**3. Results and discussion**

#### **3.1. Napiergrass (***Pennisetum purpureum* **Schumach)**

Napiergrass is a herbaceous tropical species, native to the east Africa. There are wide varia‐ tion of phenotypes in napiergrass, reflected by plant breeding due to the crossing of dwarf genotype and relative species such as pearl millet (*Pennisetum americanum*) (Ishii *et al*., 2005a, Hanna and Sollenburger, 2007). Dwarf variety of late-heading type originated from Florida, USA, via Thailand (Mukhtar *et al*., 2003) was assessed to be suitable for both grazing (Ishii *et al*., 2005b) and cut-and-carry systems among several sites of southern Kyushu, Japan (Utamy *et al*., 2011). Dwarf variety of napiergrass meets the requirement of lignocellulose for the bio‐ fuel production, because it has low lignin-content and a high herbage mass per year and per area (Rengsirikul *et al*., 2011). Therefore, we have continued to use this dwarf type of napier‐ grass for the bio-ethanol (Yasuda *et al*., 2011) and bio-hydrogen production (Shiragami *et al.*, 2012 ) in University of Miyazaki.

#### **3.2. Alkali-pretreatment**

The powdered lignocelluloses (30.0 g) were washed with MeOH to remove lipids and treat‐ ed with a 1% aqueous solution of NaOH (400 mL) at 95 ºC for 1 h (Silverstein, *et al.,* 2007). 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, the washed holocellulose was collected by centrifugation and dried.

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‐

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

Saccharides were analyzed on a high-performance liquid chromatography system (LC-20AD, Shimadzu, Kyoto, Japan) equipped with RI detector (RID-10A) using anion ex‐ change column (NH2P-50 4E; Shodex Asahipak, 250 mm in length and 4.6 mm in ID, Yoko‐ hama, Japan). Acetonitrile-water (8:2 v/v) was flowed at 1.0 mL min-1 as mobile phase. As a method to supplement LC analysis of saccarides, the amount of the reducing sugars re‐ leased by the saccharification process was analyzed by a modified Somogyi–Nelson method (Kim and Sakano, 1996) assuming the composition of sugars to be C6H12O6. The amounts of pentose were analyzed by a modified orcinol method using 5-methylresorcinol (orcinol), FeCl3 5H2O, and conc HCl (Fernell and King, 1953). Ethanol was analyzed by gas-liquid chromatography using a Shimadzu gas chromatograph (model GC–2014) and a glass col‐ umn of 5% Thermon 1000 on Sunpak-A (Shimadzu) with 2-propanol as an internal stand‐ ard. Scanning electron microscope (SEM) images were taken on a Hitachi S–4100 (Tokyo,

Napiergrass is a herbaceous tropical species, native to the east Africa. There are wide varia‐ tion of phenotypes in napiergrass, reflected by plant breeding due to the crossing of dwarf genotype and relative species such as pearl millet (*Pennisetum americanum*) (Ishii *et al*., 2005a, Hanna and Sollenburger, 2007). Dwarf variety of late-heading type originated from Florida, USA, via Thailand (Mukhtar *et al*., 2003) was assessed to be suitable for both grazing (Ishii *et al*., 2005b) and cut-and-carry systems among several sites of southern Kyushu, Japan (Utamy *et al*., 2011). Dwarf variety of napiergrass meets the requirement of lignocellulose for the bio‐ fuel production, because it has low lignin-content and a high herbage mass per year and per area (Rengsirikul *et al*., 2011). Therefore, we have continued to use this dwarf type of napier‐ grass for the bio-ethanol (Yasuda *et al*., 2011) and bio-hydrogen production (Shiragami *et al.*,

The powdered lignocelluloses (30.0 g) were washed with MeOH to remove lipids and treat‐ ed with a 1% aqueous solution of NaOH (400 mL) at 95 ºC for 1 h (Silverstein, *et al.,* 2007).

timal temperature.

**2.4. Analysis**

Japan).

**3. Results and discussion**

2012 ) in University of Miyazaki.

**3.2. Alkali-pretreatment**

**3.1. Napiergrass (***Pennisetum purpureum* **Schumach)**

**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.

Physical changes from non-pretreated lignocelluloses to alkali-pretreated lignocelluloses were studied using SEM images, as shown in Fig. 1. The fiber bundles observed in lignocel‐ luloses were unloosened by the removal of lignin to change into the thin fibers in the alkalipretreated lignocelluloses. It was expected that the accessibility of enzyme to the cellulose was increased by the alkali- pretreatment.

