials/chemicals in biomass hydrolysates such as acids, phenolics, salts, and sugar oligomers. In addi­tion, the microorganism should be robust, resistant to contamination and environmental stresses, with mini­mal metabolic by-product production. To achieve these goals, metabolic engineering, or extensive physiolog­ical reprogramming of the producing organisms may provide solutions.

MFC Reactor Designs

There are many different types of MFC bioreactors. They include single-chamber, dual-chamber, multi­chamber, membrane-less, multianode, multicathode and so on. Many MFC reactors were discussed by Du

et al. (2007). More recently, Zhou et al. (2013) reviewed some new MFC reactors and their combinations, including MFCs operated as microbial electrolysis cells (MECs) to produce bio-products such as hydrogen and methane. It should be pointed out that improvement in MFC reactor design must consider cost and mainte­nance. Complicated designs are not only costly but also prone to biofouling, causing maintenance and sus­tainability problems. A simplistic tubular MFC reactor

with convective axial flow was proposed (Zhou et al.,

2013) . To reduce cost and fouling, no membrane was used. To prevent oxygen back-diffusion into the anodic region, a substantial flow rate from the anode to the cathode is required. This means that the biofilm has to be highly efficient in the digestion of organic matter in wastewater streams. This type of design will become attractive only when robust "super-bug" biofilms are successfully engineered.

Substrates Used in MFCs

In MFCs, the substrates greatly impact their perfor­mances such as power density and Coulombic efficiency (Pant et al., 2010). The substrates range from the simple volatile fatty acids (VFAs) to complex compounds such as lignocellulosic biomass. Anaerobes evolved when the earth’s atmosphere was still anaer­obic long before aerobes evolved. Many of them lack
the metabolic pathways to utilize high-grade organic carbons such as cellulose, hemicellulose, various hex — oses and phenylpropane moieties (components of lignin). Most of the electrogenic microbes capable of DET feed only on low-grade organic carbons such as VFAs and alcohols. Only a few organisms such as R. ferrireducens (Chaudhuri and Lovley, 2003; Schroder, 2007) utilize glucose, while Geobacter and Shewanella strains cannot (Lovley, 2006a). This limits MFC power output because high-grade organic carbons are unutilized.

Certain plant materials require enzymatic maceration prior to lipid release as their volatile components are glycosidically bound. Enzymes can be either endogenous or exogenous to the biomass. For example, methyl salicylate (wintergreen oil) is an organic ester that is naturally produced by many species of plants. The plant leaves contain the precursor gaultherin and the enzyme primeverosidase; when the leaves are macerated in warm water, the endogenous enzyme acts on the gaultherin and liberates free methyl salicylate and primeverose (Handa, 2008). In the case of the exog­enous addition of enzymes, recent advances in the field of algal lipids have demonstrated the addition of com­plex mixtures of enzymes to selectively degrade cell walls in a cascade of hydrolytic reactions. Released lipids are isolated and collected for further processing (Liang et al., 2012).

Expression (Cold Pressing)

Expression or cold pressing is commonly used in the production of essential and food oils. The term expres­sion refers to any physical process in which the essential oil glands in the biomass are crushed or broken to release the oil. The resulting oil—water emulsion is typi­cally separated by centrifugation. Traditionally, cold pressing was conducted by hand; however, for large — scale commercialization, this is impractical. Thus, with the advancement of industrialization, a number of machines have been designed to achieve the same results on commercial scale. It is important to note that oils extracted using this method have a relatively short shelf life (Martinez et al., 2008).

Zeolites are natural or synthetic materials, classically defined as crystalline aluminosilicate compounds (Cundy and Cox, 2003). Zeolites can be prepared by different synthetic routes with different Si/Al molar ratios, crystal structures, and proton exchange levels. These modifica­tions favor the rationalization of catalytic properties such as pore size, hydrophobicity, strength and distribu­tion of acid sites. When designed in a positive way, all these properties can be interesting and useful for applica­tions in heterogeneous catalysis (Corma et al., 1989).

