**Bioethanol**


[28] PN-EN 16723-2:2017. Natural gas and biomethane for use in transport and biomethane for injection in the natural gas network—Part 2: Automotive fuel specifications

[29] List of upgrading plants. December 2016. IEA Bioenergy Task 37. www.iea-biogas.net

[30] Statistical Report of the European Biogas Association. 2016. http://european-biogas. eu/2016/12/21/eba-launches-6th-edition-of-the-statistical-report-of-the-european-bio

[Accessed: 2018-01-29]

52 Biofuels - State of Development

gas-association/ [Accessed: 2018-01-18]

**Chapter 4**

**Provisional chapter**

**Pretreatment Empty Fruit Bunch of Oil Palm Tree for**

**Pretreatment Empty Fruit Bunch of Oil Palm Tree for** 

Empty fruit bunch of oil palm tree (EFBOPT), solid waste of palm oil industries, is potential for raw materials of biofuel especially bioethanol production because of its cellulose and hemicellulose contents. There are four steps to produce bioethanol, called the second generation bioethanol, from EFBOPT or other lignocellulosic materials. The steps are (a) pretreatment of lignocellulose biomass into cellulose/hemicellulose, (b) hydrolysis of cellulose/hemicellulose into monosaccharides, (c) fermentation of monosaccharides into bioethanol, and (d) recovery of bioethanol from medium fermentation broth. Pretreatment steps are the key success factor to convert lignocellulosic materials into bioethanol. This paper will review EFBOPT and pretreatment steps, including physical pretreatments, physicochemical pretreatments, and biologi-

**Keywords:** bioethanol, biomass, empty fruit bunch of oil palm tree, lignocellulose,

Depletion of fossil fuels, increasing of climate changes, and improvement of world energy consumption [1] have directed towards the development of biofuel production [2]. Biofuels are able to replace petroleum-based fuels, decrease greenhouse gas emission [3], and have significant potential sustainability [4]. Biofuels can be as gaseous (methane or hydrogen) or liquid (biodiesel, biobutanol, or bioethanol) forms and are commonly generated from agricultural materials. Either agricultural commodities or agricultural waste materials can be used for developing biofuels; for example, vegetable oil [5, 6] is used for biodiesel and agricultural

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.76587

**Improving Enzymatic Saccharification**

**Improving Enzymatic Saccharification**

Sutikno Sutikno and Muhammad Kismurtono

Sutikno Sutikno and Muhammad Kismurtono

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76587

**Abstract**

cal pretreatments.

pretreatment

**1. Introduction**

#### **Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification**

DOI: 10.5772/intechopen.76587

Sutikno Sutikno and Muhammad Kismurtono Sutikno Sutikno and Muhammad Kismurtono

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76587

#### **Abstract**

Empty fruit bunch of oil palm tree (EFBOPT), solid waste of palm oil industries, is potential for raw materials of biofuel especially bioethanol production because of its cellulose and hemicellulose contents. There are four steps to produce bioethanol, called the second generation bioethanol, from EFBOPT or other lignocellulosic materials. The steps are (a) pretreatment of lignocellulose biomass into cellulose/hemicellulose, (b) hydrolysis of cellulose/hemicellulose into monosaccharides, (c) fermentation of monosaccharides into bioethanol, and (d) recovery of bioethanol from medium fermentation broth. Pretreatment steps are the key success factor to convert lignocellulosic materials into bioethanol. This paper will review EFBOPT and pretreatment steps, including physical pretreatments, physicochemical pretreatments, and biological pretreatments.

**Keywords:** bioethanol, biomass, empty fruit bunch of oil palm tree, lignocellulose, pretreatment

#### **1. Introduction**

Depletion of fossil fuels, increasing of climate changes, and improvement of world energy consumption [1] have directed towards the development of biofuel production [2]. Biofuels are able to replace petroleum-based fuels, decrease greenhouse gas emission [3], and have significant potential sustainability [4]. Biofuels can be as gaseous (methane or hydrogen) or liquid (biodiesel, biobutanol, or bioethanol) forms and are commonly generated from agricultural materials. Either agricultural commodities or agricultural waste materials can be used for developing biofuels; for example, vegetable oil [5, 6] is used for biodiesel and agricultural

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**2. Empty fruit bunch of oil palm tree**

lignin [34, 35].

EFBOPT is solid waste residue generated from palm oil factories. Fresh fruit bunches are harvested from oil palm tree (**Figure 2**) and then sterilized in a steam sterilizer for inactivating enzymes that present in pericarp and loosening fruits from bunches. The sterilized bunches are then fed into a rotary drum thresher to separate the sterilized fruit from bunches without fruit, which are called as empty fruit bunch of oil palm tree (EFBOPT). The sterilized fruits are further processed for palm oil production and the EFBOPT (**Figure 3**) are conveyed to the damping ground and left unused. It was reported that each 100 ton of fresh fruit bunches yielded 14 tons oil-rich fiber and 20–22 tons of EFBOPT [32]. EFBOPT is dry and brown; its shape is not uniform with low bulk density; and its length and width can vary from 17 to

Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification

http://dx.doi.org/10.5772/intechopen.76587

Like other lignocellulose materials, native EFBOPT fiber is mainly constructed from a complex matrix of three main polymers (**Figure 4**) [33], namely cellulose, hemicellulose, and lignin. The EFBOPT fiber consists of 44.2–50.0% cellulose, 22.0–33.5% hemicellulose, and 20.4–21.0%

Cellulose, a major constitutes of FEBOPT, is closely associated with hemicelluloses and lignin, and the separation of the cellulose from hemicelluloses and lignin requires intensive physical and chemical treatments. Cellulose is a linear polymer of D-glucopyranose units bound by β-1-4-glycosidic linkages (**Figure 5**). The successive glucose units are rotated by 180o

tive to each other to form a cellobiose unit as a repeating unit of cellulose chains ended by a hydroxyl group. The hydroxyl (OH) groups in the cellulose act as functional groups which are able to interact each other or with O-, N-, and S-groups forming hydrogen bonds. Hydrogen bonds also occur between the OH-group of cellulose and water. Through hydrogen bond, cellulose chains are packed together to set up highly crystalline microfibrils. An individual microfibril contains ten of glucan chains in a parallel orientation [36]. This microfibril fraction

**Figure 2.** Oil palm tree (A) with fresh fruit brunches (B). Source available from: http://www.thestar.com.my/~/media/ online/2014/09/07/08/01/str2\_ma\_0809\_p10a-lead-pic.ashx/?w=620&h=413&crop=1&hash=B2A67ABD156961DCE9E42C

52EA4D72C3F9503686 (accessed on February 25, 2018).

can be hydrolyzed into glucose either by enzymatic or by chemical methods [37].

rela-

57

30 cm long and 25 to 35 cm wide depending on the size of fresh fruit bunch [32].

**Figure 1.** Steps of second generation bioethanol production from agricultural waste biomass. Source available from [30]: Knauf and Moniruzzaman.

wastes [7, 8] were processed for developing methane or hydrogen gases. Meanwhile, bioethanol can be produced either from agricultural high starch/sugar-containing commodities such as corn [9] and sugarcane [10] or from agricultural solid wastes such as non-edible parts of cassava [11], banana peels [12], cocoa-pod waste [13], rice straw [14, 15], sugarcane bagasse [16–19], sorghum bagasse [20], and empty fruit bunch of oil palm tree (EFBOPT) [21–24]. Bioethanol produced from starch/sugar containing commodities is called the first generation bioethanol, and one produced from agricultural solid wastes or lignocelluloses is called the second generation bioethanol.

Among agricultural materials, oil palm tree (*Elaeis guineensis* Jacq.) is considered as a major source of biofuel. In the world, oil palm tree is planted in about 43 developing countries and plantation area increased eight time in the past four decades to over 12 million ha in 2009 [25]. In a good condition, oil palm tree can yield about 4.5 ton oil and 7–8 ton EFBOPT/ha/year. Unlike palm oil which can be processed directly into biodiesel, EFBOPT has to be pretreated before converted into bioethanol [26].

There are four steps to produce bioethanol, called the second generation bioethanol, from lignocellulosic materials [27–29] such as EFBOPT.They are (a) pretreatment of lignocellulose biomass into cellulose/hemicellulose, (b) cellulose/hemicellulose hydrolysis into monosaccharides, (c) monosaccharides fermentation into bioethanol, and (d) bioethanol recovery (**Figure 1**) [30]. The key success factor to convert EFBOPT into bioethanol is pretreatment step [31]; therefore, this paper will review the EFBOPT pretreatment and discuss recent research results which significantly enhanced enzymatic saccharifications.

## **2. Empty fruit bunch of oil palm tree**

wastes [7, 8] were processed for developing methane or hydrogen gases. Meanwhile, bioethanol can be produced either from agricultural high starch/sugar-containing commodities such as corn [9] and sugarcane [10] or from agricultural solid wastes such as non-edible parts of cassava [11], banana peels [12], cocoa-pod waste [13], rice straw [14, 15], sugarcane bagasse [16–19], sorghum bagasse [20], and empty fruit bunch of oil palm tree (EFBOPT) [21–24]. Bioethanol produced from starch/sugar containing commodities is called the first generation bioethanol, and one produced from agricultural solid wastes or lignocelluloses is called the

**Figure 1.** Steps of second generation bioethanol production from agricultural waste biomass. Source available from [30]:

Among agricultural materials, oil palm tree (*Elaeis guineensis* Jacq.) is considered as a major source of biofuel. In the world, oil palm tree is planted in about 43 developing countries and plantation area increased eight time in the past four decades to over 12 million ha in 2009 [25]. In a good condition, oil palm tree can yield about 4.5 ton oil and 7–8 ton EFBOPT/ha/year. Unlike palm oil which can be processed directly into biodiesel, EFBOPT has to be pretreated

There are four steps to produce bioethanol, called the second generation bioethanol, from lignocellulosic materials [27–29] such as EFBOPT.They are (a) pretreatment of lignocellulose biomass into cellulose/hemicellulose, (b) cellulose/hemicellulose hydrolysis into monosaccharides, (c) monosaccharides fermentation into bioethanol, and (d) bioethanol recovery (**Figure 1**) [30]. The key success factor to convert EFBOPT into bioethanol is pretreatment step [31]; therefore, this paper will review the EFBOPT pretreatment and discuss recent research results which

second generation bioethanol.

Knauf and Moniruzzaman.

56 Biofuels - State of Development

before converted into bioethanol [26].

significantly enhanced enzymatic saccharifications.

EFBOPT is solid waste residue generated from palm oil factories. Fresh fruit bunches are harvested from oil palm tree (**Figure 2**) and then sterilized in a steam sterilizer for inactivating enzymes that present in pericarp and loosening fruits from bunches. The sterilized bunches are then fed into a rotary drum thresher to separate the sterilized fruit from bunches without fruit, which are called as empty fruit bunch of oil palm tree (EFBOPT). The sterilized fruits are further processed for palm oil production and the EFBOPT (**Figure 3**) are conveyed to the damping ground and left unused. It was reported that each 100 ton of fresh fruit bunches yielded 14 tons oil-rich fiber and 20–22 tons of EFBOPT [32]. EFBOPT is dry and brown; its shape is not uniform with low bulk density; and its length and width can vary from 17 to 30 cm long and 25 to 35 cm wide depending on the size of fresh fruit bunch [32].

Like other lignocellulose materials, native EFBOPT fiber is mainly constructed from a complex matrix of three main polymers (**Figure 4**) [33], namely cellulose, hemicellulose, and lignin. The EFBOPT fiber consists of 44.2–50.0% cellulose, 22.0–33.5% hemicellulose, and 20.4–21.0% lignin [34, 35].

Cellulose, a major constitutes of FEBOPT, is closely associated with hemicelluloses and lignin, and the separation of the cellulose from hemicelluloses and lignin requires intensive physical and chemical treatments. Cellulose is a linear polymer of D-glucopyranose units bound by β-1-4-glycosidic linkages (**Figure 5**). The successive glucose units are rotated by 180o relative to each other to form a cellobiose unit as a repeating unit of cellulose chains ended by a hydroxyl group. The hydroxyl (OH) groups in the cellulose act as functional groups which are able to interact each other or with O-, N-, and S-groups forming hydrogen bonds. Hydrogen bonds also occur between the OH-group of cellulose and water. Through hydrogen bond, cellulose chains are packed together to set up highly crystalline microfibrils. An individual microfibril contains ten of glucan chains in a parallel orientation [36]. This microfibril fraction can be hydrolyzed into glucose either by enzymatic or by chemical methods [37].

**Figure 2.** Oil palm tree (A) with fresh fruit brunches (B). Source available from: http://www.thestar.com.my/~/media/ online/2014/09/07/08/01/str2\_ma\_0809\_p10a-lead-pic.ashx/?w=620&h=413&crop=1&hash=B2A67ABD156961DCE9E42C 52EA4D72C3F9503686 (accessed on February 25, 2018).

**Figure 3.** Empty fruit bunch of oil palm tree (EFBOPT) on damping ground. Source available from: https://s3-apsoutheast-2.amazonaws.com/ecostore-static-assets/Page+images/Palm+Oil+Page/Empty-fruit-bunches.jpg (accessed on February 25, 2018).

> **Figure 5.** Chemical structure of cellulose. Source: available from https://www.intechopen.com/books/cellulosefundamental-aspects/cellulose-microfibril-angle-in-wood-and-its-dynamic-mechanical-significance (accessed on

Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification

http://dx.doi.org/10.5772/intechopen.76587

59

**Figure 6.** Chemical structure of hemicellulose. Source: available from https://www.researchgate.net/figure/Chemicalstructure-of-hemicellulose-compounds-xylan-and-glucomannan-are-the-most\_fig4\_266026683 (accessed on February

February 26, 2018).

26, 2018).

