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Evaluation of a transgenic poplar as a potential biomass crop for biofuel production
Bioresource Technology 129 (2013) 639–641
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Evaluation of a transgenic poplar as a potential biomass crop for biofuel production Young Hoon Jung a,1, Hong Joo Cho a,1, Jae-Soon Lee b, Eun Woon Noh b, Ohkmae K. Park a,⇑, Kyoung Heon Kim a,⇑ a b
School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea Department of Forest Genetic Resources, Korea Forest Research Institute, Suwon 441-847, Republic of Korea
h i g h l i g h t s " A transgenic poplar (OX8) with overexpressed RabG3bCA from Arabidopsis was tested. " OX8 grows faster and taller than the wid type (WT). " OX8 was 9.8% higher in glucan content compared to WT. " Pretreated OX8 exhibited a higher enzymatic digestibility than WT.
a r t i c l e
i n f o
Article history: Received 26 October 2012 Received in revised form 8 December 2012 Accepted 10 December 2012 Available online 20 December 2012 Keywords: Transgenic poplar Biomass Biofuel Pretreatment Enzymatic digestibility
a b s t r a c t A transgenic poplar, in which the RabG3bCA gene from Arabidopsis was overexpressed, was analyzed for its biomass composition and enzymatic digestibility after chemical pretreatment. In comparison with a wild-type poplar (WT), the transgenic poplar (OX8) showed 9.8% higher glucan content. The levels of other biomass components did not differ greatly between WT and OX8. When WT and OX8 samples were pretreated by sulfuric acid (1%, w/v at 190 °C), sodium hydroxide (1%, w/v at 190 °C), or ammonia (14%, w/w at 80 °C), the washed pretreated solids of OX8 exhibited a higher enzymatic digestibility than those of WT in each chemical pretreatment. The sodium hydroxide pretreatment was the most effective among the three pretreatment processes, showing 58.7% and 69.4% of theoretical glucose yield from the saccharification of pretreated OX8 and WT, respectively. The transgenic poplar, growing faster and taller, was found to contain more glucan and have a higher enzymatic digestibility than WT. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Producing fermentable sugar at low costs is the key to developing commercially viable alternative liquid fuels such as bioethanol from lignocellulose (Lynd et al., 2008). Currently, the cost of producing fermentable sugar from lignocellulose strongly depends on the price of biomass feedstocks and cost of the pretreatment process (Bak et al., 2009; Kumar and Murthy, 2011; Lynd et al., 2008). Poplars are considered suitable dedicated energy crops, which are different from traditional crops, because of their many advantages: these trees grow fast, have a short rotation period (Sannigrahi et al., 2010), are rich in cellulose and hemicellulose
⇑ Corresponding authors. Tel.: +82 2 3290 3028; fax: +82 2 925 1970 (K.H. Kim), tel.: +82 2 3290 3458 (O.K. Park). E-mail addresses: [email protected] (O.K. Park), [email protected] (K.H. Kim). 1 Both authors contributed equally to this work. 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.074
(that are the main components used for sugar production), and require less fertilizer (Baucher et al., 2003). There have been many efforts to study transgenic poplars in order to develop new energy crops. However, among the numerous reports on transgenic poplars, only a few showed analytical results of enzymatic digestibility of transgenic poplar biomass. The reduction of lignin (Min et al., 2012; Wang et al., 2012) or xylan content (Lee et al., 2009) or the overexpression of cellulase or hemicellulases (Kaida et al., 2009) in transgenic poplars was found to increase the enzymatic digestibility. It has also been reported that a transgenic poplar with a reduced lignin content showed adverse effects such as a lower growth rate and sugar yield (Voelker et al., 2010). Therefore, for the development of transgenic plants to be used as energy crops, the systematic evaluation of transgenic plants with respect to pretreatment and saccharification is essential. In our previous work, transgenic poplars with increased cellulose contents were developed, in which the RabG3bCA gene from Arabidopsis was overexpressed (Kwon et al., 2011). The
