Keywords

2.1 Introduction

Biomass refers to any matter of plant origin, which can be processed to provide more elaborate bioenergetic and chemical forms suitable for end use. Around the world, more than 146 billion tons of residues are annually available, but only a portion (up to 100 million) of it is being used for energy and biofuel production (Ayres 2014). The rest of these residues is usually burnt or left behind, which may lead to the worsening of the greenhouse effect. A few of the most biomass-producing countries in the world are Brazil, China, Indonesia, Russia, and the USA (Table 2.1).

Table 2.1 Comparative production of crops in different countries
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Food, fiber, or wood production generates biomass, which could be useful for energy production using mature technologies. Currently, the final biomass energy produced is 50 EJ totaling 14% of the final energy used in the world, but it has a potential to be increased up to 150 EJ by 2035. A total supply of 38–45% is estimated to be originated from waste and residues from agriculture and the remaining supply would be shared by the energy crops, forestry, and residual products (IRENA 2014). According to the World Bioenergy Association (WBA 2014), the estimated potential of residues from agriculture in order to produce energy varies between 17 and 128 EJ. There is a high potential of using residues from agriculture, especially in Asia and the Americas because of the different crops highly produced in these regions. There is an enormous potential for exploiting the use of biomass in order to produce larger quantities of products with high value.

Due to the various possible sources of lignocellulosic biomass, this chapter is focused on describing the structure and characteristics of its major components, such as cellulose, hemicellulose, pectin, and lignin present in the primary and secondary cell walls. The resins, fatty acids, phenols, tannins, nitrogenous compounds, and mineral salts, e.g., calcium, potassium, and magnesium on a smaller scale, can also be found, which depends on the plant species (Neureiter et al. 2002). Subsequently, the chapter presents a description of different biomass sources that can be used for the production of bioenergy, followed by the major enzymatic systems that can degrade plant cell walls and their mechanism of action on biomass, especially in sugarcane, for the production of second-generation bioethanol.

2.2 Composition of Lignocellulosic Biomass

Cellulose (23–50 wt% of the dry matter of lignocellulosic biomass) is a linear homopolymer containing up to 15,000 units of β-d-glucose bound by β-1,4-glycosidic linkages (Nanda et al. 2015). It has reducing and nonreducing ends, and it is extremely resistant to degradation (Michelin et al. 2013). The strength of cellulose is due to many hydroxyl groups on the glucose structure that contribute to the formation of massive intramolecular bounds (linkages among glucose units of the same molecule) and intermolecular hydrogen bounds (among glucose units of adjacent molecules), which are responsible for stiffness. The intramolecular bonds are responsible for the formation of fibrils, which are highly ordered structures. According to the degree of organization of the bonds between the cellulose chains, the structure can be crystalline (highly ordered) or amorphous (less ordered). The amorphous regions can absorb water more easily and are more susceptible to enzymatic action.

Hemicellulose is a heteropolysaccharide (15–45 wt% of dry lignocellulosic material) with branched chains of monosaccharides, mainly including aldopentoses (xylose and arabinose) and aldohexoses (glucose, mannose, and galactose). This macromolecule also contains deoxyhexoses and acids, such as β-d-galacturonic acid, d-4-O-methylglucuronic acid, and β-d-glucuronic acid (Polizeli et al. 2005). The variety of bonds and branching, as well as the presence of different monomeric units, contributes to the complexity of the hemicellulosic structure and its different conformations. Unlike cellulose, hemicellulose has low molecular mass (100–200 glycosidic units) and does not contain crystalline regions, which makes it easier to hydrolyze in non-drastic conditions (Polizeli et al. 2005).

There are different types of hemicellulose, such as arabinoxylan, acetylglucuronoxylan, arabinan, arabinogalactan, xylan, galactomannan, xyloglucan, galactoglucomannan, and glucomannan. Hemicellulose is classified according to its sugar chain composition. Thus, the term hemicellulose does not denote a defined chemical compound, rather, a set of polymeric components present in fibrous plants where each component has different properties. The major hemicellulose constituent is the xylan, which is a xylose β-1,4-polymer with several branched residues (Polizeli et al. 2005). The fermentation of pentoses is not yet as developed as the processes involving glucose. Mannan is the second most abundant component of the hemicellulosic fraction being widely found in woods (gymnosperms), tubers, seeds, and grains in different compositions structure and complexity. The main chain of mannan consists of mannose residues bound by β-1,4-linkages or a combination of mannose and glucose residues associated by a same type linkage. In addition, the major chain of mannan may have side chains attached to α-1,6-galactose residues (De Marco et al. 2015).

Pectins are heterogeneous polysaccharides, which form the middle lamella being the largest component inside it. These polysaccharides are adhesive materials in the extracellular portion found in the higher plant cells located in the primary walls. The structure consists of axial bonds with acid units of α-1,4-d-galacturonic and contains molecules of galactose, arabinose, xylose, and l-rhamnose as the side chains (Polizeli et al. 2013).

