Elsevier

Bioresource Technology

Volume 146, October 2013, Pages 393-399
Bioresource Technology

Comparison of the enzymatic digestibility of physically and chemically pretreated selected line of diploid-Miscanthus sinensis Shiozuka and triploid-M. × giganteus

https://doi.org/10.1016/j.biortech.2013.07.084Get rights and content

Highlights

  • Miscanthus sinensis “Shiozuka” contained moderate sugar but low lignin contents.

  • Shiozuka was more sensitive to physical and chemical treatments than M × G.

  • Enzymatic digestibilities of the pretreated Shiozuka were higher than those of M × G.

  • Shiozuka is a promising bioenergy grass cultivar.

Abstract

The diploid Miscanthus sinensis “Shiozuka” which was selected as a high-biomass producing line, and the triploid M. × giganteus (M × G) were treated by ball milling (physical treatment) and alkaline hydrogen peroxide treatment (AHP; chemical treatment), and their structural sugar compositions and enzymatic digestibility were compared. The structural sugar content of Shiozuka was moderate and lower than that of M × G. The Klason lignin content of Shiozuka was also lower than that of M × G. However, Shiozuka was sensitive to ball milling and AHP treatment; ball milled and AHP-treated Shiozuka had higher enzymatic digestibility than ball milled and AHP-treated M × G. Shiozuka would be promising feedstock to obtain fermentable sugars with low energy consumption. Finally, enzymes for the hydrolysis of chemically treated Miscanthus were isolated from Trichoderma reesei ATCC 66589 and Penicillium pinophilum. The sugar yield could be increased by enzymatic hydrolysis of AHP-treated samples with NaOH and H2O2 and the isolated enzymes.

Introduction

The genus Miscathus including M. sinensis called “Susuki”, and M. × giganteus (M × G) (Hodkinson and Renvoize, 2001), are considered to be the bioenergy crops because they are C4 plants that can fix CO2 in high efficiency at low temperature, and less requirement of fertilizer for cultivation (Lewandowski et al., 2003, Naidu and Long, 2004, Heaton et al., 2010, Anzoua et al., 2011, Clifton-Brown et al., 2011). In particular, M × G, which is a natural sterile allotriploid (3n = 57) from the cross between diploid M. sinensis (2n = 38) and tetraploid M. sacchariflorus (4n = 76), has attracted attention and widespread investigation as a nonfood bioenergy feedstock because of its potential for a huge amount of biomass production, and its ability to maintain high photosynthetic productivity at low temperatures by unlike other C4 plants in the EU and USA (Heaton et al., 2010, Anzoua et al., 2011, Clifton-Brown et al., 2011). M × G has been commercially used as a biofuel crop of thermal power plants in the EU (Christian et al., 2008). However, M × G has some limitations, for example, its poorly establishment in cold areas at high latitudes, increased risk of susceptibility to disease because it has only a single genotype and difficulty in breeding for improved characteristics owing to its triploid nature (Stewart et al., 2009).

On the other hand, M. sinensis, which is a parent diploid species of M × G, is widespread and has been used for biomass production, traditional houses and buildings, organic fertilizer, and livestock feed for centuries in Japan (Stewart et al., 2009), although these applications are very rare now. Recently, M. sinensis has attracted interest because it exhibits high productivity in cool regions, such as Hokkaido Prefecture in Japan, although the biomass production depends on the environmental conditions and genotypes. The selection and evaluation of M. sinensis as a bioenergy crop in cold areas has been previously performed and three wild grown populations (Matsumae, Akeno, and Shiozuka) were selected as high biomass potential lines (Anzoua et al., 2011). Concretely, the biomass productions based on the dry weights of M. sinensis Matsumae, M. sinensis Akeno, and M. sinensis Shiozuka, which were harvested in Sapporo (43°04′N, 141°20′E) in 2009, were 1622 ± 692, 2653 ± 635, and 2531 ± 561 g plant−1year−1, respectively (Anzoua et al., 2011). No information is available regarding the chemical compositions, efficiency of pretreatment, and enzymatic digestibility for producing fermentable sugars in the Japanese M. sinensis selected line in terms of high-biomass production, although some useful information is available on general M. sinensis plants (Brosse et al., 2012, Hodgson et al., 2010, Huang et al., 2012, Kim et al., 2012, Yoshida et al., 2008).