**Figure 4.** Polymer structure of lignocellulosic biomass. Source [33] Quintero-Ramirez.

Hemicellulose is a polymer consisting of heteropolymers of D-xylose, D-mannose, D-glucose, D-galactose, and L-arabinose, in the form of linear and branched (**Figure 6**). Its structure is not crystalline and is, therefore, easier to hydrolyze than cellulose [38]. Hemicelluloses usually form cross-linked to other polysaccharides, proteins, or lignin. Xylans are considered to be the main interface between lignin and other carbohydrates [39].

Lignin is a cross-linked aromatic, hydrophobic, and complex polymer consisting of three different phenyl-propane precursor monomer units which are very difficult to biodegrade (**Figure 7**). Lignin is mostly observed as an integral part of the plan cell wall, embedded in a polymer matrix of cellulose and hemicellulose. Thus, lignin is the most non-biodegradable component of the plant cell wall [36].

Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification http://dx.doi.org/10.5772/intechopen.76587 59

**Figure 5.** Chemical structure of cellulose. Source: available from https://www.intechopen.com/books/cellulosefundamental-aspects/cellulose-microfibril-angle-in-wood-and-its-dynamic-mechanical-significance (accessed on February 26, 2018).

Hemicellulose is a polymer consisting of heteropolymers of D-xylose, D-mannose, D-glucose, D-galactose, and L-arabinose, in the form of linear and branched (**Figure 6**). Its structure is not crystalline and is, therefore, easier to hydrolyze than cellulose [38]. Hemicelluloses usually form cross-linked to other polysaccharides, proteins, or lignin. Xylans are considered to be the

**Figure 3.** Empty fruit bunch of oil palm tree (EFBOPT) on damping ground. Source available from: https://s3-apsoutheast-2.amazonaws.com/ecostore-static-assets/Page+images/Palm+Oil+Page/Empty-fruit-bunches.jpg (accessed on

Lignin is a cross-linked aromatic, hydrophobic, and complex polymer consisting of three different phenyl-propane precursor monomer units which are very difficult to biodegrade (**Figure 7**). Lignin is mostly observed as an integral part of the plan cell wall, embedded in a polymer matrix of cellulose and hemicellulose. Thus, lignin is the most non-biodegradable

main interface between lignin and other carbohydrates [39].

**Figure 4.** Polymer structure of lignocellulosic biomass. Source [33] Quintero-Ramirez.

component of the plant cell wall [36].

February 25, 2018).

58 Biofuels - State of Development

**Figure 6.** Chemical structure of hemicellulose. Source: available from https://www.researchgate.net/figure/Chemicalstructure-of-hemicellulose-compounds-xylan-and-glucomannan-are-the-most\_fig4\_266026683 (accessed on February 26, 2018).

**Figure 7.** Chemical structure of lignin. Source: available from http://palaeos.com/plants/glossary/images/Lignin.gif (accessed on February 26, 2018).

and pyrolysis. The chemical and physicochemical pretreatments consist of acid (hydrochloric acid, phosphoric acid, and sulfuric acid), alkali (ammonia and sodium hydroxide), explosion

**Figure 8.** Effects of pretreatment on degrading-enzyme accessibility, and bioethanol or biogas yield and productivity.

Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification

http://dx.doi.org/10.5772/intechopen.76587

61

explosion, SO2

(chlorine dioxide, nitrogen dioxide, and sulfur dioxide), oxidizing agents (hydrogen peroxide, ozone, and wet oxidation), and solvent extraction of lignin (benzene-water extraction, butanol-water extraction, ethanol-water extraction, ethylene glycol extraction, and swelling agents), and biological. Biological pretreatments commonly utilize fungi and actinomycetes as microbial producing enzymes which can degrade lignin compounds. Advantages and disadvantages of some different pretreatments are well tabulated (**Table 1**) by Brodeur et al. and

The main objective of the acid pretreatment is to solubilize lignin and hemicelluloses chemically so that the cellulose is more accessible to enzymes. Either diluted or concentrated acid can be utilized to perform this acid pretreatment. The diluted acid pretreatment method is more attractive because of less inhibitor compound formation, such as furfural, 5-hydroxymethylfurfural, phenolic acids, and aldehydes. In addition, diluted acids are less toxic, corrosive, hazardous, and corrosive, as well as more feasible for industrial scale. The diluted acid pretreatment methods have been developed in different types of reactors including percolation,

explosion, and steam explosion), gas

(ammonia fiber explosion/AFEX, CO<sup>2</sup>

Source: available from Taherzadeh and Karimi [40].

Maurya et al. [42, 43].

**3.1. Acid pretreatments**

#### **3. Pretreatments**

Pretreatment is actions given to biomass materials such as EFBOPT for enhancing the cellulose reactivity with cellulase enzymes and for increasing the yield of fermentable sugars. There are eight requirements for effective and economical pretreatment; they are (a) producing higher reactive cellulose fibers for enzymatic attachment, (b) producing less residues, (c) avoiding formation of compound inhibitors for hydrolytic enzymes and fermenting microorganisms, (d) avoiding destruction of celluloses and hemicelluloses, (e) reducing of material cost for setting upper-treatment reactors, (f) minimizing the energy demand, (g) reducing the cost of size reduction for feedstock, (h) consuming little or no chemical, and (i) using a cheap or no chemical [40]. The goal of pretreatment is to disrupt the crystallinity of cellulose, to open lignin and hemicellulose protection, to increase EFBOPT surface accessibility, and to decrease the degree of hemicellulose acetylation [41]. Pretreatment can increase significantly bioethanol or biogas yield and productivity. Effects of pretreatment on the degrading enzyme accessibility, bioethanol or biogas yield, and productivity from lignocellulosic materials were diagrammatically (**Figure 8**) shown by Taherzadeh and Karimi [40].

Based on actions given to biomass materials, pretreatment methods are classified into three groups, namely physical, chemical and physicochemical, and biological pretreatment methods [40]. The physical methods include milling (ball milling, colloid milling, hammer milling, tworoll milling, and vibrant energy milling), irradiation (electron-beam irradiation, gamma-ray irradiation, and microwave irradiation), and expansion, extrusion, high pressure, hydrothermal, Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification http://dx.doi.org/10.5772/intechopen.76587 61

**Figure 8.** Effects of pretreatment on degrading-enzyme accessibility, and bioethanol or biogas yield and productivity. Source: available from Taherzadeh and Karimi [40].

and pyrolysis. The chemical and physicochemical pretreatments consist of acid (hydrochloric acid, phosphoric acid, and sulfuric acid), alkali (ammonia and sodium hydroxide), explosion (ammonia fiber explosion/AFEX, CO<sup>2</sup> explosion, SO2 explosion, and steam explosion), gas (chlorine dioxide, nitrogen dioxide, and sulfur dioxide), oxidizing agents (hydrogen peroxide, ozone, and wet oxidation), and solvent extraction of lignin (benzene-water extraction, butanol-water extraction, ethanol-water extraction, ethylene glycol extraction, and swelling agents), and biological. Biological pretreatments commonly utilize fungi and actinomycetes as microbial producing enzymes which can degrade lignin compounds. Advantages and disadvantages of some different pretreatments are well tabulated (**Table 1**) by Brodeur et al. and Maurya et al. [42, 43].

#### **3.1. Acid pretreatments**

**3. Pretreatments**

60 Biofuels - State of Development

(accessed on February 26, 2018).

Pretreatment is actions given to biomass materials such as EFBOPT for enhancing the cellulose reactivity with cellulase enzymes and for increasing the yield of fermentable sugars. There are eight requirements for effective and economical pretreatment; they are (a) producing higher reactive cellulose fibers for enzymatic attachment, (b) producing less residues, (c) avoiding formation of compound inhibitors for hydrolytic enzymes and fermenting microorganisms, (d) avoiding destruction of celluloses and hemicelluloses, (e) reducing of material cost for setting upper-treatment reactors, (f) minimizing the energy demand, (g) reducing the cost of size reduction for feedstock, (h) consuming little or no chemical, and (i) using a cheap or no chemical [40]. The goal of pretreatment is to disrupt the crystallinity of cellulose, to open lignin and hemicellulose protection, to increase EFBOPT surface accessibility, and to decrease the degree of hemicellulose acetylation [41]. Pretreatment can increase significantly bioethanol or biogas yield and productivity. Effects of pretreatment on the degrading enzyme accessibility, bioethanol or biogas yield, and productivity from lignocellulosic materials were

**Figure 7.** Chemical structure of lignin. Source: available from http://palaeos.com/plants/glossary/images/Lignin.gif

Based on actions given to biomass materials, pretreatment methods are classified into three groups, namely physical, chemical and physicochemical, and biological pretreatment methods [40]. The physical methods include milling (ball milling, colloid milling, hammer milling, tworoll milling, and vibrant energy milling), irradiation (electron-beam irradiation, gamma-ray irradiation, and microwave irradiation), and expansion, extrusion, high pressure, hydrothermal,

diagrammatically (**Figure 8**) shown by Taherzadeh and Karimi [40].

The main objective of the acid pretreatment is to solubilize lignin and hemicelluloses chemically so that the cellulose is more accessible to enzymes. Either diluted or concentrated acid can be utilized to perform this acid pretreatment. The diluted acid pretreatment method is more attractive because of less inhibitor compound formation, such as furfural, 5-hydroxymethylfurfural, phenolic acids, and aldehydes. In addition, diluted acids are less toxic, corrosive, hazardous, and corrosive, as well as more feasible for industrial scale. The diluted acid pretreatment methods have been developed in different types of reactors including percolation,


plug flow, shrinking-bed, batch, flow-through reactor and countercurrent reactors [40]. There are two approach processes of dilute acid pretreatment methods, namely (a) high temperature (e.g., 180°C) during a short period of time and (b) lower temperature (e.g., 120°C) for

**Table 1.** Advantages and disadvantages of different pretreatment methods of lignocellulosic biomass [42, 43].

**Advantages Disadvantages**

Milling • Cellulose crystallinity and degree of

increase

• Lignin transforms

Wet oxidation • Majority of hemicellulose and lignin are solubilized

• Hemicellulose is solubilized • Yield of glucose is high

• Inhibitor compounds are avoided

Steam explosions • It is low cost

• Particle size decreases

polymerization are decreased

• Specific surface area and pore size

high hydrolysis yields [37, 44]. Other acids used for cellulosic material pretreatments are ace-

AFEX is one of the physicochemical pretreatments which treated lignocellulosic biomass with liquid ammonia at relatively moderate temperature (90–100°C) for about 30–60 min, then followed by a rapid pressure release [50]. A rapid expansion of the liquid ammonia causes swelling and physical disruption of lignocellulosic fibers and partial reduction of cellulose crystallinity. AFEX process is able to either modify or effectively decrystallization of cellulose and lignin fractions [51]. AFEX removes the least acetyl groups of biomass by deacetylation process, so that the digestibility of lignocellulosic biomass increases [52, 53]. The main advantage of the AFEX is that it does not produce inhibitors for the downstream biological processes, so water wash is not needed. AFEX is more effective for agricultural residues [54]. The AFEX process conditions (ammonia loading, temperature, blowdown pressure, moisture content of biomass, and residence time) have been optimized [55]. At the optimal conditions, AFEX can convert over 90% cellulose and hemicellulose to fermentable sugars for a broad variety of biomass materials including EFBOPT. Due to high volatility, ammonia is easy to be recovered and recycled [56], and leaving the dried biomass ready for enzymatic hydrolysis [57]. After pre-pretreatment, ammonia must be recycled in order to reduce the cost and protect the environment [56].

, formic acid, hydrochloric acid (HCl), maleic acid, oxalic acid, phosphoric

SO4

• Power and energy consumptions are high

http://dx.doi.org/10.5772/intechopen.76587

63

• Acid catalyst is needed to make process efficient with high lignin content material

• Costs of oxygen and alkaline catalyst are high

• Partial hemicellulose degrades

Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification

• Toxic compound is formed

which provides

longer retention time (30–90 min). The most widely used acid is dilute H2

tic acid, C2

**Pretreatment method**

> H4 O3

**3.2. AFEX pretreatment**

acid, and nitric acid [45–49].


**Table 1.** Advantages and disadvantages of different pretreatment methods of lignocellulosic biomass [42, 43].

plug flow, shrinking-bed, batch, flow-through reactor and countercurrent reactors [40]. There are two approach processes of dilute acid pretreatment methods, namely (a) high temperature (e.g., 180°C) during a short period of time and (b) lower temperature (e.g., 120°C) for longer retention time (30–90 min). The most widely used acid is dilute H2 SO4 which provides high hydrolysis yields [37, 44]. Other acids used for cellulosic material pretreatments are acetic acid, C2 H4 O3 , formic acid, hydrochloric acid (HCl), maleic acid, oxalic acid, phosphoric acid, and nitric acid [45–49].

