**3. Biological pretreatments**

a higher yield of compounds derived from pentoses (furfural), hexoses (5-hydroxymethylfur‐ fural) and lignin (low molecular weight phenolic compounds) that are inhibitory to subsequent fermentation by ethanologenic yeasts [26]. The mathematical concept of combined severity, which combines the various factors that define acid hydrolysis conditions (e.g. temperature, residence time, pH), allows objective comparisons between different conditions that enables the determination of optimal conditions for a given substrate [26]; however, doubts have been

184 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

Another highly effective pretreatment strategy is steam explosion, in which biomass is briefly heated to high temperatures (~200°C) under high pressure, then subjected to a rapid pressure drop that renders the biomass more penetrable by enzymes for subsequent hydrolysis [18]. In some cases, steam explosion is enhanced by the addition of an acid catalyst such as sulfuric acid [27]. For lignocellulosic agricultural residues, steam explosion under optimized condi‐ tions has been shown to be an effective pretreatment strategy for enzymatic saccharification [28]. Steam explosion has also been successfully used in combination with other physiochem‐ ical pretreatments such as acid/water impregnation of cereal straws [29]. Both of the latter studies resulted in the release of hemicellulose-derived pentose oligomers into the liquid fraction, and it was suggested that the use of ethanologenic strains capable of converting these pentoses into ethanol would further improve overall process efficiency [28]. Other assessments have suggested that the hemicellulose fraction would be more efficiently converted to other value-added products rather than ethanol using post-treatment enzyme addition or further

Organosolv is a process by which the lignin fraction is chemically modified and essentially removed from biomass using high-temperature extraction with alcohols such as methanol or ethanol or other solvents, sometimes with dilute acid (e.g. hydrochloric or sulfuric acid) as a catalyst [17]. While organosolv processes require a solvent recovery step to be economical and efficient, they provide a robust means of generating three streams of potential products: an extracted, modified lignin component, a hemicellulose-enriched aqueous phase, and a residue that is highly enriched in cellulose and an excellent substrate for the production of biofuels by enzymatic saccharification followed by bacterial or yeast fermentation. Organosolv is one of the pretreatment options that results in a fraction containing chemically modified, low molecular weight lignin components. This stream has a good deal of product potential in addition to its possible use as a fuel for combustion to provide energy to the process [7, 31]. While organosolv is particularly suited to very lignin-rich feedstocks such as wood [32], there is increasing interest in using organosolv extractions for agricultural residues such as wheat straw and dedicated biofuels crops [33]. Goh et al. [34] optimized organosolv conditions for empty palm fruit bunch using combined severity calculations, with excellent results and the

Microwave pretreatment of biomass is another option that has been reported to improve subsequent enzymatic saccharification of rice straw [35]. Microwaves have the advantage of combining very rapid heating times with a lower energy input than conventional heating strategies. This irradiative pretreatment creates localized hotspots, which open up the lignocellulose composite molecule, thereby facilitating enzyme access for saccharification and

raised about its accuracy [17].

acid hydrolysis [30].

ability to accurately predict product stream yields.

Microorganisms have evolved a capacity to modify and access lignocellulosic biomass to meet their metabolic needs. The exploitation of this capacity offers a natural, low-input means for preparing biomass for biofuels processes. Natural modification and degradation of the lignin component in particular can reduce the severity requirements of subsequent thermochemical pretreatment steps. For example, Itoh et al. [42] used a variety of lignin-degrading white-rot fungi to treat wood chips prior to extracting lignin by an organosolv method, and demon‐ strated that improved ethanol yields were obtained from the solid fraction along with a 15% savings in electricity use. Similarly, brown-rot fungal species *Coniophora puteana* and *Postia placenta* have been successfully used to improve glucose yields upon enzymatic saccharifica‐ tion of pine, acting as a complete replacement for thermomechanical pretreatments [43]. While it is clear that it is possible to exploit the metabolic capabilities of microorganisms to facilitate biofuels production, the very wide taxonomic array of microorganisms that modify or degrade lignocellulose presents a tremendous variety of choices for implementing such a strategy. Each approach carries its own advantages and challenges.

#### **3.1. Microbial consortia**

One approach for applying the power of microbial metabolism to the challenges of biofuel production involves ensiling, which is a commonly used means for enhancing the digestibility of forage and other biomass for ruminants [44, 45]. The process of ensiling exploits the capacity of naturally occurring bacteria, mostly Lactobacillaceae, to ferment the sugars within ligno‐ cellulosic residues and produce a substrate that is more easily digested by ruminal microor‐ ganisms. While these bacterial consortia lack the ability to substantially degrade the lignin component, the changes effected on the biomass can improve yields of fermentable sugars upon subsequent enzymatic hydrolysis. For example, ensiling a variety of agricultural residues, including wheat, barley, and triticale straws along with cotton stocks resulted in significant improvements in fermentable carbohydrate yields upon application of cellulosedegrading enzymes [46]. Due to limitations in the ability of ensilage to substantially modify the lignin component, this method is not normally a suitable stand-alone biological pretreat‐ ment. However, ensiling has been exploited as a means to preserve biomass for biofuels production and has been found to be a very effective, on-farm biomass pretreatment. A strain of *Lactobacillus fermentum* was highly effective in preserving sugar beet pulp cellulose and hemicellulose, and ensiling improved enzymatic saccharification by as much as 35% [47]. Ensiling has also been found to improve yields of methane in anaerobic digestion, with the added benefit of facilitating the longer-term storage of biomass (up to 1 year) while retaining the yield improvements [48, 49]. Improvements in methane yields of up to 50% have been observed with hemp and maize residue, while other crops showed little improvement [50]. However, other researchers have cautioned that the total solids loss may be overestimated for certain substrates, which may result in a misleading, apparent improvement in methane yields by ensiling [51]. Furthermore, while some studies noted above have shown that desirable carbohydrates can be preserved through ensiling, others have noted degradation of cellulose and hemicellulose of up to 10% in this relatively uncontrolled, complex process [46]. Never‐ theless, ensiling does offer the substantial benefit of biomass preservation and, importantly, it utilizes existing technology and expertise and can be performed on-farm using unmodified farm equipment. Moreover, ensiling is a relatively low-input process that is anaerobic and therefore does not require mixing and aeration. For these reasons, ensiling could easily be incorporated into an overall biorefinery process at the earliest stages of energy production.