The catalytic activity of zeolytes can derive from the properties of the cation that is present in its chemical composition. Moreover, the exchange of these cations by protons may generate different degrees of zeolite acidity, which is also an interesting property for various catalytic processes (Csicsery, 1984). In fact, acid zeolites are used in many industrial catalytic applications, mainly in the petrochemical industry.

Another interesting property is the organized and uniform pore distribution and the existence of a cav­ity system of regular molecular dimensions ranging from <1 nm to over 10 nm, depending on the solid material. This feature may bestow rather important catalytic properties to the resulting material such as selectivity.

In general, zeolites and other porous materials of similar composition and textural properties, named zeolite-like materials and zeotypes, have been prepared and used as catalysts in various chemical processes. These solid catalysts present a strong scientific appeal for green chemistry applications since they are consid­ered environmentally benign when one takes into account their chemical composition.

There are several reports regarding the use of zeolite — based catalysts for various chemical reactions. Such uses have been recently reviewed (Martinez and Corma, 2011; Rinaldi and Schuth, 2009). This versatility of uses is not only justified for their great variety in chemical composition but also because of the uniformity of their pore structure.

Due to the presence of pores and channels, catalysts based on zeolites can present size selectivity that is rarely seen in other solids. This selectivity can be observed for reactants, products and transition state in­termediates that are expected to control a given catalytic reaction (Csicsery, 1984). However, for the same reason, these solids not always perform well in catalytic processes that are dependent on one of their main chem­ical properties (e. g. acidity). This occurs mainly for pro­cesses in which the reactants have dimensions exceeding the catalytic channels and pores provided by zeolitic solid catalyst. Therefore, the structure of a zeolite cata­lyst must be idealized in order to have not only the appropriate chemical property but also the textural properties that would offer an array of pores and chan­nels that are adequate for the diffusion of the chemical reactants. The strategy to meet these two goals is a chal­lenge for the catalytic application of these solids.

The preparation of zeotype materials with mesopores (2—50 nm) appears to be the solution to avoid the mass transfer limitations of zeolites in many catalytic pro­cesses. In this sense, many efforts have been made in the scientific community to prepare zeolite-like mesopo — rous materials that are able to address this goal (Tao et al., 2006).

For applications in the esterification of fatty acids or in transesterification of oils and fats, in which large mol­ecules are directly involved in the production of bio­fuels, it is expected that, apart from their high acidity, the surface of the zeolitic and/or zeotype solids should be hydrophobic enough to promote the adsorption of the substrate on the catalyst surface. In this regard, the adsorption of polar molecules may cause deactivation of catalytic sites, such as in the case of water in esterifi­cation reactions (Helwani et al., 2009a). For example, faujasite is a highly hydrophilic zeolite that presents high levels of water adsorption. Hence, this material is barely adequate for esterification because water may

not only inactivate the catalytic sites on the solid surface but also compromise the reaction yields by interfering with the reaction mechanism (Nijhuis et al., 2002).

The MCM-41 molecular sieves have been used as an alternative to zeolite microporous materials. Since its discovery in the early 1990s, these molecular sieves have been used as catalyst in different chemical reac­tions (Beck et al., 1992; Climent et al., 1999), including in the preparation of biofuels (Twaiq et al., 2003).

Similar to the zeolites, these mesoporous compounds also have a regular and ordered distribution of pores (mesopores) across the solid catalyst, allowing their use for the conversion of larger molecules (Carmo et al., 2009). Moreover, the incorporation of metals in the structure of mesoporous solids may lead to acidic materials with special characteristics such as a higher hydrothermal stability. For example, the incorporation of aluminum ions in zeolitic materials can lead to a decrease in the Si/Al ratio and a subsequent increase in the amount of the solid acid sites, since it is well known that the lower the framework Si/Al ratio of the zeolite, the lower the strength of its acid sites, regardless of its higher density (Ma et al., 1996; Corma et al., 1989).