The biochemical structure of lignin (10–30 wt% of dry lignocellulosic material) cannot be related to sugar molecules, and it is not suitable for bioconversion or fermentative routes. However, this fraction plays a key role in the success of hydrolysis technology, since it blocks the access to cellulose. The structure of lignin has a three-dimensional polymeric structure formed by p-propylphenol units with methoxy substituent on the aromatic ring joined by ether bonds that crosslink each other. This macromolecule is originated through sinapyl, coumaric, and coniferyl alcohols.

2.3 Commonly Available Agricultural Crop Residues in the World

2.3.1 Barley and Coffee Residues

Barley (Hordeum vulgare) is one of the main cereal sources produced in the world for humans and livestock consumption. Every year, an area of around 50 million hectares is harvested, being the fourth major cereal produced in the world. The most abundant staple crops cultivated in the world are wheat (~200 million hectares), rice (~170 million hectares), and maize (~145 million hectares). Russia, Australia, Ukraine, and Canada are the major barley-producing countries (WAP 2019). Barley and beer residues are similar to other lignocellulosic materials being composed of cellulose, hemicellulose, and lignin. It is reported that the percentage of cellulose and hemicellulose in such materials is high (Table 2.2), which can be hydrolyzed and fermented aiming the production of biofuels and biochemicals.

Table 2.2 Cellulose, hemicellulose, and lignin composition of some of the most widely found lignocellulosic feedstocks in the world
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The coffee tree is a plant of permanent culture that belongs to the Rubiaceae family and to the genus Coffea such as Coffea arabica and Coffea canephora. Around 8.9 million tons of coffee are produced over an area of approximately 10.6 million hectares (FAO 2018). The major coffee producers in the world are Brazil, Vietnam, and Colombia (Al-Abdulkader et al. 2018). One ton of bark is generated per each ton of beans of coffee that are processed (Saenger et al. 2001). The coffee pulp is the main residue originated in the wet processing of the mature coffee, representing approximately 40 wt% of the dry weight of the coffee bean, which is rich in cellulose and hemicellulose (Table 2.2), proteins, minerals, and appreciable amounts of tannins, caffeine, and potassium. Currently all these raw materials could be used in developed processes to generate value-added and bulk chemicals products such as active secondary metabolites, unicellular proteins, enzymes, and organic acids (Pandey et al. 2000). Another alternative is the use of these wastes for bioenergy production.

2.3.2 Sugarcane, Maize, Rice, and Wheat Residues

Sugarcane, maize, rice, and wheat residues generate most of the lignocellulosic residues in the world (Saini et al. 2015). One of the most famous plant being used nowadays is the sugarcane (Saccharum ssp.). It is a C4 crop that has been used for a long time in the sugar production, but currently bioethanol and electricity have become the most famous value-added energy products generated through bioconversion and burning, respectively (Cotrim et al. 2018). The total harvested area for sugarcane in the world is estimated to be around 26.7 million hectares and the major producers are Brazil, India, and China. After the processing of the sugarcane, large quantities of bagasse are generated what presents good amounts of cellulose and hemicellulose. These factors make it an interesting, renewable, and cheap resource to produce biochemicals and biofuels (Bhatia and Paliwal 2011).

There are several varieties of commercial sugarcane in the world. In Brazil, the SP80-3280 variety is one of the most studied. The composition of the cell wall of SP80-3280 variety sugarcane is reported having fundamental importance for understanding the enzymes necessary for its saccharification (De Souza et al. 2013). In recent years, the search for sugarcane with high levels of sugars for first-generation bioethanol and the accumulation of biomass for second-generation bioethanol production have increased. This sugarcane is known as energy cane, which presents the particularity of having a higher fiber content, being distributed in the fractions of lignin, cellulose, and hemicellulose. The energy cane comes from genetic improvements of the sugarcane (Saccharum spontaneum) to increase the energy availability and adaptation to different cultivation environments (Tew and Cobill 2008).

Maize (Zea mays) is a well-known cereal being considered the main input in animal protein production, in human nutrition, and also in the biofuel production. The total area harvested each year in the world is estimated to be around 145 million hectares and the major maize producers in the world are the USA, China, and Brazil (FAO 2018). From Table 2.2 it can be inferred that the maize residues have small amounts of lignin, which makes it interesting for the use in the bioenergy and in the production of chemicals.

Rice consists of two main species such as Oryza glaberrima and Oryza sativa. It is a plant of the grass family and is among the products intended for human consumption, being second in importance, only behind wheat. In some parts of the world, especially in Asia, rice is staple food crop (CONAB 2018). It is harvested from over 163 million hectares in more than 100 countries (Laborte et al. 2017). The major rice producers are China, India, and Indonesia (FAO 2018). During the rice cultivation, huge amounts of rice straw are generated along with leaf blades, stems, leaf sheaths, and husks as the leftovers. These residues are one of the largest lignocellulosic wastes in the world. Among the most rice straw producers is Asia which alone generates 667.6 million tons of rice straw that consists of 91.3% of the global rice production (Saini et al. 2015). This residue is also an important source with similar contents of hemicellulose, lignin, and cellulose when compared with other organic residues, which could be better used for the production of biofuels and biochemicals.