#### **3.2. AFEX pretreatment**

**Pretreatment method**

62 Biofuels - State of Development

Ammonia recycle percolation

CO2

Acid • Yield of glucose is high

• Hemicellulose is solubilized

• It is suitable for low lignin content

• Cellulose becomes more accessible

• Formation of inhibitors is low

• Majority of lignin is removed

• Cellulose content after pretreatment is

• Herbaceous materials are most affected

• Degree of cellulose polymerization is

• Hemicelluloses are partial hydrolyzed

• Inhibitory compounds are not formed • It is non-flammability and relatively

• Recovery after extraction is easy • It is environmentally acceptable

• Chemicals are not required • Environmental conditions are mild

AFEX • Effectiveness for herbaceous material is high

biomass

Alkali • Lignin is efficiently removed

high

Biological • Energy requirements are low

reduced

explosion • Accessible surface area increase

Liquid hot water • Hemicellulose is separated from rest of feedstock

> • It is no need for catalyst • Hemicellulose is hydrolyzed

cheap

• Lignin is removed

**Advantages Disadvantages**

• Formation of inhibitors is low • Lignin structure changes

• Costs of acids are high • Acid recovery is needed

high

• Costs of corrosive resistant equipment are

• Process effectiveness decreases with increas-

• Fermentation inhibitors are formed

• Recycling of ammonia is needed

ing biomass lignin content

• Costs of ammonia are high

• Process rate is slow • Treatment rate is very low

application

• Costs of alkaline catalyst are high • Alteration of lignin structure

• Energy costs and liquid loading are high

• It is not very effective for commercial

• Pressure requirements are very high

• Energy/water input is high

(cellulose/lignin)

• Solid mass left over will need to be dealt with

AFEX is one of the physicochemical pretreatments which treated lignocellulosic biomass with liquid ammonia at relatively moderate temperature (90–100°C) for about 30–60 min, then followed by a rapid pressure release [50]. A rapid expansion of the liquid ammonia causes swelling and physical disruption of lignocellulosic fibers and partial reduction of cellulose crystallinity. AFEX process is able to either modify or effectively decrystallization of cellulose and lignin fractions [51]. AFEX removes the least acetyl groups of biomass by deacetylation process, so that the digestibility of lignocellulosic biomass increases [52, 53]. The main advantage of the AFEX is that it does not produce inhibitors for the downstream biological processes, so water wash is not needed. AFEX is more effective for agricultural residues [54]. The AFEX process conditions (ammonia loading, temperature, blowdown pressure, moisture content of biomass, and residence time) have been optimized [55]. At the optimal conditions, AFEX can convert over 90% cellulose and hemicellulose to fermentable sugars for a broad variety of biomass materials including EFBOPT. Due to high volatility, ammonia is easy to be recovered and recycled [56], and leaving the dried biomass ready for enzymatic hydrolysis [57]. After pre-pretreatment, ammonia must be recycled in order to reduce the cost and protect the environment [56].

#### **3.3. Alkali pretreatment**

Alkali pretreatment is commonly utilized ammonium, calcium, sodium, and potassium hydroxides at certain temperature and pressure. The main advantage of this pretreatment is that lignin is efficiently removed from the biomass (**Table 1**). This process eliminates acetyl and uronic acid groups at hemicelluloses; as a result, the accessibility of enzyme that degrades hemicellulose increases [58]. Xylan ester linkages on hemicellulose residues are also hydrolyzed [56]. The advantages of alkali pretreatments are able to largely improve the cellulose digestibility, solubilize lignin more effectively, exhibit less cellulose, and hemicellulose solubilization compared to the acid pretreatments [59]. Alkali pretreatments can also be performed at lower temperature, pressure, and time ranging from hours to days. NaOH solution is more effective than other alkalis [60, 61]. Alkali pretreatments were shown to be more effective on decreasing the degree of polymerization and crystallinity, increasing the internal surface area of cellulose, and disrupting the lignin structure [41].

lignin peroxidases—lignin-degrading enzymes and manganese-dependent peroxidases which show high delignification efficiency on various biomass materials [52, 67]. Some advantages of biological pretreatments include low-capital cost, low energy requirement, no chemical requirement, and mild environmental conditions (**Table 1**). However, the main drawback of the biological methods is that hydrolysis rate is very low [56]. To solve this drawback, some researches have to perform to find out isolates which have ability to delignify the

Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification

transfer properties and a liquid-like solvating power. This method can remove lignin effectively

used as an extraction solvent because of its several advantages including easy recovery after extraction, environmental acceptability, non-toxicity, non-flammability, and relatively low cost

reacts with H2

accessible to ammonia and water molecules. In this pretreatment, cellulose and hemicellulose structures disrupt so that the surface area of the substrate increases and can easily attack by

effective than ammonia expansion and produces lower inhibitors than steam explosion [70].

Liquid hot water (LHW) is one of the hydrothermal pretreatment without rapid decompression and any catalyst or chemical additions and performs under high pressure in order to maintain the water in the liquid state at high temperatures. It is usually carried out at temperature range between 170 and 230°C and pressure (5 MPa) [71]. LHW eliminates hemicellulose from biomass materials so that the cellulose is more accessible to enzymatic attack (**Table 1**). After pretreatment, the obtained slurry is able to be filtered to yield two fractions, namely a solid cellulose-enriched fraction and a liquid fraction containing high hemicellulose derived sugars. Better pH (4–7) of this pretreatment can be controlled in order to minimize the non-specific degradation of polysaccharides and also to avoid the formation of inhibitors [37]. To promote more effective contact between the biomass materials and the liquid water, three methods have been developed, namely co-current, countercurrent, and flow-through methods. In co-current method, water and biomass slurry are heated to the desired temperature and held at the pretreatment conditions for a certain residence time before being cooled. Countercurrent method is designed to move water opposite to biomass through the pretreatment system. Hot water flows through passage system over a stationary bed of biomass which hydrolyzes and dissolves biomass components and brings them out of the system [72, 73]. LHW pretreatments are generally preferred because it is required lower costs due to no need chemicals and corrosion-resistant materials for hydrolysis reactors. In addition, the LHW pretreatments produce lower concentration of the solubilized hemicellulose and lignin products due to high water input (**Table 1**). Lower formation of inhibitory components and higher

so that enzymes can digest biomass materials effectively [68]. Supercritical CO2

as a supercritical fluid. The fluid displays gas like mass

O to form carbonic acid and increases

http://dx.doi.org/10.5772/intechopen.76587

explosion pretreatment is more cost-

molecules are able to penetrate small pores

has been mostly

65

biomass materials more quickly and efficiently.

 **explosion pretreatment**

(**Table 1**) [69]. In aqueous solution, CO2

**3.7. Liquid hot water pretreatment**

hydrolysis rate. Because of their small size, CO<sup>2</sup>

the digestive enzymes. For several substrates, the CO<sup>2</sup>

explosion pretreatment utilizes CO<sup>2</sup>

**3.6. CO2**

CO2

Besides NaOH, Ca(OH)2 (lime) is another alkali widely used. It also eliminates lignin-carbohydrate ester and acetyl groups, and enhances cellulose digestibility [37]. Lime pretreatment has been proven successful for several biomass pretreatments, such as wheat straw, poplar wood, switchgrass, and corn stover [61, 62]. This pretreatment has lower reagent cost and less safety requirements compared to NaOH or KOH pretreatments. In addition, lime can be easier recovered from hydrolysate by reaction with CO2 [37]. The air/oxygen addition to the alkaline pretreatments (NaOH or lime) can increase lignin removal [59].

#### **3.4. Ammonia recycle percolation**

Ammonia recycled percolation (ARP) is an another type of ammonia-based pretreatment in which aqueous ammonia (5–15 wt %) passes through a packed of bed reactor along with biomass materials at high temperature (140–210°C) for 90 min and the rate of percolation is maintained at 5 mL/min [56, 64]. ARP can remove hemicellulose and lignin from the biomass as the liquid phase [31] although requires high liquid loading or process temperature. To reduce energy cost, soaking in aqueous ammonia (SAA) at lower temperatures (40–90°C) for longer reaction times has been used. This approach can preserve most of the glucan and xylan in the biomass samples which are then fermented using the simultaneous saccharification and cofermentation (SSCF) method [63].

#### **3.5. Biological pretreatment**

Biological pretreatments are treatments to biomass materials with microbes such as white rot fungi. Like conventional physicochemical methods, the objectives of biological pretreatments are to degrade lignin. The biological pretreatment is considered as a cheap, ecofriendly, and efficient method [64]. This method is carried out using cellulolytic and hemicellulolytic microbes, such as filamentous fungi which are ubiquitous and can be isolated from soil, living plants or lignocellulosic waste material [65, 66]. The most effective microorganisms for the pretreatment of most of the biomass materials are white-rot fungi [52], such as Ceriporia lacerata, Ceriporiopsis subvermispora, Cyathus stercoreus, Phanerochaete chrysosporium, Pleurotus ostreatus, Pycnoporus cinnabarinus, and P. chrysosporium. These fungi produce lignin peroxidases—lignin-degrading enzymes and manganese-dependent peroxidases which show high delignification efficiency on various biomass materials [52, 67]. Some advantages of biological pretreatments include low-capital cost, low energy requirement, no chemical requirement, and mild environmental conditions (**Table 1**). However, the main drawback of the biological methods is that hydrolysis rate is very low [56]. To solve this drawback, some researches have to perform to find out isolates which have ability to delignify the biomass materials more quickly and efficiently.

#### **3.6. CO2 explosion pretreatment**

**3.3. Alkali pretreatment**

64 Biofuels - State of Development

Besides NaOH, Ca(OH)2

**3.4. Ammonia recycle percolation**

**3.5. Biological pretreatment**

Alkali pretreatment is commonly utilized ammonium, calcium, sodium, and potassium hydroxides at certain temperature and pressure. The main advantage of this pretreatment is that lignin is efficiently removed from the biomass (**Table 1**). This process eliminates acetyl and uronic acid groups at hemicelluloses; as a result, the accessibility of enzyme that degrades hemicellulose increases [58]. Xylan ester linkages on hemicellulose residues are also hydrolyzed [56]. The advantages of alkali pretreatments are able to largely improve the cellulose digestibility, solubilize lignin more effectively, exhibit less cellulose, and hemicellulose solubilization compared to the acid pretreatments [59]. Alkali pretreatments can also be performed at lower temperature, pressure, and time ranging from hours to days. NaOH solution is more effective than other alkalis [60, 61]. Alkali pretreatments were shown to be more effective on decreasing the degree of polymerization and crystallinity, increasing the internal

hydrate ester and acetyl groups, and enhances cellulose digestibility [37]. Lime pretreatment has been proven successful for several biomass pretreatments, such as wheat straw, poplar wood, switchgrass, and corn stover [61, 62]. This pretreatment has lower reagent cost and less safety requirements compared to NaOH or KOH pretreatments. In addition, lime can be easier recovered from hydrolysate by reaction with CO2 [37]. The air/oxygen addition to the

Ammonia recycled percolation (ARP) is an another type of ammonia-based pretreatment in which aqueous ammonia (5–15 wt %) passes through a packed of bed reactor along with biomass materials at high temperature (140–210°C) for 90 min and the rate of percolation is maintained at 5 mL/min [56, 64]. ARP can remove hemicellulose and lignin from the biomass as the liquid phase [31] although requires high liquid loading or process temperature. To reduce energy cost, soaking in aqueous ammonia (SAA) at lower temperatures (40–90°C) for longer reaction times has been used. This approach can preserve most of the glucan and xylan in the biomass samples which are then fermented using the simultaneous saccharification and cofermentation (SSCF) method [63].

Biological pretreatments are treatments to biomass materials with microbes such as white rot fungi. Like conventional physicochemical methods, the objectives of biological pretreatments are to degrade lignin. The biological pretreatment is considered as a cheap, ecofriendly, and efficient method [64]. This method is carried out using cellulolytic and hemicellulolytic microbes, such as filamentous fungi which are ubiquitous and can be isolated from soil, living plants or lignocellulosic waste material [65, 66]. The most effective microorganisms for the pretreatment of most of the biomass materials are white-rot fungi [52], such as Ceriporia lacerata, Ceriporiopsis subvermispora, Cyathus stercoreus, Phanerochaete chrysosporium, Pleurotus ostreatus, Pycnoporus cinnabarinus, and P. chrysosporium. These fungi produce

(lime) is another alkali widely used. It also eliminates lignin-carbo-

surface area of cellulose, and disrupting the lignin structure [41].

alkaline pretreatments (NaOH or lime) can increase lignin removal [59].

CO2 explosion pretreatment utilizes CO<sup>2</sup> as a supercritical fluid. The fluid displays gas like mass transfer properties and a liquid-like solvating power. This method can remove lignin effectively so that enzymes can digest biomass materials effectively [68]. Supercritical CO2 has been mostly used as an extraction solvent because of its several advantages including easy recovery after extraction, environmental acceptability, non-toxicity, non-flammability, and relatively low cost (**Table 1**) [69]. In aqueous solution, CO2 reacts with H2 O to form carbonic acid and increases hydrolysis rate. Because of their small size, CO<sup>2</sup> molecules are able to penetrate small pores accessible to ammonia and water molecules. In this pretreatment, cellulose and hemicellulose structures disrupt so that the surface area of the substrate increases and can easily attack by the digestive enzymes. For several substrates, the CO<sup>2</sup> explosion pretreatment is more costeffective than ammonia expansion and produces lower inhibitors than steam explosion [70].

#### **3.7. Liquid hot water pretreatment**

Liquid hot water (LHW) is one of the hydrothermal pretreatment without rapid decompression and any catalyst or chemical additions and performs under high pressure in order to maintain the water in the liquid state at high temperatures. It is usually carried out at temperature range between 170 and 230°C and pressure (5 MPa) [71]. LHW eliminates hemicellulose from biomass materials so that the cellulose is more accessible to enzymatic attack (**Table 1**). After pretreatment, the obtained slurry is able to be filtered to yield two fractions, namely a solid cellulose-enriched fraction and a liquid fraction containing high hemicellulose derived sugars. Better pH (4–7) of this pretreatment can be controlled in order to minimize the non-specific degradation of polysaccharides and also to avoid the formation of inhibitors [37]. To promote more effective contact between the biomass materials and the liquid water, three methods have been developed, namely co-current, countercurrent, and flow-through methods. In co-current method, water and biomass slurry are heated to the desired temperature and held at the pretreatment conditions for a certain residence time before being cooled. Countercurrent method is designed to move water opposite to biomass through the pretreatment system. Hot water flows through passage system over a stationary bed of biomass which hydrolyzes and dissolves biomass components and brings them out of the system [72, 73]. LHW pretreatments are generally preferred because it is required lower costs due to no need chemicals and corrosion-resistant materials for hydrolysis reactors. In addition, the LHW pretreatments produce lower concentration of the solubilized hemicellulose and lignin products due to high water input (**Table 1**). Lower formation of inhibitory components and higher pentose recovery can be achieved in this LHW pretreatment compared to the steam explosion. However, this method has not yet developed at a commercial scale due to higher water demand and high energy input.