Furthermore, the catalyst hydrophobicity can also be changed by modifying the Si/Al ratio. This leads to an alteration in the ability of the solid to adsorb nonpolar molecules in the catalytic reactions such as esterification and transesterification, as well as in desorption of polar molecules such as water (Luque et al., 2007). In general, high Si/Al ratios (or low aluminum contents) leads to high solid hydrophobicity. Thus, since the Si/Al ratio modifies the acidity and hydrophobicity of the catalyst, its influence on the catalytic properties is subtle, mainly in esterification reactions.

The presence of water is an important factor in the conversion outcome of esterification reactions. The equi­librium constant for ester formation is very low (3.38 for the reaction of acetic acid with ethanol in nonpolar sol­vents) (Corma et al., 1989). So, to obtain high ester yields, the reaction must be displaced toward the products, for example, by the continuous removal of water from the system. Furthermore, the reaction can be shifted toward the products when working with a large excess of reagents.

In order to segregate the water from the reaction envi­ronment, it is necessary to work with high reaction rates and this can be achieved with homogeneous acid cata­lysts such as sulfuric acid. However, for solid catalysts such as the zeolitic materials and zeotypes, the proper balance between strength and density of the acid sites, suitable for a good catalytic performance and water segregation, is often difficult to achieve. The rate of reac­tions catalyzed by zeolite catalysts and other solid mate­rials is usually very low compared to that of sulfuric acid (Ma et al., 1996).

Ajaikumar and Pandurangan (2007) prepared Al — MCM-41 materials with different Si/Al ratios (29, 52, 74 and 110) and used these solids in the esterification of acetic and propionic acids with various alcohols (1-hexanol, 2-ethyl-1-isoamyl alcohol and cyclohexanol). With a small addition of aluminum, which translates into a high Si/Al ratio of 110, these authors observed a higher hydrophobicity and a higher catalyst hydrother­mal stability of the material concerning the amount of water formed during esterification. Furthermore, the use of more hydrophobic solid materials prevented the subsequent hydrolysis of the ester formed. On the other hand, solid catalysts with lower Si/Al ratio promoted lower levels of alcohol dehydration, which can also be favored at high temperature. As a result, the selectivity of the catalytic reaction is improved toward the ester pro­duction as the accumulation of possible by-products (etherified and dehydrated compounds) is decreased. Hence, the hydrophobicity achieved at higher Si/Al ratios was an important factor for the best catalytic performance (catalytic efficiency), whereas the use of low aluminum contents led to more selective catalytic systems.

Corma et al. (1989) reported that strong Bron — sted—Lowry acid sites are required for the catalytic esterification of acetic acid since they are able to proton — ate the acetic acid carbonyl group. Working with proton — ated faujazite zeolite after dealuminization, these authors observed that some dealuminized HY zeolites with Si/Al ratio less than or equal to 15 had better cata­lytic performance. The strong acid sites present in that solid, which correspond to those aluminum vacant sites or sites occupied by one aluminum atom and the respec­tive nearest neighbors, are more active for the catalytic esterification of fatty acids. Zeolites with high Si/Al ratio presented a more hydrophobic surface and this hydrophobicity became the predominant factor for the equilibrium shifting toward the production of alkyl esters. Also, the higher the aluminum content of the zeolite, the higher the observed adsorption effect.

Carmo et al. (2009) also prepared solids based on Al-MCM-41 to investigate the relationship between the high availability of acid sites, introduced by increasing the aluminum content in the mesoporous solid, and the degree of esterification of palmitic acid with meth­anol, ethanol and isopropanol. However, these authors restricted their work to solids with low Si/Al ratios (8, 16 and 32) whose hydrophobicity was much smaller than the solids described in the previous work. This and most of the data already available in the literature pro­pose the utilization of solid catalysts for esterification reactions. In general, these studies have been motivated by the technological challenge of developing a suitable catalytic system to convert vegetable oils or animal fats of high acid number in biodiesel. Hence, by the catalytic esterification of their fatty acid content, these materials would be neutralized and become suitable for transes­terification in alkaline media.