Wheat is considered a staple food and cereal in all over the world (Shewry 2009; Balkovič et al. 2014). The common wheat, Triticum aestivum, is the most widely grown in the world, over an arable land about 220.4 million hectares (FAO 2018). During the wheat harvesting process, straw is generated at a rate of 2.5–7.5 tons/hectare annually under rigorous farming conditions. Cellulose, hemicelluloses, and lignin make up the most part of the agricultural residues in a similar rate. However, wheat straw presents pectin and proteins, whereas rice straw has silica within its tissues (Sarkar et al. 2012).

2.3.3 Cotton

Cotton is a staple fiber found across the world. It belongs to the genus Gossypium within the Malvaceae family. Cellulose is the main component of the cotton bolls. It is estimated that 25 million tons of cotton are annually produced in the world over an area that estimates about 2.5% of the total arable land at a global scale. The cotton cultivation area in the world is accounted around 33.6 million hectares per year and the major cotton producers are China, India, and the USA (Daisy et al. 2018). After harvesting the cotton, huge amounts of residues are left in the field, such as side branches, stalks, bolls, seeds with adhering cotton lint, leaves, etc. (Huang et al. 2012). The total amount of residues (e.g., cotton straw, cotton sticks, cotton wood) generated from the cotton harvesting can range from 5 to 7 tons/hectare. The quality of these materials that are generated from the cotton harvesting presents a high variability as commonly observed in crop wastes. However, these residues are also important sources of lignin, cellulose, and hemicellulose as shown in Table 2.2.

2.3.4 Oil Palm Residues

Oil palm is a vegetable oil extracted from the mesocarp of Elaeis guineensis. Palm oil is a common cooking ingredient in Brazil, Africa, and Asia. Oil palm trees present a highly efficient land use with a large yield when compared with other crops used for oil extraction. It is estimated that a total harvested area of about 17 million hectares of mature oil palm plantation produces a total of 62.6 million tons of oil palm per year. The major oil palm producers are Indonesia, Malaysia, and Nigeria (EPOA 2018). Europe is the major importer of oil palm for using it as a precursor for biodiesel production (Soeriaatmadja and Leong 2018). In addition to its massive potentials for biodiesel production, all the oil palm residues generated after the oil extraction could also be used to produce the bioethanol since it is composed of cellulose, hemicellulose, and lignin (Table 2.2).

2.3.5 Potato Residues

Potato (Solanum tuberosum) is a staple food crop cultivated in most countries across the world. Potatoes are considered the fifth largest crop for human consumption after wheat, rice, maize, and barley. In 2016, the total world potato production and harvested area were estimated at 376 million tons and 19 million hectares, respectively (FAO 2018). The three largest potato producers in the world are China, India, and Russia. Potato processing industry generates massive amounts of organic wastes. It is estimated that one quarter of the total weight of potato is obtained as wastes from the food processing plants. These wastes can be used, for example, as a carbon source for yeast during alcohol fermentation to produce bioethanol. The potato peel waste contains adequate amount of starch, hemicellulose, cellulose, lignin (Table 2.2), and residual carbohydrates, which make potato peel a suitable feedstock for ethanol production (Ojewumi et al. 2018).

2.3.6 Soybean Residues

The soybean (Glycine max) is known to be widely cultivated because of its comestible bean that presents several different uses. The worldwide soybean production was estimated around 337 million tons in 2017–2018 (WASDE 2018). The major producers of soybean in the world are the USA, Brazil, and Argentina. Based on the global production, it is possible to affirm that these three countries are responsible by approximately 85% of the total soybean production (FAO 2015). During the recovery of the protein from the soybean seeds, large amounts of residues are generated. The residues generated are composed of 16 wt% cellulose and 23 wt% hemicellulose (Table 2.2). Previous studies have estimated that approximately 10,000 tons per year of these by-products are generated and only a small portion is used for animal feeding, while the rest is mainly disposed in the environment (Heck et al. 2002).

2.4 Enzymatic Saccharification of Lignocellulosic Residues

2.4.1 Cellulases

Cellulases are glycosyl hydrolases that act in the cleavage of cellulose and can be classified into endoglucanases (EG), exoglucanases (CBH), and β-glucosidases (BG) (Payne et al. 2015; Wei and Mcdonald 2016). Some cellulases are organized in complexes called cellulosomes. These structures are produced by anaerobic bacteria (Artzi et al. 2017) and fungi (Haitjema et al. 2017) as a strategy to improve the hydrolysis efficiency. They are composed of a complex scaffolding as structural subunit and various enzymatic subunits. Many cellulases and hemicellulases are attached to structures called carbohydrate binding module (CBM). These structures have the property to attach the enzyme to the substrate. CBMs are not necessary to the hydrolytic function and there is no evidence that they improve the action of cellulases (Várnai et al. 2014).

