**2.1. Chemical components of herbaceous lignocellulose**

The lignocellulosic materials were cut, dried, and powdered until the 70 % of the particles became in a range of 32-150 µm in length to promote the cellulase- saccharification and to reduce varying in components in each experiment. The lignin-contents in lignocelluloses were determined as follows. The powdered lignocelluloses (30.0 g) was washed with MeOH and treated with a 1% aqueous solution of NaOH (400 mL) at 95 ºC for 1 h (Silverstein, *et al.*, 2007; Yasuda *et al.*, 2011; Yasuda *et al.*, 2012). After centrifugation at 10,000 rpm for 10 min to separate the precipitates, the supernatant solution was neutralized to pH 5.0 by a dilute HCl solution to give the lignin as a dark brown precipitate. The lignin-contents of napiergrass, rice straw, silvergrass, and bamboo were determined to be 14.9, 18.2, 21.7, and 26.2 wt%, re‐ spectively.

The holocellulose (cellulose and hemicellulose) was isolated as a pale yellow precipitate by the above centrifugation. The saccharide components of holocellulose were determined ac‐ cording to the methods published by the National Renewable Energy Laboratory (NREL) as follows (Sluiter *et al.*, 2010). Sulfuric acid (72%) was added to holocellulose and then diluted with water until the concentration of sulfuric acid became 4%. This was heated at 121 ºC for 1 h in a grass autoclave (miniclave, Büchi AG, Switerland). HPLC analysis of the hydroly‐ zate showed that holocellulose mainly composed of glucose and xylose along with the small amounts of arabinose and galactose. The ash component in lignocelluloses was obtained by the burning of the lignocelluloses (2.0 g) in an electric furnace (KBF784N1, Koyo, Nara, Ja‐ pan) for 2 h at 850 ºC. Chemical components of lignocelluloses are shown in Table 1.

#### **2.2. Saccarification**

As has been previously reported (Yasuda *et al*., 2011; Yasuda *et al.*, 2012), a cellulase from *Acremonium cellulolyticum* (Acremozyme, Kyowa Kasei, Osaka, Japan) was selected by the comparison in activity with other cellulase such as Meycellase (Kyowa Kasei), a cellulase from *Trichoderma viride* (Wako Chemicals, Osaka, Japan) and a cellulase from *Aspergillus ni‐ ger* (Fluka Japan, Tokyo). The cellulase activity of Acremozyme was determined by the method of the breakdown of filter paper (Yasuda *et al*., 2012). At first, cellulase activity was

#### Effectiveness of Lignin-Removal in Simultaneous Saccharification and Fermentation for Ethanol Production from Napiergrass, Rice Straw, Silvergrass, and Bamboo with Different Lignin-Contents http://dx.doi.org/10.5772/54194 93

defined as 10,000 units when two sheets of filter papers (1 cm×1 cm) degraded at pH 5.0 and 45 °C by the cellulase for 150 min. The filter papers were entirely degraded in 114 min by 10 mg of Acremozyme. Thus, cellulase activity of Acremozyme was determined to be 1320 units mg–1 according to the following equation: cellulase activity (units mg–1) = 150×10,000/ (a×b) where a and b denoted weight of cellulase in mg and period in min required for the degradation, respectively.


a) The amounts of components derived from 100 g of lignocellulose.

b) The values in the parenthesis are the amounts (g) of hexose and pentose derived from 100 g of lignocelluloses.

c) Referred from Yasuda *et al*., 2012.

2011; Lin *et al*., 2011a; Lin *et al*., 2010b; Kai *et al.*, 2010; Anderson *et al*., 2008) have been inter‐ ested in napiergrass (*Pennisetum purpureum* Schumach) which is herbaceous lignocellulose with its low lignin- content. During our investigations on bioethanol production, it was found that the alkali-pretreatment of napiergrass enhances scarcely the ethanol yield where‐ as the alkali-pretreatment of silvergrass (*Miscanthus sinensis* Anderss) remarkably enhances the ethanol yield (Yasuda *et al*., 2011). Here, we compared the effectiveness of lignin-remov‐ al between napiergrass and other lingocelluloses with different lignin-contents (rice straw, silvergrass, and bamboo) in order to evaluate the availability of non-pretreated napiergrass

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

The lignocellulosic materials were cut, dried, and powdered until the 70 % of the particles became in a range of 32-150 µm in length to promote the cellulase- saccharification and to reduce varying in components in each experiment. The lignin-contents in lignocelluloses were determined as follows. The powdered lignocelluloses (30.0 g) was washed with MeOH and treated with a 1% aqueous solution of NaOH (400 mL) at 95 ºC for 1 h (Silverstein, *et al.*, 2007; Yasuda *et al.*, 2011; Yasuda *et al.*, 2012). After centrifugation at 10,000 rpm for 10 min to separate the precipitates, the supernatant solution was neutralized to pH 5.0 by a dilute HCl solution to give the lignin as a dark brown precipitate. The lignin-contents of napiergrass, rice straw, silvergrass, and bamboo were determined to be 14.9, 18.2, 21.7, and 26.2 wt%, re‐