The taxonomically broadly distributed white- and brown-rot fungi have developed a variety of means to access and degrade lignocellulose over their long evolutionary history, and their powerful metabolism has been exploited for industrial applications in recent decades. For example, lignin-degrading fungi were noted to have a brightening effect on kraft pulp derived from hardwoods, with savings in bleaching chemicals and potentially decreased environmen‐ tal impact on paper mill operations [56]. This "biobleaching" was developed further using well-known fungi, such as *Trametes versicolor* [57, 58] and *Phanerochaete chrysosporium* [59, 60]. Similar approaches were used to decolorize and detoxify pulp mill effluent and black liquor [61-63]. In addition, white-rot fungi have been exploited for their ability to decrease energy requirements in pulp manufacturing. This process, known as biopulping, softens the woody substrate and substantially decreases mill electricity requirements for mechanical pulp manufacture [64, 65]. The required scale of industrial pulp manufacture and the applicability of white-rot fungi in providing manufacturing benefits led to the development of feasible means of applying white-rot fungi to biomass on an industrially-relevant scale [66]. This twoauger system featured a wood chip decontamination step and an inoculation step, followed by incubation at ambient temperatures in large chip piles with forced aeration. A series of outdoor trials of this method each featured the treatment of ~36 tonnes of softwood chips with the biopulping fungus *Ceriporiopsis subvermispora* for two weeks. The results were energy savings of around 30% in subsequent pulping, which is slightly higher than was observed in

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More recently, wood-degrading fungi have been investigated for their ability to assist in processing biomass for biofuels production. Again, with the tremendous variety of woodrotting species and feedstocks available, there is a wide array of strategies reported for biological pretreatment. One very promising approach used rice straw as feedstock, treated with the white-rot fungus *Pleurotus ostreatus* (oyster mushroom) followed by AFEX [67]. This strategy resulted in significant reductions in the severity of the required pretreatment along with improved glucose yields upon enzyme treatment - and produced edible mushrooms as a by-product. Another study found that the incubation time required for *Pleurotus ostreatus* to improve enzymatic saccharification with rice hulls was decreased from 60 days to 18 days by pretreating the rice hulls with hydrogen peroxide prior to fungal inoculation [68]. Similarly, preconditioning of softwood using various white-rot fungi resulted in degradation and modification of the lignin, although significant cellulose loss was also observed [69]. Never‐ theless, improved glucose yields were observed by enzymatic saccharification of softwood treated with *Stereum hirsutum* compared to untreated controls, which was attributed to an increase in the pore size of the substrate [69]. Other studies have exploited the selective lignin degradation ability of the white-rot fungus *Echinodontium taxodii* to enhance enzymatic saccharification of water hyacinth in combination with dilute acid pretreatment [70], or of woody substrates without subsequent thermochemical pretreatment [71]. Biological pretrea‐ ment has also been shown to improve biogas yields from agricultural residues via anaerobic digestion [72]. A tremendous variety of other approaches to biological pretreatment has been

reported to be successful on many different lignocellulosic substrates [73, 74].

bench-scale trails [66].

*3.2.1. Species and systems investigated*

#### **3.2. Lignin-degrading fungi**

The earliest colonization of land by plants began around 450 million years ago. The evolu‐ tionary innovation that facilitated their spread and success in the non-marine environment was lignification, which provided protection from ultraviolet radiation, structural rigidity and eventually protection from coevolved pathogens and herbivores [52]. The complexity of the phenylpropanoid polymer also provided a carbon sink as land plants fixed atmospheric CO2 into degradation-resistant lignin. The vast coal reserves whose combustion have contributed to the recent spike in atmospheric CO2 concentrations trace their origins to the Carboniferous period (~350-300 million years ago), when lignin was not effectively decomposed [52]. Near the end of the Carboniferous period, saprophytic fungi of the class Agaricomycetes evolved the ability to degrade the lignin component of plant biomass, which contributed to a substantial decline in organic carbon burial to the extent that little coal formation occurs today [53, 54]. The large majority of fungal species that are capable of wood decay are known as "white-rot" fungi, which degrade all of the major wood polymers. Approximately 6% of wood decay species are "brown-rot" fungi, which evolved from white-rot fungi and selectively degrade the cellulose and hemicellulose fraction of wood, leaving a lignin-rich residue that is a major contributor to soil carbon in forest ecosystems [55].

The taxonomically broadly distributed white- and brown-rot fungi have developed a variety of means to access and degrade lignocellulose over their long evolutionary history, and their powerful metabolism has been exploited for industrial applications in recent decades. For example, lignin-degrading fungi were noted to have a brightening effect on kraft pulp derived from hardwoods, with savings in bleaching chemicals and potentially decreased environmen‐ tal impact on paper mill operations [56]. This "biobleaching" was developed further using well-known fungi, such as *Trametes versicolor* [57, 58] and *Phanerochaete chrysosporium* [59, 60]. Similar approaches were used to decolorize and detoxify pulp mill effluent and black liquor [61-63]. In addition, white-rot fungi have been exploited for their ability to decrease energy requirements in pulp manufacturing. This process, known as biopulping, softens the woody substrate and substantially decreases mill electricity requirements for mechanical pulp manufacture [64, 65]. The required scale of industrial pulp manufacture and the applicability of white-rot fungi in providing manufacturing benefits led to the development of feasible means of applying white-rot fungi to biomass on an industrially-relevant scale [66]. This twoauger system featured a wood chip decontamination step and an inoculation step, followed by incubation at ambient temperatures in large chip piles with forced aeration. A series of outdoor trials of this method each featured the treatment of ~36 tonnes of softwood chips with the biopulping fungus *Ceriporiopsis subvermispora* for two weeks. The results were energy savings of around 30% in subsequent pulping, which is slightly higher than was observed in bench-scale trails [66].