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Y.H. Jung et al. / Bioresource Technology 129 (2013) 639–641
overexpression of RabG3bCA, which is known to promote xylem development in Arabidopsis, was found to cause increased stem growth and possibly promote the synthesis of cellulose in the transgenic poplars (Kwon et al., 2011). In this study, to evaluate the effectiveness of using transgenic poplar as biomass for bioethanol production, whole trees of transgenic poplar were subjected to various chemical pretreatments and enzymatic saccharification. 2. Experimental 2.1. Preparation of transgenic poplar A hybrid poplar clone, Poplar alba Poplar tremula var. glandulosa BH1, was genetically transformed as explained in our previous study (Kwon et al., 2011). In brief, the RabG3bCA gene construct related to secondary wall formation during wood development was transferred to poplar cells. The transformed poplar cells were regenerated by transferring the calli to woody plant medium, and they were further planted in soil and grown in a greenhouse at 28–30 °C. A wild-type poplar (WT) and a transgenic poplar (OX8) were dried, dissected, and then milled using a cutting mill (MF 10; IKA, Staufen, Germany), and poplar particles smaller than 1 mm were collected for experiments. 2.2. Pretreatment of poplars Three kinds of catalysts including sulfuric acid (Daejung Chemicals & Metals, Siheung, Korea), sodium hydroxide (Sigma–Aldrich, St. Louis, MO), and ammonia (Junsei, Tokyo, Japan) were used for the pretreatment of poplar. For aqueous ammonia pretreatment, poplar particles were incubated in 14% (w/w) of aqueous ammonia solution for 10 h at 80 °C in a constant temperature water bath (Biofree, Seoul, Korea). For sulfuric acid or sodium hydroxide pretreatment, poplar particles at a final solids concentration of 10% (w/v) in the pretreatment solution containing 1% (w/v) of each catalyst were pretreated at 190 °C in a microwave digester (Milestone, Shelton, CT). The temperature of the poplar slurry was raised to 190 °C within 3 min, and there was no holding time at the desired temperature. After the pretreatment, the vessels were moved into ice water for quenching the reaction. The pretreated biomass was filtered through a filtration cloth (22–25 lm; Calbiochem, La Jolla, CA) and washed with deionized distilled water until the liquor pH reached 6–7, and the washed solids were dried in a vacuum-drying oven at 45 °C for 3 days. 2.3. Enzymatic hydrolysis of pretreated poplars Both untreated and pretreated poplars were digested following the National Renewable Energy Laboratory (NREL) procedure to compare their pretreatment results (Selig et al., 2008). To analyze the enzymatic digestibility of pretreated poplars, the enzymatic hydrolysis mixture containing poplar biomass at a final biomass solids concentration of 1% (w/v) in 10 ml of 0.05 M citrate buffer (pH 4.8) with 60 FPU of Accellerase 1000 (Genencor, Rochester, NY) per g of glucan in poplar with tetracycline (0.4 mg/ml) and cycloheximide (0.3 mg/ml) was incubated at 50 °C for 72 h. 2.4. Analyses of biomass composition and sugars Compositional analysis was performed following the NREL procedure (Sluiter et al., 2008). In brief, 0.3 g of dried biomass was predigested using 72% (w/w) sulfuric acid solution, diluted to 4%, and decomposed at 121 °C for 1 h. The biomass slurry was filtered through a crucible, and the solid residues on the crucible were transferred into a furnace (Daihan LabTech, Namyangju, Korea) at 575 °C for 3 h. The acid-insoluble lignin content was determined
