Recent Advances in Bioconversion of Lignocellulose to Biofuels and Value Added Chemicals within the Biorefinery Concept

Recent Advances in Bioconversion of Lignocellulose to Biofuels and Value Added Chemicals within the Biorefinery Concept covers the latest developments on biorefineries, along with their potential use for the transformation of residues into a broad range of more valuable products. Within this context, the book discusses the enzymatic conversion process of lignocellulosic biomass to generate fuels and other products in a unified approach. It focuses on new approaches to increase enzymatic production by microorganisms, the action of microbial inhibitors, and strategies for their removal.

Furthermore, it outlines the benefits of this integrated approach for generating value-added products and the benefits to social and economic aspects, circular bio economy, HUBs and perspectives.

• Covers the mechanisms of enzymatic conversion of biomass into value-added products
• Discusses bioproducts derived from lignocellulose and their applications
• Includes discussions on design, development and the technologies needed for the sustainable manufacture of materials and chemicals
• Offers a techno-economic evaluation of biorefineries for integrated sustainability assessments
• Discusses the socioeconomic and cultural-economic perspectives of the lignocellulosic biorefinery
• Presents a virtual biorefinery as an integrated approach to evaluate the lignocellulose production chain

• Language: English
• Publisher: Elsevier Science
• Release date: May 7, 2020
• ISBN: 9780128182246

Book preview

EE0008256.

1

Introduction

Leonora Rios de Souza Moreira¹, Cristiane Sanchez Farinas², Eduardo de Aquino Ximenes³ and Edivaldo Ximenes Ferreira Filho¹,    ¹Laboratory of Enzymology, Cellular Biology Department, University of Brasília, Brasília, Brazil,    ²Brazilian Agricultural Research Corporation – Embrapa Instrumentation, São Carlos, Brazil,    ³Laboratory of Renewable Resources Engineering (LORRE), Purdue University, West Lafayette, IN, United States

Abstract

In an actual scenario of great concern about sustainability, the biorefinery concept is a viable path to accomplish the effective bioconversion of lignocellulosic biomass into higher value-added products, such as chemicals, animal feed, novel materials, biofuels, and electricity. The enzymatic hydrolysis of feedstocks addresses key aspects needed for that, such as the reduction on pollution and production of value-added products from lignocellulosic feedstock. This book presents the latest developments on biorefineries, and their potential use for transformation of residues that otherwise would be wasted into a broad range of more valuable products.

Keywords

Biorefinery; lignocellulose; bioproducts; lignocellulose-degrading enzymes; agroindustrial residue

Chapter Outline

Outline

Acknowledgments 4

References 4

The world dependence on fossil fuels is becoming unsustainable (Mohan et al., 2016), bringing the critical need for potential alternative to the petroleum-based refinery (Fazard et al., 2017). Thus renewable sources of energy have been massively studied in recent years to address that. The growing global commitment to replacing fossil fuels with renewable raw materials, aiming at reducing greenhouse gas emissions, has led to a huge interest in technologies for converting lignocellulosic biomass into bioproducts and energy. Moreover, the pollution generated by the agricultural and urban residues is a major environmental concern. The concept of biorefinery not only meets the needs of exploitation and valorization of biomass from agroindustrial and urban waste, but it also integrates biomass conversion processes and equipment to produce fuels, power, heat, and value-added chemicals and biomass materials (Fig. 1–1). Through a concept of biorefinery, hubs can be created to become centers for the supply of biomass and valuable associated bioproducts, enhancing ecological awareness, while also addressing critical social and economic aspects, and therefore effectively contributing to the need for a more sustainable world (Silva et al., 2017).

Figure 1–1 The potential of biorefinery as a tool to produce value-added bioproducts.

Furthermore, the compilation of data about this subject is an important tool for research and biotechnological center to develop further work in related areas. In a current scenario of great concern about sustainability, the biorefinery concept is a viable path to accomplish the effective bioconversion of lignocellulosic biomass into higher value-added products, such as chemicals, animal feed, novel materials, biofuels, and electricity. Biofuels are produced from biological route using a renewable substrate and have become a potential option to reduce environmental pollution and dependence on fossil fuels.

