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Enhanced production of glucose and xylose with partial dissolution of corn stover in ionic liquid, 1-Ethyl-3-methylimidazolium acetate
Bioresource Technology 114 (2012) 720–724
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Enhanced production of glucose and xylose with partial dissolution of corn stover in ionic liquid, 1-Ethyl-3-methylimidazolium acetate Feng Xu a, Yong-Cheng Shi b, Donghai Wang a,⇑ a b
Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, United States Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, United States
a r t i c l e
i n f o
Article history: Received 18 January 2012 Received in revised form 5 March 2012 Accepted 8 March 2012 Available online 14 March 2012 Keywords: Ionic liquid pretreatment Crystallinity Enzymatic hydrolysis Hemicellulose
a b s t r a c t Partial dissolution of corn stover in ionic liquid (IL) was employed for pretreating biomass and achieving enhanced glucose and xylose yield. The xylan recovery in solid fraction after IL pretreatment decreased as temperature increased. Xylose yield was significantly increased at a relatively low temperature (110 °C) compared with the high-temperature (over 130 °C) pretreatment, which resulted in a significant xylan degradation. A maximum total sugar yield of 88.0%, with glucose yield of 93.9% and xylose yield of 75.9% based on untreated biomass, was obtained at 110 °C for 3 h pretreatment without complete dissolution of biomass. A sugar yield of 78.0% was obtained with IL pretreatment at 70 °C for 24 h. Synchrotron wide-angle X-ray diffraction was employed to investigate the crystalline structure of biomass. Both cellulose crystallinity and remaining lignin amount were correlated with cellulose digestibility. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The production of biofuels and bio-based products from lignocellulosic biomass is promising because biomass is a sustainable source of mixed sugars. To date, a number of processing methods have been developed to overcome the natural recalcitrance of biomass and convert the carbohydrates in biomass to monosaccharides that could be utilized by microbial fermentation (Mosier et al., 2005). Ionic liquid (IL) is an ideal solvent for biomass processing because it can effectively dissolve cellulose by replacing the inter- and intra-molecular hydrogen bonds of cellulose with its anion (Swatloski et al., 2002). Recently, IL with different anions has been used for biomass pretreatment (Lee et al., 2009). Complete dissolution of biomass, such as corn stover and switchgrass, has been reported to achieve high digestibility of cellulose at 160 °C for 3 h, and glucose recovery after hydrolysis was over 90%, which was significantly higher than that of ammonia fiber explosion (AFEX) pretreated biomass (about 82% of glucose yield) (Li et al., 2011). However, hemicellulose, an important pentose source, is considerably dissolved in ionic liquid and cannot be completely recovered by simply adding water to IL, eventually resulting in an increase in the cost of downstream separation. Another study of pretreatment using a mixture of IL ([C2mim][OAc]) and water showed enhanced digestibility of both cellulose and hemicellulose (Fu and Mazza, 2011). The pre-
⇑ Corresponding author. Tel.: +1 785 532 2919; fax: +1 785 5325825. E-mail address: [email protected] (D. Wang). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.023
treated biomass with high digestibility (over 90%) was obtained at the pretreatment condition of over 150 °C and 3.6 h, but about 20% of cellulose and 40% of xylan were lost during IL pretreatment. As a result, the highest sugar recovery (predicted at 71%) after enzymatic hydrolysis was lower than that (78% of total sugar) using AFEX pretreatment (Li et al., 2011), which preserves most structural sugars in solid fraction. It would be attractive to maximize the amount of polysaccharides (cellulose and hemicellulose) in insoluble fraction for further simultaneous enzymatic hydrolysis and fermentation because both glucose and xylose could be utilized for ethanol production (Ho et al., 1998). It was previously reported that wood (maple) was pretreated using IL at a temperature as low as 50 °C (Lee et al., 2009), but the digestibility of xylan after pretreatment was not investigated. In this study, we hypothesized that biomass structure could be disrupted when the carbohydrates are partially dissolved by ionic liquid pretreatment at relatively low temperatures, meaning that much more hemicellulose could be reserved in solid fraction for further hydrolysis without significantly lowering cellulose digestibility. Furthermore, the IL pretreatment at a lower temperature leads to less degradation of IL (Meine et al., 2010), possibly resulting in an enhanced reusability. Generally, IL with a chloride anion exhibits higher viscosity than that with an acetate anion (Swatloski et al., 2002), and relatively high temperature is required for carbohydrate dissolution in IL with a chloride anion. Therefore, 1-Ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an acetatecontaining IL, was selected for processing biomass at a relatively low temperature. With a pronounced increase in total sugar yield (including both glucose and xylose), we expected that the cost of
F. Xu et al. / Bioresource Technology 114 (2012) 720–724
ionic liquid pretreatment to decrease because of the lower energy input. To date, a detailed study of the effect of IL pretreatment on digestibility of both cellulose and xylan in corn stover without full dissolution has not been reported yet. This study provides a detailed examination of compositional and structural changes of corn stover to understand how low-temperature IL pretreatment enhances the digestibility of biomass. 2. Methods 2.1. Materials Corn stover used in this study was harvested at the Kansas State University (KSU) Agronomy Research Farm in 2008. After being ground with a cutting mill (SM 2000, Retsch Inc., Newtown, PA, USA) to <1 mm particle size, the sample with moisture content of 6.5% was sealed in plastic bag at 4 °C until further use. The composition analysis was conducted by the procedure from National Renewable Energy Laboratory. The content of glucan, xylan, and lignin were 37.9%, 19.9%, and 16.7%, respectively. AccelleraseÒ 1500, an enzyme complex including cellulase and b-glucosidase (Endoglucanase activity: 2200–2900 CMC U/g, one CMC U unit of activity liberates 1 lmol of reducing sugars [expressed as glucose equivalents] in 1 min under specific assay conditions of 50 °C and pH 4.8), and AccelleraseÒ XY, a hemicellulose enzyme complex (activity: 20000–30000 ABXU/g), were generously provided by Genencor (Rochester, NY). IL, 1-Ethyl-3-methylimidazolium acetate, was purchased from Sigma (Sigma–Aldrich, Inc., St. Louis, MO, USA). All chemicals used in this research were of analytical grade and purchased from Sigma.