by measuring its weight difference before and after burning in the furnace. The flow-through from the crucible was neutralized and centrifuged at 16,000 rpm for 5 min and filtered using a 0.2lm syringe filter (Chrom Tech, Apple Valley, MN). The filtrate was analyzed for carbohydrates including glucose, xylose, galactose, arabinose, and mannose by an Agilent 1100 high performance liquid chromatography (HPLC; Agilent Technologies, Waldbronn, Germany) system equipped with a refractive index detector (G1362A; Agilent Technologies Waldbronn, Germany) and SP0810 column (Pb2+ form, 300 mm 8.0 mm 7 lm; Shodex, Showa Denko, Kawasaki, Japan). All analyses were conducted in triplicate. 3. Results and discussion 3.1. Biomass composition of wild-type and transgenic poplars In a previous study comparing WT and OX8, significant increases in stem length, stem thickness, and total weight were exhibited in the transgenic plants (Kwon et al., 2011). As shown in Table 1, the biomass of the WT was composed of 41.0% glucan, 13.8% xylan, 6.1% galactan, 4.4% arabinan, 4.8% mannan, 23.5% acid-insoluble lignin, and 2.3% ash on a dry-weight basis. The transgenic poplar, OX8, consisted of 45.0% glucan, 13.6% xylan, 6.3% galactan, 4.6% arabinan, 4.9% mannan, 23.2% lignin, and 1.6% ash. The glucan content of OX8 was found to be 9.8% higher than that of the WT, but the other components in OX8 were found in comparatively lower amounts. Most studies on transgenic poplars with increased cellulose contents reported that the changes in cellulose content resulted from genetic alteration in the biosynthesis of lignin (Voelker et al., 2010; Wang et al., 2012). 3.2. Pretreatment of wild-type and transgenic poplars Sulfuric acid, sodium hydroxide, and ammonia were applied to the pretreatment of WT and OX8 (Jung et al., 2011; Kim et al., 2002; Ko et al., 2009; Macdonald et al., 1983). The insoluble biomass samples of the pretreated WT and OX8 were washed with water and analyzed for their compositions (Table 2). The xylan and lignin recovery yields after pretreatment were not significantly different between WT and OX8. Considering the higher content of glucan in the untreated OX8, the glucan recovery yields in sulfuric acid- or ammonia-pretreated OX8 were unexpectedly lower than those of WT, thereby indicating that the glucan of transgenic plants did not show higher solubilization in the sulfuric acid and ammonia pretreatments. The insoluble solids of the pretreated WT and OX8 were washed with water and hydrolyzed for 72 h using 60 FPU cellulase/g glucan in biomass. The enzymatic digestibility of the untreated OX8 (i.e., 43.0%) was higher than that of the untreated WT (i.e., 34.8%), implying that the overexpression of the RabG3bCA gene promoted the enzymatic digestibility of the untreated poplar biomass. The higher enzymatic digestibility in the OX8 when compared to the WT was also observed when the poplars were pretreated by three Table 1 Biomass compositions of the wild-type (WT) and transgenic (OX8) poplars (g each component/100 g total dry biomass). Biomass component
WT
OX8
Glucan Xylan Galactan Arabinan Mannan Acid-insoluble lignin Ash
41.0 ± 0.6 13.8 ± 0.3 6.1 ± 0.2 4.4 ± 0.9 4.8 ± 0.4 23.5 ± 0.5 2.3 ± 0.1
45.0 ± 2.3 13.6 ± 0.5 6.3 ± 0.1 4.6 ± 1.0 4.9 ± 0.3 23.2 ± 0.2 1.6 ± 0.3
Y.H. Jung et al. / Bioresource Technology 129 (2013) 639–641 Table 2 Recovery of biomass components after washing the pretreated wild-type (WT) and transgenic (OX8) poplars (g each component/100 g total dry untreated biomass).