Endo-β-1,4-glucanases (EC 3.2.1.4) hydrolyze the glycosidic bonds mainly at the amorphous regions of cellulose (Fig. 2.1). They are distributed along 16 out of 134 glycoside hydrolase (GH) families and are characterized by shorter loops. Thus, this feature enables the active site fissures to reach accessible sites of cellulose chain. The endoglucanases act in the interior of cellulose, resulting in long-chain oligomers. When compared with other cellulases, they have a faster dissociation rate and show the best cellulose liquefaction results, thus decreasing the chain length and consequently the viscosity (Badieyan et al. 2012; Boyce and Walsh 2015; Juturu and Wu 2014).

Fig. 2.1
figure 1

Cellulase action on cellulose crystalline and crystal structure. Endoglucanase (EG) in blue at the amorphous region with a CBM attached to the crystalline region releasing smaller cellulose chains. Cellobiohydrolase (CBH) in orange is attached to the crystalline terminal releasing cellobiose. β-Glucosidase (BG) in yellow cleaves the cellobiose to glucose. Auxiliary activity 9 enzyme (AA9) in purple acts on the crystalline region leaving a rupture in the cellulose chain. Swollenin (SWO) in pink acts between the cellulose chains resulting in a less crystalline structure

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Exoglucanases or cellobiohydrolases act in the end of crystalline cellulose chains to release cellobiose (Fig. 2.1). They act efficiently in cellulose hydrolysis and are divided into EC 3.2.1.91 (acting in the nonreducing ends) and EC 32.1.176 (acting in the reducing ends). These enzymes have long loops usually forming tunnels around the catalytic residues; thus the substrate is directed along these tunnels to encounter the active site (Obeng et al. 2017; Wilson and Kostylev 2012).

β-Glucosidases (BGs, EC 3.2.1.21) break down cellobiose to glucose (Fig. 2.1). Several β-glucosidases are also able to catalyze the reverse reaction by transglycosylation in which glucose molecules are transferred to another glucose molecule or cellobiose to yield different oligosaccharides. β-Glucosidases are susceptible to product inhibition, especially at high-biomass conditions. Thus, it is one of the biggest bottlenecks for the total cellulose conversion to glucose. The addition of large amounts of β-glucosidases in enzymatic cocktails has the extra goal to reduce the product inhibition exhibited by cellobiose on cellobiohydrolases and endoglucanases (Andríc et al. 2010; Singhania et al. 2013).

The most reported cause to product inhibition on fungal β-glucosidases is via competitive inhibition, but it is also described as one of the noncompetitive and mixed mechanisms. The majority of β-glucosidases belong to the 1 and 3 GH families, but they are also found in families 5, 9, and 30. The enzymes belonging to family GH1 are 10 to 1000-fold more tolerant to glucose when compared to GH3 β-glucosidases. The structural analysis shows that glucose tolerance is strongly correlated with active site accessibility (Andríc et al. 2010; De Giuseppe et al. 2014; Singhania et al. 2013).

2.4.2 Auxiliary Activity 9 Enzyme and Swollenin Protein

Auxiliary activity 9 (AA9) enzyme is a fungal lytic polysaccharide monooxygenase (LPMO), a cooper-dependent enzyme that is added to the most commercial cocktails. AA9 boost the action of cellulases in lignocellulosic material hydrolysis. It promotes the oxidative cleavage of glyosidic bounds of C1 or C4, thus disturbing cellulose crystallinity and giving access to canonical cellulases, consequently improving the overall hydrolysis yield (Fig. 2.1). The oxidation promoted by LPMO is dependent on an electron donor, e.g., ascorbic acid, cellobiose dehydrogenase, and lignin or photocatalytic systems. The oxidation occurs only in the presence of O2 or H2O2 as the co-substrate (Bertini et al. 2018; Möllers et al. 2017; Müller et al. 2018; Walton and Davies 2016).

Swollenins (SWOs) are nonenzymatic proteins with high homology to plant expansins. This class of proteins has the property to cause deagglomeration of crystalline cellulose. SWOs act in the breakage of hydrogen bonds between the cellulose chains, thus loosening the structure, increasing the accessibility of canonical cellulases and enzymatic hydrolysis (Fig. 2.1). SWOs can also disrupt hemicellulose, promoting a better solubilization and acting synergistically with hemicellulases (Cosgrove 2000; Gourlay et al. 2013; Kang et al. 2013; Kim et al. 2014; Santos et al. 2017).

2.4.3 Hemicellulases

Hemicellulases are classified as the class of enzymes responsible for depolymerizing the hemicellulose. In order to achieve the complete degradation of hemicelluloses, the action of several enzymes belonging to GH families is important. The most significant enzymes of these complex are discussed as follows (Polizeli et al. 2005; Saha 2003).