Wet oxidation pretreatments affect all three main components of lignocellulosic materials, namely, cellulose, hemicellulose, and lignin. The hemicelluloses are extensively hydrolyzed to low molecular weight sugars that become soluble in water. Lignin is cleaved and oxidized, and cellulose is partly degraded and becomes highly susceptible to enzymatic hydrolysis. Addition of some alkaline compounds, such as sodium carbonate make easier to hydrolyze hemicellulose components and also minimizes the formation inhibitor compounds, such as

Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification

The main advantage of wet oxidation is efficient lignin removal and less inhibitor formations (**Table 1**). The main disadvantage of this method is requirements for high temperature and

ments cause to high costs of maintenance and also require large-scale reactors. Therefore,

Empty fruit bunch of oil palm trees have been described in terms of their physical and chemical characteristics. As solid waste products of palm oil factories, physical EFBOPT characteristics are brown, not uniform, low bulk density, varying in length from 17 to 30 cm, and varying in width from 25 to 35 cm depending on the size of fresh fruit bunch. EFBOPT fiber is mainly constructed from a complex matrix of three main polymers, namely, cellulose, hemicellulose, and lignin. The main chemical component of EFBOPT is cellulose (44.2–50.0%) and the others are hemicellulose (22.0–33.5%) and lignin (20.4–21.0%) which should be removed through

There are three kinds of pretreatment methods, namely, physical, chemical, and physicochemical and biological pretreatment methods. The various pretreatment methods for biomass materials have been described to improve enzymatic saccharifications and ethanol production. Each method has its advantages and disadvantages. There is no one treatment method yielding 100% conversion of biomass into fermentable sugars. There is always a loss of biomass materials, which affects the final yield and increases the cost of finished product, i.e., bioethanol. Although a combination of two or more pretreatment methods has indicated promising results, we still feel that there is a need for extensive researches in this area so that either a new efficient treatment method is discovered or an existing method is upgraded to yield better results. Predictive models will enable the selection, design, optimization, and

The authors are thankful to: (a) The Minister of Research, Technology, and Higher Education (Kementerian Riset, Teknologi, dan Pendidikan Tinggi), Indonesia, (b) Lampung University (Universitas Lampung), Lampung, Indonesia, and (c) Millennium Challenge Account Indonesia

process control of pretreatment methods that are suitable for biomass materials.

application of this process is limited in large-scale pretreatment of biomass materials.

O2

http://dx.doi.org/10.5772/intechopen.76587

. These require-

67

pressure maintenances and the presence of strong oxidizing agents such as H<sup>2</sup>

furan-based degradation products [89].

**4. Conclusions and future perspectives**

**Acknowledgements**

(MCA-Indonesia) for their supports.

pretreatments before the cellulose is hydrolyzed enzymatically.

#### **3.8. Milling pretreatment**

Milling pretreatment is commonly used for reducing size biomass and altering the inherent ultrastructure of biomass and degree of crystallinity so that the biomass can be easier accessed by cellulase enzymes [74]. This pretreatment is performed prior to enzymatic hydrolysis or even other chemical pretreatment processes [74, 75, 76, 77]. There are several kinds of milling, such as ball, tow roll, hammer, colloid, and vibro energy millings [40]. Milling is able to improve susceptibility to enzymatic hydrolysis because of reducing the biomass size [78], decreasing the biomass crystallinity, [79] and increasing the biomass area.

#### **3.9. Steam explosion pretreatment**

Steam explosion pretreatment is a treatment with high pressure saturated steam for few seconds (30 s) to several minutes (20 min), and then pressure is suddenly reduced. These pretreatments are the most commonly used for treating biomass materials [80, 81]. Steam explosion—typically a combination of mechanical forces and chemical—is able to hydrolyze (autohydrolyze) acetyl groups of hemicellulose. At high temperatures (160–260°C), autohydrolysis occurs and produces acetic acid from acetyl groups of the biomass materials [82, 83]. In addition, water is also able to act as an acid at high temperatures. Reducing pressure suddenly produces explosive decompression so that biomass fibers separate each other. This process is able to degrade hemicellulose and lignin because of explosive decompression, and thus increase the potential of cellulose hydrolysis [82].

Steam explosion processes have several advantages compared to other pretreatment methods (**Table 1**). The advantages include high sugar recovery, less hazardous process chemicals and conditions, lower environmental impact, lower capital investment, possibility of using larger chip size, no acid catalyst additions except for softwoods, more efficient in energy usage, and significant improvement in enzymatic hydrolysis as well as its feasibility at industrial scale [84]. The main drawbacks of steam explosion pretreatment are the partial degradation of hemicelluloses producing inhibitor compounds that can affect the enzymatic hydrolysis and fermentation process [85, 86]. Thus, an inhibitor compound separation becomes necessary (e.g., addition of activated charcoal, over liming, and ion exchange) and will increase the overall process cost [31, 87].

#### **3.10. Wet oxidation pretreatment**

Wet oxidation is treated biomass materials with water and air/oxygen at temperatures higher than 120°C for 30 min [88]. This wet oxidation pretreatment is suitable for pretreatment of biomass materials containing high lignin content. The most effective parameters in the wet oxidation process are temperature, reaction time, and oxygen pressure [87]. This pretreatment yields acid compounds from the hydrolytic processes and oxidative reactions of biomass materials.

Wet oxidation pretreatments affect all three main components of lignocellulosic materials, namely, cellulose, hemicellulose, and lignin. The hemicelluloses are extensively hydrolyzed to low molecular weight sugars that become soluble in water. Lignin is cleaved and oxidized, and cellulose is partly degraded and becomes highly susceptible to enzymatic hydrolysis. Addition of some alkaline compounds, such as sodium carbonate make easier to hydrolyze hemicellulose components and also minimizes the formation inhibitor compounds, such as furan-based degradation products [89].

The main advantage of wet oxidation is efficient lignin removal and less inhibitor formations (**Table 1**). The main disadvantage of this method is requirements for high temperature and pressure maintenances and the presence of strong oxidizing agents such as H<sup>2</sup> O2 . These requirements cause to high costs of maintenance and also require large-scale reactors. Therefore, application of this process is limited in large-scale pretreatment of biomass materials.

#### **4. Conclusions and future perspectives**

pentose recovery can be achieved in this LHW pretreatment compared to the steam explosion. However, this method has not yet developed at a commercial scale due to higher water

Milling pretreatment is commonly used for reducing size biomass and altering the inherent ultrastructure of biomass and degree of crystallinity so that the biomass can be easier accessed by cellulase enzymes [74]. This pretreatment is performed prior to enzymatic hydrolysis or even other chemical pretreatment processes [74, 75, 76, 77]. There are several kinds of milling, such as ball, tow roll, hammer, colloid, and vibro energy millings [40]. Milling is able to improve susceptibility to enzymatic hydrolysis because of reducing the biomass size [78],

Steam explosion pretreatment is a treatment with high pressure saturated steam for few seconds (30 s) to several minutes (20 min), and then pressure is suddenly reduced. These pretreatments are the most commonly used for treating biomass materials [80, 81]. Steam explosion—typically a combination of mechanical forces and chemical—is able to hydrolyze (autohydrolyze) acetyl groups of hemicellulose. At high temperatures (160–260°C), autohydrolysis occurs and produces acetic acid from acetyl groups of the biomass materials [82, 83]. In addition, water is also able to act as an acid at high temperatures. Reducing pressure suddenly produces explosive decompression so that biomass fibers separate each other. This process is able to degrade hemicellulose and lignin because of explosive decompression,

Steam explosion processes have several advantages compared to other pretreatment methods (**Table 1**). The advantages include high sugar recovery, less hazardous process chemicals and conditions, lower environmental impact, lower capital investment, possibility of using larger chip size, no acid catalyst additions except for softwoods, more efficient in energy usage, and significant improvement in enzymatic hydrolysis as well as its feasibility at industrial scale [84]. The main drawbacks of steam explosion pretreatment are the partial degradation of hemicelluloses producing inhibitor compounds that can affect the enzymatic hydrolysis and fermentation process [85, 86]. Thus, an inhibitor compound separation becomes necessary (e.g., addition of activated charcoal, over liming, and ion exchange) and will increase the overall process cost [31, 87].

Wet oxidation is treated biomass materials with water and air/oxygen at temperatures higher than 120°C for 30 min [88]. This wet oxidation pretreatment is suitable for pretreatment of biomass materials containing high lignin content. The most effective parameters in the wet oxidation process are temperature, reaction time, and oxygen pressure [87]. This pretreatment yields acid compounds from the hydrolytic processes and oxidative reactions of bio-

decreasing the biomass crystallinity, [79] and increasing the biomass area.

and thus increase the potential of cellulose hydrolysis [82].

demand and high energy input.

**3.9. Steam explosion pretreatment**

**3.10. Wet oxidation pretreatment**

mass materials.

**3.8. Milling pretreatment**

66 Biofuels - State of Development

Empty fruit bunch of oil palm trees have been described in terms of their physical and chemical characteristics. As solid waste products of palm oil factories, physical EFBOPT characteristics are brown, not uniform, low bulk density, varying in length from 17 to 30 cm, and varying in width from 25 to 35 cm depending on the size of fresh fruit bunch. EFBOPT fiber is mainly constructed from a complex matrix of three main polymers, namely, cellulose, hemicellulose, and lignin. The main chemical component of EFBOPT is cellulose (44.2–50.0%) and the others are hemicellulose (22.0–33.5%) and lignin (20.4–21.0%) which should be removed through pretreatments before the cellulose is hydrolyzed enzymatically.

There are three kinds of pretreatment methods, namely, physical, chemical, and physicochemical and biological pretreatment methods. The various pretreatment methods for biomass materials have been described to improve enzymatic saccharifications and ethanol production. Each method has its advantages and disadvantages. There is no one treatment method yielding 100% conversion of biomass into fermentable sugars. There is always a loss of biomass materials, which affects the final yield and increases the cost of finished product, i.e., bioethanol. Although a combination of two or more pretreatment methods has indicated promising results, we still feel that there is a need for extensive researches in this area so that either a new efficient treatment method is discovered or an existing method is upgraded to yield better results. Predictive models will enable the selection, design, optimization, and process control of pretreatment methods that are suitable for biomass materials.

#### **Acknowledgements**

The authors are thankful to: (a) The Minister of Research, Technology, and Higher Education (Kementerian Riset, Teknologi, dan Pendidikan Tinggi), Indonesia, (b) Lampung University (Universitas Lampung), Lampung, Indonesia, and (c) Millennium Challenge Account Indonesia (MCA-Indonesia) for their supports.

#### **Author details**

Sutikno Sutikno1 \* and Muhammad Kismurtono2

\*Address all correspondence to: sutiknolampung@fp.unila.ac.id

1 Department of Agricultural Product Technology, Lampung University, Indonesia

2 Research Unit for Natural Product Technology, The Indonesian Institute of Sicence, Indonesia

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Pretreatment Empty Fruit Bunch of Oil Palm Tree for Improving Enzymatic Saccharification

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**Author details**

68 Biofuels - State of Development

Sutikno Sutikno1

Indonesia

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\* and Muhammad Kismurtono2 \*Address all correspondence to: sutiknolampung@fp.unila.ac.id

1 Department of Agricultural Product Technology, Lampung University, Indonesia

2 Research Unit for Natural Product Technology, The Indonesian Institute of Sicence,

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**Chapter 5**

) or

year<sup>−</sup><sup>1</sup>

**Provisional chapter**

**Conversion of High Biomass/Bagasse from Sorghum**

**and Bermuda Grass into Second-Generation** 

**Conversion of High Biomass/Bagasse from Sorghum** 

Erick Heredia-Olea, Sergio O. Serna-Saldivar,

Erick Heredia-Olea, Sergio O. Serna-Saldivar, Esther Perez-Carrillo and Jesica R. Canizo

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Esther Perez-Carrillo and Jesica R. Canizo

http://dx.doi.org/10.5772/intechopen.75064

**Bioethanol**

**Abstract**

bioethanol dry t<sup>−</sup><sup>1</sup>

bioethanol

**and Bermuda Grass into Second-Generation Bioethanol**

Sorghum (Sorghum bicolor) and Bermuda (Cynodon dactylon) grass are examples of annual and perennial forage crops produced throughout the globe. These crops should be harvested at the peak of biomass production when the levels of lignin are relatively low. The high biomass sorghum, sweet sorghum bagasse (2 cuts or crops year<sup>−</sup><sup>1</sup>

rich in cellulose and hemicellulose can be efficiently transformed into bioethanol using second-generation technologies consisting of milling, pretreatment (chemical and/or enzymatic) and fermentation with microorganisms capable of transforming C5/C6 sugars to obtain ethanol. An alternative process contemplates the extrusion aimed toward the physical disruption of cell walls minimizing the use of considerable amounts of water and chemicals commonly used during pretreatment. Extruded feedstocks treated with fiber-degrading enzyme cocktails had conversion efficiencies between 60 and 78% of the hemicellulose and cellulose similar to the ones achieved after acid/enzyme hydrolyses. The chief advantages of this continuous process are that hydrolysates are practically free of enzymes and yeast inhibitors. These feedstocks can produce up to 310 L anhydrous

Bermuda grass capable of yielding up to 50, 60 and 27 tons of dry forage ha<sup>−</sup><sup>1</sup>

and have a great potential for widespread use.