WT_H2SO4 WT_NaOH WT_NH3 OX8_H2SO4 OX8_NaOH OX8_NH3
Glucan
Xylan
Acid-insoluble lignin
25.4 ± 0.3 28.4 ± 0.5 31.4 ± 0.5 23.0 ± 0.3 30.7 ± 0.6 31.1 ± 0.5
3.2 ± 0.0 7.6 ± 0.0 9.4 ± 0.4 2.7 ± 0.0 7.9 ± 0.2 8.9 ± 0.2
17.9 ± 0.2 14.8 ± 0.1 15.0 ± 0.3 16.8 ± 0.1 14.5 ± 0.1 14.3 ± 0.5
different chemical pretreatment methods (Supplementary data in Fig. S1). When dilute sulfuric acid (1%, w/v) was used at 190 °C for pretreatment, WT and OX8 exhibited 54.4% and 61.9% enzymatic digestibilities, respectively. When sodium hydroxide (1%, w/v) was applied, the difference in enzymatic digestibility between WT and OX8 was greater, with 58.7% and 69.4% digestibility, respectively. In the case of aqueous ammonia pretreatment, the enzymatic digestibilities of WT and OX8 were 53.5% and 61.3%, respectively. The glucose yields from 100 g of WT and OX8 using sodium hydroxide pretreatment and enzymatic hydrolysis with 60 FPU Accellerase 1000/g glucan were 18.5 and 23.7 g glucose, respectively. Therefore, the transgenic plant, OX8, showed superiority in pretreatment effectiveness for all three catalysts, showing higher total glucose yields than WT. In previous studies on transgenic poplars, reduced lignin contents did not correspond to a higher enzymatic digestibility (Voelker et al., 2010; Wang et al., 2012). These results can be attributed to the fact that cellulose accessibility to enzyme is more critical than lignin removal for the enzymatic digestibility of cellulose in lignocellulose (Jung et al., 2011; Rollin et al., 2011). Therefore, lignin reduction does not always enable a higher enzymatic digestibility. 4. Conclusions In this study, it was found that the overexpression of RabG3bCA in OX8, inducing phenotypic changes such as taller and thicker transgenic plants, resulted in an increased cellulose content and a higher enzymatic digestibility of pretreated transgenic poplar when compared to the wild type. For the development of transgenic energy crops, plants should be modified to improve fermentable sugar yields, and transgenic plants should be evaluated using appropriate pretreatment and saccharification methods. Acknowledgements This work was supported by grants from the Advanced Biomass R&D Center of Korea (2011-0031353) and the Pioneer Research Center Program (2011-0002327) funded by the Korean Government (MEST). Grant support was also received from the Next-Generation BioGreen 21 Program (PJ0080522011 and PJ008103032011) through the Rural Development Administration. Facility support at Korea University Food Safety Hall for the Institute of Biomedical Science and Food Safety is also acknowledged.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2012. 12.074. References Bak, J.S., Ko, J.K., Han, Y.H., Lee, B.C., Choi, I.-G., Kim, K.H., 2009. Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment. Bioresour. Technol. 100 (3), 1285–1290. Baucher, M., Halpin, C., Petit-Conil, M., Boerjan, W., 2003. Lignin: genetic engineering and impact on pulping. Crit. Rev. Biochem. Mol. Biol. 38 (4), 305– 350. Jung, Y.H., Kim, I.J., Han, J.-I., Choi, I.-G., Kim, K.H., 2011. Aqueous ammonia pretreatment of oil palm empty fruit bunches for ethanol production. Bioresour. Technol. 102 (20), 9806–9809. Kaida, R., Kaku, T., Baba, K., Oyadomari, M., Watanabe, T., Nishida, K., Kanaya, T., Shani, Z., Shoseyov, O., Hayashi, T., 2009. Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood. Mol. Plant 2 (5), 904–909. Kim, K.H., Tucker, M.P., Nguyen, Q.A., 2002. Effects of pressing lignocellulosic biomass on sugar yield in two-stage dilute-acid hydrolysis process. Biotechnol. Prog. 18 (3), 489–494. Ko, J.K., Bak, J.S., Jung, M.W., Lee, H.J., Choi, I.