YT ¼
721
Y G CG þ Y X CX 100% CG þ CX
where YT, YG, and YX are total sugar yield, glucan (glucose) yield (%), and xylan (xylose) yield (%), respectively; the glucan yield, as well as the xylan yield, was calculated as the recovery of glucan (or xylan) after enzymatic hydrolysis based on the corresponding polysaccharides content in the untreated biomass; CG and CX are the percentages of glucan and xylan in untreated biomass. 2.4. Wide-angle X-ray diffraction (WAXD) WAXD was carried out at the advance polymers beamline (X27-C) at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (Upton, NY). Details on the beamline system set up have been reported elsewhere (Chu and Hsiao, 2001). The wavelength was 0.1371 nm. Crystallinity (RCr, relative mass crystallinity, presenting a percentage of crystalline cellulose in biomass) was estimated from integrated diffraction intensity profile as the ratio of the total crystal peak diffraction intensity to the total diffraction intensity. A peak-fitting process was employed with Igor Pro 6.20 (WaveMetrics Inc. Lake Oswego, OR). Cellulose crystallinity (CCr, percentage of crystalline part in cellulose) was calculated using the following formula:
CCr ¼
RCr 100% C cell
where Ccell (%) is the cellulose content of an examined sample. 3. Results and discussion 3.1. Effects of pretreatment temperature
2.2. IL pretreatment Corn stover (150 mg, dry powder) was mixed with 4.85 g IL to prepare a 3% solution in a 25 ml autoclave vial. The vial was then transferred to either a water bath or a sand bath, depending on the temperature required, for incubation. For the study of pretreatment temperature in the range from 50 to 160 °C, the pretreatment duration was 3 h; for the study of pretreatment time in the range from 3 to 24 h, the pretreatment temperature was 70 °C. After pretreatment, about 20 ml of distilled water was added into the IL solvent to precipitate cellulose. The supernatant was removed after centrifuging at 8000g for 15 min, and the solid fraction was further washed with 20 ml distilled water three times and collected for further study. 2.3. Enzymatic hydrolysis Enzymatic hydrolysis was conducted with the pretreated sample at 2% solids concentration (grams dry weight per 100 mL) in 50 mM sodium acetate buffer (pH 5.00) with the addition of 40 lg/mL of tetracycline and 30 lg/mL of cycloheximide. The enzymes, AccelleraseÒ 1500 and AccelleraseÒ XY, were used at the recommended dosages, 0.5 mL per gram cellulose and 0.1 mL per gram cellulose, respectively. Vials were incubated in a water bath at 50 °C and agitation of 140 rpm. Progress of the reaction was monitored by interval taking 50 lL supernatant to a 1.5 mL centrifuge tube containing 450 lL distilled water, followed by holding the tube in a boiling water bath for 5 min to quench the reaction. Two replicates were obtained for each point. Sugar analysis was conducted with high-performance liquid chromatography (HPLC) equipped with reflective index detector (RID 10A, Shimadzu, MD, USA) and a Rezex RPM-monosaccharide column (Phenomenex, CA, USA) operated at 80 °C. Total sugar recovery was calculated using the following formula:
Six different temperatures ranging from 50 to 160 °C were used to compare the sugar yield after IL pretreatment and enzymatic hydrolysis. Glucose and xylose production as a function of time during enzymatic hydrolysis is shown in Fig. 1. Solid powder could be observed even after 3 h incubation at 110 °C, suggesting that the corn stover was partially dissolved at the relatively low temperature. No significant solid was found with pretreatment at either 130 or 160 °C after 3 h. After applying water to precipitate cellulose, the solid fraction from either 130 or 160 °C formed gel-like regenerated cellulose, which was significantly different from the solid fraction (biomass fiber) from the other conditions of lower temperature. Fig. 1A shows that about 90% of glucose released at 72 h of hydrolysis was obtained at 10 h. Similar results were reported that 90% of glucose released at 72 h could be obtained at 24 h after complete dissolution of switchgrass at 160 °C (Li et al., 2010). In the current study, partial dissolution of corn stover also effectively increased hydrolysis rate at a relatively low temperature, indicating that complete dissolution of corn stover in IL is not necessary and cellulose becomes easily digestible with biomass disruption by IL. For hydrolysis extent after 72 h, the glucose yield increased as the pretreatment temperature increased to 130 °C, and 94.4% of cellulose was recovered as glucose in hydrolysate after IL pretreatment at 130 °C, suggesting that the selected IL is effective in corn stover pretreatment for the following cellulose–glucose conversion. However, the xylose yield after 130 °C pretreament was 55.8%, which is significantly lower than the yield after 110 °C pretreatment (93.9% of glucose, 75.0% of xylose, and 88.0% of total sugar). The results from composition analysis (Table 1) show that the xylan recovery in solid fraction after IL pretreatment decreased as temperature increased, indicating that the degraded xylan, which could not be easily recovered by applying water, increased with increasing temperature. The increasing dissolution of xylan
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Fig. 1. Effects of pretreatment temperature on glucose (A) and xylose (B) yield. (: untreated; j: 50 °C; N: 70 °C; h: 90 °C; ⁄: 110 °C; d: 130 °C; s: 160 °C).