The high aluminum content solid catalysts (Si/Al molar ratio of 8) prepared by Carmo et al. (2009) showed relatively modest palmitic acid conversion values. The highest value achieved in this study was 79 wt% of methyl ester. The authors did not report the effect of hydrophobicity on reaction conversion but only the increased effect of aluminum incorporation in the cata­lytic activity of the resulting solids.

Ma et al. (1996) used different zeolitic solids (zeolite ZSM-5 and three HY zeolites with Si/Al molar ratios of 30, 5.1 and 9.3) to evaluate the relationship between the solid hydrophobicity and its aluminum content with the observed catalytic efficiency in the preparation of ethyl, n-butyl, isopentyl and benzyl acetates; ethyl and n-butyl benzoates and dioctyl phthalates. For all the solid catalysts used in this study, a high selectivity for the expected ester was observed without any forma­tion of ether derivatives. The increase in selectivity with decreasing aluminum content was also reported by Corma et al. (1989).

Insoluble inorganic salts and other inorganic solids based on transition metals can also be used as acid catalysts for transesterification. The application of so­dium molybdate (Na2MoO4) and sodium tungstate (Na2WO4) has been recently reported as efficient cata­lysts for biodiesel production under relatively mild experimental conditions (Nakagaki et al., 2008; Santos et al., 2011). In these studies, soybean oil (0.7 mg/g KOH of acid number), degummed soybean oil contain­ing 180 ppm of phosphatides (1.0 mg/g of KOH), and waste cooking oil (1.5 mg/g KOH) were transesterified with methanol (methanolysis). At 65—80 °C using a 54:1 methanol:oil molar ratio and 5 wt% catalyst for

3— 5 h, both catalysts reached conversions higher than 92 wt% regardless of the feedstock used for methanoly — sis. The catalytic activity of these compounds was attrib­uted to the presence of molybdenum(VI) or tungsten(VI) strong Lewis acid sites that are probably able to polarize the methanol O—H bond leading to intermediate species that possibly have high nucleophilic character.

The heterogeneous nature of the above-mentioned catalysts was investigated through their reuse in several consecutive reaction cycles. Both compounds could be reused for at least three catalytic cycles. However, part of the solid catalysts was lost during the recycling pro­cesses due to their reduced particle size and noticeable adherence to the walls of the reaction vessel. To circum­vent these problems, both molybdenum (Bail et al., 2013) and tungsten (Santos et al., 2011) compounds were het — erogenized in silica obtained by the sol—gel process and used in esterification of stearic and oleic acids. Im­provements were observed in the catalysts’ recovery and reuse and a good catalytic activity was obtained in the first and subsequent recycling stages. Similarly, zir — conia impregnated with tungsten oxide (ZrO2/WO2) was also investigated as an acid catalyst for both esteri­fication and transesterification reactions with methanol (Lopez et al., 2007).

In general oxidative depolymerization of lignin is carried out to produce aromatics with an increase in oxygen-containing groups, mostly aldehydes. The pro­duction of vanillin (3-methoxy-4-hydroxybenzaldehyde) by oxidative depolymerization of lignin, mainly from black liquor of sulfite pulping is the most well-known pro­cess. This commercial process is typically performed at 160—175 ° C under alkaline conditions using a copper catalyst by Borregard in Norway. Especially softwood lignin is yielding relatively higher amounts of vanillin as compared to hardwood lignin where syringaldehyde may prevail (Evju, 1979).