**Lignocelluloses**

**Lignocelluloses**

Napiergrass (192)

Rice straw (203)

Silvergrass (175)

Bamboo (224)

c) SSF time until the CO2 evolution ceased.

determined by averaging the data of seven experiments.

**Table 3.** The lignin removal effects on SSF processe

**PTa)** *TSA/h b)*

a) Pretreatment (PT). NO: non-treatment, AL: lignin removal by alkali-pretreatment. b) Saccharification time when the total yield of saccharides reached the maximum.

a) Theoretical amounts of ethanol obtained from glucan in lignocellulose (1 g). b) Pretreatment (PT). NO: non-treatment, AL: lignin removal by alkali-pretreatment.

e) Yield of ethanol based on the amounts of hexose occurring in lignocelluloses.

d) The amounts of products per 1 g of lignocellulosewhen the SSF reaction reached the maximum. Data were

**Table 2.** The lignin removal effects on saccharification processe

**(EtOH/mg g-1) a) PT b)** *TSSF /h c)*

d) Yields were based on the amounts of hexose and pentose occurring in lignocelluloses.

Napiergrass NO 120 215 (57.3) 91 (34.3) 307 (48.1)

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

Rice straw NO 120 192 (48.4) 51 (18.0) 244 (35.8)

Silvergrass NO 120 122 (35.7) 39 (34.2) 161 (35.3)

Bamboo NO 120 69 (15.7) 19 (6.3) 88 (11.9)

c) The amounts of products per 1 g of lignocellulosewhen the total yield of saccharides reached the maximum.

**Product c)/mg g-1 (Yield/%) d)**

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

**Hexose Pentose Total**

**Product d)/mg g-1**

**Hexose Pentose EtOH (Yield/%) e)**

AL 120 328 (87.5) 90 (34.0) 419 (65.7)

AL 120 325 (81.9) 125 (44.0) 451 (66.2)

AL 120 178 (52.0) 75 (65.8) 253 (55.5)

AL 120 180 (41.0) 118 (39.3) 297 (40.2)

NO 24 18±5.2 99±1.6 102±3.5 (53.2)

AL 96 38±5.3 125±5.0 121±4.6 (63.1)

NO 24 20±8.0 102±6.5 96±5.9 (47.3)

AL 192 27±7.2 152±6.2 139±1.4 (68.5)

NO 24 13±2.2 48±7.4 41±9.4 (23.5)

AL 96 12±3.4 93±3.5 72±4.3 (41.2)

NO 24 6±5.1 18±5.8 34±1.7 (15.2)

AL 96 22±4.3 111±1.5 78±5.6 (34.8)

*ESA*

97

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

1.36

1.85

1.57

3.39

*ESSF*

1.18

1.45

1.77

2.28

#### **3.3. Lignin-removal effect on saccharification**

The saccharification of alkali-pretreated lignocelluloses (holocellulose, 10.0 g) was per‐ formed with Acremozyme (1.0 g) in an acetate buffer (60 mL, pH 5.0) under vigorous shak‐ ing at 45 °C. The amounts of saccharides obtained from 1 g of alkali-pretreated napiergrass, rice straw, silvergrass, and bamboo were transformed to the amounts per 1.0 g of the alkaliuntreated samples by multiplication with 0.573, 0.613, 0.410, and 0.665 g g-1 which were the contents of holocellulose. Table 2 summarizes the amounts of hexose and pentose after the saccharification reaction for the time (*T*SA) to reach the maximum yields. In the cases of na‐ piergrass and rice straw, the hexose yields (87.5 and 81.9 %) reached almost maximum yields whereas the pentose yields were still low. The largest amount of reducing saccharide was 451 mg obtained from 1.0 g of rice straw.

In order to examine the effectiveness of alkali-pretreatment, the saccharification of the nonpretreated lignocelluloses (10.0 g) was performed under conditions similar to the case of al‐ kali-pretreated lignocelluloses. The largest amount of reducing saccharide was 307 mg g-1 obtained from non-pretreated napiergrass. Figure 2 shows the time-conversions of the sac‐ charification reactions of non-pretreated and alkali-pretreated lignocelluloses. In all cases, the yields of saccharides from the alkali-pretreated lignocelluloses were higher than those from the non-pretreated lignocelluloses. The ratios (*E*SA) of saccharide yields from the alkalipretreated lignocelluloses to those from the non-pretreated lignocelluloses were used as a measure of the effectiveness of the lignin-removal on the saccharification process. The *E*SA values are listed in Table 2.