Xylan, a xylose β-1,4-polymer, is a major constituent of hemicellulose. For the degradation of xylan, two main enzymes are necessary, namely, endo-β-1,4-xylanase and β-1,4-xylosidase (Fig. 2.2). Endo-β-1,4-xylanases (EC 3.2.18) or endoxylanases catalyze the hydrolysis of xylan to xylooligosaccharides. This enzyme acts in the internal backbone of xylan promoting the hydrolysis of β-1,4-glycosidic linkages. Exo-β-1,4-xylosidase (EC 3.2.1.37) or β-1,4-xylosidase acts in the nonreducing ends of xylan structure or xylooligosaccharides including xylobiose, thus releasing d-xylose monomers (Burlacu et al. 2016; Heinen et al. 2018; Polizeli et al. 2005).

Fig. 2.2
figure 2

Simplified structures of hemicelluloses with ramifications and the enzymes that act in their hydrolysis. Acetyl substitutions cleaved by acetyl xylan esterase are shown in pink. Arabinan removed from xylan chain by arabinofuranosidase is shown in yellow. Ferulic acid detached to arabinan by feruloyl esterase is shown in green. 4-O-Methyl-d-glucuronic acid removed by α-glucuronidase is shown in blue. The nonreductive end of an oligosaccharide cleaved by β-xylanase to obtain xylose monomers is shown in purple. The xylan backbone cleaved in the interior regions by endoxylanase releasing xylooligosaccharides is shown by the non-highlighted region

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The removal of the ramifications attached to xylan chain is promoted by a complex of enzymes. The most important enzymes are α-l-arabinofuranosidase, α-glucuronidase, acetyl xylan esterase, and feruloyl esterase (Fig. 2.2). α-l-Arabinofuranosidase (EC 3.2.1.55) or arabinofuranosidase cleaves the nonreductive end of the arabinan in the positions O-2 or O-3 releasing oligomers (Juturu and Wu 2013). α-Glucuronidase (EC 3.2.1.139) hydrolyses the 1-2-glycosidic bound between xylan and the side chain of 4-O-methyl-d-glucuronic acid, thus releasing glucuronic acid (Shallom and Shoham 2003). Acetyl xylan esterase (EC3.1.1.72) removes the acetyl substitutions attached on xylose releasing acetic acid (Tenkanen et al. 1992). Feruloyl esterase (EC3.1.1.73) or feruloyl xylan esterase hydrolyses the ester bond between ferulic acid and arabinose (Dilokpimol et al. 2016).

The mannan-degrading enzymes are β-mannosidase (EC 3.2.1.25), β-mannanase (EC 3.2.1.78), and β-glucosidase (3.2.1.21). There are also additional enzymes such as α-galactosidase (EC 3.2.1.22) and acetyl mannan esterase (EC 3.1.1.6) required to remove side-chain substituents, thus creating more sites for enzymatic hydrolysis (Fig. 2.3a–f) (Moreira and Filho 2008). According the Carbohydrate-Active Enzymes Database (CAZy), the mannanases are classified into GH families 5, 26, and 113 (Cantarel et al. 2009; Cruz 2013). These GH families have double-displacement mechanism holding anomeric configuration (De Marco et al. 2015).

Fig. 2.3
figure 3

Structures of mannans and pectins. Zymography of mannanase activity. (a) Mannan, (b) glucomannan, (e) galactomannan, and (f) galactoglucomannan. The mannan is hydrolyzed by Fig. 2.3 (continued) β-mannanase, while α-galactosidase releases galactose and acetyl mannan esterase releases acetyl groups. The mannobiose and glucomannobiose are hydrolyzed by (c) β-mannosidase and (d) β-glucosidase, to generate the monosaccharides mannose and glucose. SDS-PAGE (12%) and zymography of mannanase activity in crude extract, stained with Coomassie Brilliant Blue G-250 (g) and 0.1% Congo red (h). Molecular weight (MW), crude extract (CE) of soybean husk, crude extract concentrated (C) tenfold 30.000 NMWC in the concentration system QuixStand Benchtop (GE Healthcare), crude extract ultrafiltered (U) obtained by the same system as above. (i) R = H (PG) and CH3 (PMG); (j) PE and (k) R = H (PGL) and CH3 (PL). The scissors indicate the pectinases acting on the pectic substrates. Polygalacturonase (PG), polymethylgalacturonase (PMG), pectinesterase (PE), pectate lyase (PGL), and pectin lyase (PL)

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The complete hydrolysis of the mannan chain demands the synergy of exo-acting and endo-acting hydrolases together with accessory enzymes to reach complete hydrolysis. The heterosynergy is the synergistic action between main-chain and side-chain enzymes (β-mannanase and α-galactosidase) and homosynergy between two main-chain enzymes (β-mannosidase and β-mannanase) or between two side-chain enzymes (acetyl mannan esterase and α-galactosidase) reported in the degradation of mannan (Moreira and Filho 2008).

It is possible to visualize the presence and the size of the β-mannanase following the molecular markers using the technique of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Multiple bands stained for β-mannanase activity (Fig. 2.3g, h) can be detected using these techniques, which migrate at molecular masses ranging from 20 to 50 kDa. The variety of forms is usually described for hemicellulases from bacteria and fungi as the result of posttranslational modifications and differential messenger RNA (mRNA) processing. These mannanases can be isozymes, allozymes, and products of different alleles in the same gene or same molecules with different posttranscriptional modifications (De Marco et al. 2015).

