Components of herbaceous lignocellolosic materials
Second-generation biofuels from lignocellulosic materials have gained much attention since the lignocelluloses are not in competition with food sources and animal feed and will provide a new sustainable energy sources alternative to petroleum-based fuels (Galbe and Zacchi, 2007). Bioethanol production from herbaceous lignocellulose such as corn stover (Ryu and Karim, 2011), rice straw (Ko at al., 2009), sweet sorghum bagasse (Cardoba et al., 2010), switchgrass (Keshwani and Cheng, 2009), bamboo (Sathitsuksanoh at al., 2010), wheat straw (Talebnia et al., 2010), alfalfa stems (González-García at al., 2010), and silvergrass (Guo et al., 2008) has been extensively developed through a variety of processes combining the biological saccharification and fermentation steps with the pre-treatment methods. In almost all processes, the pretreatments to remove the lignin components and to promote an enzymatic digestibility of cellulosic components are carried out by the use of energy and cost which are frequently higher than those of bio-fuels gained (Alvira et al., 2010). If lignocelluloses with low lignin-content are selected, the operation to remove the lignin might be excluded from the bio-ethanol process.
Among the many kinds of lignocelluloses, therefore, we (Yasuda et al., 2011; Yasuda
2. Materials and methods
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%, respectively.
The holocellulose (cellulose and hemicellulose) was isolated as a pale yellow precipitate by the above centrifugation. The saccharide components of holocellulose were determined according 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 hydrolyzate 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, Japan) for 2 h at 850 ºC. Chemical components of lignocelluloses are shown in Table 1.
|Napiergrass c)||57.3 (37.5 : 26.5)||14.9||12.7||15.1|
|Rice straw||61.3 (39.7 : 28.4)||18.2||17.7||2.8|
|Silvergrass||41.0 (34.2 : 11.4)||21.7||4.0||33.3|
|Bamboo||66.5 (43.9 : 30.0)||26.2||1.4||5.9|
|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
As has been previously reported (Yasuda et al., 2011; Yasuda et al., 2012), a cellulase from
The saccharification of the powdered cellulosic materials (10.0 g) was performed with Acremozyme (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)
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
Saccharides were analyzed on a high-performance liquid chromatography system (LC-20AD, Shimadzu, Kyoto, Japan) equipped with RI detector (RID-10A) using anion exchange column (NH2P-50 4E; Shodex Asahipak, 250 mm in length and 4.6 mm in ID, Yokohama, 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 released 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 column of 5% Thermon 1000 on Sunpak-A (Shimadzu) with 2-propanol as an internal standard. Scanning electron microscope (SEM) images were taken on a Hitachi S–4100 (Tokyo, Japan).
3. Results and discussion
3.1. Napiergrass (
Napiergrass is a herbaceous tropical species, native to the east Africa. There are wide variation of phenotypes in napiergrass, reflected by plant breeding due to the crossing of dwarf genotype and relative species such as pearl millet (
The powdered lignocelluloses (30.0 g) were washed with MeOH to remove lipids and treated 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.
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 lignocelluloses were unloosened by the removal of lignin to change into the thin fibers in the alkali-pretreated lignocelluloses. It was expected that the accessibility of enzyme to the cellulose was increased by the alkali- pretreatment.
3.3. Lignin-removal effect on saccharification
The saccharification of alkali-pretreated lignocelluloses (holocellulose, 10.0 g) was performed with Acremozyme (1.0 g) in an acetate buffer (60 mL, pH 5.0) under vigorous shaking 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 alkali-untreated 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 (
In order to examine the effectiveness of alkali-pretreatment, the saccharification of the non-pretreated lignocelluloses (10.0 g) was performed under conditions similar to the case of alkali-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 saccharification 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 (
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
|Napiergrass||NO||120||215 (57.3)||91 (34.3)||307 (48.1)||1.36|
|AL||120||328 (87.5)||90 (34.0)||419 (65.7)|
|Rice straw||NO||120||192 (48.4)||51 (18.0)||244 (35.8)||1.85|
|AL||120||325 (81.9)||125 (44.0)||451 (66.2)|
|Silvergrass||NO||120||122 (35.7)||39 (34.2)||161 (35.3)||1.57|
|AL||120||178 (52.0)||75 (65.8)||253 (55.5)|
|Bamboo||NO||120||69 (15.7)||19 (6.3)||88 (11.9)||3.39|
|AL||120||180 (41.0)||118 (39.3)||297 (40.2)|
|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 lignocellulose
d) Yields were based on the amounts of hexose and pentose occurring in lignocelluloses.
|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 lignocellulose
e) Yield of ethanol based on the amounts of hexose occurring in lignocelluloses.
After the SSF, the pentose remained in the solution, although the hexose was consumed by the fermentation with
Also, the SSF process was applied to the non-pretreated lignocelluloses. The time- conversions 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 lignocelluloses 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 (
It is noteworthy that the SSF of alkali-pretreated lignocelluloses was remarkably slowed down in all cases. In the fermentation by
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 because of the larger
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 lignocellulose with higher lignin contents. In the case of napiegrass with low lignin- content, ethanol was produced in 102 mg g-1 and 121 mg g-1 from napiergrass through the SSF without and with alkali-pretreatment, respectively. Taking into consideration the low effectiveness of lignin-removal in ethanol yield, the retardation of fermentation rate, the loss of nutrients for the fermentation by
Moreover, the fermentation of the pentose remaining in SSF is important subject. We (Yasuda et al., 2012) started the pentose fermentation using a recombinant