#### **3.4. Effectiveness of lignin-removal on Simultaneous Saccharification and Fermentation (SSF)**

Ethanol was produced through a simultaneous saccharification and fermentation process (SSF) under optimal conditions as follows (Yasuda, *et al*., 2012). Acremozyme (133 mg) in an acetate buffer solution (3.0 mL, pH 5.0) and the cell suspension (0.16 mL) of *S. cerevisiae* were added to the suspension of alkali-pretreated lignocelluloses (1.33 g) in an acetate buffer solu‐ tion (5.0 mL, pH 5.0). The mixture was reacted at 35 °C under vigorous stirring until the CO2 evolution ceased. The amounts of the products were transformed to the amounts per 1.0 g of the alkali-unpretreated lignocelluloses by the dividing by 1.33 and multiplication with 0.573 (napiergrass), 0.613 (rice straw), 0.410 (silvergrass), and 0.665 g g-1 (bamboo). Table 3 lists the amounts of ethanol and the recovered hexose and pentose which were determined by aver‐ aging the data of seven experiments. The maximum ethanol yield in SSF of alkali-pretreated lignocelluloses was 139 mg g-1 from rice straw.

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


a) Pretreatment (PT). NO: non-treatment, AL: lignin removal by alkali-pretreatment.

b) Saccharification time when the total yield of saccharides reached the maximum.

c) The amounts of products per 1 g of lignocellulosewhen the total yield of saccharides reached the maximum.

d) Yields were based on the amounts of hexose and pentose occurring in lignocelluloses.

**Table 2.** The lignin removal effects on saccharification processe

Physical changes from non-pretreated lignocelluloses to alkali-pretreated lignocelluloses were studied using SEM images, as shown in Fig. 1. The fiber bundles observed in lignocel‐ luloses were unloosened by the removal of lignin to change into the thin fibers in the alkalipretreated lignocelluloses. It was expected that the accessibility of enzyme to the cellulose

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

The saccharification of alkali-pretreated lignocelluloses (holocellulose, 10.0 g) was per‐ formed with Acremozyme (1.0 g) in an acetate buffer (60 mL, pH 5.0) under vigorous shak‐ ing at 45 °C. The amounts of saccharides obtained from 1 g of alkali-pretreated napiergrass, rice straw, silvergrass, and bamboo were transformed to the amounts per 1.0 g of the alkaliuntreated samples by multiplication with 0.573, 0.613, 0.410, and 0.665 g g-1 which were the contents of holocellulose. Table 2 summarizes the amounts of hexose and pentose after the saccharification reaction for the time (*T*SA) to reach the maximum yields. In the cases of na‐ piergrass and rice straw, the hexose yields (87.5 and 81.9 %) reached almost maximum yields whereas the pentose yields were still low. The largest amount of reducing saccharide

In order to examine the effectiveness of alkali-pretreatment, the saccharification of the nonpretreated lignocelluloses (10.0 g) was performed under conditions similar to the case of al‐ kali-pretreated lignocelluloses. The largest amount of reducing saccharide was 307 mg g-1 obtained from non-pretreated napiergrass. Figure 2 shows the time-conversions of the sac‐ charification reactions of non-pretreated and alkali-pretreated lignocelluloses. In all cases, the yields of saccharides from the alkali-pretreated lignocelluloses were higher than those from the non-pretreated lignocelluloses. The ratios (*E*SA) of saccharide yields from the alkalipretreated lignocelluloses to those from the non-pretreated lignocelluloses were used as a measure of the effectiveness of the lignin-removal on the saccharification process. The *E*SA

**3.4. Effectiveness of lignin-removal on Simultaneous Saccharification and Fermentation**

Ethanol was produced through a simultaneous saccharification and fermentation process (SSF) under optimal conditions as follows (Yasuda, *et al*., 2012). Acremozyme (133 mg) in an acetate buffer solution (3.0 mL, pH 5.0) and the cell suspension (0.16 mL) of *S. cerevisiae* were added to the suspension of alkali-pretreated lignocelluloses (1.33 g) in an acetate buffer solu‐ tion (5.0 mL, pH 5.0). The mixture was reacted at 35 °C under vigorous stirring until the CO2 evolution ceased. The amounts of the products were transformed to the amounts per 1.0 g of the alkali-unpretreated lignocelluloses by the dividing by 1.33 and multiplication with 0.573 (napiergrass), 0.613 (rice straw), 0.410 (silvergrass), and 0.665 g g-1 (bamboo). Table 3 lists the amounts of ethanol and the recovered hexose and pentose which were determined by aver‐ aging the data of seven experiments. The maximum ethanol yield in SSF of alkali-pretreated

was increased by the alkali- pretreatment.