#### *3.2.1. Species and systems investigated*

upon subsequent enzymatic hydrolysis. For example, ensiling a variety of agricultural residues, including wheat, barley, and triticale straws along with cotton stocks resulted in significant improvements in fermentable carbohydrate yields upon application of cellulosedegrading enzymes [46]. Due to limitations in the ability of ensilage to substantially modify the lignin component, this method is not normally a suitable stand-alone biological pretreat‐ ment. However, ensiling has been exploited as a means to preserve biomass for biofuels production and has been found to be a very effective, on-farm biomass pretreatment. A strain of *Lactobacillus fermentum* was highly effective in preserving sugar beet pulp cellulose and hemicellulose, and ensiling improved enzymatic saccharification by as much as 35% [47]. Ensiling has also been found to improve yields of methane in anaerobic digestion, with the added benefit of facilitating the longer-term storage of biomass (up to 1 year) while retaining the yield improvements [48, 49]. Improvements in methane yields of up to 50% have been observed with hemp and maize residue, while other crops showed little improvement [50]. However, other researchers have cautioned that the total solids loss may be overestimated for certain substrates, which may result in a misleading, apparent improvement in methane yields by ensiling [51]. Furthermore, while some studies noted above have shown that desirable carbohydrates can be preserved through ensiling, others have noted degradation of cellulose and hemicellulose of up to 10% in this relatively uncontrolled, complex process [46]. Never‐ theless, ensiling does offer the substantial benefit of biomass preservation and, importantly, it utilizes existing technology and expertise and can be performed on-farm using unmodified farm equipment. Moreover, ensiling is a relatively low-input process that is anaerobic and therefore does not require mixing and aeration. For these reasons, ensiling could easily be incorporated into an overall biorefinery process at the earliest stages of energy production.

186 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

The earliest colonization of land by plants began around 450 million years ago. The evolu‐ tionary innovation that facilitated their spread and success in the non-marine environment was lignification, which provided protection from ultraviolet radiation, structural rigidity and eventually protection from coevolved pathogens and herbivores [52]. The complexity of the phenylpropanoid polymer also provided a carbon sink as land plants fixed atmospheric CO2 into degradation-resistant lignin. The vast coal reserves whose combustion have contributed to the recent spike in atmospheric CO2 concentrations trace their origins to the Carboniferous period (~350-300 million years ago), when lignin was not effectively decomposed [52]. Near the end of the Carboniferous period, saprophytic fungi of the class Agaricomycetes evolved the ability to degrade the lignin component of plant biomass, which contributed to a substantial decline in organic carbon burial to the extent that little coal formation occurs today [53, 54]. The large majority of fungal species that are capable of wood decay are known as "white-rot" fungi, which degrade all of the major wood polymers. Approximately 6% of wood decay species are "brown-rot" fungi, which evolved from white-rot fungi and selectively degrade the cellulose and hemicellulose fraction of wood, leaving a lignin-rich residue that is a major

**3.2. Lignin-degrading fungi**

contributor to soil carbon in forest ecosystems [55].

More recently, wood-degrading fungi have been investigated for their ability to assist in processing biomass for biofuels production. Again, with the tremendous variety of woodrotting species and feedstocks available, there is a wide array of strategies reported for biological pretreatment. One very promising approach used rice straw as feedstock, treated with the white-rot fungus *Pleurotus ostreatus* (oyster mushroom) followed by AFEX [67]. This strategy resulted in significant reductions in the severity of the required pretreatment along with improved glucose yields upon enzyme treatment - and produced edible mushrooms as a by-product. Another study found that the incubation time required for *Pleurotus ostreatus* to improve enzymatic saccharification with rice hulls was decreased from 60 days to 18 days by pretreating the rice hulls with hydrogen peroxide prior to fungal inoculation [68]. Similarly, preconditioning of softwood using various white-rot fungi resulted in degradation and modification of the lignin, although significant cellulose loss was also observed [69]. Never‐ theless, improved glucose yields were observed by enzymatic saccharification of softwood treated with *Stereum hirsutum* compared to untreated controls, which was attributed to an increase in the pore size of the substrate [69]. Other studies have exploited the selective lignin degradation ability of the white-rot fungus *Echinodontium taxodii* to enhance enzymatic saccharification of water hyacinth in combination with dilute acid pretreatment [70], or of woody substrates without subsequent thermochemical pretreatment [71]. Biological pretrea‐ ment has also been shown to improve biogas yields from agricultural residues via anaerobic digestion [72]. A tremendous variety of other approaches to biological pretreatment has been reported to be successful on many different lignocellulosic substrates [73, 74].

Exploitation of fungal metabolic activity for industrial purposes can take a variety of forms. For white- and brown-rot fungi, the mode of cultivation can have an effect on the results obtained, and the choice of cultivation conditions depends on the desired outcomes. In general, fungi can be cultivated under solid-state conditions (solid-state fermentation, or SSF), or using submerged fermentation (SmF). SSF involves culturing the fungus on the substrate under relatively low moisture conditions (~60-70%), while SmF uses liquid cultures of the fungus coincubated with the normally insoluble substrate. Early pulp biobleaching experiments used SmF of white-rot fungi such as *Trametes versicolor*, which featured the advantage of shorter incubation times than SSF [75], but suffered the drawback that very large fermentation vessels would be required for industrial-scale treatments. Many white-rot fungi grow well and perform the desired metabolism under solid-state conditions. For example, species of the genera *Trametes*, *Phanerochaete*, and *Pycnoporus* preferentially removed color and chemical oxygen demand from olive mill wastewaters and pulp mill black liquors under SSF cultivation conditions [61, 76, 77]. SSF using white-rot fungi has also been used to modify the lignin in agricultural residues, such as wheat straw, for biofuels processes [78].

to the cleavage of covalent bonds in both lignin and cellulose [86]. Hydrogen peroxide is commonly produced through the action of fungal redox enzymes, such as glyoxal oxidase,

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Another redox enzyme produced by a wide variety of wood-degrading fungi (as well as plants) is laccase, a multicopper oxidase. Laccase acts by removing a single electron from its substrate, which is typically a low-molecular weight compound (mediator) that can diffuse into the densely packed lignocellulose molecule and initiate free radical-mediated reactions leading to the depolymerisation of the substrate. The white-rot fungus *Pycnoporus cinnabarinus* uses laccase in combination with a secondary metabolite, 3-hydroxyanthranilic acid, to effect lignin depolymerisation [87, 88]. Laccase has been used in combination with a wide variety of chemical mediators to effect lignin degradation in wood pulp, with excellent results [89-91].