Other researchers used hydrogen peroxide for oxida­tive depolymerization. Kraft lignin was treated at 90 °C by a biomimetic system, using hemin as a catalyst and hydrogen peroxide as an oxidizing agent, which mimics the catalytic mechanism of lignin peroxidase. Rela­tively high yields of vanillin 19%, vanillic acid 9%, 2-methoxyphenol 2% and 4-hydroxybenzaldehyde 2% were obtained (Suparno et al., 2005). Xiang and Lee (2000) found that alkaline peroxide treatment of lignin at 80—160 °C yields mainly low molecular weight organic acids (up to 50%) with only traces of aromatics, which are rapidly degraded by hydrogen peroxide.

Sales et al. (2004, 2007) studied the alkaline oxidation of sugarcane soda lignin with a continuous fluid bed with a palladium chloride PdCl3.3H2O/g-A^Os catalyst at 100—250 °C and 2—10 bar partial oxygen pressure. Total aldehyde yield on lignin was 12%. Zakzeski et al.

(2010) reported other predominantly catalytic lignin oxidation processes yielding aromatic aldehydes and acids, which do not exceed 10% on lignin basis. Howev­er, lignin model compounds show in some catalytic pro­cesses good conversions, which are promising to further develop catalytic strategies for lignin depolymerization in a biorefinery concept.

Voitl and Rudolf von Rohr (2010) studied a process for producing vanillin and methyl vanillate from kraft lignin by acidic oxidation in aqueous methanol with H3PMo12O40 as a homogeneous catalyst in the presence of 10 bar oxygen. A stable yield of 3.5 wt% vanillin and

3.5 wt% methyl vanillate can be obtained together with 60 wt% of oligomeric products in the extract. The mono­mers can be effectively separated using organic solvent nanofiltration (Werhan et al., 2012).

A wide range of phytochemicals have long been used for dietary or neutraceutical purposes (Rao, 2012; Wang and Weller, 2006). Several vitamin homologs or precur­sors, such as b-carotene (for vitamin A), tocopherol (for vitamin E), and ascorbic acid (vitamin C), are widely pro­duced from plants. Numerous nutraceutical phytochem­icals, such as anthocyanin and flavonoids, are known for antioxidant or other bioactivities. Commonly used food or feed additives include lutein, canthaxanthin and b-carotene as dietary or coloring agent, astaxanthin for aquaculture such as salmon farming, essential oils, menthol, camphor, caffeine, tannin, capsaicin, wood fla­vor or liquid smoke (water-diluted bio-oil; Venderbosch
and Prins, 2010; Di Blasi et al., 2010) for flavor or aroma, anthocyanins as antimicrobial agents (Chattopadhyay et al., 2008), papain and bromelain for meat processing, lecithin for emulsification, and dietary fibers. Figure 20.3 shows representative dietary phytochemicals.

The chemical industry relies on simpler molecules to build complex compounds, which are used in a variety of applications. Among these organic compounds, ethylene is the building block with the highest demand, being used to manufacture "everyday" products such as polyethylene terephthalate (PET bottles), polyester, anti­freeze, and others (Ungerer et al., 2012). Ethylene (or ethene) is the second simplest unsaturated hydrocarbon, it consists of two carbons, with a double bond, and four hydrogens (H2C=CH2). It is one of the products of pyrolysis, and has been used as a fuel since the early nineteenth century as one of constituents of the gaseous fuel in gas lamps. Ethylene is the most important com­pound in the chemical industry in terms of market volume, it has a heat of combustion higher than that of gasoline or diesel and can be used as a transportation fuel or to produce electric energy in stationary plants. Currently, ethylene is a petroleum derivative produced through steam cracking. It reached a production of 100 million metric tones in 2005, accounting for 30% of all petrochemical commodities (McCoy et al., 2006; Saini and Sigman, 2012). The fluctuation in crude oil prices over the last few years (EIADOE, 2012), the imminent threat of peak oil (Nashawi et al., 2010), and the exis­tence of biological pathways for its production coupled with the ease of harvesting a gas like ethylene, make this chemical a good target for the development of a sus­tainable biological production system.