Alkaline hydrogen peroxide (AHP) treatment with a stabilizer has been used as an environmentally friendly method for paper pulp bleaching. Without stabilizer, hydrogen peroxide is decomposed to highly reactive oxygen species (superoxide and hydroxyl radicals) as per the following equation (Gould, 1984, Gould, 1985, Selig et al., 2009):H2O2H++HOO-,H2O2+HOO-HO+O2-+H2O

The H2O2-derived radicals are thought to contribute to the depolymerization and decomposition of lignin to low-molecule-weight compounds. Hence, AHP treatment without stabilizer has been investigated as a pretreatment for enzymatic hydrolysis of lignocellulosic biomass such as wheat straw, rice straw, sugarcane bagasse, and corn stover (Banerjee et al., 2012, Gould, 1984, Monte et al., 2011, Selig et al., 2009). AHP treatment of M × G has also been previously reported (Huyen et al., 2010, Wang et al., 2010). However, information about the AHP treatment of Miscanthus has not been enough, and there is no information about the AHP treatment of M. sinensis.

Cellulases are key enzymes in the biorefinery process based on sugar platform. The costs of cellulases including the purchase cost of commercial cellulases or their production cost, contribute to a large proportion of the total costs of biorefinery process such as the bioethanol production (Fujimoto et al., 2008). On-site production of cellulases is considered to be a solution for reducing the cost of cellulases, including the purchase cost of and transportation costs for distribution (Fujimoto et al., 2008, Hideno et al., 2012). Trichoderma reesei and its mutant strains have been widely investigated (Zhang et al., 2006) as promising hyper cellulase-producing fungi. However, these strains have been known to have low β-glucosidase activity with respect to the activity of a cellulose-hydrolyzing enzyme such as cellobiohydrolases (CBHs) (Zhang et al., 2006). Moreover, various enzymes are required for enzymatic hydrolysis of feedstock such as rice straw, Miscanthus sp., since these materials contain not only cellulose but also various polysaccharide (e.g., xylan, arabinan), which is called hemicellulose and cover cellulose. The supplementation including β-glucosidase and other enzymes, which have synergy effect with CBH of T. reesei, is required.

In this study, the enzymatic digestibility of physically and chemically pretreated M. sinensis Shiozuka and M × G were compared to uncover the possibility of using M. sinensis Shiozuka as material for fermentable sugars, and to develop high efficiency pretreatment and saccharification of Miscanthus. The M. sinensis Shiozuka from wild populations grown in Japan, and M × G were cultivated in Sapporo in Japan, and harvested materials were used. The components of these samples were compared, and the ball milling (physical treatment) and alkaline-peroxide treatment (chemical treatment) of these samples were carried out and evaluated by enzymatic hydrolysis using commercial cellulase. Finally, a suitable enzyme was prepared for the enzymatic hydrolysis of Miscanthus by cultivating T. reesei and Penicillium pinophilum, which are cellulase-producing filamentous fungi.

Section snippets

M. sinensis Shiozuka and M × G

M. sinensis Shiozuka, which were selected for high biomass-potential from various wild populations (Anzoua et al., 2011), and M × G were cultivated in Sapporo (43°04′N, 141°20′E), harvested in November 2011, and used for this study. The sample was manually cut to approximately 1–2 cm, and pre-milled using ABSOLUTE3 (OSAKA CHEMICAL CO., Ltd., Osaka, Japan. Speed: High, VARIABLE: 0; 30 s). The pre-milled sample was air-dried at 60 °C over night, and electrically sieved for 30 min, and materials that

Comparison of the main composition of Miscanthus

M. sinensis Shiozuka was selected as the representative high biomass production M. sinensis, and was compared with M × G cultivated in 2011. The main components of M. sinensis Shiozuka and M × G cultivated in 2011 are shown in Table 2. The glucose content of Shiozuka cultivated in 2011 was lower and the Klason lignin content was slightly higher than the contents recorded in the 2010 cultivated line. M × G had higher glucose and Klason lignin contents than all other samples. In a previous study, M × G

Conclusion

To determine the efficiencies of pretreatment and enzymatic digestibility of Miscanthus, the chemical composition and the efficiency of ball milling and AHP treatment for enzymatic hydrolysis were compared in the 3 selected lines of M. sinensis and M × G. Moreover, suitable cellulases were prepared for the enzymatic hydrolysis of AHP-treated Miscanthus. Shiozuka, which showed the highest biomass production in Sapporo, was a promising feedstock material for obtaining fermentable sugar by enzymatic

Acknowledgements

This work was supported by the Shorai Foundation for Science and Technology, and by JSPS Kakenhi Grant-in-Aid for Young Scientist (B) (Grant No. 22780292). The authors thank Dr. Seiya Watanabe (Ehime University) for supplying the commercial enzyme.

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