Table 1 Effects of pretreatment time/temperature on the recovery of biomass components.a 3h
Lignin (%) Xylan (%) Cellulose (%) a
70 °C
50 °C
70 °C
90 °C
110 °C
130 °C
160 °C
6h
9h
24 h
76.2 89.9 98.9
69.2 87.1 98.2
48.9 82.6 97.0
43.0 78.2 96.6
26.0 59.4 95.8
11.1 15.0 89.0
67.2 87.0 97.8
61.2 85.3 97.6
52.4 81.7 96.6
All percentages were calculated on the initial content of the components in untreated biomass.
in IL would not only reduce sugar yield but also increase the cost of downstream recovery. As temperature increased from 50 to 110 °C, the increase in xylose yield, as well as the increase in glucose yield, could be a result of biomass disruption by IL pretreatment. Xylose degradation became more significant at a higher temperature (over 110 °C), which eventually decreased xylose recovery. After pretreatment at 160 °C, xylan recovery in solid fraction and the xylose yield after enzymatic hydrolysis were only 15.0% and 14.3%, respectively. It was reported that xylan could be degraded to furfural or 5-hydroxymethyl-2-furaldehyde (HMF) with IL,1-ethyl-3methylimidazolium hydrogen sulfate ([EMIM][HSO4]) (Lima et al., 2009), which could be one of the reasons that explain the xylan loss after IL pretreatment. Further study is required to understand the mechanism of xylan degradation in IL. Notably, glucose recovery decreased as the pretreatment temperature increased to 160 °C. The reduced recovery of glucose (Table 1) after IL pretreatment suggested that cellulose degradation (11%) was also significant at the temperature of 160 °C. Also noteworthy is that cellulose digestibility was enhanced to over 90% with IL pretreatment at 90 °C for 3 h whereas 48.9% of lignin was left in solid fraction (Fig. 1A, Table 1). Further study is being conducted to understand the structural changes of the remaining lignin.
3.2. Effects of pretreatment time To further understand the profile of IL pretreatment at low temperature, the study of the effect of pretreatment time on biomass digestibility was conducted at 70 °C. As the pretreatment duration increased, both glucose and xylose yield at 72 h of hydrolysis increased (Fig. 2) and lignin content decreased (Table 1). It was reported that the increased lignin extraction because of an increased pretreatment time is one of the reasons explaining the enhanced sugar yield (Lee et al., 2009). Glucose loss, which was less than 4% even after 24 h pretreatment, increased as the duration increased (Table 1).
After 24 h pretreatment, the glucose, xylose, and total sugar yields were 84.9%, 64.8%, and 78.0%, respectively (Fig. 2), which was comparable to the yields of AFEX pretreatment (82.0%, 72.2%, and 78.4%, respectively) (Li et al., 2011). Biomass solid could still be observed even after 24 h pretreatment, suggesting that a complete dissolution of corn stover in IL is not necessary; therefore, the IL ([C2mim][OAc]) used is effective for pretreatment of corn stover at 70 °C. A relatively low temperature could not only reduce energy input but also increase xylan recovery as proved in this study. In addition, further study of low temperature IL pretreatment, such as pretreatment at 50 °C, coupled with the developing IL-tolerant enzymes/microbes (Nakashima et al., 2011), could make possible simultaneous carbohydrates extraction and saccharification in a single system.
3.3. Structural change of corn stover with IL pretreatment It is well accepted that pretreatment changes biomass structure rather than mass loss, resulting in change in digestibility (Mosier et al., 2005). However, it is challenging to correlate only one structural parameter of biomass with its digestibility. Numerous studies of biomass conversion have argued about the relationship between crystallinity and rate/extent of enzymatic hydrolysis (Hall et al., 2010; Mansfield et al., 1999). An important reason could be that the calculation, employed by most of the studies, was based on the weight of biomass not on that of cellulose, which added other amorphous components, such as hemicellulose and lignin as well as extractives, into the consideration of crystallinity. As a result, crystallinity could change even without a change in cellulose structure, and cellulose digestibility could even increase as crystallinity increases (Zhao et al., 2008), It might not be appropriate to use crystallinity change to explain digestibility change when the increase in crystallinity is a result of lignin removal, and it is uncertain how cellulose structure has been changed (Kim et al., 2003). We suggest using cellulose crystallinity (the percentage of crystalline part in cellulose, not total mass) to study crystallinity change.