The presence of manganese in woody substrates is exploited by lignin-degrading fungi through the production of the enzyme manganese peroxidase (MnP). The importance of MnP in lignin degradation is illustrated by its presence in the genomes of white-rot fungi and absence in the non-lignin-degrading brown-rot fungi [92], as well as by the inability of MnPdeficient mutants of *Trametes versicolor* to delignify hardwood-derived kraft pulp [93]. MnP is a heme-containing enzyme with a catalytic cycle that is typical of heme peroxidases, but is uniquely selective for Mn2+ as its preferred electron donor [94]. The oxidation of Mn2+, which is accompanied by the reduction of hydrogen peroxide to water, results in the formation of Mn3+. The latter ion is a powerful, diffusible oxidant that is chelated by organic acids such as oxalate produced as a secondary metabolite of the fungus [94]. This highly reactive ion interacts with a wide variety of substrates, including phenols, non-phenolic aromatics, carboxylic acids, and unsaturated fatty acids, producing further ROS and resulting in lignocellulose bond cleavage through oxidative mechanisms [94]. Like laccase, MnP has found application as a delignifying enzyme for pine wood [95] as well as kraft pulp [96, 97]. Peroxidases related to MnP, including lignin peroxidase (LiP) and versatile peroxidase (VP) are also produced by a variety of wood-degrading fungi and play an important role in lignin degradation [98].

Cellobiose dehydrogenase (CDH) is a unique enzyme containing both a heme and a flavin cofactor [99]. CDH is produced by a wide range of fungal species, including both lignindegrading organisms and fungi that are incapable of degrading lignin [100, 101]. CDH catalyzes the two-electron oxidation of a narrow range of β(1,4)-linked sugar molecules, principally cellobiose, and transfers these electrons to a very wide array of substrates, includ‐ ing metals such as ferric, cupric, or manganic ions, iron-containing proteins (e.g. cytochrome c), quinones, and other large and small molecules [102, 103]. The diversity of reduced substrates has led to much speculation regarding the role of CDH in lignocellulose degradation; roles have been postulated in the degradation of both cellulose [104] and lignin [105]. The reduction of cupric and ferric ions by CDH and the production of hydrogen peroxide by lignin-degrading fungi suggests that CDH may be involved in sustaining hydroxyl radical-based Fenton's chemistry, with many possible secondary reactions leading to lignocellulose bond cleavage [106]. The role of CDH in lignin-degrading basidiomycetes was addressed by generating mutants of *Trametes versicolor* that did not produce the enzyme, suggesting that CDH plays a role in cellulose degradation, with a more minor role in lignin degradation [107, 108]. Similarly,

pyranose-2 oxidase, and aryl-alcohol oxidase [15].

Despite relatively long incubation times, SSF offers an inexpensive and effective means of fungal cultivation that can also be used for the production of potentially valuable fungal enzymes [79-81]. Fungal enzymes produced by SSF have been used to enhance methane production by anaerobic digestion [82]. Alternatively, fungal lignocellulose modifying enzymes produced by SSF have been used to improve the ruminal digestibility of agricultural residues [83]. However, for SSF to work efficiently with white- or brown-rot fungi requires a decontamination step to allow the fungi to establish on the residues. In lab-scale studies, this is usually accomplished by autoclaving the residues prior to inoculation [84, 85]. While this is necessary at the research scale to establish with certainty the effects of the inoculated fungus on the substrate, autoclaving is in itself a form of pretreatment and is not feasible on an industrial scale. This is a limitation of SSF for application on the large scale that would be required for biological pretreatment of agricultural residues for biofuels production.

#### *3.2.2. Enzymatic mechanisms of fungal lignocellulose degradation*

The mechanisms that saprophytic wood degrading fungi have evolved to access their difficult growth substrate can be divided into two categories: oxidative mechanisms and hydrolytic mechanisms. These two groups of enzymes and chemicals act together in various combinations to effect the degradation of lignocellulose by different organisms.

#### *3.2.2.1. Oxidative mechanisms*

Due to the highly compact, complex nature of lignocellulose, enzymes cannot effectively penetrate this molecule to interact with their substrates. To overcome this limitation, wooddegrading fungi use chemical means to access the recalcitrant substrate. The production of reactive oxygen species (ROS) is a recurring theme in fungal lignocellulose degradation [86]. Specifically, since wood contains sufficient redox-active iron, fungal production of hydrogen peroxide will produce hydroxyl radicals via the Fenton reaction [86]. Hydroxyl radicals (•OH) are extremely powerful oxidizing agents that can catalyze highly non-specific reactions leading to the cleavage of covalent bonds in both lignin and cellulose [86]. Hydrogen peroxide is commonly produced through the action of fungal redox enzymes, such as glyoxal oxidase, pyranose-2 oxidase, and aryl-alcohol oxidase [15].