**Keywords:** high biomass sorghum, sorghum bagasse, Bermuda grass, second-generation

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.75064

#### **Conversion of High Biomass/Bagasse from Sorghum and Bermuda Grass into Second-Generation Bioethanol Conversion of High Biomass/Bagasse from Sorghum and Bermuda Grass into Second-Generation Bioethanol**

DOI: 10.5772/intechopen.75064

Erick Heredia-Olea, Sergio O. Serna-Saldivar, Esther Perez-Carrillo and Jesica R. Canizo Erick Heredia-Olea, Sergio O. Serna-Saldivar, Esther Perez-Carrillo and Jesica R. Canizo

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75064

#### **Abstract**

Sorghum (Sorghum bicolor) and Bermuda (Cynodon dactylon) grass are examples of annual and perennial forage crops produced throughout the globe. These crops should be harvested at the peak of biomass production when the levels of lignin are relatively low. The high biomass sorghum, sweet sorghum bagasse (2 cuts or crops year<sup>−</sup><sup>1</sup> ) or Bermuda grass capable of yielding up to 50, 60 and 27 tons of dry forage ha<sup>−</sup><sup>1</sup> year<sup>−</sup><sup>1</sup> rich in cellulose and hemicellulose can be efficiently transformed into bioethanol using second-generation technologies consisting of milling, pretreatment (chemical and/or enzymatic) and fermentation with microorganisms capable of transforming C5/C6 sugars to obtain ethanol. An alternative process contemplates the extrusion aimed toward the physical disruption of cell walls minimizing the use of considerable amounts of water and chemicals commonly used during pretreatment. Extruded feedstocks treated with fiber-degrading enzyme cocktails had conversion efficiencies between 60 and 78% of the hemicellulose and cellulose similar to the ones achieved after acid/enzyme hydrolyses. The chief advantages of this continuous process are that hydrolysates are practically free of enzymes and yeast inhibitors. These feedstocks can produce up to 310 L anhydrous bioethanol dry t<sup>−</sup><sup>1</sup> and have a great potential for widespread use.

**Keywords:** high biomass sorghum, sorghum bagasse, Bermuda grass, second-generation bioethanol

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

The family of true grasses, botanically known as *Gramineae* or *Poaceae*, is a large group of genus and species commercially grown and distributed practically in all places around the globe. These monocotyledonous flowering plants are the most common sources of food for mankind and forages for domestic animals. The value of these plants is that they produce kernels rich in starch and protein that constitute the main staple for most cultures around the globe. Furthermore, these grasses provide most of the fiber consumed by ruminant domestic animals that provide mankind with milk and meat. The family contains about 10,000 domesticated and wild species, which represent the fifth largest plant family. Grasslands make up one-fifth of the vegetation cover of the globe and are considered as one of the chief sources of fibrous rich feedstocks. Annual and perennial grasses are classed into three broad categories: bunch-type, stoloniferous and rhizomatous. The success of the various types of grasses is mainly attributed to their morphological and physiological diversity. According to the physiological activity and more specifically to the photosynthetic pathways for carbon fixation, grasses are divided into C3 or C4 plants. The C4 plants have a photosynthetic pathway that particularly adapts them to hot climates and atmospheres low in CO2 [1]. The commercial cereal grains are annual whereas most of the grasses that provide forages perennial. The chemical composition of these forages is affected by genotype, maturity and soil fertility. However, independently of the source, these feedstocks are highly attractive for biorefineries because of their abundance, relative low cost and quality of the fiber that it can be successfully converted into second-generation bioethanol, lactic acid, or other high value chemicals.

short perennial high biomass sorghums can grow to a height of 6 m tall, depending on the genotype and growing conditions. This capacity has been boosted by intensive plant breeding programs focused in the design of new genotypes that can be effectively converted into second-generation ethanol [5, 6]. Sorghum is resistant to both abiotic and biotic stress factors such as drought, soil salinity and alkalinity and insects. Besides, this cultivar possesses one of the best rates of carbon incorporation (50 g m−2 d−1), which promotes its fast growth and

and therefore, it can be produced twice or even three times throughout the year instead of only one crop obtained with sugarcane. Bermuda grass (*Cynodon dactylon*) is also a C4 perennial season forage crop, mainly used in the United States and northern of Mexico for ruminant feed and soil remediation from animal wastes. This grass has a short growth period and is usually cut monthly during the spring and summer seasons. The grass is highly susceptible to cold temperatures so it passes the winter inactive. The optimum growth conditions are 24–37°C maintaining a well biomass yield under water-stress conditions [8]. Together with these characteristics, the Bermuda grass is composed of a high amount of holocellulose

The aim of this review is to describe conversion technologies of sweet sorghum bagasse, high biomass sorghum and perennial grasses like Bermuda into second-generation fuel bioethanol.

Among annual cultivars, the high biomass and sweet sorghums offer the most efficient and fastest means of producing large quantities of usable biomass [1, 3, 6, 10], whereas the perennial Bermuda grass is a good example of a forage crop which can be effectively converted into second-generation bioethanol using currently available technologies. High yielding sweet sorghums planted in good soils and with adequate agronomic practices are able to produce 3500 L of anhydrous ethanol ha−1 from sweet juice, 3000 from the spent bagasse and 500 L from the starchy kernels (a total of about 7000 L) after 4 months of sowing, whereas high biomass

Amosson et al. [6] estimated dry yields of high biomass sorghum planted on dryland and irrigated areas in 8 and 18.5 t ha−1. Likewise, Habyarrimana et al. [11] evaluated the biomass yield and drought resistance in field conditions of Italian sorghum genotypes. This study which evaluated 75 lines and two hybrids concluded that tropical sorghum landraces yielded total aboveground dry biomasses of 33–51 t ha−1 under irrigation and 20–29 t ha−1 under rain-fed conditions. Dryland and irrigated high biomass sorghum planted in the high plains of Texas was capable of yielding from 2400 to 5600 L ethanol ha−1, respectively, assuming that one ton of dry biomass yields approximately 300 L of second-generation ethanol. The estimated cost of producing 1 ton of dry weight on the dryland and irrigated lands was estimated at 76.6 and

**2. Ethanol production from high biomass or sorghum bagasse and** 

utilization [7]. The sorghum growth cycle regularly lasts 3–5 months,

Conversion of High Biomass/Bagasse from Sorghum and Bermuda Grass into Second…

http://dx.doi.org/10.5772/intechopen.75064

79

enhanced rate of CO2

**Bermuda grass**

82 US dollars, respectively [6].

(>50%) and low amounts of lignin (up to 20%) [9].

dedicated crops up to 5600 L of second-generation ethanol ha−1.

According to the Renewable Fuels Association, the world biorefineries generated more than 100 billion L of bioethanol in 2016. The USA is the major producer with approximately 58% of the present production (57.7 billion L) followed by Brazil with 27% of the total production. The contemporary fuel ethanol production is based chiefly on maize and sugarcane [2] and the use of these feedstocks triggers concerns related to food security especially as world population surpasses 7600 million people [3]. More biofuels production is particularly expected in the USA where the Energy Independence Act mandates the manufacturing of 136 billion L of bioethanol and biodiesel by year 2022. In order to meet these expectations, biorefineries will convert lignocellulosic biomass to energy using forages and dedicated energy crops such as high biomass sorghum, switchgrass and Miscanthus, which could be planted in marginal zones or alternatively serve to protect ecosystems especially from soil erosion.

The C4 sorghum (*Sorghum bicolor* L. Moench) plant has been identified as one of the best potential bioenergy crops. This member of the Gramineae family poses considerable potential as a dedicated lignocellulosic crop because of its broad genetic diversity, which provides plant breeders the opportunity to develop high biomass types adapted to different environments under dryland or irrigated conditions or sweet sorghums varieties, which can be effectively converted into first- and second-generation bioethanol [4]. The annual or short perennial high biomass sorghums can grow to a height of 6 m tall, depending on the genotype and growing conditions. This capacity has been boosted by intensive plant breeding programs focused in the design of new genotypes that can be effectively converted into second-generation ethanol [5, 6]. Sorghum is resistant to both abiotic and biotic stress factors such as drought, soil salinity and alkalinity and insects. Besides, this cultivar possesses one of the best rates of carbon incorporation (50 g m−2 d−1), which promotes its fast growth and enhanced rate of CO2 utilization [7]. The sorghum growth cycle regularly lasts 3–5 months, and therefore, it can be produced twice or even three times throughout the year instead of only one crop obtained with sugarcane. Bermuda grass (*Cynodon dactylon*) is also a C4 perennial season forage crop, mainly used in the United States and northern of Mexico for ruminant feed and soil remediation from animal wastes. This grass has a short growth period and is usually cut monthly during the spring and summer seasons. The grass is highly susceptible to cold temperatures so it passes the winter inactive. The optimum growth conditions are 24–37°C maintaining a well biomass yield under water-stress conditions [8]. Together with these characteristics, the Bermuda grass is composed of a high amount of holocellulose (>50%) and low amounts of lignin (up to 20%) [9].

**1. Introduction**

78 Biofuels - State of Development

chemicals.

erosion.

The family of true grasses, botanically known as *Gramineae* or *Poaceae*, is a large group of genus and species commercially grown and distributed practically in all places around the globe. These monocotyledonous flowering plants are the most common sources of food for mankind and forages for domestic animals. The value of these plants is that they produce kernels rich in starch and protein that constitute the main staple for most cultures around the globe. Furthermore, these grasses provide most of the fiber consumed by ruminant domestic animals that provide mankind with milk and meat. The family contains about 10,000 domesticated and wild species, which represent the fifth largest plant family. Grasslands make up one-fifth of the vegetation cover of the globe and are considered as one of the chief sources of fibrous rich feedstocks. Annual and perennial grasses are classed into three broad categories: bunch-type, stoloniferous and rhizomatous. The success of the various types of grasses is mainly attributed to their morphological and physiological diversity. According to the physiological activity and more specifically to the photosynthetic pathways for carbon fixation, grasses are divided into C3 or C4 plants. The C4 plants have a photosynthetic

pathway that particularly adapts them to hot climates and atmospheres low in CO2

commercial cereal grains are annual whereas most of the grasses that provide forages perennial. The chemical composition of these forages is affected by genotype, maturity and soil fertility. However, independently of the source, these feedstocks are highly attractive for biorefineries because of their abundance, relative low cost and quality of the fiber that it can be successfully converted into second-generation bioethanol, lactic acid, or other high value

According to the Renewable Fuels Association, the world biorefineries generated more than 100 billion L of bioethanol in 2016. The USA is the major producer with approximately 58% of the present production (57.7 billion L) followed by Brazil with 27% of the total production. The contemporary fuel ethanol production is based chiefly on maize and sugarcane [2] and the use of these feedstocks triggers concerns related to food security especially as world population surpasses 7600 million people [3]. More biofuels production is particularly expected in the USA where the Energy Independence Act mandates the manufacturing of 136 billion L of bioethanol and biodiesel by year 2022. In order to meet these expectations, biorefineries will convert lignocellulosic biomass to energy using forages and dedicated energy crops such as high biomass sorghum, switchgrass and Miscanthus, which could be planted in marginal zones or alternatively serve to protect ecosystems especially from soil

The C4 sorghum (*Sorghum bicolor* L. Moench) plant has been identified as one of the best potential bioenergy crops. This member of the Gramineae family poses considerable potential as a dedicated lignocellulosic crop because of its broad genetic diversity, which provides plant breeders the opportunity to develop high biomass types adapted to different environments under dryland or irrigated conditions or sweet sorghums varieties, which can be effectively converted into first- and second-generation bioethanol [4]. The annual or

[1]. The

The aim of this review is to describe conversion technologies of sweet sorghum bagasse, high biomass sorghum and perennial grasses like Bermuda into second-generation fuel bioethanol.

#### **2. Ethanol production from high biomass or sorghum bagasse and Bermuda grass**

Among annual cultivars, the high biomass and sweet sorghums offer the most efficient and fastest means of producing large quantities of usable biomass [1, 3, 6, 10], whereas the perennial Bermuda grass is a good example of a forage crop which can be effectively converted into second-generation bioethanol using currently available technologies. High yielding sweet sorghums planted in good soils and with adequate agronomic practices are able to produce 3500 L of anhydrous ethanol ha−1 from sweet juice, 3000 from the spent bagasse and 500 L from the starchy kernels (a total of about 7000 L) after 4 months of sowing, whereas high biomass dedicated crops up to 5600 L of second-generation ethanol ha−1.

Amosson et al. [6] estimated dry yields of high biomass sorghum planted on dryland and irrigated areas in 8 and 18.5 t ha−1. Likewise, Habyarrimana et al. [11] evaluated the biomass yield and drought resistance in field conditions of Italian sorghum genotypes. This study which evaluated 75 lines and two hybrids concluded that tropical sorghum landraces yielded total aboveground dry biomasses of 33–51 t ha−1 under irrigation and 20–29 t ha−1 under rain-fed conditions. Dryland and irrigated high biomass sorghum planted in the high plains of Texas was capable of yielding from 2400 to 5600 L ethanol ha−1, respectively, assuming that one ton of dry biomass yields approximately 300 L of second-generation ethanol. The estimated cost of producing 1 ton of dry weight on the dryland and irrigated lands was estimated at 76.6 and 82 US dollars, respectively [6].

The short rotational sweet sorghums yield high sugar in their stalks, whereas the high biomass sorghums developed for second-generation bioethanol are mainly composed of fibrous chemical compounds. In terms of conversion into ethanol, the extracted sweet juice can be easily and highly efficiently converted into bioethanol leaving the spent bagasse as other potential feedstock for second-generation ethanol manufacture. Vencor Green [12] reported that the cost of ethanol production from sweet sorghum juice was 20% lower than either sugarcane or corn. According to Serna Saldivar et al. [1], the mature stems of sweet sorghum contain about 73% moisture and 27% solids which are manly comprised of structural and non-structural carbohydrates. Approximately 13% of the solids are non-structural carbohydrates composed of the disaccharide sucrose and monosaccharides glucose and fructose, in variable amounts according to cultivar, maturity stage and harvesting season [13, 14]. The sweet sorghum cultivars are classified based on their juice sugar composition into sugar and syrup types. The first is rich in sucrose while the second in glucose and fructose. According to the same authors, Wray, Keller and H173 sweet sorghum cultivars harvested post-anthesis yielded an average of 10, 7 and 4 t ha−1 of fermentable sugars. Other studies [15, 16] reported sugar yields varying from 4.5 to 18 t ha−1. These sugars are considerably highly fermentable and able to yield approximately 46% ethanol after 48 h fermentation. Therefore, the conceivable production of anhydrous ethanol fluctuates from 1000 to 8000 L.

slightly higher compared to the value (11.1% dwb) reported by Prasad et al. [7]. Importantly, lignin is directly related to plant maturity and thus inversely correlated to the proneness of the

High biomass sorghum2 27–52 17–23 6.2–8.1 Sweet sorghum bagasse3 31–34 18–25 10–18 Bermuda grass<sup>4</sup>,5 31–35 26–30 14–23

**Table 1.** Chemical composition of high biomass sorghum, sweet sorghum bagasse and Bermuda grass1

**Cellulose Hemicellulose Lignin**

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According to Lee et al. [21], the Bermuda grass contains 30.4, 22.6, 4.9 and 23.2% of glucans, xylans, arabinans and lignin, respectively. On the other hand, Canizo [22] determined a similar composition of the potentially fermentable sugars but a slightly lower lignin content (**Table 1**).