Moreover, biorefineries have a positive impact on the world’s social and economic aspects considering that they present new job opportunities. A study carried out in Thailand (Gheewala et al., 2011) showed that the maximization of biomass utilization at the level of the biorefinery complex resulted in a number of benefits, including greenhouse gases emissions reduction, enhanced living conditions for sugarcane farmers, and employees of the biorefinery and economic benefits, particularly with regard to profit and income generation. In this sense, biorefineries are a strategic path to achieving sustainable growth. The development of this industry may combine sustainable economic growth, new jobs generation, and increasing the competitiveness of developing countries that can become hubs for feedstocks.

The lignocellulosic biorefinery concept is a viable alternative for the effective conversion of biomass feedstock. Among the lignocellulose conversion technologies, the enzymatic process is a promising candidate because it represents an extremely efficient, specific, environment friendly, and sustainable biomass management approach. In such a concept the use of enzymes for lignocellulose hydrolysis is an essential step. However, although major progress has been made in recent years (Silva et al., 2017), there are still challenges to overcome involving the enzymatic conversion of renewable lignocellulosic biomass (predominantly comprised of cellulose, hemicellulose, and lignin) to biofuel and value-added products. They include improving hydrolysis with efficient enzymes, reduced production cost, and novel technology for substrate handling.

Moreover, these enzymes can also be part of a new paradigm for a sustainable bioeconomy. Optimal design of the process of lignocellulose conversion is achieved by choosing an effective technology for each step based on a specific objective. Within this same line of reasoning, several factors affect the enzymatic step of lignocellulose conversion, including enzyme ratios, synergistic cooperation between enzymes, substrate loadings, enzyme loading, enzyme, and microbial inhibitors, nonspecific adsorption of enzymes on lignin and use of surfactants. Also, the pretreatment step of lignocellulose is a key process for enhancing enzymes accessibility and their further hydrolytic action during the saccharification step (Silva et al., 2017).

Within the context of waste biorefinery, enzymes can also be part of a holistic approach of lignocellulosic biomass management, integrating remediation and resource recovery through closed-loop bioprocesses cascade, enabling the shift toward circular and low-carbon bioeconomy. In this circular economy model, lignocellulosic materials are recovered, recycled, and used repeatedly (Fig. 1–2). This holistic approach must deal with some parameters, such as feedstock storage and handling; pretreatment, saccharification, and fermentation to ethanol and other value-added products; water and solid recovery; as well as wastewater treatment. Mohan et al. (2016) proposed waste biorefinery models toward sustainable circular bioeconomy based on closed-loop approach, wherein waste is valorized through a cascade of various biotechnological processes addressing the circular economy. In this case, we must consider enzymes as also part of holistic biorefinery approach with hybrid-integrated strategies for multipurpose applications. Also, the strategy of biorefineries as hubs strategically located in regions of intense agricultural activity and integrated with the largest agricultural producers would have an economic impact, minimizing the cost of acquiring, transporting, and storing lignocellulosic waste (Silva et al., 2017; Kim and Dale, 2015).

Figure 1–2 An overview of the link between biorefinery and circular bioeconomy.

This present book brings the latest developments on biorefineries, and their potential use for the transformation of residues that otherwise would be wasted, into a broad range of more valuable products. Moreover, it will provide updated information for professionals of different fields in the area, as well as for those who are not directly involved but have interest in it. Thus the target audience includes agriculture and process engineers, microbiologists, biochemists, researchers, industry sector, nonscientists interested in the field, and early commercial adopters seeking to enable conversion of biomass fibers into value-added products. In addition, this book addresses the following topics: the mechanisms of enzymatic conversion of biomass into value-added products; bioproducts derived from lignocellulose and their applications on design, development, and implementation of processes; technologies for the sustainable manufacture of materials, chemicals; techno-economic evaluation of biorefinery for integrated sustainability assessments; the socioeconomic and cultural-economic perspectives on lignocellulosic biorefinery; and virtual biorefinery as an integrated approach to evaluate the lignocellulose production chain.