Exploitation of fungal metabolic activity for industrial purposes can take a variety of forms. For white- and brown-rot fungi, the mode of cultivation can have an effect on the results obtained, and the choice of cultivation conditions depends on the desired outcomes. In general, fungi can be cultivated under solid-state conditions (solid-state fermentation, or SSF), or using submerged fermentation (SmF). SSF involves culturing the fungus on the substrate under relatively low moisture conditions (~60-70%), while SmF uses liquid cultures of the fungus coincubated with the normally insoluble substrate. Early pulp biobleaching experiments used SmF of white-rot fungi such as *Trametes versicolor*, which featured the advantage of shorter incubation times than SSF [75], but suffered the drawback that very large fermentation vessels would be required for industrial-scale treatments. Many white-rot fungi grow well and perform the desired metabolism under solid-state conditions. For example, species of the genera *Trametes*, *Phanerochaete*, and *Pycnoporus* preferentially removed color and chemical oxygen demand from olive mill wastewaters and pulp mill black liquors under SSF cultivation conditions [61, 76, 77]. SSF using white-rot fungi has also been used to modify the lignin in

188 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

Despite relatively long incubation times, SSF offers an inexpensive and effective means of fungal cultivation that can also be used for the production of potentially valuable fungal enzymes [79-81]. Fungal enzymes produced by SSF have been used to enhance methane production by anaerobic digestion [82]. Alternatively, fungal lignocellulose modifying enzymes produced by SSF have been used to improve the ruminal digestibility of agricultural residues [83]. However, for SSF to work efficiently with white- or brown-rot fungi requires a decontamination step to allow the fungi to establish on the residues. In lab-scale studies, this is usually accomplished by autoclaving the residues prior to inoculation [84, 85]. While this is necessary at the research scale to establish with certainty the effects of the inoculated fungus on the substrate, autoclaving is in itself a form of pretreatment and is not feasible on an industrial scale. This is a limitation of SSF for application on the large scale that would be

required for biological pretreatment of agricultural residues for biofuels production.

The mechanisms that saprophytic wood degrading fungi have evolved to access their difficult growth substrate can be divided into two categories: oxidative mechanisms and hydrolytic mechanisms. These two groups of enzymes and chemicals act together in various combinations

Due to the highly compact, complex nature of lignocellulose, enzymes cannot effectively penetrate this molecule to interact with their substrates. To overcome this limitation, wooddegrading fungi use chemical means to access the recalcitrant substrate. The production of reactive oxygen species (ROS) is a recurring theme in fungal lignocellulose degradation [86]. Specifically, since wood contains sufficient redox-active iron, fungal production of hydrogen peroxide will produce hydroxyl radicals via the Fenton reaction [86]. Hydroxyl radicals (•OH) are extremely powerful oxidizing agents that can catalyze highly non-specific reactions leading

*3.2.2. Enzymatic mechanisms of fungal lignocellulose degradation*

to effect the degradation of lignocellulose by different organisms.

*3.2.2.1. Oxidative mechanisms*

agricultural residues, such as wheat straw, for biofuels processes [78].

Another redox enzyme produced by a wide variety of wood-degrading fungi (as well as plants) is laccase, a multicopper oxidase. Laccase acts by removing a single electron from its substrate, which is typically a low-molecular weight compound (mediator) that can diffuse into the densely packed lignocellulose molecule and initiate free radical-mediated reactions leading to the depolymerisation of the substrate. The white-rot fungus *Pycnoporus cinnabarinus* uses laccase in combination with a secondary metabolite, 3-hydroxyanthranilic acid, to effect lignin depolymerisation [87, 88]. Laccase has been used in combination with a wide variety of chemical mediators to effect lignin degradation in wood pulp, with excellent results [89-91].

The presence of manganese in woody substrates is exploited by lignin-degrading fungi through the production of the enzyme manganese peroxidase (MnP). The importance of MnP in lignin degradation is illustrated by its presence in the genomes of white-rot fungi and absence in the non-lignin-degrading brown-rot fungi [92], as well as by the inability of MnPdeficient mutants of *Trametes versicolor* to delignify hardwood-derived kraft pulp [93]. MnP is a heme-containing enzyme with a catalytic cycle that is typical of heme peroxidases, but is uniquely selective for Mn2+ as its preferred electron donor [94]. The oxidation of Mn2+, which is accompanied by the reduction of hydrogen peroxide to water, results in the formation of Mn3+. The latter ion is a powerful, diffusible oxidant that is chelated by organic acids such as oxalate produced as a secondary metabolite of the fungus [94]. This highly reactive ion interacts with a wide variety of substrates, including phenols, non-phenolic aromatics, carboxylic acids, and unsaturated fatty acids, producing further ROS and resulting in lignocellulose bond cleavage through oxidative mechanisms [94]. Like laccase, MnP has found application as a delignifying enzyme for pine wood [95] as well as kraft pulp [96, 97]. Peroxidases related to MnP, including lignin peroxidase (LiP) and versatile peroxidase (VP) are also produced by a variety of wood-degrading fungi and play an important role in lignin degradation [98].

Cellobiose dehydrogenase (CDH) is a unique enzyme containing both a heme and a flavin cofactor [99]. CDH is produced by a wide range of fungal species, including both lignindegrading organisms and fungi that are incapable of degrading lignin [100, 101]. CDH catalyzes the two-electron oxidation of a narrow range of β(1,4)-linked sugar molecules, principally cellobiose, and transfers these electrons to a very wide array of substrates, includ‐ ing metals such as ferric, cupric, or manganic ions, iron-containing proteins (e.g. cytochrome c), quinones, and other large and small molecules [102, 103]. The diversity of reduced substrates has led to much speculation regarding the role of CDH in lignocellulose degradation; roles have been postulated in the degradation of both cellulose [104] and lignin [105]. The reduction of cupric and ferric ions by CDH and the production of hydrogen peroxide by lignin-degrading fungi suggests that CDH may be involved in sustaining hydroxyl radical-based Fenton's chemistry, with many possible secondary reactions leading to lignocellulose bond cleavage [106]. The role of CDH in lignin-degrading basidiomycetes was addressed by generating mutants of *Trametes versicolor* that did not produce the enzyme, suggesting that CDH plays a role in cellulose degradation, with a more minor role in lignin degradation [107, 108]. Similarly, a recent study with the non- lignin-degrading ascomycete *Neurospora crassa* revealed that deletion of the gene encoding CDH resulted in vastly decreased cellulase activity, and that the oxidation of cellobiose was coupled to the reductive activation of copper-containing polysac‐ charide monooxygenases [109]. These studies strongly suggest a role for CDH in supporting cellulose catabolism by fungi, with the latter study in particular providing a highly plausible mechanism for the *in vivo* function of CDH.