F. Xu et al. / Bioresource Technology 114 (2012) 720–724
723
Fig. 2. Effects of pretreatment time on sugar yield. Pretreatment temperature was 70 °C.
Fig. 3. The correlation between cellulose digestibility and cellulose crystallinity (CCr) (), as well as lignin recovery in solid fraction (j). Cellulose digestibility was calculated as the percentage of released glucose yield from recovered cellulose in pretreated biomass.
In this study, cellulose digestibility was significantly correlated to both lignin removal and cellulose crystallinity (Fig. 3). As the pretreatment temperature increase from 50 to 160 °C, the cellulose crystallinity, as well as the lignin recovery, decreased, and the cellulose digestibility increased. A similar trend was observed when the pretreatment time increased from 3 to 24 h at 70 °C. Therefore, the changes of both cellulose crystallinity and lignin content contributed to the increase in digestibility. It is well accepted that lignin removal could reduce the irreversible absorption of cellulase on lignin (Berlin et al., 2005). In addition, the removal of amorphous components, including lignin and hemicellulose, could increase the accessible area for cellulose–enzyme interaction (Zhang and Lynd, 2004) that further enhanced cellulose digestibility. Similar to other pretreatments, such as diluted acid or alkali pretreatment, the disruption of biomass by chemical attack makes polysaccharides more reactive; however, it is not similar to most of the other pretreatments that IL actually effectively de-crystallized cellulose (Binder and Raines, 2010). Notably is the transition of cellulose from type I to II (See Supplementary Material). Native cellulose (type I) has typical diffraction peaks at 1.70 and 2.52 nm1, respectively, whereas regenerated cellulose (type II) has typical diffraction peak at 2.26 nm1 (Zhang et al., 2005). As the pretreatment temperature or time increased, the (0 0 2) and (0 2 0) reflections waned and
the (1 1 0) reflection increased as reported (Zhang et al., 2005). The reflection peaks of (0 2 0) and (1 1 0) overlapped with a partial dissolution at relatively low temperature, possibly suggesting that both cellulose I and II coexisted in recovered solid fraction. It was reported that the structure of cellulose II is more stable than that of cellulose I (Kolpak and Blackwell, 1976), but more research is needed to determine whether the transition is favorable to digestibility or not since the changes of crystallinity and/or crystal size of cellulose, that accompany the transition during IL pretreatment, may also affect digestibility.
4. Conclusion Partially dissolving corn stover in IL at a relatively low temperature (110 °C) for 3 h, with higher xylan recovery (75.9%), was employed to achieve glucose yield of 93.9% and total sugar yield of 88.0%. It was demonstrated that about 78% of structural sugar was recovered with IL pretreatment at a lower temperature (70 °C) for 24 h. Structural investigation revealed that both cellulose crystallinity and lignin content contributed to the increase in digestibility of biomass. Further study is being conducted at lower temperatures to develop a system of simultaneous IL pretreatment and enzymatic hydrolysis.
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Acknowledgements We thank Drs. Jun Wang, Benjamin S. Hsiao, and Lixia Rong for their assistance in WAXD studies. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. This is contribution no. 12-208-J from the Kansas Agricultural Experiment Station. 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. 03.023. References Berlin, A., Gilkes, N., Kurabi, A., Bura, R., Tu, M., Kilburn, D., Saddler, J., 2005. Weak lignin-binding enzymes. Appl. Biochem. Biotechnol. 121, 163–170. Binder, J.B., Raines, R.T., 2010. Fermentable sugars by chemical hydrolysis of biomass. PNAS 107, 4516–4521. Chu, B., Hsiao, B., 2001. Small-angle x-ray scattering of polymers. Chem. Rev. 101, 1727–1762. Fu, D., Mazza, G., 2011. Optimization of processing conditions for the pretreatment of wheat straw using aqueous ionic liquid. Bioresour. Technol. 102, 8003–8010. Hall, M., Bansal, P., Lee, J., Realff, M., Bommarius, A., 2010. Cellulose crystallinity – a key predictor of the enzymatic hydrolysis rate. FEBS J. 9999. Ho, N.W.Y., Chen, Z., Brainard, A.P., 1998. Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl. Environ. Microbiol. 64, 1852. Kim, T., Kim, J., Sunwoo, C., Lee, Y., 2003. Pretreatment of corn stover by aqueous ammonia. Bioresour. Technol. 90, 39–47.