**3.3. Lignin-removal effect on saccharification**

was 451 mg obtained from 1.0 g of rice straw.

lignocelluloses was 139 mg g-1 from rice straw.

values are listed in Table 2.

**(SSF)**


a) Theoretical amounts of ethanol obtained from glucan in lignocellulose (1 g).

b) Pretreatment (PT). NO: non-treatment, AL: lignin removal by alkali-pretreatment.

c) SSF time until the CO2 evolution ceased.

d) The amounts of products per 1 g of lignocellulosewhen the SSF reaction reached the maximum. Data were determined by averaging the data of seven experiments.

e) Yield of ethanol based on the amounts of hexose occurring in lignocelluloses.

**Table 3.** The lignin removal effects on SSF processe

was low, irrespective of higher content of hexose probably because of poor accessibility of

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

99

Also, the SSF process was applied to the non-pretreated lignocelluloses. The time- conver‐ sions of CO2-evolution were compared between non-pretreated and the alkali-pretreated lignocelluloses, as shown in Fig. 3. The yields of ethanol from non- pretreated lignocellulo‐ ses were lower compared with the cases from alkali-pretreated lignocelluloses. Among the non-pretreated lignocelluloses, the largest amount of ethanol was 102 mg g-1 obtained from napiergrass. The enhanced effect of SSF yields by alkali-pretreatment was evaluated by the ratio (*E*SSF) of ethanol yields from the alkali-pretreated lignocelluloses to those from non-pre‐

(A) (B)

(C) (D)

CO2

**Figure 3.** CO2-evolution in the SSF of napiergrass (A), rice straw (B), silvergrass (C), and bamboo (D) for the non-treat‐ ed lignocelluloses (●) and the alkali-pretreated lignocelluloses (△). The amounts of CO<sup>2</sup> from alkali-pretreated ligno‐ celluloses was transformed to the amounts per 1 g of the alkali-unpretreated samples by multiplication with 0.573

It is noteworthy that the SSF of alkali-pretreated lignocelluloses was remarkably slowed down in all cases. In the fermentation by *S. cerevisiae* of the alkali-pretreated lignocelluloses, a nitrogen-source and a mineral were thought to be insufficient, since the aminoacids and

evolution/mL

CO2

evolution/mL

0 24 48 72 96

Reaction time/h

0 24 48 72 96 120 144 168 192

Reaction time/h

the enzyme to holocellulosic components of bamboo (Yamashita *et al*., 2010).

treated lignocelluloses. The *E*SSF values are listed in Table 3.

CO2

evolution/mL

CO2

evolution/mL

0 24 48 72 96

(napiergrass), 0.613 (rice straw), 0.410 (silvergrass), and 0.665 g g-1 (bamboo).

Reaction time/h

0 24 48 72 96

Reaction time/h

**Figure 2.** Time conversion of the saccharification of napiergrass (A), rice straw (B), silvergrass (C), and bamboo (D) for the non-pretreated lignocelluloses (●) and the alkali-pretreated lignocelluloses (△). The amounts of sugar from the alkali-pretreated lignocelluloses were transformed to the amounts per 1 g of the alkali-unpretreated samples by mul‐ tiplication with 0.573 (napiergrass), 0.613 (rice straw), 0.410 (silvergrass), and 0.665 g g-1 (bamboo).

After the SSF, the pentose remained in the solution, although the hexose was consumed by the fermentation with *S. cerevisiae*. The amounts of pentose was compared between SSF and cellulase-saccharification processes under the optimized conditions. The amounts of pentose formed in SSF were larger than those in saccharification, except for the case of bamboo (Ta‐ ble 2 and 3). Therefore, the SSF process accelerated the hydrolysis of cellulosic components compared to the saccharification process. The consumption of saccharides by fermentation with *S. cerevisiae* might move the equilibrium to the product side in the hydrolysis of cellulo‐ sic components to saccharides with Acremozyme. In the case of bamboo, the ethanol yield was low, irrespective of higher content of hexose probably because of poor accessibility of the enzyme to holocellulosic components of bamboo (Yamashita *et al*., 2010).