The most common occurrence of ethylene in nature is as a hormone found in vascular plants, where it is asso­ciated with many effects such as defoliation, responses to temperature stress, mechanical injury, and for promoting fruit ripening (Abeles, 1972). In addition to vascular plants, many other plants and algae have been shown to be able to produce ethylene and even if it not found in animals, this gaseous hormone has been shown to induce regulatory responses in inverte­brate and mammalian cells (Perovic et al., 2001).

The most common biosynthetic pathway for ethylene production is the Yang cycle that occurs in plants, where it is produced from methionine in a three-step reaction, having S-adenosylmethionine (AdoMet) and 1-aminocyclopropane-1-carboxylic acid (ACC) as precursors. However, the cellular response to this hormone occurs at very low concentrations, a characteristic that, together with the fast and easy diffusion of this gas into plant tissues, makes the con­version of AdoMet to ACC, catalyzed by ACC syn­thase, and from ACC to ethylene (ACC oxidase) a tightly regulated process. Both enzymes are multigenic with differential regulation through distinct promoters and operators for groups of genes of the same enzyme (Nakatsuka et al., 1998). The methionine used in this pathway is recycled through the Yang cycle (Taiz and Zeiger, 2002; Wang et al., 2002).

The pretreatment methods cause physical and/or chemical changes in the lignocellulosic biomass; thus, pretreatment technologies are usually classified into physical, chemical, physicochemical, and biological. For the purposes of classification, steam and water are excluded from being considered chemical agents for pretreatment since extraneous chemicals are not added to the biomass (Mosier et al., 2005). This chapter focuses on chemical, physical and physicochemical pretreatments but a brief description of biological treatments are include in order to contrast and compare them.

Biological Pretreatments

Biological pretreatments employ microorganisms mainly brown, white and soft-rot fungi, which degrade lignin, hemicellulose and cellulose in small proportion (Alvira et al., 2010). Recently, this approach has received renewed attention as biological pretreatments have several advantages over conventional physical/chemi — cal pretreatment methods, such as they are considered as environmentally friendly, low capital cost, low energy, no chemicals requirement, and mild environ­mental conditions (Saritha and Lata, 2011). However, the main drawbacks to develop biological methods are the low hydrolysis rate obtained in most biological materials and the relatively long time of the pretreat­ment compared to physical/chemical methods. Conse­quently, more space and longer processes are required, which increase the operating costs (Alvira et al., 2010; Saritha and Lata, 2011).

The white-rot fungi are able to decompose all wood fractions, including lignin because they produce various enzymes involved in lignin degradation such as lignin peroxidase, laccase, manganese peroxidase, versatile peroxidase, and H2O2-forming enzymes such as glyoxal oxidase and aryl alcohol oxidase. White-rot fungi also produce cellulases, xylanases and other hemicellulases that are required in the hydrolysis. Almost all

white-rot fungi produce manganese peroxidase and lac — case, but only some of them produce lignin peroxidase (Isroi et al., 2011).

Several white-rot fungi such as Phanerochaete chryso- sporium, Ceriporia lacerata, Cyathus stercolerus, Ceriporiop — sis subvermispora, Pycnoporus cinnarbarinus, Pleurotus ostreaus, Dichomitus squalens, Coriolus versicolor, Tricho — derma reesei, Aspergillus terreus, Aspergillus awamori, Bjer — kandera adusta, Phlebia tremellosus, Fusarium proliferatum, and Pleurotus florida have been examined on different lignocellulosic biomass (Alvira et al., 2010; Cui et al., 2012; Isroi et al., 2011; Kuhar et al., 2008; Pinto et al., 2012; Saritha and Lata, 2011; Wan and Li, 2011). Recently, some bacterial laccases have also been characterized from Azospirillum lipoferum and Bacillus subtilis (Saritha and Lata, 2011). However, they face three major chal­lenges associated with lignin structure: (1) the lignin polymer is large; therefore, ligninolytic systems must be extracellular, (2) lignin structure comprises interunit carbon—carbon and ether bonds; therefore, the degrada­tion mechanism must be oxidative rather than hydrolyt­ic, and (3) lignin polymer is stereoirregular, therefore the ligninolytic agents must be much less specific than degradative enzymes (Isroi et al., 2011).