In conclusion, this book edition intends to show an integrated concept of biorefinery with enzymes generating value-added products; as well as related to social and economic aspects, circular bioeconomy, hubs, and perspectives.

Acknowledgments

This work was supported by Hatch Act 10677, Purdue University Agricultural Research Programs and the Department of Agricultural and Biological Engineering and Department of Energy—DOE EE0008256. The authors also would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES PrInt program, Process 88887.364337/2019-00), the Brazilian Agricultural Research Corporation (Embrapa), the Brazilian National Council for Scientific and Technological Development (CNPq, Processes 401182/2014-2 and 303614/2017-0), and the São Paulo Research Foundation (FAPESP, Process 2016/10636-8) for financial support.

References

1. Fazard S, Mandegari MA, Guo M, Haigh KF, Shah N, Görgens JF. Multi-product biorefineries from lignocelluloses: a pathway to the revitalisation of the sugar industry?. Biotechnol Biofuels. 2017;10:87.

2. Gheewala SH, Bonnet S, Prueksakorn K, Nilsalab P. Sustainability assessment of a biorefinery complex in Thailand. Sustainability. 2011;3:518–530.

3. Kim S, Dale BE. All biomass is local: the cost, the volume produced, and global warming impact of cellulosic biofuels depend strongly on logistics and local conditions. Biofuels, Bioprod Biorefin. 2015;9:422–434.

4. Mohan SV, Nikhil GN, Chiranjeevi P, et al. Waste biorefinery models towards sustainable circular bioeconomy: Critical review and future perspectives. Bioresour Technol. 2016;215:2–12.

5. Silva COG, Vaz RP, Filho EXF. Bringing plant cell wall-degrading enzymes into the lignocellulosic biorefinery concept. Biofuels, Bioprod Biorefin. 2017;12(2):277–289.

2

Enzymatic path to bioconversion of lignocellulosic biomass

Samkelo Malgas¹, Lithalethu Mkabayi¹, Brian N. Mathibe¹, Mariska Thoresen¹, Mpho S. Mafa¹, ², Marilize Le Roes-Hill³, Willem Heber (Emile) van Zyl⁴ and Brett I. Pletschke¹,    ¹Enzyme Science Programme (ESP), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa,    ²Protein Structure-Function Research Unit (PSFRU), School of Molecular and Cell Biology, Wits University, Johannesburg, South Africa,    ³Institute of Biomedical and Microbial Biotechnology, Cape Peninsula University of Technology, Cape Town, South Africa,    ⁴Department of Microbiology, Stellenbosch University, Matieland, South Africa

Abstract

Lignocellulosic biomass is an abundant, carbon neutral, renewable resource that holds potential for the production of biofuels and fine chemicals. First, the lignocellulosic biomass feedstock is required to go through a recalcitrance-reducing step (pretreatment). Enzyme cocktails consisting of cellulolytic or hemicellulolytic (or both) activities are then used to precisely break down the polysaccharides, cellulose, and hemicellulose, respectively, into simple sugars suitable for several bioprocesses, such as fermentation to ethanol. Currently, the enzymes, particularly the glycoside hydrolases, used industrially to valorize lignocellulosic biomass into fine chemicals, are faced with numerous challenges. These challenges include biomass recalcitrance, slow rates of association to the substrate (i.e., the formation of a catalytically competent complex), and the heterogeneous nature of biomass—these limit the overall reaction rate and, as a result, the process remains prohibitively expensive—compared to the production of petroleum-based fine chemicals. It is reported that even a twofold reduction in the cost of enzymatic valorization of lignocellulosic biomass could make a big difference to the economics of renewable fuels and chemicals production from lignocellulose. This review details consolidated and new strategies to enhance enzymatic activity on lignocellulosic biomass, including approaches such as enzyme synergism, prospecting for novel and superior enzymes, enzyme immobilization and recycling, detoxification of enzyme inhibitors from reaction slurries, use of additives, and designing multifunctional enzymes (i.e., chimeras and cellulosomes).