Also, the SSF process was applied to the non-pretreated lignocelluloses. The time- conver‐ sions of CO2-evolution were compared between non-pretreated and the alkali-pretreated lignocelluloses, as shown in Fig. 3. The yields of ethanol from non- pretreated lignocellulo‐ ses were lower compared with the cases from alkali-pretreated lignocelluloses. Among the non-pretreated lignocelluloses, the largest amount of ethanol was 102 mg g-1 obtained from napiergrass. The enhanced effect of SSF yields by alkali-pretreatment was evaluated by the ratio (*E*SSF) of ethanol yields from the alkali-pretreated lignocelluloses to those from non-pre‐ treated lignocelluloses. The *E*SSF values are listed in Table 3.

Yields of saccharides/ mg g

Yields of saccharides/ mg g



0 24 48 72 96 120

(C) (D)

Saccharification time/h Saccharification time/h


Yields of saccharides/ mg g

Yields of saccharides/ mg g


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

(B)

Saccharification time/h Saccharification time/h

**Figure 2.** Time conversion of the saccharification of napiergrass (A), rice straw (B), silvergrass (C), and bamboo (D) for the non-pretreated lignocelluloses (●) and the alkali-pretreated lignocelluloses (△). The amounts of sugar from the alkali-pretreated lignocelluloses were transformed to the amounts per 1 g of the alkali-unpretreated samples by mul‐

After the SSF, the pentose remained in the solution, although the hexose was consumed by the fermentation with *S. cerevisiae*. The amounts of pentose was compared between SSF and cellulase-saccharification processes under the optimized conditions. The amounts of pentose formed in SSF were larger than those in saccharification, except for the case of bamboo (Ta‐ ble 2 and 3). Therefore, the SSF process accelerated the hydrolysis of cellulosic components compared to the saccharification process. The consumption of saccharides by fermentation with *S. cerevisiae* might move the equilibrium to the product side in the hydrolysis of cellulo‐ sic components to saccharides with Acremozyme. In the case of bamboo, the ethanol yield

tiplication with 0.573 (napiergrass), 0.613 (rice straw), 0.410 (silvergrass), and 0.665 g g-1 (bamboo).

0 24 48 72 96 120

0 24 48 72 96 120

0 24 48 72 96 120

**Figure 3.** CO2-evolution in the SSF of napiergrass (A), rice straw (B), silvergrass (C), and bamboo (D) for the non-treat‐ ed lignocelluloses (●) and the alkali-pretreated lignocelluloses (△). The amounts of CO<sup>2</sup> from alkali-pretreated ligno‐ celluloses was transformed to the amounts per 1 g of the alkali-unpretreated samples by multiplication with 0.573 (napiergrass), 0.613 (rice straw), 0.410 (silvergrass), and 0.665 g g-1 (bamboo).

It is noteworthy that the SSF of alkali-pretreated lignocelluloses was remarkably slowed down in all cases. In the fermentation by *S. cerevisiae* of the alkali-pretreated lignocelluloses, a nitrogen-source and a mineral were thought to be insufficient, since the aminoacids and the mineral were removed from lignocelluloses by alkali-pretreatment and the additional nutrients were not added in the SSF process (Alfenore *et al*., 2003). Moreover, the fermenta‐ tion process was affected by the inhibitory materials derived from the alkali-pretreatment since *T*SA of both non-pretreated and the alkali-pretreated lignocelluloses were almost same (Alvira, 2010).

ethanol was produced in 102 mg g-1 and 121 mg g-1 from napiergrass through the SSF with‐ out and with alkali-pretreatment, respectively. Taking into consideration the low effective‐ ness of lignin-removal in ethanol yield, the retardation of fermentation rate, the loss of nutrients for the fermentation by *S. cerevisiae*, and the cost of lignin-removal, we concluded that ethanol production from napiergrass should be performed through the SSF process without the alkali-pretreatment. For example, Inoue and his coworkers (Hideno *et al.*, 2009) have recently proposed the enzymatic saccharification of rice straw treated by a wet disk milling method without chemical pretreatment. Even so, the development of a pretreatment method with low energy and low cost to enhance saccharification yields by the structural change of cellulosic components rather than lignin-removal are desired for economically viable bio-ethanol production. In our group, the development of more efficient pretreatment method other than alkali-pretreatment to produce effectively bioethanol from napiergrass is

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

101

Moreover, the fermentation of the pentose remaining in SSF is important subject. We (Yasu‐ da *et al*., 2012) started the pentose fermentation using a recombinant *Escherichia coli* KO11. Pentose fermentation by *E. coli* KO11 produced additionally 31.4 mg g-1 of ethanol. Under the optimized conditions, the combination of the SSF and KO11 fermentation processes re‐ sulted in the production of 144 mg g–1 of ethanol from the non-pretreated napiergrass pow‐ der. The ethanol yield was 44.2% of the theoretical yield based on the hexose (375 mg) and