-G., Kim, T.H., Kim, K.H., 2009. Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes. Bioresour. Technol. 100 (19), 4374–4380. Kumar, D., Murthy, G.S., 2011. Impact of pretreatment and downstream processing technologies on economics and energy in cellulosic ethanol production. Biotechnol. Biofuels 4 (27), 1–19. Kwon, S.I., Cho, H.J., Lee, J.-S., Jin, H., Shin, S.-J., Kwon, M., Noh, E.W., Park, O.K., 2011. Overexpression of constitutively active Arabidopsis RabG3b promotes xylem development in transgenic poplars. Plant Cell Environ. 34 (12), 2212– 2224. Lee, C., Teng, Q., Huang, W., Zhong, R., Ye, Z.-H, 2009. Down-regulation of PoGT47C expression in poplar results in a reduced glucuronoxylan content and an increased wood digestibility by cellulase. Plant Cell Physiol. 50 (6), 1075–1089. Lynd, L.R., Laser, M.S., Bransby, D., Dale, B.E., Davison, B., Hamilton, R., Himmel, M., Keller, M., McMillan, J.D., Sheehan, J., Wyman, C.E., 2008. How biotech can transform biofuels. Nat. Biotechnol. 26 (2), 169–172. Macdonald, D.G., Bakhshi, N.N., Mathews, J.F., Roychowdhury, A., Bajpai, P., MooYoung, M., 1983. Alkali treatment of corn stover to improve sugar production by enzymatic hydrolysis. Biotechnol. Bioeng. 25 (8), 2067–2076. Min, D., Li, Q., Jameel, H., Chiang, V., Chang, H.-M., 2012. The cellulase-mediated saccharification on wood derived from transgenic low-lignin lines of black cottonwood (Populus trichocarpa). Appl. Biochem. Biotechnol. 168 (4), 947– 955. Rollin, J.A., Zhu, Z., Sathitsuksanoh, N., Zhang, Y.-H.P., 2011. Increasing cellulose accessibility is more important than removing lignin: a comparison of cellulose solvent-based lignocellulose fractionation and soaking in aqueous ammonia. Biotechnol. Bioeng. 108 (1), 22–30. Sannigrahi, P., Tuskan, G.A., Ragauskas, A.J., 2010. Poplar as a feedstock for biofuels: a review of compositional characteristics. Biofuels Bioprod. Bioref. 4 (2), 209– 226. Selig, M., Weiss, N., Ji, Y., 2008. Laboratory Analytical Procedure: Enzymatic Saccharification of Lignocellulosic Biomass. National Renewable Energy Laboratory, Golden, CO. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Laboratory Analytical Procedure: Determination of Structural Carbohydrates and Lignin in Biomass. National Renewable Energy Laboratory, Golden, CO. Voelker, S.L., Lachenbruch, B., Meinzer, F.C., Jourdes, M., Ki, C., Patten, A.M., Davin, L.B., Lewis, N.G., Tuskan, G.A., Gunter, L., Decker, S.R., Selig, M.J., Sykes, R., Himmel, M.E., Kitin, P., Shevchenko, O., Strauss, S.H., 2010. Antisense downregulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar. Plant Physiol. 154 (2), 874–886. Wang, H., Xue, Y., Chen, Y., Li, R., Wei, J., 2012. Lignin modification improves the biofuel production potential in transgenic Populus tomentosa. Ind. Crop Prod. 37 (1), 170–177.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Evaluation of a transgenic poplar as a potential biomass crop for biofuel production Young Hoon Jung a,1, Hong Joo Cho a,1, Jae-Soon Lee b, Eun Woon Noh b, Ohkmae K. Park a,⇑, Kyoung Heon Kim a,⇑ a b
School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea Department of Forest Genetic Resources, Korea Forest Research Institute, Suwon 441-847, Republic of Korea
h i g h l i g h t s " A transgenic poplar (OX8) with overexpressed RabG3bCA from Arabidopsis was tested. " OX8 grows faster and taller than the wid type (WT). " OX8 was 9.8% higher in glucan content compared to WT. " Pretreated OX8 exhibited a higher enzymatic digestibility than WT.