As mentioned before, one of the most main draw­backs of biological pretreatments is the time of the pre­treatment; reported time of treatment is between 7 and 60 days (Giles et al., 2011; Mahalaxmi et al., 2010; Wan and Li, 2011). After 18 days of pretreatment, C. subver­mispora effectively delignified corn stover, switchgrass, and hardwood with glucose yields during enzymatic hydrolysis that reached 56.50%, 37.15%, and 24.21%, respectively (Wan and Li, 2011). Also, glucose yield after 21 days of pretreatment with Poria subvermispora and Irpex lacteus reached 69% and 66% of cellulose available in the wheat straw, respectively, with an ethanol yield of 62% in both cases (Salvachua et al., 2011).

Biological pretreatment has also been used before py­rolysis of biomass to produce fuel. The biological pre­treatment of corn stover can optimize the thermal decomposition, decrease the reaction temperature and reduce the gas contamination (SOx), making the biomass pyrolysis more efficient and environmentally friendly. Biological pretreatment can decrease the activa­tion energy and reacting temperature of the hemicellu — lose and cellulose pyrolysis (up to 36 °C), shorten the temperature range of the active pyrolysis (up to 14 °C), and increase the thermal decomposition rate (Isroi et al., 2011; Yang et al., 2011).

A cost-competitive biological pretreatment of lignocel — lulose requires continuous studying and testing more mi­croorganisms for their ability to delignify the plant material quickly and efficiently (Saritha and Lata, 2011). Also, integrated methods, such as, cotreatment with organic solvents, diluted acids, supercritical CO2 and ionic liquids (ILs); mutation breeding and crossbreeding of fungal mycelia to obtain engineering strains; and inte­gration of fungal pretreatment with simultaneous saccharification and fermentation to produce biofuels and value-added products should be studied (Tian et al., 2012).

In the presence of excess sugar, yeast, especially S. cere — visiae, has a strong tendency to undergo alcoholic fermen­tation, even when oxygen is available in excess (Van Diken and Scheffers, 1986). In the biochemical pathway for
carbohydrate metabolism in most yeasts, two modes of disaccharide metabolism exist. While extracellular hydro­lysis of sucrose to glucose and fructose followed by trans­port of these monosaccharides into the cell is the most common method for sucrose metabolism in yeast, trans­port of disaccharides by proton—sugar symport followed by intracellular hydrolysis occurs in maltose and lactose metabolism (Weusthuis et al., 1994). However, hydrolysis of sucrose can occur either intracellularly or extracellu­larly in S. cerevisiae (Santos et al., 1982), followed by the phosphorylation of glucose to glucose-6-phosphate, which is subsequently catabolized to pyruvate via the Embden—Meyerhof—Parnas pathway (Figure 7.2). Although most of the synthesized pyruvate is decarboxy — lated to acetaldehyde (ethanal) by PDC followed by the reduction of acetaldehyde to ethanol by ADH, a small proportion of the pyruvate is converted to fusel alcohols such as isobutanol (Figures 7.1 and 7.2; Table 7.1).