ent peroxidases, which function to degrade the cellulosic/hemicellulosic and lignin compo‐ nents of the cell wall, respectively. This research provided the groundwork for more comprehensive analyses of the genome [118], transcriptome and secretome of *Phanerochaete chrysosporium* [119-122]. These studies highlighted hundreds of wood-degrading genes that were upregulated when *P. chrysosporium* was grown in cellulose-rich medium, including

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Complementary to this research on white-rot fungi was a genome/transcriptome/proteome study on the brown-rot fungus *Postia placenta* [123]. Despite an abundance of similarities between *P. chrysosporium* and *P. placenta*, there were notably fewer glycoside hydrolases expressed by *P. placenta*, such as extracellular cellulases (e.g. endo-(1,4)-β-glucanases), highlighting the mechanistic differences between white- and brown-rot fungi. This work was followed by transcriptomic and proteomic studies investigating the biomass-degrading activity of *Phanerochaete carnosa* [124, 125]. Despite the overall similarity of the transcriptome composition among *P. carnosa* and *P. chrysosporium*, the most abundant transcripts in *P. carnosa* grown on wood substrates (hardwood and softwood) were peroxidases and oxidases involved in lignin degradation [124], whereas *P. chrysosporium* grown only on hardwood revealed only a few highly expressed lignin-degrading enzymes [121]. The differing expression of lignocellulosic enzymes in response to different woody substrates was also explored by examining gene expression patterns in both *P. placenta* and *P. chrysosporium* on hardwood and softwood species [126]. The results of this study strongly suggest that both species of fungi alter their gene expression patterns to degrade wood with different structural characteristics.

In addition to helping uncover the fundamental biochemical machinery involved in biomass degradation, these genomic, transcriptomic and proteomic studies of biomass-degrading fungi have also identified hundreds of target enzymes that could be utilized industrially for bioenergy production, with unique enzyme cocktails suited for specific substrates (e.g. hardwoods vs. softwoods). Several commercial enzymes are commonly used to degrade lignocellulosic residue into fermentable sugars (e.g. Celluclast and Novozyme 188). These 'omic' studies have identified hundreds of fungal glycoside hydrolases that may supplement or completely replace these industry standards. Pre-treatment strategies may also take advantage of the numerous lignin-modifying enzymes identified from biomass-degrading fungi, including lignin and manganese-dependent peroxidases, which have the potential to reduce the severity of thermomechanical and thermochemical pretreatment processes.

The explosion of 'omic' data for a wide variety of lignocellulose-degrading fungi [117, 123-125] along with the development of sophisticated tools for annotating fungal genomes [127] will continue to add to our understanding of the mechanisms of fungal decay of lignocellulose. Furthermore, increased knowledge of fungal decay mechanisms can aid in the development of strains with improved characteristics. For example, a major limitation to the application of fungal strains for biological pretreatment is the degradation of the desired carbohydrates (cellulose and hemicellulose) for fungal metabolism [73, 74]. Creating or selecting strains that lack the ability to degrade these carbohydrates while retaining the ability to degrade and

almost 200 genes encoding enzymes of unknown function [122].

*3.2.2.4. Exploiting fungal mutants for biological pretreatment*

### *3.2.2.2. Hydrolytic mechanisms*

Complementing the degradative power of the redox chemistry catalyzed by the enzymes produced by lignocellulose-degrading fungi is a suite of enzymes that act by adding a water molecule to glycosidic bonds, resulting in bond cleavage and depolymerization. In contrast to the redox enzymes, these hydrolytic enzymes recognize and act on specific glycosidic linkages, releasing sugar molecules that can be utilized as an energy source to support fungal metabo‐ lism. Cellulose degradation is catalyzed by the synergistic action of three classes of hydrolytic cellulase enzymes: endo-(1,4)-β-glucanase (endocellulase), cellobiohydrolase (exocellulase), and β-glucosidase [110]. Endocellulases catalyze the cleavage of cellulose chains internally at amorphous regions, while exocellulases remove cellobiose units from the ends of cellulose chains. β-glucosidases are extracellular, cell wall-associated or intracellular enzymes that cleave cellobiose into glucose, which also supports exocellulase activity by relieving endproduct inhibition [110]. The redundancy in cellulase genes in fungi is at least partially explained by the fact that different exocellulase enzymes preferentially attack the reducing or non-reducing end of a cellulose chain. This has the effect of exposing new sites for exocellulases of the opposite specificity and also generates new amorphous regions to be acted upon by endocellulases [110, 111]. Hemicellulose degradation is effected by the activity of a wide range of hydrolytic enzymes, including endo-xylanases; endo-α-L-arabinase; endo-mannanase, βgalactosidase, and an array of corresponding β-glucosidases [112]. In addition, covalent bonds within lignocellulose are hydrolyzed by cinnamoyl or feruloyl esterases, which cleave the ester bond between polymerized lignin subunits and the hemicellulose within the composite molecule [113, 114]. Complementary cellulase activity by these various "accessory enzymes" is shown on complex substrates by the improvement in enzymatic saccharification observed when enzymes such as xylanase, pectinase, and feruloyl esterase are added to cellulase cocktails [115, 116].