a r t i c l e
i n f o
Article history: Received 26 October 2012 Received in revised form 8 December 2012 Accepted 10 December 2012 Available online 20 December 2012 Keywords: Transgenic poplar Biomass Biofuel Pretreatment Enzymatic digestibility
a b s t r a c t A transgenic poplar, in which the RabG3bCA gene from Arabidopsis was overexpressed, was analyzed for its biomass composition and enzymatic digestibility after chemical pretreatment. In comparison with a wild-type poplar (WT), the transgenic poplar (OX8) showed 9.8% higher glucan content. The levels of other biomass components did not differ greatly between WT and OX8. When WT and OX8 samples were pretreated by sulfuric acid (1%, w/v at 190 °C), sodium hydroxide (1%, w/v at 190 °C), or ammonia (14%, w/w at 80 °C), the washed pretreated solids of OX8 exhibited a higher enzymatic digestibility than those of WT in each chemical pretreatment. The sodium hydroxide pretreatment was the most effective among the three pretreatment processes, showing 58.7% and 69.4% of theoretical glucose yield from the saccharification of pretreated OX8 and WT, respectively. The transgenic poplar, growing faster and taller, was found to contain more glucan and have a higher enzymatic digestibility than WT. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Producing fermentable sugar at low costs is the key to developing commercially viable alternative liquid fuels such as bioethanol from lignocellulose (Lynd et al., 2008). Currently, the cost of producing fermentable sugar from lignocellulose strongly depends on the price of biomass feedstocks and cost of the pretreatment process (Bak et al., 2009; Kumar and Murthy, 2011; Lynd et al., 2008). Poplars are considered suitable dedicated energy crops, which are different from traditional crops, because of their many advantages: these trees grow fast, have a short rotation period (Sannigrahi et al., 2010), are rich in cellulose and hemicellulose
⇑ Corresponding authors. Tel.: +82 2 3290 3028; fax: +82 2 925 1970 (K.H. Kim), tel.: +82 2 3290 3458 (O.K. Park). E-mail addresses: [email protected] (O.K. Park), [email protected] (K.H. Kim). 1 Both authors contributed equally to this work. 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.074
(that are the main components used for sugar production), and require less fertilizer (Baucher et al., 2003). There have been many efforts to study transgenic poplars in order to develop new energy crops. However, among the numerous reports on transgenic poplars, only a few showed analytical results of enzymatic digestibility of transgenic poplar biomass. The reduction of lignin (Min et al., 2012; Wang et al., 2012) or xylan content (Lee et al., 2009) or the overexpression of cellulase or hemicellulases (Kaida et al., 2009) in transgenic poplars was found to increase the enzymatic digestibility. It has also been reported that a transgenic poplar with a reduced lignin content showed adverse effects such as a lower growth rate and sugar yield (Voelker et al., 2010). Therefore, for the development of transgenic plants to be used as energy crops, the systematic evaluation of transgenic plants with respect to pretreatment and saccharification is essential. In our previous work, transgenic poplars with increased cellulose contents were developed, in which the RabG3bCA gene from Arabidopsis was overexpressed (Kwon et al., 2011). The
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Y.H. Jung et al. / Bioresource Technology 129 (2013) 639–641
overexpression of RabG3bCA, which is known to promote xylem development in Arabidopsis, was found to cause increased stem growth and possibly promote the synthesis of cellulose in the transgenic poplars (Kwon et al., 2011). In this study, to evaluate the effectiveness of using transgenic poplar as biomass for bioethanol production, whole trees of transgenic poplar were subjected to various chemical pretreatments and enzymatic saccharification. 2. Experimental 2.1. Preparation of transgenic poplar A hybrid poplar clone, Poplar alba Poplar tremula var. glandulosa BH1, was genetically transformed as explained in our previous study (Kwon et al., 2011). In brief, the RabG3bCA gene construct related to secondary wall formation during wood development was transferred to poplar cells. The transformed poplar cells were regenerated by transferring the calli to woody plant medium, and they were further planted in soil and grown in a greenhouse at 28–30 °C. A wild-type poplar (WT) and a transgenic poplar (OX8) were dried, dissected, and then milled using a cutting mill (MF 10; IKA, Staufen, Germany), and poplar particles smaller than 1 mm were collected for experiments. 