Keywords

Cellulolytic; glycosyl hydrolases; hemicellulolytic; inhibitors; lignin; lignocellulose; LPMOs; synergism

Chapter Outline

Outline

2.1 Introduction 6

2.2 Lignocellulose 6

2.2.1 Cellulose 7

2.2.2 Hemicellulose 8

2.2.3 Pectic polysaccharides 8

2.2.4 Lignin 8

2.3 Enzymes involved in lignocellulose deconstruction (cellulases, hemicellulases, pectinases, ligninases, and lytic polysaccharide monooxygenases) 8

2.4 Application of carbohydrate-active enzymes in bioconversion of lignocellulosic biomass 11

2.4.1 Extraction and modification of phenolic antioxidants 13

2.5 Prospecting for novel superior enzymes 15

2.6 Improvement of the catalytic performance of current glycoside hydrolases 16

2.6.1 Targeted mutagenesis 16

2.6.2 Chimeric enzymes and cellulosomes 17

2.7 Enzyme recycling 18

2.7.1 Enzyme immobilization 19

2.7.2 Readsorption of enzymes on fresh substrate 19

2.7.3 Membrane filtration 19

2.8 Enzyme synergism 20

2.9 Pretreatment of lignocellulose to improve its enzymatic degradability 21

2.9.1 Ionic liquid pretreatment 21

2.10 Addition of surfactants 22

2.11 Detoxification of pretreated lignocellulose slurries 22

2.12 Conclusion and future perspectives 23

References 24

Further reading 32

2.1 Introduction

Lignocellulosic biomass is an abundant, carbon neutral, renewable resource that holds potential for the production of biofuels and fine chemicals. First, the lignocellulosic biomass feedstock is required to go through a recalcitrance-reducing step (pretreatment) (Weiss et al., 2017). Enzyme cocktails consisting of cellulolytic or hemicellulolytic (or both) activities are then used to precisely break down the polysaccharides, cellulose, and hemicellulose, respectively, into simple sugars suitable for several bioprocesses, such as fermentation to ethanol (Cota et al., 2013). Currently, the enzymes, particularly the glycoside hydrolases (GHs), used industrially to valorize lignocellulosic biomass into fine chemicals, are faced with numerous challenges. These challenges include biomass recalcitrance, slow rates of association to the substrate (i.e., the formation of a catalytically competent complex), and the heterogeneous nature of biomass—these limit the overall reaction rate and, as a result, the process remains prohibitively expensive—compared to the production of petroleum-based fine chemicals (Banerjee et al., 2016; Rizk et al., 2012). It is reported that even a twofold reduction in the cost of enzymatic valorization of lignocellulosic biomass could make a big difference to the economics of renewable fuels and chemicals production from lignocellulose. This review details consolidated and new strategies to enhance enzymatic activity on lignocellulosic biomass, including approaches such as enzyme synergism, prospecting for novel and superior enzymes, enzyme immobilization and recycling, detoxification of enzyme inhibitors from reaction slurries, use of additives, and designing multifunctional enzymes (i.e., chimeras and cellulosomes).

2.2 Lignocellulose

Lignocellulose is the major structural component of plant cell walls and is predominantly composed of three biopolymers, that is, cellulose (35%–50%), hemicellulose (20%–35%), and lignin (5%–30%), along with minor traces of pectins, proteins, extractives, and ash (Chandel et al., 2018; Lee et al., 2014). Fig. 2–1 illustrates the structures of various polysaccharides typically found in lignocellulosic biomass. These polymers are intertwined with each other to form a meshwork of nonuniform three-dimensional structures, where the relative compositions and morphologies vary according to the plant species (Van Dyk and Pletschke, 2012). Lignocellulosic biomass arises from (1) forest residues (hardwoods and softwoods); (2) agricultural residues (sugarcane bagasse and corn stover); (3) municipal residues (paper waste); (4) dedicated energy crops (terrestrial and aquatic); and (5) various grasses (Lee et al., 2014; Van Dyk and Pletschke, 2012).