KIV is an important precursor for valine biosynthesis, which is also shared by isobutanol production. KIV biosynthesis is initiated by the condensation of two pyruvate molecules to 2-acetolactate, which is catalyzed by acetolactate synthase (ILV2 + ILV6; Figure 7.2). Notably, ILV6 is the regulatory subunit of acetolactate synthase and an enhancer of ILV2 catalytic activity
(Chen et al., 2011). The 2-acetolactate is reduced to

2,3- dihydroxyisovalerate via catalysis by acetohydrox — yacid reductoisomerase (ILV5), the precursor for KIV biosynthesis (Figure 7.2; Velasco et al., 1993). Thus, KIV is produced through catalysis of 2,3-dihydroxyisovalerate by dihydroxyacid dehydratase (ILV3). Further, the bidirec­tional conversion between KIV and valine is catalyzed by aminotransferases (Bat1 and Bat2). While aminotransferase Bat1 is present in the mitochondrial matrix of S. cerevisiae, aminotransferase Bat2 is present in the cytosol (Kispal et al., 1996; Chen et al., 2011). Next, KIV is decarboxylated by PDC, a KDC, to isobutyraldehyde and subsequently, reduced to isobutanol by ADHs (Figure 7.2).

Biomethane (CH4) production from microalgal biomass is of interest because the efficiency of algal biomass production per hectare is estimated to be 5—30 times greater than that of the terrestrial crop plants (Sheehan et al., 1998). Golueke and Oswald (1959) pub­lished one of the first feasibility studies using microalgae for CH4 production and concluded that the process was feasible (Golueke and Oswald, 1959). There are two well-established methods of CH4 production: (1) harvest of an algal polyculture from a wastewater treatment pond, or (2) axenic growth of specific algae at a bench scale (Asinari Di San Marzano et al., 1982; Yen and Brune, 2007). The digestion process is described in

Figure 10.9. It begins with bacterial hydrolysis of the algal biomass. Organic polymers, such as lipids, carbo­hydrates, and proteins, are first broken down to soluble derivatives, which are further fractionated into carbon dioxide, hydrogen, ammonia, and organic acids by acidogenic bacteria. Acetogenic bacteria then convert these resulting organic acids into acetic acid, along with additional ammonia, hydrogen, and carbon diox­ide. Finally, methanogens convert these products to methane and carbon dioxide. Regardless of operating conditions and species, the proportion of methane in the biogas produced for the majority of studies falls in the range 69—75%. Anaerobic digestion is an effective process for biological oxygen demand removal, but it is not effective for nutrient removal. Thus, there is a need for further treatment of effluent from anaerobic di­gesters before it can be discharged into the environment. The nutrient-rich digestate also produced can be used as fertilizer. This process of converting microalgae to CH4 is dependent on several key metrics, namely (1) pH, (2) retention time, (3) mixing, (4) composition of the biomass and (5) composition of the surrounding milieu.

One of the most important factors influencing CH4 biogas production from algal biomass has been reported to be pH. At high pH, due to high alkalinity from NH3 release, the gas production will shift toward CH4. The oxidation state of the biomass also affects biogas quality, which in turn drives the proportion of methane released (Sialve et al., 2009). Due to lowered content of sulfated amino acids, the microalgal biomass digestion releases a lower amount of hydrogen sulfide than do other types of organic substrates (Becker, 1988). The composition of the microalgal feedstock also affects biomethane yields. The relatively high lipid, starch, and protein contents and the absence of lignin make microalgae an ideal candidate for efficient biomethane production via fermentation in biogas plants. Theoretically, higher

cellular lipid contents will result in higher methane yields. Thus lipid-rich microalgae make attractive sub­strates for anaerobic digestion, as they have a higher gas production potential when compared to carbohy­drates and proteins (Li et al., 2002; Cirne et al., 2007). The hydraulic and solid retention time is another key metric in the anaerobic process. The hydraulic and solid retention time is a measure of the average length of the time that a soluble compound remains in a constructed bioreactor. Retention times should be sufficiently high to allow active bacterial populations (e. g. methanogens) to remain in the reactor yet not limit hydrolysis, which is considered to be the rate-limiting step in the overall con­version of complex substrates to methane. Moreover, optimal loading rates and hydraulic retention times must be enhanced to ensure efficient conversion of organic matter, and will depend on algal substrate composition and accessibility.