2.2. Pretreatment of poplars Three kinds of catalysts including sulfuric acid (Daejung Chemicals & Metals, Siheung, Korea), sodium hydroxide (Sigma–Aldrich, St. Louis, MO), and ammonia (Junsei, Tokyo, Japan) were used for the pretreatment of poplar. For aqueous ammonia pretreatment, poplar particles were incubated in 14% (w/w) of aqueous ammonia solution for 10 h at 80 °C in a constant temperature water bath (Biofree, Seoul, Korea). For sulfuric acid or sodium hydroxide pretreatment, poplar particles at a final solids concentration of 10% (w/v) in the pretreatment solution containing 1% (w/v) of each catalyst were pretreated at 190 °C in a microwave digester (Milestone, Shelton, CT). The temperature of the poplar slurry was raised to 190 °C within 3 min, and there was no holding time at the desired temperature. After the pretreatment, the vessels were moved into ice water for quenching the reaction. The pretreated biomass was filtered through a filtration cloth (22–25 lm; Calbiochem, La Jolla, CA) and washed with deionized distilled water until the liquor pH reached 6–7, and the washed solids were dried in a vacuum-drying oven at 45 °C for 3 days. 2.3. Enzymatic hydrolysis of pretreated poplars Both untreated and pretreated poplars were digested following the National Renewable Energy Laboratory (NREL) procedure to compare their pretreatment results (Selig et al., 2008). To analyze the enzymatic digestibility of pretreated poplars, the enzymatic hydrolysis mixture containing poplar biomass at a final biomass solids concentration of 1% (w/v) in 10 ml of 0.05 M citrate buffer (pH 4.8) with 60 FPU of Accellerase 1000 (Genencor, Rochester, NY) per g of glucan in poplar with tetracycline (0.4 mg/ml) and cycloheximide (0.3 mg/ml) was incubated at 50 °C for 72 h. 2.4. Analyses of biomass composition and sugars Compositional analysis was performed following the NREL procedure (Sluiter et al., 2008). In brief, 0.3 g of dried biomass was predigested using 72% (w/w) sulfuric acid solution, diluted to 4%, and decomposed at 121 °C for 1 h. The biomass slurry was filtered through a crucible, and the solid residues on the crucible were transferred into a furnace (Daihan LabTech, Namyangju, Korea) at 575 °C for 3 h. The acid-insoluble lignin content was determined
by measuring its weight difference before and after burning in the furnace. The flow-through from the crucible was neutralized and centrifuged at 16,000 rpm for 5 min and filtered using a 0.2lm syringe filter (Chrom Tech, Apple Valley, MN). The filtrate was analyzed for carbohydrates including glucose, xylose, galactose, arabinose, and mannose by an Agilent 1100 high performance liquid chromatography (HPLC; Agilent Technologies, Waldbronn, Germany) system equipped with a refractive index detector (G1362A; Agilent Technologies Waldbronn, Germany) and SP0810 column (Pb2+ form, 300 mm 8.0 mm 7 lm; Shodex, Showa Denko, Kawasaki, Japan). All analyses were conducted in triplicate. 3. Results and discussion 3.1. Biomass composition of wild-type and transgenic poplars In a previous study comparing WT and OX8, significant increases in stem length, stem thickness, and total weight were exhibited in the transgenic plants (Kwon et al., 2011). As shown in Table 1, the biomass of the WT was composed of 41.0% glucan, 13.8% xylan, 6.1% galactan, 4.4% arabinan, 4.8% mannan, 23.5% acid-insoluble lignin, and 2.3% ash on a dry-weight basis. The transgenic poplar, OX8, consisted of 45.0% glucan, 13.6% xylan, 6.3% galactan, 4.6% arabinan, 4.9% mannan, 23.2% lignin, and 1.6% ash. The glucan content of OX8 was found to be 9.8% higher than that of the WT, but the other components in OX8 were found in comparatively lower amounts. Most studies on transgenic poplars with increased cellulose contents reported that the changes in cellulose content resulted from genetic alteration in the biosynthesis of lignin (Voelker et al., 2010; Wang et al., 2012). 