Figure 2–1 A schematic representation of plant cell wall polysaccharides: (A) cellulose, (B) hemicelluloses [(B1) glucuronoarabinoxylan and (B2) galactoglucomannan], and (C) pectin/arabinogalactan. Black residues represent reducing ends of the corresponding polysaccharide fibrils and lines represent glycosidic linkages joining sugar resides.

2.2.1 Cellulose

Cellulose is the most abundant and principal component of lignocellulosic biomass, comprising approximately 35%–50% of the total biomass (Kumar et al., 2016; Kumar and Murthy, 2013; Peciulyte et al., 2014). Cellulose is a homopolysaccharide composed of parallel and unbranched β-1,4-D-glucopyranosyl units (Bayer et al., 1998; Teeri, 1997). The polymer is not only connected by intra- and intermolecular hydrogen bonds to form a microcrystalline structure (George and Sabapathi, 2015; Moon et al., 2011) but also has less ordered or amorphous regions (Horn et al., 2012; Park et al., 2010).

2.2.2 Hemicellulose

Hemicellulose is a term used to describe noncellulosic and nonpectic polysaccharides in plant cell walls. Hemicelluloses are the second most common polysaccharides on the Earth and constitute about 20%–30% of the dry weight of lignocellulose. There are currently four groups of polysaccharides (xylans, mannans, mixed linked β-glucans, and xyloglucans) that are regarded as hemicellulose and are classified according to the predominant type of sugar residue present in their backbones (Dey and Roy, 2018; Van Dyk and Pletschke, 2012). The building blocks/monomers of these polysaccharides are pentoses (D-xylose and L-arabinose), hexoses (D-mannose, D-galactose, and D-glucose), hexuronic acids (4-O-methyl-D-glucuronic acid, D-glucuronic acid, and D-galacturonic acid), and acetyl groups, as well as small amounts of L-rhamnose and L-fucose (Bochicchio and Reicher, 2003; Van Dyk and Pletschke, 2012; Xu et al., 2018).

2.2.3 Pectic polysaccharides

Pectic polysaccharides make up to 35% of the primary cell wall in dicotyledonous plants and nongraminaceous (nongrass) monocots, 2%–10% of grass primary walls, and up to 5% of wood tissues. Pectic polysaccharides are divided into five classes, that is, homogalacturonan (HG), xylogalacturonan, apiogalacturonan, and rhamnogalacturonan I and II (Wolf et al., 2009). HG consists of a linear α-1,4-linked galactoronic acid backbone with a degree of polymerization of about 100 units. HG is generally acetylated at C-2 and/or C-3 and methyl-esterificated at C-6 (Wolf et al., 2009).

2.2.4 Lignin

Lignin accounts for about 5%–30% of the dry material in various feedstocks (Kumar et al., 2016). It is a highly hydrophobic and complex polyphenol that forms a protective sheath around the polysaccharides through various ester and ether linkages (Horn et al., 2012; Perez et al., 2002). The monomeric composition of lignin comprises three phenyl propionic alcohols, including (1) phenylpropanoids p-hydroxyphenyl (coumaryl alcohol), (2) guaiacyl (coniferyl alcohol), and (3) syringyl monolignols (synapyl alcohol) (Karp et al., 2013; Ramos, 2003). Depending on the plant species, the relative composition and distribution of monolignols may vary (Ramos, 2003).

2.3 Enzymes involved in lignocellulose deconstruction (cellulases, hemicellulases, pectinases, ligninases, and lytic polysaccharide monooxygenases)

The complexity of lignocellulose in terms of composition, impenetrability due to high lignin content, hemicellulose sheathing, as well as high degree of crystallinity of particularly the cellulose portion, renders it recalcitrant toward microbial degradation (Obeng et al., 2017). The intertwined nature of the cellulose, hemicellulose, and lignin components in lignocellulose necessitates that a large repertoire of carbohydrate-active enzymes synergistically act in succession to deconstruct this complex substrate (Guerriero et al., 2015; Obeng et al., 2017). Variable composition of lignocellulose within plants and between different plants adds another layer of complexity for microorganisms to decompose lignocellulose. It is thus not surprising that 45 and 89 GHs were secreted by Trichoderma reesei and Aspergillus niger, respectively, when cultivated on sugarcane bagasse. However, a subset of hydrolyzes was identified in both groups, which form the core of deconstructive enzymes required for conversion of particularly the cellulose, hemicellulose, and pectin fractions to reducing sugars (Borin et al., 2015).