2.4.4 Pectin-Degrading Enzyme System

Pectinases (EC 3.2.1.15) are a set of complex enzymes that degrade pectin in plant cell wall (Fig. 2.3i–k). Pectin-degrading enzymes are capable of degrading pectic substances per de-esterification (esterases) or depolymerization (lyases and hydrolases) reactions (Tariq and Latif 2012) and include polygalacturonases (PGs), pectinesterases (PEs), pectate lyases (PGLs), and pectin lyases (PLs) (Ahlawat et al. 2009; Polizeli et al. 2013). Polygalacturonases (PGs) are hydrolases, which cleave α-1,4-glycosidic linkage in homopolygalacturon backbone. They are classified into GH family 28 according to the Carbohydrate-Active Enzymes Database (CAZy) classification.

Endopolygalacturonases (EC 3.2.1.15) randomly attack the α-1,4-glycosidic bonds of the polysaccharide chain, thus releasing galacturonic acid oligomers. On the other hand, exopolygalacturonase type I (EC 3.2.1.67) acts in the nonreducing end and the d-galacturonic acid is hydrolyzed. Di-galacturonate is released as a result of the action of exopolygalacturonase type II (EC 3.2.1.82) in the nonreducing end of polygalacturonic acid (Khan et al. 2013). Pectinesterases (EC 3.1.1.11) are esterases that catalyze the methoxyl group of pectin forming pectic acid (Amin et al. 2017). Endopectin lyases (EC 4.2.2.10) accomplish trans-elimination reaction of pectin. Endopectate lyases (EC 4.2.2.2) and exopectate lyases (EC 4.2.2.9) cleave internal and nonreducing end of α-1,4-polygalacturonic acid, respectively, via β-elimination reaction (Yang et al. 2018).

2.4.5 Delignifying Enzymes

Delignifying enzymes are used for biomass pretreatment to remove lignin prior to bioconversion of biomass. As the lignin is removed or ruptured, the hemicellulose and cellulose fibrils become exposed. Thus, the glycoside hydrolases can have a better access to the substrate. In addition, the delignifying enzymes can remove some inhibitors (mainly phenolics), which interfere in the fermentation process (Kudanga and Roes-Hill 2014).

The most important delignifying enzymes are the laccases (EC1.10.3.2). They are multi-copper enzymes crucial to lignin degradation and the removal of phenolics. They can be divided into different enzyme families according to Sirim et al. (2011) as shown in Table 2.3. Laccases act by promoting the oxidation of one electron in a wide variety of substrates, notably the lignin phenylpropanoids (Mate and Alcalde 2017). Other important enzymes are the heme-peroxidases. They catalyze hydrogen peroxide-dependent oxidative degradation of lignin of phenolic (as manganese peroxidase-EC1.11.1.13) and in the non-phenolics (as lignin peroxidase-EC1.11.1.14) substrates (Plácido and Capareda 2015).

Table 2.3 Classification of laccase families, sequences, and structures
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2.5 Bioconversion of Biomass to Second-Generation Bioethanol

Among several alternative biomasses described in this chapter, the production of bioethanol from sugarcane (Saccharum spp.) is very advantageous. Sugarcane is rich in sucrose and allows the fermentation of this sugar by microorganisms resulting in first-generation (1G) bioethanol. On the other hand, the resulting lignocellulosic biomass (bagasse) can be used to produce second-generation (2G) bioethanol. To improve each step of the 2G ethanol production, it is necessary to know the composition of lignocellulosic residues.

Although the main targets for 2G ethanol production are sugarcane in Brazil (Polizeli et al. 2011, 2016, 2017) and corn in the USA (Li et al. 2018), there are studies reporting other biomasses, which were previously described in this chapter such as barley (Yang et al. 2015) and coffee (Nguyen et al. 2019). The key elements in the production of 2G bioethanol from sugarcane bagasse are shown in Fig. 2.4 and discussed below.

Fig. 2.4
figure 4

Scheme of the 2G bioethanol production process including pretreatment and bioconversion of lignocellulosic biomass

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2.6 Pretreatment of Lignocellulosic Biomass

Pretreatment of biomass may contribute to enhance the cell wall hydrolysis. It alters the chemical composition and physical structure of the substrates, since it aims to separate the carbohydrates from the lignin matrix. This process of separation benefits the enzymatic access to the cellulose and hemicellulose. The presence of lignin allows the unproductive binding and inactivation of the cellulases, thus making the high 2G ethanol yield practically impossible (Srinorakutara et al. 2013).