3.2. Pretreatment of wild-type and transgenic poplars Sulfuric acid, sodium hydroxide, and ammonia were applied to the pretreatment of WT and OX8 (Jung et al., 2011; Kim et al., 2002; Ko et al., 2009; Macdonald et al., 1983). The insoluble biomass samples of the pretreated WT and OX8 were washed with water and analyzed for their compositions (Table 2). The xylan and lignin recovery yields after pretreatment were not significantly different between WT and OX8. Considering the higher content of glucan in the untreated OX8, the glucan recovery yields in sulfuric acid- or ammonia-pretreated OX8 were unexpectedly lower than those of WT, thereby indicating that the glucan of transgenic plants did not show higher solubilization in the sulfuric acid and ammonia pretreatments. The insoluble solids of the pretreated WT and OX8 were washed with water and hydrolyzed for 72 h using 60 FPU cellulase/g glucan in biomass. The enzymatic digestibility of the untreated OX8 (i.e., 43.0%) was higher than that of the untreated WT (i.e., 34.8%), implying that the overexpression of the RabG3bCA gene promoted the enzymatic digestibility of the untreated poplar biomass. The higher enzymatic digestibility in the OX8 when compared to the WT was also observed when the poplars were pretreated by three Table 1 Biomass compositions of the wild-type (WT) and transgenic (OX8) poplars (g each component/100 g total dry biomass). Biomass component
WT
OX8
Glucan Xylan Galactan Arabinan Mannan Acid-insoluble lignin Ash
41.0 ± 0.6 13.8 ± 0.3 6.1 ± 0.2 4.4 ± 0.9 4.8 ± 0.4 23.5 ± 0.5 2.3 ± 0.1
45.0 ± 2.3 13.6 ± 0.5 6.3 ± 0.1 4.6 ± 1.0 4.9 ± 0.3 23.2 ± 0.2 1.6 ± 0.3
Y.H. Jung et al. / Bioresource Technology 129 (2013) 639–641 Table 2 Recovery of biomass components after washing the pretreated wild-type (WT) and transgenic (OX8) poplars (g each component/100 g total dry untreated biomass).
WT_H2SO4 WT_NaOH WT_NH3 OX8_H2SO4 OX8_NaOH OX8_NH3
Glucan
Xylan
Acid-insoluble lignin
25.4 ± 0.3 28.4 ± 0.5 31.4 ± 0.5 23.0 ± 0.3 30.7 ± 0.6 31.1 ± 0.5
3.2 ± 0.0 7.6 ± 0.0 9.4 ± 0.4 2.7 ± 0.0 7.9 ± 0.2 8.9 ± 0.2
17.9 ± 0.2 14.8 ± 0.1 15.0 ± 0.3 16.8 ± 0.1 14.5 ± 0.1 14.3 ± 0.5
different chemical pretreatment methods (Supplementary data in Fig. S1). When dilute sulfuric acid (1%, w/v) was used at 190 °C for pretreatment, WT and OX8 exhibited 54.4% and 61.9% enzymatic digestibilities, respectively. When sodium hydroxide (1%, w/v) was applied, the difference in enzymatic digestibility between WT and OX8 was greater, with 58.7% and 69.4% digestibility, respectively. In the case of aqueous ammonia pretreatment, the enzymatic digestibilities of WT and OX8 were 53.5% and 61.3%, respectively. The glucose yields from 100 g of WT and OX8 using sodium hydroxide pretreatment and enzymatic hydrolysis with 60 FPU Accellerase 1000/g glucan were 18.5 and 23.7 g glucose, respectively. Therefore, the transgenic plant, OX8, showed superiority in pretreatment effectiveness for all three catalysts, showing higher total glucose yields than WT. In previous studies on transgenic poplars, reduced lignin contents did not correspond to a higher enzymatic digestibility (Voelker et al., 2010; Wang et al., 2012). These results can be attributed to the fact that cellulose accessibility to enzyme is more critical than lignin removal for the enzymatic digestibility of cellulose in lignocellulose (Jung et al., 2011; Rollin et al., 2011). Therefore, lignin reduction does not always enable a higher enzymatic digestibility. 4. Conclusions In this study, it was found that the overexpression of RabG3bCA in OX8, inducing phenotypic changes such as taller and thicker transgenic plants, resulted in an increased cellulose content and a higher enzymatic digestibility of pretreated transgenic poplar when compared to the wild type. For the development of transgenic energy crops, plants should be modified to improve fermentable sugar yields, and transgenic plants should be evaluated using appropriate pretreatment and saccharification methods. Acknowledgements This work was supported by grants from the Advanced Biomass R&D Center of Korea (2011-0031353) and the Pioneer Research Center Program (2011-0002327) funded by the Korean Government (MEST). Grant support was also received from the Next-Generation BioGreen 21 Program (PJ0080522011 and PJ008103032011) through the Rural Development Administration. Facility support at Korea University Food Safety Hall for the Institute of Biomedical Science and Food Safety is also acknowledged.
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