This study was done as a part of the project entitled "Research and Development of Catalyt‐ ic Process for Efficient Conversion of Cellulosic Biomass into Biofuels and Chemicals" through Special Funds for Education and Research from the Ministry of Education, Culture,

, Tomoko Matsumoto2

1 Department of Applied Chemistry, Faculty of Engineering, University of Miyazaki, Ga‐

2 Center for Collaborative Research and Community Cooperation, University of Miyazaki,

3 Department of Animal and Grassland Sciences, Faculty of Agriculture, University of Miya‐

and Yasuyuki Ishii3

, Tsutomu Shiragami1

,

pentose (265 mg) derived from 1 g of dry powdered napiergrass.

now in progress.

**Acknowledgements**

**Author details**

Masahide Yasuda1

Kazuhiro Sugamoto1

Sports, Science, and Technology of Japan.

kuen-Kibanadai Nishi, Miyazaki, Japan

Gakuen-Kibanadai Nishi, Miyazaki, Japan

zaki, Gakuen-Kibanadai Nishi, Miyazaki, Japan

, Keisuke Takeo1

, Yoh-ichi Matsushita1

#### **3.5. Availability of napiergrass as raw materials for ethanol production**

In the cases of rice straw, silvergrass, and bamboo with relatively high lignin-contents (18.2– 26.2 wt%), the lignin-removal was effective for both saccharification and SSF processes be‐ cause of the larger *E*SA (1.57–3.39) and *E*SSF values (1.45–2.28). However, in the case of napier‐ grass with low lignin-content (14.9 wt%), the *E*SSF value was small (1.18). Figure 4 shows the plots of the *E*SSF values against the lignin-contents of lignocelluloses. As the lignin-contents increased, the *E*SSF values gradually increased. From the extrapolation of a fitting line of the plots, it is assumed that the *E*SSF values at 13.4 wt% of lignin-content will reach 1.0 which means no enhancement effect of lignin-removal. Thus, it was elucidated that the alkali-treat‐ ment was effective for lignocelluloses with higher lignin content than 13.4 wt%, but was not effective as the pretreatment of lignocelluloses with lower lignin content than 13.4 wt%.

**Figure 4.** Dependence of *E*SSF on the lignin contents in the SSF of napiergrass (A), rice straw (B), silvergrass (C), and bamboo (D). The plots showed that the *E*SSF value became 1.0 at 13.4 wt% of lignin content.

#### **4. Conclusion**

In general, the alkali-pretreatment increases the accessibility of enzymes to the cellulose by the lignin-removal. Therefore alkali-pretreatment is effective for saccarification of the ligno‐ cellulose with higher lignin contents. In the case of napiegrass with low lignin- content, 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 101

ethanol was produced in 102 mg g-1 and 121 mg g-1 from napiergrass through the SSF with‐ out and with alkali-pretreatment, respectively. Taking into consideration the low effective‐ ness of lignin-removal in ethanol yield, the retardation of fermentation rate, the loss of nutrients for the fermentation by *S. cerevisiae*, and the cost of lignin-removal, we concluded that ethanol production from napiergrass should be performed through the SSF process without the alkali-pretreatment. For example, Inoue and his coworkers (Hideno *et al.*, 2009) have recently proposed the enzymatic saccharification of rice straw treated by a wet disk milling method without chemical pretreatment. Even so, the development of a pretreatment method with low energy and low cost to enhance saccharification yields by the structural change of cellulosic components rather than lignin-removal are desired for economically viable bio-ethanol production. In our group, the development of more efficient pretreatment method other than alkali-pretreatment to produce effectively bioethanol from napiergrass is now in progress.

Moreover, the fermentation of the pentose remaining in SSF is important subject. We (Yasu‐ da *et al*., 2012) started the pentose fermentation using a recombinant *Escherichia coli* KO11. Pentose fermentation by *E. coli* KO11 produced additionally 31.4 mg g-1 of ethanol. Under the optimized conditions, the combination of the SSF and KO11 fermentation processes re‐ sulted in the production of 144 mg g–1 of ethanol from the non-pretreated napiergrass pow‐ der. The ethanol yield was 44.2% of the theoretical yield based on the hexose (375 mg) and pentose (265 mg) derived from 1 g of dry powdered napiergrass.