Production of lignocellulosic anhydrous ethanol consists of five sequential steps: milling of the feedstock, chemical or physical pretreatment, enzyme catalysis or saccharification, fermentation of resulting C6 and C5 sugars and ethanol distillation-dehydration [4]. Cellulose and hemicellulose upon hydrolysis yield the C6 and C5 fermentable sugars, respectively. The effectiveness of the process is strongly affected by the availability of cellulose and hemicellulose which must

The lignocellulosic residue is usually milled in hammer or rotary mills with the objectives of reducing the particle size and disrupt cell walls so fiber components are more prone to subsequent chemical, enzymatic and fermentation treatments. Then, the ground feedstock is hydrolyzed using chemicals such as acid, alkalis and ammonia and/or a set of fiber-degrading enzymes. Generally, the chemical or physical hydrolysis precedes the enzymatic in order to

Extrusion cooking and steam explosion are two alternative types of physical pretreatments that are used to expose cellulose and hemicellulose associated with lignified cell walls. The first has been effectively used to disrupt fiber components especially hemicellulose minimizing the use of water [23, 24]. Heredia-Olea [25] effectively employed a thermoplastic twin extruder to modify the fiber structure of ground sorghum bagasse. Results indicated that that thermoplastic extrusion additionally reduced the particle size of the ground feedstock

be separated from lignin, which hinders rate of hydrolysis and therefore ethanol yields.

other fiber components to hydrolysis and ethanol production.

**2.2. Second-generation ethanol production**

**Crop Composition (%)**

Composition and yields are expressed on dry matter basis.

Stenberg et al. [27], Heredia-Olea et al. [20].

1

2

3

4

5

Nagaiah et al. [17].

Canizo et al. [22].

Lee et al. [9].

*2.2.1. Pretreatments and sugars yields*

further release C6 and C5 sugars [4].

On the other hand, an established perennial Bermuda grass field is capable of producing from 6 to 27 t ha−1 year−1 of dry forage [8], which can be converted into 1200–5400 L fuel ethanol. The large yield variability is due to water availability, nitrogen fertilization, soil fertility and number of monthly cuts during the year. Generally, the Bermuda grass is cut monthly except during the cold season of the year.

#### **2.1. Second-generation ethanol from high biomass or sorghum bagasse and Bermuda grass**

#### *2.1.1. Fiber composition*

The high biomass forage or sorghum bagasse leftover after juice extraction of sweet cultivars is a fiber-rich feedstock with some variation in composition according to intrinsic and extrinsic factors such as genotype, maturity or degree of lignification and climatic conditions.

Nagaiah et al. [17] determined the structural composition of six high biomass sorghums differing in plant height (3.9–6.2 m) and yield (53.6–90.5 t of fresh stalks). The cultivars contained from 27 to 52% cellulose, 17 to 23% hemicellulose and 6.2 to 8.1% lignin (**Table 1**). According to Woods [18], from 12 to 17% (average 15%), 15% of the total sweet sorghum plant weight is constituted by the fibrous portion. Typically, the sweet sorghum harvested at optimum time yields half juice and half bagasse after milling or crushing the stalks. The spent bagasse with 52% moisture contains 5.4% residual fermentable sugar, 17% cellulose, 11.9% hemicellulose and 8.5% lignin [19, 20]. Heredia-Olea et al. [20], evaluating the fiber and structural carbohydrate composition of sweet sorghum bagasse, concluded that the main structural carbohydrates were cellulose derived β-glucans and xylans and arabinans related to hemicellulose. The quantity of lignin of 13.5% (dwb) was similar to that assayed by Gnansounou et al. [19] and


**Table 1.** Chemical composition of high biomass sorghum, sweet sorghum bagasse and Bermuda grass1 .

slightly higher compared to the value (11.1% dwb) reported by Prasad et al. [7]. Importantly, lignin is directly related to plant maturity and thus inversely correlated to the proneness of the other fiber components to hydrolysis and ethanol production.

According to Lee et al. [21], the Bermuda grass contains 30.4, 22.6, 4.9 and 23.2% of glucans, xylans, arabinans and lignin, respectively. On the other hand, Canizo [22] determined a similar composition of the potentially fermentable sugars but a slightly lower lignin content (**Table 1**).

#### **2.2. Second-generation ethanol production**

#### *2.2.1. Pretreatments and sugars yields*

4

5

Canizo et al. [22].

Lee et al. [9].

The short rotational sweet sorghums yield high sugar in their stalks, whereas the high biomass sorghums developed for second-generation bioethanol are mainly composed of fibrous chemical compounds. In terms of conversion into ethanol, the extracted sweet juice can be easily and highly efficiently converted into bioethanol leaving the spent bagasse as other potential feedstock for second-generation ethanol manufacture. Vencor Green [12] reported that the cost of ethanol production from sweet sorghum juice was 20% lower than either sugarcane or corn. According to Serna Saldivar et al. [1], the mature stems of sweet sorghum contain about 73% moisture and 27% solids which are manly comprised of structural and non-structural carbohydrates. Approximately 13% of the solids are non-structural carbohydrates composed of the disaccharide sucrose and monosaccharides glucose and fructose, in variable amounts according to cultivar, maturity stage and harvesting season [13, 14]. The sweet sorghum cultivars are classified based on their juice sugar composition into sugar and syrup types. The first is rich in sucrose while the second in glucose and fructose. According to the same authors, Wray, Keller and H173 sweet sorghum cultivars harvested post-anthesis yielded an average of 10, 7 and 4 t ha−1 of fermentable sugars. Other studies [15, 16] reported sugar yields varying from 4.5 to 18 t ha−1. These sugars are considerably highly fermentable and able to yield approximately 46% ethanol after 48 h fermentation. Therefore, the conceiv-

On the other hand, an established perennial Bermuda grass field is capable of producing from 6 to 27 t ha−1 year−1 of dry forage [8], which can be converted into 1200–5400 L fuel ethanol. The large yield variability is due to water availability, nitrogen fertilization, soil fertility and number of monthly cuts during the year. Generally, the Bermuda grass is cut monthly except

The high biomass forage or sorghum bagasse leftover after juice extraction of sweet cultivars is a fiber-rich feedstock with some variation in composition according to intrinsic and extrinsic factors such as genotype, maturity or degree of lignification and climatic conditions.

Nagaiah et al. [17] determined the structural composition of six high biomass sorghums differing in plant height (3.9–6.2 m) and yield (53.6–90.5 t of fresh stalks). The cultivars contained from 27 to 52% cellulose, 17 to 23% hemicellulose and 6.2 to 8.1% lignin (**Table 1**). According to Woods [18], from 12 to 17% (average 15%), 15% of the total sweet sorghum plant weight is constituted by the fibrous portion. Typically, the sweet sorghum harvested at optimum time yields half juice and half bagasse after milling or crushing the stalks. The spent bagasse with 52% moisture contains 5.4% residual fermentable sugar, 17% cellulose, 11.9% hemicellulose and 8.5% lignin [19, 20]. Heredia-Olea et al. [20], evaluating the fiber and structural carbohydrate composition of sweet sorghum bagasse, concluded that the main structural carbohydrates were cellulose derived β-glucans and xylans and arabinans related to hemicellulose. The quantity of lignin of 13.5% (dwb) was similar to that assayed by Gnansounou et al. [19] and

able production of anhydrous ethanol fluctuates from 1000 to 8000 L.

**2.1. Second-generation ethanol from high biomass or sorghum bagasse and** 

during the cold season of the year.

**Bermuda grass**

*2.1.1. Fiber composition*

80 Biofuels - State of Development

Production of lignocellulosic anhydrous ethanol consists of five sequential steps: milling of the feedstock, chemical or physical pretreatment, enzyme catalysis or saccharification, fermentation of resulting C6 and C5 sugars and ethanol distillation-dehydration [4]. Cellulose and hemicellulose upon hydrolysis yield the C6 and C5 fermentable sugars, respectively. The effectiveness of the process is strongly affected by the availability of cellulose and hemicellulose which must be separated from lignin, which hinders rate of hydrolysis and therefore ethanol yields.

The lignocellulosic residue is usually milled in hammer or rotary mills with the objectives of reducing the particle size and disrupt cell walls so fiber components are more prone to subsequent chemical, enzymatic and fermentation treatments. Then, the ground feedstock is hydrolyzed using chemicals such as acid, alkalis and ammonia and/or a set of fiber-degrading enzymes. Generally, the chemical or physical hydrolysis precedes the enzymatic in order to further release C6 and C5 sugars [4].

Extrusion cooking and steam explosion are two alternative types of physical pretreatments that are used to expose cellulose and hemicellulose associated with lignified cell walls. The first has been effectively used to disrupt fiber components especially hemicellulose minimizing the use of water [23, 24]. Heredia-Olea [25] effectively employed a thermoplastic twin extruder to modify the fiber structure of ground sorghum bagasse. Results indicated that that thermoplastic extrusion additionally reduced the particle size of the ground feedstock and exposed cellulose which was more susceptible to fiber-degrading enzymes. The extruded feedstock treated with the kit of fiber-degrading enzymes had conversion efficiencies of 77.5 and 60 of cellulose and hemicellulose, respectively. These conversion rates are comparable to rates attained when acid and enzyme hydrolyses. The main benefits of the continuous extrusion process are that hydrolysates are virtually free of yeast inhibitors such as acetic acid, furfural and hydroxymethyl furfural [1, 24].

indicated that the different acid pretreatments produced similar quantities of fermentable carbohydrates. These pretreatments liberated from 56 to 57% of the total sugars present in the sorghum bagasse (390–415 mg sugar g−1 bagasse) and from 44 to 61 mg total inhibitors g−1 (**Table 2**). Among the three pretreatments, the HCl treatment was the best alternative due to its relatively lower hydrolysis time (less energy expenditure) and satisfactory yield of C5 and

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On the other hand, the use of strong alkalies breaks ester bonds of cross-linked lignin and xylans producing an enriched cellulose and hemicellulose fraction. The preferred alkalis are sodium hydroxide, ammonia and calcium hydroxide or lime. The alkaline pretreatments are regularly conducted at relatively lower temperatures, pressures and times compared to other technologies. The main withdraw of chemical treatments is the production of known yeast inhibitors divided into three categories: organic acids (i.e. acetic, formic and levulinic), furans (furfural and 5 hydroxymethylfurfural) and phenolics such as *p*-hydroxybenzoic acid released from lignin moieties [28]. The concentration of these hydrosoluble compounds is known to upset cell physiology and viability and thus the efficacy of ethanologenic microorganisms. According to Amartey and Jeffries [30], the removal of these inhibitors before fermentation

Wang and Cheng investigated the efficiency of lime or calcium hydroxide pretreatment in order to enhance reducing sugar recovery in coastal Bermuda grass [31]. These authors studied the effects of temperature (21–121°C) and lime loadings (0.02–0.2 g/g of dry biomass) followed by biocatalysis with cellulases and cellobiases. The best pretreatment combinations removed approximately 20% of the original total lignin content, which was over twice more than that from the untreated counterpart. The best total fermentable sugar yield for the lime pretreated Bermuda grass (100°C, 15 min, and 0.1 g lime g−1 dry biomass) was 78% of the theoretical maximum. Moreover, this specific pretreatment converted 87%, and 68% of the

> **Extruded + enzymatic hydrolysis**

55.6 80.6 67.5 40.9 89.2

**Table 2.** Sugars and second generation ethanol generated from sorghum bagasse or Bermuda grass after different

**H2 SO4 hydrolysis** **H2 SO4**

 **hydrolysis + enzymatic hydrolysis**

**Compound (%) Sweet sorghum bagasse4 Bermuda grass5**

Glucose 52.2 82.2 66.5 20.9 90.5 Xylose 53.3 75.7 94.1 66.6 84.6 Arabinose 42.6 50.2 28.7 90.0 90.0

Sugars yield = total sugars (mg/g)/[1.11× glucans (mg/g) + 1.14 (xylans (mg/g) + arabinans (mg/g))].

**HCl hydrolysis + enzymatic hydrolysis**

The compounds are percentage of sweet sorghum bagasse or Bermuda grass.

C6 fermentable sugars.

can reduce approximately 25% the production cost.

**HCl hydrolysis**

Total sugar yield3

Results are in dry matter basis.

.

Heredia et al. [25].

Canizo et al. [22].

pretreatments1,2

1

2

3

4

5

Steam explosion consists of placing the feedstock in a pressurized reactor where steam is injected. The reactor's operation temperature is in the range of 170–210°C [26, 27]. After a 2–10 min holding cycle, the blown down valve suddenly opens and the resulting pressure variation disrupts the fiber matrix [27]. Sipos et al. [26] achieved an extraction of 89–92% of cellulose associated with sweet sorghum after the use of this technology. The same authors documented an impregnation process for ground sorghum bagasse with up to 3% w/w SO<sup>2</sup> in plastic bags for up to 30 min. The SO2 impregnated bagasse prior to steam pretreatment improved the subsequent saccharification step [27]. The ammonia fiber explosion process known as AFEX is a novel pretreatment that disrupts the internal fiber structure and molecular characteristics of the raw material without the production of liquid. Lee et al. studied the effectiveness in terms of fermentable sugars generation of autohydrolysis or AFEX applied to coastal Bermuda grass [9]. The AFEX process conducted at 100°C for 30 min yielded 94.8% sugars of the theoretical possible value, whereas autohydrolysis at 170°C for 1 h yielded just 55%. The study clearly demonstrated that the proposed AFEX pretreatment enhanced significantly the enzymatic accessibility of the Bermuda grass.