Kolpak, F., Blackwell, J., 1976. Determination of the structure of cellulose II. Macromolecules 9, 273–278. Lee, S.H., Doherty, T.V., Linhardt, R.J., Dordick, J.S., 2009. Ionic liquid mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis. Biotechnol. Bioeng. 102, 1368–1376. Li, C., Cheng, G., Balan, V., Kent, M.S., Ong, M., Chundawat, S.P.S., Da Costa Sousa, L., Melnichenko, Y.B., Dale, B.E., Simmons, B.A., 2011. Influence of physicochemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover. Bioresour. Technol. 102, 6928–6936. Li, C., Knierim, B., Manisseri, C., Arora, R., Scheller, H.V., Auer, M., Vogel, K.P., Simmons, B.A., Singh, S., 2010. Comparison of dilute acid and ionic liquid pretreatment of switchgrass: biomass recalcitrance, delignification and enzymatic saccharification. Bioresour. Technol. 101, 4900–4906. Lima, S., Neves, P., Antunes, M.M., Pillinger, M., Ignatyev, N., Valente, A.A., 2009. Conversion of mono/di/polysaccharides into furan compounds using 1-alkyl-3methylimidazolium ionic liquids. Appl. Catal. A 363, 93–99. Mansfield, S.D., Mooney, C., Saddler, J.N., 1999. Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol. Prog. 15, 804–816. Meine, N., Benedito, F., Rinaldi, R., 2010. Thermal stability of ionic liquids assessed by potentiometric titration. Green Chem. 12, 1711–1714. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686. Nakashima, K., Yamaguchi, K., Taniguchi, N., Arai, S., Yamada, R., Katahira, S., Ishida, N., Takahashi, H., Ogino, C., Kondo, A., 2011. Direct bioethanol production from cellulose by the combination of cellulase-displaying yeast and ionic liquid pretreatment. Green Chem. 13, 2948–2954. Swatloski, R.P., Spear, S.K., Holbrey, J.D., Rogers, R.D., 2002. Dissolution of cellose with ionic liquids. JACS 124, 4974–4975. Zhang, H., Wu, J., Zhang, J., He, J., 2005. 1-Allyl-3-methylimidazolium chloride room temperature ionic liquid: a new and powerful nonderivatizing solvent for cellulose. Macromolecules 38, 8272–8277. Zhang, Y., Lynd, L., 2004. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol. Bioeng. 88, 797–824. Zhao, X.B., Wang, L., Liu, D.H., 2008. Peracetic acid pretreatment of sugarcane bagasse for enzymatic hydrolysis: a continued work. J. Chem. Technol. Biotechnol. 83, 950–956.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Enhanced production of glucose and xylose with partial dissolution of corn stover in ionic liquid, 1-Ethyl-3-methylimidazolium acetate Feng Xu a, Yong-Cheng Shi b, Donghai Wang a,⇑ a b
Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, United States Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, United States
a r t i c l e
i n f o
Article history: Received 18 January 2012 Received in revised form 5 March 2012 Accepted 8 March 2012 Available online 14 March 2012 Keywords: Ionic liquid pretreatment Crystallinity Enzymatic hydrolysis Hemicellulose
a b s t r a c t Partial dissolution of corn stover in ionic liquid (IL) was employed for pretreating biomass and achieving enhanced glucose and xylose yield. The xylan recovery in solid fraction after IL pretreatment decreased as temperature increased. Xylose yield was significantly increased at a relatively low temperature (110 °C) compared with the high-temperature (over 130 °C) pretreatment, which resulted in a significant xylan degradation. A maximum total sugar yield of 88.0%, with glucose yield of 93.9% and xylose yield of 75.9% based on untreated biomass, was obtained at 110 °C for 3 h pretreatment without complete dissolution of biomass. A sugar yield of 78.0% was obtained with IL pretreatment at 70 °C for 24 h. Synchrotron wide-angle X-ray diffraction was employed to investigate the crystalline structure of biomass. Both cellulose crystallinity and remaining lignin amount were correlated with cellulose digestibility. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The production of biofuels and bio-based products from lignocellulosic biomass is promising because biomass is a sustainable source of mixed sugars. To date, a number of processing methods have been developed to overcome the natural recalcitrance of biomass and convert the carbohydrates in biomass to monosaccharides that could be utilized by microbial fermentation (Mosier et al., 2005). Ionic liquid (IL) is an ideal solvent for biomass processing because it can effectively dissolve cellulose by replacing the inter- and intra-molecular hydrogen bonds of cellulose with its anion (Swatloski et al., 2002). Recently, IL with different anions has been used for biomass pretreatment (Lee et al., 2009). Complete dissolution of biomass, such as corn stover and switchgrass, has been reported to achieve high digestibility of cellulose at 160 °C for 3 h, and glucose recovery after hydrolysis was over 90%, which was significantly higher than that of ammonia fiber explosion (AFEX) pretreated biomass (about 82% of glucose yield) (Li et al., 2011). However, hemicellulose, an important pentose source, is considerably dissolved in ionic liquid and cannot be completely recovered by simply adding water to IL, eventually resulting in an increase in the cost of downstream separation. Another study of pretreatment using a mixture of IL ([C2mim][OAc]) and water showed enhanced digestibility of both cellulose and hemicellulose (Fu and Mazza, 2011). The pre-
⇑ Corresponding author. Tel.: +1 785 532 2919; fax: +1 785 5325825. E-mail address: [email protected] (D. Wang). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.023