All enzymes are assigned an Enzyme Commission (EC) number as part of the uniform nomenclature for enzyme names based on the reactions they catalyze (McDonald and Tipton, 2014). Lignocellulose deconstructing enzymes comprise members of the larger GHs—predominantly EC 2.4.1.xxx, EC3.2.1.xxx activities; glycosyltransferases—EC 2.4.x.xxx—large structural overlap with GHs enzymes; polysaccharide lyases—EC 4.2.2.xxx; carbohydrate esterases—few representatives in EC 2.xxx EC 3.xxx, EC 4.xxx; and auxiliary activities (AAs—mostly in EC 1.xxx) (Lairson et al., 2007).

Key enzymes required for the hydrolysis of microcrystalline cellulose are endoglucanases (EGs), exoglucanases [cellobiohydrolases (CBHs)], and β-glucosidases (Van Dyk and Pletschke, 2012). The EGs are responsible for random hydrolysis of amorphous cellulose regions, and the opening up and generation of reducing and nonreducing cellulose chain ends in more crystalline cellulose regions (Payne et al., 2015). CBHs are processive enzymes with specificity for either the reducing (CBHI) or nonreducing (CBHII) ends to release cellobiose moieties from crystalline cellulose. The cellobiose and smaller oligosaccharides generated by the synergistic activity of EGs and CBHs are eventually converted to glucose with the aid of β-glucosidases (Obeng et al., 2017; Magrey et al., 2018).

The hemicelluloses in plant material are much more heterogeneous with xylans and mannans dominating in hard- and softwoods, respectively. Endoxylanases and xylosidases attack the xylan chains with α-L-arabinofuranosidases, α-D-glucuronidases, and acetylesterases removing arabinose and glucuronic acid sugar and acetyl moieties from the xylan chains (Kumar et al., 2017; Obeng et al., 2017; Shahi et al., 2016). In the case of mannan, endomannanases and mannosidases are responsible for the hydrolysis of mannan chains, with α-galactosidases removing substituent sugars from the mannan chains (De Moura et al., 2015; Willats et al., 2006; Yamabhai et al., 2016).

Pectins are often found in the cell walls of fruits, vegetables and, cereals and, like hemicellulose, are intertwined with cellulose fibers. Enzymes required for pectin deconstruction are pectinesterases (these enzymes remove the methoxyl groups), polygalacturonases, poly- and polymethyl-galacturonate lyases (enzymes that are key in the softening and ripening of fruits) (Amin et al., 2019; Jayani et al., 2005; Kohli et al., 2015; Kohli and Gupta, 2015).

To date, 16 AAs families have been reported. These AA families include redox enzymes that are known to be involved in lignin breakdown (AA1-AA8), which also include the lytic polysaccharide monooxygenase (LPMO) AA9-AA11 and AA13-AA16 families (Filiatrault-Chastel et al., 2019; Levasseur et al., 2013). The well-known multicopper oxidases (AA1—laccases, ferroxidases, and laccase-like multicopper oxidases; EC 1.10.3.2) and peroxidases (AA2—manganese peroxidases, lignin peroxidases, and versatile peroxidases; EC 1.11.1.x) are enzymes that have been the focus of numerous studies over the past century and therefore have been extensively reviewed (Andler et al., 2018; Chio et al., 2019; Feofilova and Mysyakina, 2016; Guan et al., 2018). Family AA3 (glucose–methanol–choline oxidoreductases) was recently reviewed by Sützl et al. (2018) and includes cellobiose dehydrogenase (EC 1.1.99.18), aryl alcohol oxidase/glucose oxidase (EC 1.1.3.7/1.1.3.4), alcohol oxidase (EC 1.1.3.13), and pyranose oxidase (EC 1.1.3.10). Of these enzyme groups, it is believed that cellobiose dehydrogenase plays a key role in enhancing the biocatalytic activity of LPMOs (Kracher and Ludwig, 2016).