#### *3.2.2.3. Fungal enzyme discovery, production, and application*

Tremendous progress has been made in the last decade concerning the genetic mechanisms underlying plant biomass degradation and modification by microbes, specifically ascomyce‐ tous and basidiomycetous fungi. Key to these advancements was the complete genome sequencing of several biomass-degrading fungi, including *Phanerochaete chrysosporium*, *Phanerochaete carnosa*, *Postia placenta and Trametes versicolor*. The first basidiomycete genome to be sequenced and analyzed, *Phanerochaete chrysosporium*, revealed a tremendous diversity of genes encoding enzymes involved in wood degradation [117]. Among these genes were approximately 240 carbohydrate-active enzymes and several lignin and manganese-depend‐ ent peroxidases, which function to degrade the cellulosic/hemicellulosic and lignin compo‐ nents of the cell wall, respectively. This research provided the groundwork for more comprehensive analyses of the genome [118], transcriptome and secretome of *Phanerochaete chrysosporium* [119-122]. These studies highlighted hundreds of wood-degrading genes that were upregulated when *P. chrysosporium* was grown in cellulose-rich medium, including almost 200 genes encoding enzymes of unknown function [122].

a recent study with the non- lignin-degrading ascomycete *Neurospora crassa* revealed that deletion of the gene encoding CDH resulted in vastly decreased cellulase activity, and that the oxidation of cellobiose was coupled to the reductive activation of copper-containing polysac‐ charide monooxygenases [109]. These studies strongly suggest a role for CDH in supporting cellulose catabolism by fungi, with the latter study in particular providing a highly plausible

190 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

Complementing the degradative power of the redox chemistry catalyzed by the enzymes produced by lignocellulose-degrading fungi is a suite of enzymes that act by adding a water molecule to glycosidic bonds, resulting in bond cleavage and depolymerization. In contrast to the redox enzymes, these hydrolytic enzymes recognize and act on specific glycosidic linkages, releasing sugar molecules that can be utilized as an energy source to support fungal metabo‐ lism. Cellulose degradation is catalyzed by the synergistic action of three classes of hydrolytic cellulase enzymes: endo-(1,4)-β-glucanase (endocellulase), cellobiohydrolase (exocellulase), and β-glucosidase [110]. Endocellulases catalyze the cleavage of cellulose chains internally at amorphous regions, while exocellulases remove cellobiose units from the ends of cellulose chains. β-glucosidases are extracellular, cell wall-associated or intracellular enzymes that cleave cellobiose into glucose, which also supports exocellulase activity by relieving endproduct inhibition [110]. The redundancy in cellulase genes in fungi is at least partially explained by the fact that different exocellulase enzymes preferentially attack the reducing or non-reducing end of a cellulose chain. This has the effect of exposing new sites for exocellulases of the opposite specificity and also generates new amorphous regions to be acted upon by endocellulases [110, 111]. Hemicellulose degradation is effected by the activity of a wide range of hydrolytic enzymes, including endo-xylanases; endo-α-L-arabinase; endo-mannanase, βgalactosidase, and an array of corresponding β-glucosidases [112]. In addition, covalent bonds within lignocellulose are hydrolyzed by cinnamoyl or feruloyl esterases, which cleave the ester bond between polymerized lignin subunits and the hemicellulose within the composite molecule [113, 114]. Complementary cellulase activity by these various "accessory enzymes" is shown on complex substrates by the improvement in enzymatic saccharification observed when enzymes such as xylanase, pectinase, and feruloyl esterase are added to cellulase

Tremendous progress has been made in the last decade concerning the genetic mechanisms underlying plant biomass degradation and modification by microbes, specifically ascomyce‐ tous and basidiomycetous fungi. Key to these advancements was the complete genome sequencing of several biomass-degrading fungi, including *Phanerochaete chrysosporium*, *Phanerochaete carnosa*, *Postia placenta and Trametes versicolor*. The first basidiomycete genome to be sequenced and analyzed, *Phanerochaete chrysosporium*, revealed a tremendous diversity of genes encoding enzymes involved in wood degradation [117]. Among these genes were approximately 240 carbohydrate-active enzymes and several lignin and manganese-depend‐

mechanism for the *in vivo* function of CDH.

*3.2.2.2. Hydrolytic mechanisms*

cocktails [115, 116].

*3.2.2.3. Fungal enzyme discovery, production, and application*

Complementary to this research on white-rot fungi was a genome/transcriptome/proteome study on the brown-rot fungus *Postia placenta* [123]. Despite an abundance of similarities between *P. chrysosporium* and *P. placenta*, there were notably fewer glycoside hydrolases expressed by *P. placenta*, such as extracellular cellulases (e.g. endo-(1,4)-β-glucanases), highlighting the mechanistic differences between white- and brown-rot fungi. This work was followed by transcriptomic and proteomic studies investigating the biomass-degrading activity of *Phanerochaete carnosa* [124, 125]. Despite the overall similarity of the transcriptome composition among *P. carnosa* and *P. chrysosporium*, the most abundant transcripts in *P. carnosa* grown on wood substrates (hardwood and softwood) were peroxidases and oxidases involved in lignin degradation [124], whereas *P. chrysosporium* grown only on hardwood revealed only a few highly expressed lignin-degrading enzymes [121]. The differing expression of lignocellulosic enzymes in response to different woody substrates was also explored by examining gene expression patterns in both *P. placenta* and *P. chrysosporium* on hardwood and softwood species [126]. The results of this study strongly suggest that both species of fungi alter their gene expression patterns to degrade wood with different structural characteristics.

In addition to helping uncover the fundamental biochemical machinery involved in biomass degradation, these genomic, transcriptomic and proteomic studies of biomass-degrading fungi have also identified hundreds of target enzymes that could be utilized industrially for bioenergy production, with unique enzyme cocktails suited for specific substrates (e.g. hardwoods vs. softwoods). Several commercial enzymes are commonly used to degrade lignocellulosic residue into fermentable sugars (e.g. Celluclast and Novozyme 188). These 'omic' studies have identified hundreds of fungal glycoside hydrolases that may supplement or completely replace these industry standards. Pre-treatment strategies may also take advantage of the numerous lignin-modifying enzymes identified from biomass-degrading fungi, including lignin and manganese-dependent peroxidases, which have the potential to reduce the severity of thermomechanical and thermochemical pretreatment processes.