treated biomass with high digestibility (over 90%) was obtained at the pretreatment condition of over 150 °C and 3.6 h, but about 20% of cellulose and 40% of xylan were lost during IL pretreatment. As a result, the highest sugar recovery (predicted at 71%) after enzymatic hydrolysis was lower than that (78% of total sugar) using AFEX pretreatment (Li et al., 2011), which preserves most structural sugars in solid fraction. It would be attractive to maximize the amount of polysaccharides (cellulose and hemicellulose) in insoluble fraction for further simultaneous enzymatic hydrolysis and fermentation because both glucose and xylose could be utilized for ethanol production (Ho et al., 1998). It was previously reported that wood (maple) was pretreated using IL at a temperature as low as 50 °C (Lee et al., 2009), but the digestibility of xylan after pretreatment was not investigated. In this study, we hypothesized that biomass structure could be disrupted when the carbohydrates are partially dissolved by ionic liquid pretreatment at relatively low temperatures, meaning that much more hemicellulose could be reserved in solid fraction for further hydrolysis without significantly lowering cellulose digestibility. Furthermore, the IL pretreatment at a lower temperature leads to less degradation of IL (Meine et al., 2010), possibly resulting in an enhanced reusability. Generally, IL with a chloride anion exhibits higher viscosity than that with an acetate anion (Swatloski et al., 2002), and relatively high temperature is required for carbohydrate dissolution in IL with a chloride anion. Therefore, 1-Ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an acetatecontaining IL, was selected for processing biomass at a relatively low temperature. With a pronounced increase in total sugar yield (including both glucose and xylose), we expected that the cost of
F. Xu et al. / Bioresource Technology 114 (2012) 720–724
ionic liquid pretreatment to decrease because of the lower energy input. To date, a detailed study of the effect of IL pretreatment on digestibility of both cellulose and xylan in corn stover without full dissolution has not been reported yet. This study provides a detailed examination of compositional and structural changes of corn stover to understand how low-temperature IL pretreatment enhances the digestibility of biomass. 2. Methods 2.1. Materials Corn stover used in this study was harvested at the Kansas State University (KSU) Agronomy Research Farm in 2008. After being ground with a cutting mill (SM 2000, Retsch Inc., Newtown, PA, USA) to <1 mm particle size, the sample with moisture content of 6.5% was sealed in plastic bag at 4 °C until further use. The composition analysis was conducted by the procedure from National Renewable Energy Laboratory. The content of glucan, xylan, and lignin were 37.9%, 19.9%, and 16.7%, respectively. AccelleraseÒ 1500, an enzyme complex including cellulase and b-glucosidase (Endoglucanase activity: 2200–2900 CMC U/g, one CMC U unit of activity liberates 1 lmol of reducing sugars [expressed as glucose equivalents] in 1 min under specific assay conditions of 50 °C and pH 4.8), and AccelleraseÒ XY, a hemicellulose enzyme complex (activity: 20000–30000 ABXU/g), were generously provided by Genencor (Rochester, NY). IL, 1-Ethyl-3-methylimidazolium acetate, was purchased from Sigma (Sigma–Aldrich, Inc., St. Louis, MO, USA). All chemicals used in this research were of analytical grade and purchased from Sigma.
YT ¼
721
Y G CG þ Y X CX 100% CG þ CX
where YT, YG, and YX are total sugar yield, glucan (glucose) yield (%), and xylan (xylose) yield (%), respectively; the glucan yield, as well as the xylan yield, was calculated as the recovery of glucan (or xylan) after enzymatic hydrolysis based on the corresponding polysaccharides content in the untreated biomass; CG and CX are the percentages of glucan and xylan in untreated biomass. 2.4. Wide-angle X-ray diffraction (WAXD) WAXD was carried out at the advance polymers beamline (X27-C) at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (Upton, NY). Details on the beamline system set up have been reported elsewhere (Chu and Hsiao, 2001). The wavelength was 0.1371 nm. Crystallinity (RCr, relative mass crystallinity, presenting a percentage of crystalline cellulose in biomass) was estimated from integrated diffraction intensity profile as the ratio of the total crystal peak diffraction intensity to the total diffraction intensity. A peak-fitting process was employed with Igor Pro 6.20 (WaveMetrics Inc. Lake Oswego, OR). Cellulose crystallinity (CCr, percentage of crystalline part in cellulose) was calculated using the following formula:
CCr ¼
RCr 100% C cell
where Ccell (%) is the cellulose content of an examined sample. 3. Results and discussion 3.1. Effects of pretreatment temperature