The most employed chemical pretreatment employed for second-generation ethanol production is acid hydrolysis because it is relatively cheap, releases significant quantities of sugars and improves the susceptibility of disrupted fiber components to the next stage of the process consisting of treating the biomass with fiber-degrading enzymes [28]. The major advantage and disadvantage of acid hydrolysis is that it enhances cell wall delignification and generates relevant quantities of known yeast inhibitors. Sulfuric, hydrochloric, hydrofluoric or acetic acids have been used. The process involves adding diluted acid solution (0.1–10% mass fraction) to the milled feedstock followed by pressure-cooking in a reactor. The major control points of acid hydrolysis are the strength of the acid solution, the pressure and temperature and the processing time.

Several investigators have researched the effectiveness of acid hydrolysis of sweet sorghum bagasse. Kurian et al. [29] pressure-cooked sorghum bagasse with sulfuric acid at a concentration of 5 g kg−1 for half an hour and obtained an extract with 92 g L−1 of total sugars, whereas Ban et al. [28] treated the same raw material at a solid-liquid ratio of 10% with 80 g phosphoric acid L−1 at 120°C for 80 min. These authors reported 302 g kg−1 bagasse of reducing sugars after applying this specific acid treatment. Recently, Heredia-Olea et al. [20] researched through surface response methodologies two different acid pretreatments (sulfuric or hydrochloric acid) and one blend of these acids on ground sweet sorghum bagasse harvested post-anthesis. Resulting acid hydrolysates were treated with calcium hydroxide in order to detoxify hydrolysates. The sweet sorghum bagasse free of the sweet juice contained 41.2% cellulose and 24.5% hemicellulose. The response variables were production of C5 and C6 sugars and the three major inhibitors of yeast (acetic acid, 5-hydroxymethylfurfural, and furfural). Results indicated that the different acid pretreatments produced similar quantities of fermentable carbohydrates. These pretreatments liberated from 56 to 57% of the total sugars present in the sorghum bagasse (390–415 mg sugar g−1 bagasse) and from 44 to 61 mg total inhibitors g−1 (**Table 2**). Among the three pretreatments, the HCl treatment was the best alternative due to its relatively lower hydrolysis time (less energy expenditure) and satisfactory yield of C5 and C6 fermentable sugars.

On the other hand, the use of strong alkalies breaks ester bonds of cross-linked lignin and xylans producing an enriched cellulose and hemicellulose fraction. The preferred alkalis are sodium hydroxide, ammonia and calcium hydroxide or lime. The alkaline pretreatments are regularly conducted at relatively lower temperatures, pressures and times compared to other technologies. The main withdraw of chemical treatments is the production of known yeast inhibitors divided into three categories: organic acids (i.e. acetic, formic and levulinic), furans (furfural and 5 hydroxymethylfurfural) and phenolics such as *p*-hydroxybenzoic acid released from lignin moieties [28]. The concentration of these hydrosoluble compounds is known to upset cell physiology and viability and thus the efficacy of ethanologenic microorganisms. According to Amartey and Jeffries [30], the removal of these inhibitors before fermentation can reduce approximately 25% the production cost.

Wang and Cheng investigated the efficiency of lime or calcium hydroxide pretreatment in order to enhance reducing sugar recovery in coastal Bermuda grass [31]. These authors studied the effects of temperature (21–121°C) and lime loadings (0.02–0.2 g/g of dry biomass) followed by biocatalysis with cellulases and cellobiases. The best pretreatment combinations removed approximately 20% of the original total lignin content, which was over twice more than that from the untreated counterpart. The best total fermentable sugar yield for the lime pretreated Bermuda grass (100°C, 15 min, and 0.1 g lime g−1 dry biomass) was 78% of the theoretical maximum. Moreover, this specific pretreatment converted 87%, and 68% of the


1 The compounds are percentage of sweet sorghum bagasse or Bermuda grass.

2 Results are in dry matter basis.

3 Sugars yield = total sugars (mg/g)/[1.11× glucans (mg/g) + 1.14 (xylans (mg/g) + arabinans (mg/g))].

4 Heredia et al. [25].

5 Canizo et al. [22].

and exposed cellulose which was more susceptible to fiber-degrading enzymes. The extruded feedstock treated with the kit of fiber-degrading enzymes had conversion efficiencies of 77.5 and 60 of cellulose and hemicellulose, respectively. These conversion rates are comparable to rates attained when acid and enzyme hydrolyses. The main benefits of the continuous extrusion process are that hydrolysates are virtually free of yeast inhibitors such as acetic acid,

Steam explosion consists of placing the feedstock in a pressurized reactor where steam is injected. The reactor's operation temperature is in the range of 170–210°C [26, 27]. After a 2–10 min holding cycle, the blown down valve suddenly opens and the resulting pressure variation disrupts the fiber matrix [27]. Sipos et al. [26] achieved an extraction of 89–92% of cellulose associated with sweet sorghum after the use of this technology. The same authors documented an impregnation process for ground sorghum bagasse with up to 3% w/w SO<sup>2</sup>

improved the subsequent saccharification step [27]. The ammonia fiber explosion process known as AFEX is a novel pretreatment that disrupts the internal fiber structure and molecular characteristics of the raw material without the production of liquid. Lee et al. studied the effectiveness in terms of fermentable sugars generation of autohydrolysis or AFEX applied to coastal Bermuda grass [9]. The AFEX process conducted at 100°C for 30 min yielded 94.8% sugars of the theoretical possible value, whereas autohydrolysis at 170°C for 1 h yielded just 55%. The study clearly demonstrated that the proposed AFEX pretreatment enhanced signifi-

The most employed chemical pretreatment employed for second-generation ethanol production is acid hydrolysis because it is relatively cheap, releases significant quantities of sugars and improves the susceptibility of disrupted fiber components to the next stage of the process consisting of treating the biomass with fiber-degrading enzymes [28]. The major advantage and disadvantage of acid hydrolysis is that it enhances cell wall delignification and generates relevant quantities of known yeast inhibitors. Sulfuric, hydrochloric, hydrofluoric or acetic acids have been used. The process involves adding diluted acid solution (0.1–10% mass fraction) to the milled feedstock followed by pressure-cooking in a reactor. The major control points of acid hydrolysis are the strength of the acid solution, the pressure and temperature

Several investigators have researched the effectiveness of acid hydrolysis of sweet sorghum bagasse. Kurian et al. [29] pressure-cooked sorghum bagasse with sulfuric acid at a concentration of 5 g kg−1 for half an hour and obtained an extract with 92 g L−1 of total sugars, whereas Ban et al. [28] treated the same raw material at a solid-liquid ratio of 10% with 80 g phosphoric acid L−1 at 120°C for 80 min. These authors reported 302 g kg−1 bagasse of reducing sugars after applying this specific acid treatment. Recently, Heredia-Olea et al. [20] researched through surface response methodologies two different acid pretreatments (sulfuric or hydrochloric acid) and one blend of these acids on ground sweet sorghum bagasse harvested post-anthesis. Resulting acid hydrolysates were treated with calcium hydroxide in order to detoxify hydrolysates. The sweet sorghum bagasse free of the sweet juice contained 41.2% cellulose and 24.5% hemicellulose. The response variables were production of C5 and C6 sugars and the three major inhibitors of yeast (acetic acid, 5-hydroxymethylfurfural, and furfural). Results

impregnated bagasse prior to steam pretreatment

furfural and hydroxymethyl furfural [1, 24].

82 Biofuels - State of Development

in plastic bags for up to 30 min. The SO2

and the processing time.

cantly the enzymatic accessibility of the Bermuda grass.

**Table 2.** Sugars and second generation ethanol generated from sorghum bagasse or Bermuda grass after different pretreatments1,2 .

glucan and xylan into glucose and xylose, respectively. A microwave-assisted alkali pretreatment of coastal Bermuda grass followed by enzyme hydrolysis proved to be an effective technology to improve fermentable sugars. Pretreatments were performed by immersing the feedstock in different dilute alkalis and microwaving the resulting slurries at 250 W for 5–20 min [32]. Sodium hydroxide was the most effective alkali for microwaving of the coastal Bermuda grass. Approximately 87% glucose and 59% xylose yields were achieved after the hydrolysate previously treated with 2% NaOH and microwave treated for 10 min was treated with the fiber-degrading enzymes. For pretreated Bermuda grass, the hypothetical yields of ethanol based on glucose or glucose and xylose were 147 and 208 L ton−1, respectively.

C5 and C6 sugars. The most important and applicable are strains of *Saccharomyces cerevisiae*, *Kluyveromyces marxianus*, *Pichia stipitis*, *Klebsiella oxytoca*, *Klebsiella planticola*, *Candida shehatae*, *Flammulina velutipes* and *Issatchenkia orientalis.* These microorganisms have been innoculated

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Heredia-Olea et al. [20] fermented detoxified sweet sorghum bagasse worts obtained after acid and/or enzyme treatments with a genetically-engineered *S. cerevisiae* or *I. orientalis* which were modified so they were able to metabolize C5 and C6 sugars. *S. cerevisiae* and *I. orientalis* were able to produce 18.4 and 20.9 g ethanol∙100 g−1 dry sweet sorghum bagasse, respectively, and were capable of fermenting 64.4 and 73.3% of the fermentable sugars. Remarkably, most of the bioethanol was produced during the first day of fermentation. Likewise, Ballesteros et al. [35] obtained 16.2 g ethanol L−1(10% solids) from sweet sorghum bagasse hydrolyzates fermented with *K. marxianus*, whereas Kurian et al. [29] working with *P. stipitis* produced 38.7 g ethanol L−1

Although similar pretreatments have been also applied to Bermuda grass (alkaline, hot water, ozonolysis, steam explosion), the diluted acid hydrolysis had better effectiveness in terms of releasing monomeric sugars, specially xylose [23]. Canizo et al. [22] concluded that H2

hydrolysis (121°C, 75 min, 1.25% acid concentration, and 12.5 solid to liquid fraction) released 20.9% glucose, 66.6% xylose and 90% arabinose. Likewise, Ballesteros et al [35] used a hydrolysis scheme with more acid concentration (1.5% w/w) and time (90 min) achieving 66 and 52% xylose and glucose yield, respectively. Wang and Cheng studied the efficiency of lime pretreatment and enzyme hydrolysis in terms of reducing sugar production and bioethanol yield of costal Bermuda grass [31]. The highest total reducing sugar production of 449.8 mg/g of raw biomass was attained using the pretreatment conditions of 0.1 g of lime/g of dry biomass, 100°C, and 15 min. Fermentation tests of the hydrolysates indicated that more than 99% glucose was converted by the yeast with ethanol yields of approximately 95% of the theoretical maximum. Assuming that the annual production of coastal Bermuda Grass was 14.8 tons/ ha, on the basis of the theoretical ethanol yield from raw Bermuda grass of 167 L/dry ton of biomass for only glucose fermentation or 273 L/dry ton of biomass for co-fermentation of glucose and xylose, could be 2056–2755 L/ha. Redding et al. [36] employed higher temperatures (140°C for 30 min) achieving hydrolysis rates of 83% of total xylose and 28.3% glucose. A significant improvement in fermentable carbohydrate yield from Bermuda grass is documented after using a fiber-degrading enzyme complex [23]. Likewise, Canizo et al. [22] hydrolyzed Bermuda grass solids after separating the acid pretreated hydrolysate using 27 FPU celullase g−1 cellulose, 0.6% β-glycosidase and 0.5% xylanase for 168 h at 50°C, pH 4.8 and 150 rpm and concluded that the dual technology released 90.1% of glucose from cellulose and 18% xylose from hemicellulose. Thus, the dual treatment yielded 89.2% of the total sugars from the Bermuda grass cell walls (**Table 2**). Sun and Cheng [37] used 25 FPU g−1 cellulase plus 75 IU g−1 cellobiase to increase approximately 20% the yield of glucose. Likewise, Redding et al. [36] used 40 FPU g−1 cellulase plus 70 IU g−1 cellobiase in order to obtain 95% of the theo-

With the estimated ethanol yield obtained by Canizo et al. [22] is feasible to attain a theoretical amount of 180 mg of ethanol g−1 of dried Bermuda grass. This means a theoretical ethanol

SO<sup>4</sup>

85

alone or in blends at temperatures around 37°C and pH varying from 5.2 to 6 [1, 32].

with a theoretical conversion of 82.5%.

retical fermentable sugar yield.

yield between 1.08 and 4.86 t ha−1 year−1 (**Figure 1**).

Heredia-Olea et al. [20] assayed the quantities of furfural, hydroxymethyl furfural and acetic acid produced after diverse acid hydrolyses. These compounds were generated in higher extents in hydrolysates obtained with higher acid concentrations and processed for longer periods of time. The principal inhibitor was acetic acid which was freed from hemicellulose covalently bound by acetic moieties to lignin [33, 34]. The different sorts of acids broke these linkages generating this acid inhibitor. Detoxification strategies are commercially used to lower inhibitors. The most usual approaches are the use of calcium hydroxide (lime) or activated carbon which entraps phenolic compounds from lignin, ion-exchange resins and enzymes such as laccase [4]. Heredia-Olea et al. [20] successfully detoxified with lime different sorts of acid hydrolysates obtained from sorghum bagasse. Results indicated that the lime treatment removed 19% of the acetic acid and 38% of the hydroxymethylfurfural.