LPMOs are currently drawing a lot of attention, mainly because of their synergistic effect on cellulase activity (Johansen, 2016). LPMOs are copper-dependent enzymes found to exhibit enhanced activity in the presence of an external electron donor (e.g., ascorbic acid, lignin, and cellobiose dehydrogenase) and require the presence of hydrogen peroxide or dioxygen (Eijsink et al., 2019; Johansen, 2016; Marjamaa and Kruus, 2018). In terms of substrate specificity, these enzymes have been shown to be active toward a range of substrates: chitin, cellulose, starch, cellodextrins, glucomannan, and xyloglucan (Eijsink et al., 2019; Filiatrault-Chastel et al., 2019; Johansen, 2016). This substrate specificity seems to be delimited to specific AA families, for example, the fungal AA9 family of LPMOs is active on cellulose, xyloglucans, glucomannans, cello-oligosaccharides, and β-glucans, while the AA10 family (bacterial and viral) and AA15 family (invertebrates) are active only on chitin and cellulose (Filiatrault-Chastel et al., 2019). It is believed that LPMOs form part of the first wave of attack on the lignocellulose polymer where they catalyze the cleavage of glycosidic linkages, making the rest of the lignocellulose polymers accessible to other enzymes (Johansen, 2016). Even though there have been extensive studies on LPMOs, there are still some challenges being faced in the functional characterization of LPMOs, an aspect that has been extensively reviewed and highlighted by Eijsink et al. (2019).

Other AA families, AA4 (vanillyl alcohol oxidase), AA5 (radical-copper oxidase), AA6 (1,4-benzoquinone reductase), AA7 (glucooligosaccharide oxidase), and AA8 (iron reductase domain), have been briefly reviewed by Levasseur et al. (2013). These enzymes can be considered to be part of the second wave of attack: vanillyl alcohol oxidase (EC1.1.3.38) catalyzes the conversion of phenolic compounds that are released during the breakdown of lignin; the radical-copper oxidases, glyoxal oxidase (EC 1.1.3.-), convert aldehydes to carboxylic acids and galactose oxidase (EC 1.1.3.9) oxidizes carbohydrates and primary alcohols with the reduction of dioxygen to hydrogen peroxide; 1,4-benzoquinone reductase (EC 1.6.5.6) degrades aromatic compounds; glucooligosaccharide oxidase (EC 1.1.3.-) catalyzes two half reactions by oxidizing reducing sugars to a lactone followed by spontaneous hydrolysis to yield a corresponding acid; and while the physiological functions of iron reductases are unknown, it is hypothesized that they are involved in the generation of reactive hydroxyl radicals (Levasseur et al., 2013). As more genome sequences are becoming available, these will hopefully assist in the identification of new enzymes and developing a full picture of the enzymes potentially involved in lignocellulose degradation.

2.4 Application of carbohydrate-active enzymes in bioconversion of lignocellulosic biomass

The interest in cellulosic ethanol was renewed in the early 2000s in an attempt to extend the limited production of first-generation bioethanol from sugar and starchy feedstocks to more abundant nonfood lignocellulosic feedstocks. The large variety of lignocellulose feedstocks available is complemented with large combinations of pretreatment technologies to sugar streams, which through several conversion technologies can be converted to biofuels and biochemicals. Utilization of lignocellulosic biomass necessitates expensive pretreatment and the process is inherently capital intensive. Although lignocellulosic feedstocks are considered abundant and cheap, their low density and high transport expenditure add to the overall biofuel production costs. After capital and feedstock costs that can represent more than 50% of the total costs, enzymes are considered the third most expensive commodity required for cellulosic ethanol production (Stephen et al., 2012). Unless the costs of these three items are drastically reduced, second-generation cellulosic ethanol will not be competitive with first-generation ethanol as initially