2.2. IL pretreatment Corn stover (150 mg, dry powder) was mixed with 4.85 g IL to prepare a 3% solution in a 25 ml autoclave vial. The vial was then transferred to either a water bath or a sand bath, depending on the temperature required, for incubation. For the study of pretreatment temperature in the range from 50 to 160 °C, the pretreatment duration was 3 h; for the study of pretreatment time in the range from 3 to 24 h, the pretreatment temperature was 70 °C. After pretreatment, about 20 ml of distilled water was added into the IL solvent to precipitate cellulose. The supernatant was removed after centrifuging at 8000g for 15 min, and the solid fraction was further washed with 20 ml distilled water three times and collected for further study. 2.3. Enzymatic hydrolysis Enzymatic hydrolysis was conducted with the pretreated sample at 2% solids concentration (grams dry weight per 100 mL) in 50 mM sodium acetate buffer (pH 5.00) with the addition of 40 lg/mL of tetracycline and 30 lg/mL of cycloheximide. The enzymes, AccelleraseÒ 1500 and AccelleraseÒ XY, were used at the recommended dosages, 0.5 mL per gram cellulose and 0.1 mL per gram cellulose, respectively. Vials were incubated in a water bath at 50 °C and agitation of 140 rpm. Progress of the reaction was monitored by interval taking 50 lL supernatant to a 1.5 mL centrifuge tube containing 450 lL distilled water, followed by holding the tube in a boiling water bath for 5 min to quench the reaction. Two replicates were obtained for each point. Sugar analysis was conducted with high-performance liquid chromatography (HPLC) equipped with reflective index detector (RID 10A, Shimadzu, MD, USA) and a Rezex RPM-monosaccharide column (Phenomenex, CA, USA) operated at 80 °C. Total sugar recovery was calculated using the following formula:
Six different temperatures ranging from 50 to 160 °C were used to compare the sugar yield after IL pretreatment and enzymatic hydrolysis. Glucose and xylose production as a function of time during enzymatic hydrolysis is shown in Fig. 1. Solid powder could be observed even after 3 h incubation at 110 °C, suggesting that the corn stover was partially dissolved at the relatively low temperature. No significant solid was found with pretreatment at either 130 or 160 °C after 3 h. After applying water to precipitate cellulose, the solid fraction from either 130 or 160 °C formed gel-like regenerated cellulose, which was significantly different from the solid fraction (biomass fiber) from the other conditions of lower temperature. Fig. 1A shows that about 90% of glucose released at 72 h of hydrolysis was obtained at 10 h. Similar results were reported that 90% of glucose released at 72 h could be obtained at 24 h after complete dissolution of switchgrass at 160 °C (Li et al., 2010). In the current study, partial dissolution of corn stover also effectively increased hydrolysis rate at a relatively low temperature, indicating that complete dissolution of corn stover in IL is not necessary and cellulose becomes easily digestible with biomass disruption by IL. For hydrolysis extent after 72 h, the glucose yield increased as the pretreatment temperature increased to 130 °C, and 94.4% of cellulose was recovered as glucose in hydrolysate after IL pretreatment at 130 °C, suggesting that the selected IL is effective in corn stover pretreatment for the following cellulose–glucose conversion. However, the xylose yield after 130 °C pretreament was 55.8%, which is significantly lower than the yield after 110 °C pretreatment (93.9% of glucose, 75.0% of xylose, and 88.0% of total sugar). The results from composition analysis (Table 1) show that the xylan recovery in solid fraction after IL pretreatment decreased as temperature increased, indicating that the degraded xylan, which could not be easily recovered by applying water, increased with increasing temperature. The increasing dissolution of xylan
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Fig. 1. Effects of pretreatment temperature on glucose (A) and xylose (B) yield. (: untreated; j: 50 °C; N: 70 °C; h: 90 °C; ⁄: 110 °C; d: 130 °C; s: 160 °C).
Table 1 Effects of pretreatment time/temperature on the recovery of biomass components.a 3h
Lignin (%) Xylan (%) Cellulose (%) a
70 °C
50 °C
70 °C
90 °C
110 °C
130 °C
160 °C
6h
9h
24 h
76.2 89.9 98.9
69.2 87.1 98.2
48.9 82.6 97.0
43.0 78.2 96.6
26.0 59.4 95.8
11.1 15.0 89.0
67.2 87.0 97.8
61.2 85.3 97.6
52.4 81.7 96.6
All percentages were calculated on the initial content of the components in untreated biomass.
in IL would not only reduce sugar yield but also increase the cost of downstream recovery. As temperature increased from 50 to 110 °C, the increase in xylose yield, as well as the increase in glucose yield, could be a result of biomass disruption by IL pretreatment. Xylose degradation became more significant at a higher temperature (over 110 °C), which eventually decreased xylose recovery. After pretreatment at 160 °C, xylan recovery in solid fraction and the xylose yield after enzymatic hydrolysis were only 15.0% and 14.3%, respectively. It was reported that xylan could be degraded to furfural or 5-hydroxymethyl-2-furaldehyde (HMF) with IL,1-ethyl-3methylimidazolium hydrogen sulfate ([EMIM][HSO4]) (Lima et al., 2009), which could be one of the reasons that explain the xylan loss after IL pretreatment. Further study is required to understand the mechanism of xylan degradation in IL. Notably, glucose recovery decreased as the pretreatment temperature increased to 160 °C. The reduced recovery of glucose (Table 1) after IL pretreatment suggested that cellulose degradation (11%) was also significant at the temperature of 160 °C. Also noteworthy is that cellulose digestibility was enhanced to over 90% with IL pretreatment at 90 °C for 3 h whereas 48.9% of lignin was left in solid fraction (Fig. 1A, Table 1). Further study is being conducted to understand the structural changes of the remaining lignin.