#### **2.3. Enzymatic hydrolysis and fermentation**

The enzyme hydrolysis of fiber is one of the fundamental steps for second-generation alcohol production. Normally, this biocatalysis is performed in chemically treated biomass or alternatively and less frequently with ground untreated fiber. This last process is more recommended for feedstocks low in lignin and has the advantages of saving energy, processing time. Besides the sole utilization of enzymes without the chemical pretreatment is less harmful for the environment. There are several enzymes normally utilized to convert cellulose and hemicellulose into fermentable carbohydrates. They consist of blends of endo and exocellulases, cellobiase, hemicellulases, pectinases, xylanases, B-glucosidase and others [4]. Cellulose is more effectively hydrolyzed by the synergistic activity of endo and exo-acting enzymes (exoglucanases). Nowadays, it is common practice to employ enzyme kits consisting of seven or more cell wall degrading enzymes which act synergistically. Heredia-Olea et al. [25] investigated the efficacy of two concentrations of a fiber-degrading enzyme kit supplemented directly to ground sorghum bagasse and concluded that the higher concentration produced about 20% more fermentable sugars. The main soluble carbohydrates generated during the enzymatic treatment were glucose and xylose. Moreover, the thermoextruded feedstock treated with the fiber enzyme complex was effective when applied in a SSF system. This combination yielded hydrolysates with high amounts of fermentable sugars employing less energy and processing time.

Production of ethanol from physically, chemically and/or enzymatically treated hydrolysates is feasible with the utilization of osmotolerant and C5 (pentose) yeast or bacterial strains. The recent advances in biotechnology has generated numerous genetically modified or engineered yeast and bacteria proficient of fermenting hydrolyzates containing significant amounts of C5 and C6 sugars. The most important and applicable are strains of *Saccharomyces cerevisiae*, *Kluyveromyces marxianus*, *Pichia stipitis*, *Klebsiella oxytoca*, *Klebsiella planticola*, *Candida shehatae*, *Flammulina velutipes* and *Issatchenkia orientalis.* These microorganisms have been innoculated alone or in blends at temperatures around 37°C and pH varying from 5.2 to 6 [1, 32].

glucan and xylan into glucose and xylose, respectively. A microwave-assisted alkali pretreatment of coastal Bermuda grass followed by enzyme hydrolysis proved to be an effective technology to improve fermentable sugars. Pretreatments were performed by immersing the feedstock in different dilute alkalis and microwaving the resulting slurries at 250 W for 5–20 min [32]. Sodium hydroxide was the most effective alkali for microwaving of the coastal Bermuda grass. Approximately 87% glucose and 59% xylose yields were achieved after the hydrolysate previously treated with 2% NaOH and microwave treated for 10 min was treated with the fiber-degrading enzymes. For pretreated Bermuda grass, the hypothetical yields of ethanol based on glucose or glucose and xylose were 147 and 208 L ton−1, respectively.

Heredia-Olea et al. [20] assayed the quantities of furfural, hydroxymethyl furfural and acetic acid produced after diverse acid hydrolyses. These compounds were generated in higher extents in hydrolysates obtained with higher acid concentrations and processed for longer periods of time. The principal inhibitor was acetic acid which was freed from hemicellulose covalently bound by acetic moieties to lignin [33, 34]. The different sorts of acids broke these linkages generating this acid inhibitor. Detoxification strategies are commercially used to lower inhibitors. The most usual approaches are the use of calcium hydroxide (lime) or activated carbon which entraps phenolic compounds from lignin, ion-exchange resins and enzymes such as laccase [4]. Heredia-Olea et al. [20] successfully detoxified with lime different sorts of acid hydrolysates obtained from sorghum bagasse. Results indicated that the lime

treatment removed 19% of the acetic acid and 38% of the hydroxymethylfurfural.

The enzyme hydrolysis of fiber is one of the fundamental steps for second-generation alcohol production. Normally, this biocatalysis is performed in chemically treated biomass or alternatively and less frequently with ground untreated fiber. This last process is more recommended for feedstocks low in lignin and has the advantages of saving energy, processing time. Besides the sole utilization of enzymes without the chemical pretreatment is less harmful for the environment. There are several enzymes normally utilized to convert cellulose and hemicellulose into fermentable carbohydrates. They consist of blends of endo and exocellulases, cellobiase, hemicellulases, pectinases, xylanases, B-glucosidase and others [4]. Cellulose is more effectively hydrolyzed by the synergistic activity of endo and exo-acting enzymes (exoglucanases). Nowadays, it is common practice to employ enzyme kits consisting of seven or more cell wall degrading enzymes which act synergistically. Heredia-Olea et al. [25] investigated the efficacy of two concentrations of a fiber-degrading enzyme kit supplemented directly to ground sorghum bagasse and concluded that the higher concentration produced about 20% more fermentable sugars. The main soluble carbohydrates generated during the enzymatic treatment were glucose and xylose. Moreover, the thermoextruded feedstock treated with the fiber enzyme complex was effective when applied in a SSF system. This combination yielded hydrolysates with high amounts of fermentable sugars employing less energy and processing time. Production of ethanol from physically, chemically and/or enzymatically treated hydrolysates is feasible with the utilization of osmotolerant and C5 (pentose) yeast or bacterial strains. The recent advances in biotechnology has generated numerous genetically modified or engineered yeast and bacteria proficient of fermenting hydrolyzates containing significant amounts of

**2.3. Enzymatic hydrolysis and fermentation**

84 Biofuels - State of Development

Heredia-Olea et al. [20] fermented detoxified sweet sorghum bagasse worts obtained after acid and/or enzyme treatments with a genetically-engineered *S. cerevisiae* or *I. orientalis* which were modified so they were able to metabolize C5 and C6 sugars. *S. cerevisiae* and *I. orientalis* were able to produce 18.4 and 20.9 g ethanol∙100 g−1 dry sweet sorghum bagasse, respectively, and were capable of fermenting 64.4 and 73.3% of the fermentable sugars. Remarkably, most of the bioethanol was produced during the first day of fermentation. Likewise, Ballesteros et al. [35] obtained 16.2 g ethanol L−1(10% solids) from sweet sorghum bagasse hydrolyzates fermented with *K. marxianus*, whereas Kurian et al. [29] working with *P. stipitis* produced 38.7 g ethanol L−1 with a theoretical conversion of 82.5%.

Although similar pretreatments have been also applied to Bermuda grass (alkaline, hot water, ozonolysis, steam explosion), the diluted acid hydrolysis had better effectiveness in terms of releasing monomeric sugars, specially xylose [23]. Canizo et al. [22] concluded that H2 SO<sup>4</sup> hydrolysis (121°C, 75 min, 1.25% acid concentration, and 12.5 solid to liquid fraction) released 20.9% glucose, 66.6% xylose and 90% arabinose. Likewise, Ballesteros et al [35] used a hydrolysis scheme with more acid concentration (1.5% w/w) and time (90 min) achieving 66 and 52% xylose and glucose yield, respectively. Wang and Cheng studied the efficiency of lime pretreatment and enzyme hydrolysis in terms of reducing sugar production and bioethanol yield of costal Bermuda grass [31]. The highest total reducing sugar production of 449.8 mg/g of raw biomass was attained using the pretreatment conditions of 0.1 g of lime/g of dry biomass, 100°C, and 15 min. Fermentation tests of the hydrolysates indicated that more than 99% glucose was converted by the yeast with ethanol yields of approximately 95% of the theoretical maximum. Assuming that the annual production of coastal Bermuda Grass was 14.8 tons/ ha, on the basis of the theoretical ethanol yield from raw Bermuda grass of 167 L/dry ton of biomass for only glucose fermentation or 273 L/dry ton of biomass for co-fermentation of glucose and xylose, could be 2056–2755 L/ha. Redding et al. [36] employed higher temperatures (140°C for 30 min) achieving hydrolysis rates of 83% of total xylose and 28.3% glucose. A significant improvement in fermentable carbohydrate yield from Bermuda grass is documented after using a fiber-degrading enzyme complex [23]. Likewise, Canizo et al. [22] hydrolyzed Bermuda grass solids after separating the acid pretreated hydrolysate using 27 FPU celullase g−1 cellulose, 0.6% β-glycosidase and 0.5% xylanase for 168 h at 50°C, pH 4.8 and 150 rpm and concluded that the dual technology released 90.1% of glucose from cellulose and 18% xylose from hemicellulose. Thus, the dual treatment yielded 89.2% of the total sugars from the Bermuda grass cell walls (**Table 2**). Sun and Cheng [37] used 25 FPU g−1 cellulase plus 75 IU g−1 cellobiase to increase approximately 20% the yield of glucose. Likewise, Redding et al. [36] used 40 FPU g−1 cellulase plus 70 IU g−1 cellobiase in order to obtain 95% of the theoretical fermentable sugar yield.

With the estimated ethanol yield obtained by Canizo et al. [22] is feasible to attain a theoretical amount of 180 mg of ethanol g−1 of dried Bermuda grass. This means a theoretical ethanol yield between 1.08 and 4.86 t ha−1 year−1 (**Figure 1**).

Sweet and biomass dedicated sorghums can yield up to 60 tons of dry forage ha−1 year −1,

These lignocellulosic feedstocks can be converted into bioethanol using the conventional process of milling and pretreatment (chemical and/or enzymatic) with the aim of producing both C5 and C6 sugars in preparation for fermentation with yeast or genetically modified microorganisms capable of fermenting these sugars. After fermentation, the fermented broth is distilled in order to obtain high concentrated ethanol, which is further dehydrated in order to get anhydrous alcohol. An alternative process contemplates the use of thermoplastic extrusion aimed toward the physical disruption of the cell walls of the biomass minimizing the use of considerable amounts of water and chemicals commonly used during pretreatment. These feedstocks can yield up to 310 L anhydrous ethanol dry t−1 and have a great potential for general use.

\*, Esther Perez-Carrillo<sup>1</sup>

Conversion of High Biomass/Bagasse from Sorghum and Bermuda Grass into Second…

http://dx.doi.org/10.5772/intechopen.75064

87

and Jesica R. Canizo2

whereas the perennial Bermuda grass up to 20 tons of dry forage ha−1 year.

, Sergio O. Serna-Saldivar1

1 Centro de Biotecnología-FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de

2 INTA EEA Manfredi, Departamento de Producción Animal, Balcarce, Argentina

[1] Serna-Saldivar SO, Chuck Hernandez C, Perez Carrillo E, Heredia Olea E. Sorghum as a multifunctional crop for the production of fuel ethanol: Current status and future trends. In: Pinheiro Lima MA, Policastro Natalence AP, editors. Bioethanol. London: InTech;

[2] Renewable Fuels Association. The Industry-Statistics. Available from: http://www.etha-

[3] FAOSTAT. Cereal production. In: FAOSTAT; Rome Italy. 2018. Available from: http://

[4] Serna-Saldivar SO, Rooney WL. Production and supply logistics of sweet sorghum as an energy feedstock. Chapter 8. In: Wang L, editor. Sustainable Bioenergy Production. Boca

[5] Rooney W, Blumenthal J, Bean B, Mullet JE. Designing sorghum as a dedicated bioen-

[6] Amosson S, Girase J, Bean B, Rooney W, Becker J. Economic Analysis of Biomass Sorghum for Biofuels Production in the Texas High Plains. AgroLife Extension and Research, Texas

ergy feedstock. Biofuels, Bioproducts and Biorefining. 2007;**1**(2):147-157

nolrfa.org/wp-content/uploads/2017/02/Ethanol-Industry-Outlook-2017.pdf

\*Address all correspondence to: sserna@itesm.mx

Monterrey, Monterrey, N.L., Mexico

**Author details**

Erick Heredia-Olea1

**References**

2012. pp. 55-74

faostat.fao.org/

A&M System; 2011

Raton, FL: Taylor & Francis; 2013

**Figure 1.** General schemes for production of second-generation ethanol from high biomass sorghum, sweet sorghum bagasse or Bermuda grass.

#### **3. Conclusions**

Both biomass sorghum and Bermuda grass adapted to tropical, subtropical and temperate agriculture regions of the world can be effectively converted into second-generation bioethanol due to the composition of the fiber rich in cellulose and hemicellulose and low in lignin. Sweet and biomass dedicated sorghums can yield up to 60 tons of dry forage ha−1 year −1, whereas the perennial Bermuda grass up to 20 tons of dry forage ha−1 year.

These lignocellulosic feedstocks can be converted into bioethanol using the conventional process of milling and pretreatment (chemical and/or enzymatic) with the aim of producing both C5 and C6 sugars in preparation for fermentation with yeast or genetically modified microorganisms capable of fermenting these sugars. After fermentation, the fermented broth is distilled in order to obtain high concentrated ethanol, which is further dehydrated in order to get anhydrous alcohol. An alternative process contemplates the use of thermoplastic extrusion aimed toward the physical disruption of the cell walls of the biomass minimizing the use of considerable amounts of water and chemicals commonly used during pretreatment. These feedstocks can yield up to 310 L anhydrous ethanol dry t−1 and have a great potential for general use.

#### **Author details**

Erick Heredia-Olea1 , Sergio O. Serna-Saldivar1 \*, Esther Perez-Carrillo<sup>1</sup> and Jesica R. Canizo2

\*Address all correspondence to: sserna@itesm.mx

1 Centro de Biotecnología-FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de Monterrey, Monterrey, N.L., Mexico

2 INTA EEA Manfredi, Departamento de Producción Animal, Balcarce, Argentina

#### **References**

**Figure 1.** General schemes for production of second-generation ethanol from high biomass sorghum, sweet sorghum

Both biomass sorghum and Bermuda grass adapted to tropical, subtropical and temperate agriculture regions of the world can be effectively converted into second-generation bioethanol due to the composition of the fiber rich in cellulose and hemicellulose and low in lignin.

bagasse or Bermuda grass.

86 Biofuels - State of Development

**3. Conclusions**


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http://dx.doi.org/10.5772/intechopen.75064

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**Section 4**

**Biodiesel**


**Section 4**