The holocellulose (cellulose and hemicellulose) was isolated as a pale yellow precipitate by the above centrifugation. The saccharide components of holocellulose were determined ac‐ cording to the methods published by the National Renewable Energy Laboratory (NREL) as follows (Sluiter *et al.*, 2010). Sulfuric acid (72%) was added to holocellulose and then diluted with water until the concentration of sulfuric acid became 4%. This was heated at 121 ºC for 1 h in a grass autoclave (miniclave, Büchi AG, Switerland). HPLC analysis of the hydroly‐ zate showed that holocellulose mainly composed of glucose and xylose along with the small amounts of arabinose and galactose. The ash component in lignocelluloses was obtained by the burning of the lignocelluloses (2.0 g) in an electric furnace (KBF784N1, Koyo, Nara, Ja‐

pan) for 2 h at 850 ºC. Chemical components of lignocelluloses are shown in Table 1.

As has been previously reported (Yasuda *et al*., 2011; Yasuda *et al.*, 2012), a cellulase from *Acremonium cellulolyticum* (Acremozyme, Kyowa Kasei, Osaka, Japan) was selected by the comparison in activity with other cellulase such as Meycellase (Kyowa Kasei), a cellulase from *Trichoderma viride* (Wako Chemicals, Osaka, Japan) and a cellulase from *Aspergillus ni‐ ger* (Fluka Japan, Tokyo). The cellulase activity of Acremozyme was determined by the method of the breakdown of filter paper (Yasuda *et al*., 2012). At first, cellulase activity was

as the raw materials of bio-ethanol.

**2. Materials and methods**

spectively.

**2.2. Saccarification**

**2.1. Chemical components of herbaceous lignocellulose**

**Table 1.** Components of herbaceous lignocellolosic materials

The saccharification of the powdered cellulosic materials (10.0 g) was performed with Acre‐ mozyme (1.0 g) in an acetate buffer (60 mL, pH 5.0) under vigorous shaking at 45 °C. At the given saccharification time, the portion was taken from the reaction mixture and centrifuged at 12,000 rpm. The supernatant solutions were subjected to analysis for saccharides. The amounts of the reducing saccharides obtained from the saccharification reactions at 30, 40, and 45 °C were almost the same.

#### **2.3. Simultaneous Saccharification and Fermentation (SSF)**

*Saccharomyces cerevisiae* NBRC 2044 was grown at 30 ºC for 24 h in a basal medium (initial pH 5.5) consisting of glucose (20.0 g L–1), peptone (1.0 g L–1, Difco), yeast extract (1.0 g L–1), NaHPO4 (1.0 g L–1), and MgSO4 (3.0 g L–1). After incubation for 24 h, the cell suspension of *S. cerevisiae* was obtained. The grown culture of *S. cerevisiae* showed a cell density of 7.7×107 cells mL–1.

The suspension of cellulosic materials (1.33 g) in an acetate buffer solution (5 mL, pH 5.0) was introduced into the test tube (100 mL) and was autoclaved at 121 ºC for 20 min. After cooling the autoclaved suspension of cellulosic materials, the cell suspension (0.16 mL) of *S. cerevisiae* and the Acremozyme cellulase (133 mg) in an acetate buffer solution (3 mL, pH 5.0) were added (Yasuda *et al*., 2012). The glucan contents were determined to be 436, 475, 410, and 525 mg in non-treated cellulosic materials (1.33 g) of napiergrass, rice straw, silvergrass, and bamboo, respectively. In the case of alkali-treated cellulosic materials (1.33 g), 761 (na‐ piergrass), 774 (rice straw), 999 (silvergrass), and 790 mg (bamboo) of the glucan contents were included. The reaction vessel was connected by tube to messcylinder set in a waterbath to collect the evolved CO2 gas. The reaction progress was monitored by the volume of CO2. Thus, the simultaneous saccharification and fermentation (SSF) process was performed by stirring vigorously the reaction mixture with a magnetic stirrer at 34 °C, which is the op‐ timal temperature.

The resulting lignin-removed holocellulose was isolated by centrifugation of the solution at 10,000 rpm for 10 min. Lignin remained in the alkali solution. The precipitate was washed by dispersion in water to remove the contaminated lignin. After the pH-adjustment to 7.0,

Effectiveness of Lignin-Removal in Simultaneous Saccharification and Fermentation for Ethanol Production from

Napiergrass, Rice Straw, Silvergrass, and Bamboo with Different Lignin-Contents

http://dx.doi.org/10.5772/54194

95

**Figure 1.** SEM images of non-treated (NO) and alkali-pretreated (AL) napiergrass (A), rice straw (B), silvergrass (C), and

bamboo (D). The SEM images were taken under the magnification of 200.

the washed holocellulose was collected by centrifugation and dried.