#### *3.2.2.4. Exploiting fungal mutants for biological pretreatment*

The explosion of 'omic' data for a wide variety of lignocellulose-degrading fungi [117, 123-125] along with the development of sophisticated tools for annotating fungal genomes [127] will continue to add to our understanding of the mechanisms of fungal decay of lignocellulose. Furthermore, increased knowledge of fungal decay mechanisms can aid in the development of strains with improved characteristics. For example, a major limitation to the application of fungal strains for biological pretreatment is the degradation of the desired carbohydrates (cellulose and hemicellulose) for fungal metabolism [73, 74]. Creating or selecting strains that lack the ability to degrade these carbohydrates while retaining the ability to degrade and modify lignin would provide a means to avoid this drawback of fungal pretreatment. Early studies with strains that were deficient in the production of cellulase met with only moderate success, with substantial degradation of cellulose observed [128, 129]. This is probably attributable to the high degree of redundancy in fungal cellulases, with large numbers of genes contributing to the hydrolytic degradation of cellulose and hemicellulose in various species [15]. More recently, we have applied a strain of *Trametes versicolor* that is unable to produce cellobiose dehydrogenase (CDH) to the pretreatment of canola residue, and found that the strain was proficient in lignin degradation but was unable to catabolize the cellulose [107]. Xylose within the substrate appeared to have been utilized to support the greatly decreased fungal growth compared to the wild-type strain. Furthermore, we found that the application of a fungal cell wall-degrading enzyme cocktail (glucanex; a concentrated supernatant of a SmF culture of *Trichoderma harzianum*) to the fungus-treated biomass resulted in the release of fungal cell wall-associated glucose [107]. Biological pretreatment with *T. versicolor* therefore had the overall effect of converting some of the xylose within the substrate to glucose, which is more easily fermented by ethanologenic yeasts.

little carbohydrate degradation, low costs for nutrients, a reasonable storage time, ability to compete with endogenous microbiota, decreased thermomechanical pretreatment severity, improved yields of glucose upon enzymatic saccharification, and a lack of production of compounds inhibitory to fermenting organisms. These criteria underscore the major limita‐ tions of biological pretreatment, the most important of which are the propensity of the organisms to degrade the carbohydrate component, their inability to establish growth on unsterilized biomass, and the relatively long incubation times that are required. These limitations are related to the ecological niche that these saprophytic fungi fill in nature. That is, they have evolved to access and utilize those plant carbohydrates that are difficult for other microorganisms to access. For this reason, these fungi typically appear at the end of an ecological succession of organisms that degrade decaying wood and are often ill equipped to compete with the faster-growing molds and bacteria that access the more easily degraded plant carbohydrates [137]. While it may be possible using reverse genetics tools and/or strain selection to limit carbohydrate degradation by pretreatment fungi [107], it is likely that such strains will be even less able to compete with endogenous microorganisms; therefore, estab‐ lishment on recently harvested biomass will remain a challenge. Some sort of treatment of the biomass to suppress the growth of endogenous molds prior to inoculation with the pretreat‐

Pretreatment of Lignocellulosic Biomass Using Microorganisms: Approaches, Advantages, and Limitations

http://dx.doi.org/10.5772/55088

193

The unavoidable expense of the pre-inoculation treatment can be compensated by taking advantage of a potential benefit of biological pretreatment that has received very little attention: wood-degradingfungimaymodifythelignincomponentsufficientlytoprovidepositivebenefits for particle compression of agricultural biomass during densification. Densification (briquet‐ ting or pelleting) of biomass aims to increase the bulk density of agricultural residues far be‐ yondwhatisachievablebybaling,anditisanessentialstepforprovidingbiomasswithsufficient caloric density for efficient transportation [138]. The production of biomass pellets provides a substrate that is suitable for conversion into biofuels through microbial processes or gasifica‐ tion[139,140],orcanbecombusteddirectlytoproduceenergy[141].Awidevarietyofagricultur‐ alfeedstocksissuitableforpelleting[142];however,untreatedbiomassisverydifficulttodensify and, without pretreatment, produces weak, powdery pellets that are expensive to produce and cannotwithstandthephysicalrigorsoftransportation.Ligninactsasanaturalbinderthatprovides strength and durability to biomass pellets, and pretreatment of the biomass is required in order to release lignin fragments during compaction and produce pellets with the desired characteris‐ tics [139, 143]. A number of options are available to prepare biomass for pelleting, with two very promisingmethodsbeingmicrowaveheatingandradiofrequencyheating.Bothofthesemethods provide a number of advantages over conventional heating, particularly regarding treatment times [144-146]. We have found that a very brief microwave treatment of a variety of agricultur‐ al feedstocks suppresses the growth of endogenous molds and bacteria sufficiently for inoculat‐ ed white-rot fungi to establish growth on these substrates. Moreover, canola residue treated with *Trametes versicolor* produces pellets with excellent compaction characteristics and durability (Canam, Town, and Dumonceaux, unpublished). Such pellets would retain the thermochemi‐ calpretreatmentbenefitsaffordedbythefungalpretreatmentintermsofenzymaticsaccharifica‐ tion [107], but would offer vastly increased transportation efficiency in a full-scale biorefinery

ment fungi will likely be necessary.

scenario.

Studies such as these also provide biological data regarding the role of the genes that are downregulated in the mutant strains. This reverse genetics approach is a powerful method for investigating gene function, and in the current genomic era reverse genetics tools can often be applied in the known context of the entire genome of the fungus. Gene silencing by RNA interference (RNAi) is a common method for down-regulating genes in a variety of model systems [130, 131], and the recent demonstration of RNAi mechanisms in the model white-rot fungus *Phanerochaete chrysosporium* [132] suggests that RNAi could be used for targeted downregulation of specific genes in species that are useful for biological pretreatment. The availa‐ bility of convenient gene silencing transformation vectors for ascomycetes such as pSilent [133] and pTroya [134] as well as pHg/pSILBAγ for basidiomycetes [135] will greatly facilitate the investigation of gene function and may also result in the development of modified strains featuring enhanced properties for biological pretreatment of lignocellulosic substrates for biofuels production.