Some effects of the pretreatments are (a) breakage of the lignin structure and its connection with cellulose and hemicellulose, (b) improvement in the access of cellulases to cellulose due to the removal of hemicellulose and lignin, and (c) decrease in the degree of polymerization of cellulose and its crystallinity (van Dyk and Pletschke 2012; Nanda et al. 2014). According to Buckeridge et al. (2019), this step makes it possible to break down several covalent bonds between lignin and cellulose or hemicellulose, thus making the lignocellulosic biomass more accessible to the enzymatic attack.

There are broad ranges of pretreatments available, such as acidic, basic, or thermal. The acid pretreatment consists of the use of concentrated or diluted acids, associated with temperatures above 120 °C to decrease the rigidity of lignocellulosic biomass and increase the accessibility to the cellulose. In this type of pretreatment, hemicellulose is removed. The alkaline pretreatment removes lignin from the biomass and causes less damage to the sugars. Thermal pretreatment, also known as autohydrolysis, uses water, high temperature, and high pressure to break the lignocellulosic matrix (Michelin et al. 2014; Polizeli et al. 2015).

Each of these pretreatments has a different peculiarity, causing different chemical modifications as mentioned above. During pretreatment of the cellulosic residues, inhibitory compounds may be formed, such as furfural, hydroxymethylfurfural (HMF), phenolic compounds, and organic acids. These compounds have an impact on the action of hydrolytic enzymes, as well as on the microorganisms involved in the fermentative process (Srinorakutara et al. 2013; van Dyk and Pletschke 2012).

The surfactants are used as additives in order to avoid the adsorption of enzymes to their substrate in an unproductive way and to enhance the hydrolysis. The surface active agents are used in the bioconversion in the following ways: (a) during pretreatment, (b) during enzymatic saccharification, and (c) for the recycling of enzymes after plant cell wall degradation (Giese et al. 2012; van Dyk and Pletschke 2012).

2.7 Obtaining an Enzymatic Consortium

In order to achieve a sustainable 2G bioethanol production, the key to increase the efficiency of the saccharification is the thorough understanding of the plant cell wall structure since enzymatic degradation of lignocellulosic biomass is required to yield most of the fermentable sugars. According to Buckeridge and de Souza (2014), plant cell walls show regions of polymers that relate with other polymer chains, such as xyloglucan and cellulose. The interaction among the macromolecules of cell walls of biomass makes its total hydrolysis impossible for endoglucanases to attack glycosidic bonds of cellulose. Moreover, there is a need for the formulation of efficient enzymatic cocktails with enzymes with endo/exo characters as well as branching and debranching feature. The synergism between the enzymes responsible for cell wall hydrolysis considers that the structure of the polysaccharides present in the lignocellulosic biomass may play an important role in the kinetic hydrolysis properties (Mohanram et al. 2013; De Souza et al. 2013; Buckeridge and de Souza 2014).

De Souza et al. (2013) proposed a theoretical scheme composed of the enzymes necessary for the saccharification of the cell wall of sugarcane, which included hemicellulases, cellulases, β-galactosidases, lichenases, and other accessory enzymes. Commercial enzymatic cocktails available for the depolymerization of lignocellulosic materials are loosely defined as a complex mixture containing about 80–200 proteins, including hemicellulases, cellulases, and accessory enzymes (Mohanram et al. 2013). To improve the performance of enzyme cocktails, it took years of bioprospecting studies, optimization of strains through genetic engineering for the production of enzymes, and development of pretreatment strategies since these are the major technical challenges in an efficient 2G ethanol production process.

The enzymes act in synergy for substrate degradation for ethanol production. van Dyk and Pletschke (2012) as well as Li et al. (2014) have demonstrated that the understanding of synergism is of fundamental importance to clarify the mechanism of the action of the enzymes alone and the interaction between them. The enzymatic saccharification is beneficial when compared to other methods because it is a more specific and cleaner process besides the reduction of the formation of inhibitory compounds. During the enzymatic hydrolysis of lignocellulosic biomass, oligosaccharides, disaccharides, and monomers are formed. They cause the inhibition of β-glucosidase and β-xylosidase associated with the saccharification as previously mentioned in this chapter. High concentrations of these sugars may have an impact on the hydrolytic efficiency of the lignocellulosic residue (van Dyk and Pletschke 2012).

For the bioconversion of lignocellulosic residue to fermentable sugars, it is necessary to decrease the cost of enzymes, in addition to studies aiming to enhance the efficiency of the enzymes used in the cell wall hydrolysis. In this way, an effective tool to reduce the cost of the various hydrolytic enzymes would be their immobilization. This process leads to an economy in the process of producing biofuels besides allowing the recovery and reuse of the enzymes (Borges et al. 2014; Maitan-Alfenas et al. 2015).

2.8 Fermentation for the Production of Second-Generation Bioethanol

Alcoholic fermentation consists of the transformation of the sugars from the hydrolysate into ethanol, CO2, and energy due to the catalytic action of the yeast or bacteria. There are two modes of operations for bioethanol production of lignocellulosic biomass, namely, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) (Fig. 2.4b). Efforts at process integration are still constrained by obstacles. There is a need of industrial microorganisms capable to assimilate pentoses and hexoses as well as the control in the formation and elimination of inhibitory compounds resulting from the whole process. Another factor that must be studied is the standardization of the ideal conditions for the integration of two or more processes to be simultaneously performed in a single bioreactor (Ramos et al. 2015).