3.2. Effects of pretreatment time To further understand the profile of IL pretreatment at low temperature, the study of the effect of pretreatment time on biomass digestibility was conducted at 70 °C. As the pretreatment duration increased, both glucose and xylose yield at 72 h of hydrolysis increased (Fig. 2) and lignin content decreased (Table 1). It was reported that the increased lignin extraction because of an increased pretreatment time is one of the reasons explaining the enhanced sugar yield (Lee et al., 2009). Glucose loss, which was less than 4% even after 24 h pretreatment, increased as the duration increased (Table 1).
After 24 h pretreatment, the glucose, xylose, and total sugar yields were 84.9%, 64.8%, and 78.0%, respectively (Fig. 2), which was comparable to the yields of AFEX pretreatment (82.0%, 72.2%, and 78.4%, respectively) (Li et al., 2011). Biomass solid could still be observed even after 24 h pretreatment, suggesting that a complete dissolution of corn stover in IL is not necessary; therefore, the IL ([C2mim][OAc]) used is effective for pretreatment of corn stover at 70 °C. A relatively low temperature could not only reduce energy input but also increase xylan recovery as proved in this study. In addition, further study of low temperature IL pretreatment, such as pretreatment at 50 °C, coupled with the developing IL-tolerant enzymes/microbes (Nakashima et al., 2011), could make possible simultaneous carbohydrates extraction and saccharification in a single system.
3.3. Structural change of corn stover with IL pretreatment It is well accepted that pretreatment changes biomass structure rather than mass loss, resulting in change in digestibility (Mosier et al., 2005). However, it is challenging to correlate only one structural parameter of biomass with its digestibility. Numerous studies of biomass conversion have argued about the relationship between crystallinity and rate/extent of enzymatic hydrolysis (Hall et al., 2010; Mansfield et al., 1999). An important reason could be that the calculation, employed by most of the studies, was based on the weight of biomass not on that of cellulose, which added other amorphous components, such as hemicellulose and lignin as well as extractives, into the consideration of crystallinity. As a result, crystallinity could change even without a change in cellulose structure, and cellulose digestibility could even increase as crystallinity increases (Zhao et al., 2008), It might not be appropriate to use crystallinity change to explain digestibility change when the increase in crystallinity is a result of lignin removal, and it is uncertain how cellulose structure has been changed (Kim et al., 2003). We suggest using cellulose crystallinity (the percentage of crystalline part in cellulose, not total mass) to study crystallinity change.
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Fig. 2. Effects of pretreatment time on sugar yield. Pretreatment temperature was 70 °C.
Fig. 3. The correlation between cellulose digestibility and cellulose crystallinity (CCr) (), as well as lignin recovery in solid fraction (j). Cellulose digestibility was calculated as the percentage of released glucose yield from recovered cellulose in pretreated biomass.
In this study, cellulose digestibility was significantly correlated to both lignin removal and cellulose crystallinity (Fig. 3). As the pretreatment temperature increase from 50 to 160 °C, the cellulose crystallinity, as well as the lignin recovery, decreased, and the cellulose digestibility increased. A similar trend was observed when the pretreatment time increased from 3 to 24 h at 70 °C. Therefore, the changes of both cellulose crystallinity and lignin content contributed to the increase in digestibility. It is well accepted that lignin removal could reduce the irreversible absorption of cellulase on lignin (Berlin et al., 2005). In addition, the removal of amorphous components, including lignin and hemicellulose, could increase the accessible area for cellulose–enzyme interaction (Zhang and Lynd, 2004) that further enhanced cellulose digestibility. Similar to other pretreatments, such as diluted acid or alkali pretreatment, the disruption of biomass by chemical attack makes polysaccharides more reactive; however, it is not similar to most of the other pretreatments that IL actually effectively de-crystallized cellulose (Binder and Raines, 2010). Notably is the transition of cellulose from type I to II (See Supplementary Material). Native cellulose (type I) has typical diffraction peaks at 1.70 and 2.52 nm1, respectively, whereas regenerated cellulose (type II) has typical diffraction peak at 2.26 nm1 (Zhang et al., 2005). As the pretreatment temperature or time increased, the (0 0 2) and (0 2 0) reflections waned and
the (1 1 0) reflection increased as reported (Zhang et al., 2005). The reflection peaks of (0 2 0) and (1 1 0) overlapped with a partial dissolution at relatively low temperature, possibly suggesting that both cellulose I and II coexisted in recovered solid fraction. It was reported that the structure of cellulose II is more stable than that of cellulose I (Kolpak and Blackwell, 1976), but more research is needed to determine whether the transition is favorable to digestibility or not since the changes of crystallinity and/or crystal size of cellulose, that accompany the transition during IL pretreatment, may also affect digestibility.
4. Conclusion Partially dissolving corn stover in IL at a relatively low temperature (110 °C) for 3 h, with higher xylan recovery (75.9%), was employed to achieve glucose yield of 93.9% and total sugar yield of 88.0%. It was demonstrated that about 78% of structural sugar was recovered with IL pretreatment at a lower temperature (70 °C) for 24 h. Structural investigation revealed that both cellulose crystallinity and lignin content contributed to the increase in digestibility of biomass. Further study is being conducted at lower temperatures to develop a system of simultaneous IL pretreatment and enzymatic hydrolysis.
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