The pentoses are fermented by yeasts of the genus Candida, Komagataella, and Meyerozyma, but studies with these yeasts are still limited. The hexoses are assimilated by yeasts Saccharomyces cerevisiae, which have high fermentative efficiency. This efficiency is due to the rapid growth of yeasts as well as the ability to metabolize sugars. In addition, they have the ability to produce and consume ethanol and are tolerant to high concentrations of this compound and resistant to low levels of oxygen. Another bacterial producer of bioethanol, Zymomonas mobilis, has shown promising results for the large-scale production of bioethanol.

2.9 Conclusions

The dependence on nonrenewable and highly polluting energy sources has led to seek alternative fuel sources. Clean energy has the advantage to lower CO2 emissions, providing energy capable of reducing the production of greenhouse gases, maintaining environmental sustainability, mitigating climate change, and stimulating the global economy. Thus, the growing demand for biofuels has motivated the use of biological materials, specifically photosynthetic organisms, as the raw materials.

It is known that billions of tons of plant-based residues are obtained across the world. However, only a small fraction of this organic waste is used for bioenergy and bioproducts. Lignocellulosic biomass is mainly constituted by a polysaccharide-lignin complex with varied composition depending on the plant variety. The deconstruction of this intricate complex is one of the main bottlenecks in the conversion of biomass to biofuels. The development of novel technologies is necessary to make the hydrolysis and saccharification economically viable. Genetic modifications of plants resulting in a vegetal with increased content of biodegradable and fermentable saccharides are some of the perspectives. Another focus in plants is to increase the fiber content, as the energy cane, considering 2G ethanol production from sugarcane.

Another problem confronted with 2G ethanol production is the biochemical structure of the lignin because it blocks the access to cellulose. Various pretreatments of biomass alter the chemical/physical structure of the plant cell walls, thus separating the polysaccharides from lignin and allowing better enzymatic access to cellulose, hemicellulose, and pectin. One of the problems of such pretreatments is the formation of inhibitory compounds as furfural, hydroxymethylfurfural, phenolic compounds, and organic acids, which inhibit enzymatic action and microbial activity during bioconversion. Hydrothermal pretreatment and steam explosion are alternative strategies to acid, alkaline, and other pretreatments. Delignifying enzymes may contribute as a biomass pretreatment for bioethanol production since the removal of lignin exposes hemicellulose and cellulose fibrils, and consequently, glycoside hydrolase can have better access to the substrates.

In all cases, it is important to have an in-depth and detailed knowledge of the structure of the biomass cell constituents and how they interact aiming at the production of fermentable sugars. Immunofluorescence techniques using antibodies against the macromolecules of the biomass cell wall have given excellent know-how, and a glycolic code of sugarcane walls has also been reported in the literature.

Enzymatic saccharification of raw or pretreated biomass for the liberation of fermentable sugars by yeast is advantageous when compared to the chemical methods because of being a selective, cleaner, and less energy-intensive process, which produces less amounts of inhibitory biological compounds. For this purpose, commercial enzyme cocktails obtained from recombinant microorganisms, such as bacteria and filamentous fungi, are used. Enzymatic hydrolysis process is largely successful in many cases, but problems, such as the viability of the strain, mycotoxins, contamination, genetic instability, reproducibility, inhibition of the enzyme by end products, and other molecular complications, have been reported. Some proposed alternatives include bioprospecting studies of mesophilic and thermophilic strains and optimization of strains through genetic engineering for the production and thermostabilization of enzymes.

Efficient enzymatic cocktails include the cellulases, hemicellulases, pectinases, ligninases, and, accessory enzymes and proteins, such as auxiliary activity 9 and swollenin. Synergism is expected between the hydrolitic enzymes, auxiliary activity enzymes, and swollenins because they can act together favoring the oxidative cleavage of glycosidic bonds and deagglomeration of crystalline cellulose, thus giving access to cellulases and facilitating enzymatic hydrolysis. Besides, the debranching of hemicellulose is favored by the use of enzymatic cocktail. The addition of ligninolytic enzymes in the cocktail helps in removing some phenolic compounds that may interfere in the final fermentation process. Usually, the combinatory action of enzymes reverts to higher levels of fermentable sugars. Thus, the understanding of the synergic mechanisms between the hydrolytic enzymes is fundamental to improve the saccharification process.

A perturbing factor for large-scale bioconversion studies, which still needs attention, is the high cost of commercial hydrolytic enzymes. An alternative could be the immobilization or co-immobilization of the enzymes. This process sometimes results in enzymatic hyperactivation and allows various reuses of the biocatalysts (enzyme and support). Furthermore, as may be concluded, various studies must be developed aiming to achieve the total success of the lignocellulosic biomass conversion in fermentable sugars. Besides, it is a clean process with many advantages that need recognition.