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Effect of dimethyl sulfoxide on ionic liquid 1-ethyl-3-methylimidazolium acetate pretreatment of eucalyptus wood for enzymatic hydrolysis
Bioresource Technology 140 (2013) 90–96
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Effect of dimethyl sulfoxide on ionic liquid 1-ethyl-3-methylimidazolium acetate pretreatment of eucalyptus wood for enzymatic hydrolysis Long Wu a, Seung-Hwan Lee b,⇑, Takashi Endo a,⇑ a b
Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
h i g h l i g h t s DMSO significantly reduces the viscosity of [EMIM]OAc. DMSO–[EMIM]OAc pretreatment has a mild effect on the composition of biomass. [EMIM]OAc improves biomass digestibility by disrupting cellulose crystal structures. DMSO has little influence on the interaction between cellulose and the ionic liquid. Proper use of DMSO facilitates the pretreatment without lowering hydrolysis yields.
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Article history: Received 22 January 2013 Received in revised form 17 April 2013 Accepted 18 April 2013 Available online 27 April 2013 Keywords: Lignocellulose Biorefinery Recalcitrance Crystallinity Saccharification
a b s t r a c t Ground eucalyptus wood was pretreated with 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc)– dimethyl sulfoxide (DMSO) solutions with different mixing ratios under various conditions. The changes in the composition and structure of the biomass were investigated; and the enzymatic hydrolysis performance of the pretreated biomass was evaluated. [EMIM]OAc–DMSO pretreatment had a relatively mild effect on the composition of the biomass, but excessively high pretreatment temperatures led to massive loss of xylan after pretreatment. The enzymatic digestibility of the biomass was significantly improved with increased pretreatment temperature. X-ray diffraction analysis revealed that the disruption of cellulose crystal structure by [EMIM]OAc at a sufficiently high temperature was primarily responsible for the remarkable improvement in the digestibility. Appropriate addition of DMSO could help minimize the consumption of [EMIM]OAc without impairing the performance of the ionic liquid, and contribute to the improvement in pretreatment efficiency due to the viscosity reduction effect on the pretreatment liquor. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Sugar-platform based biorefineries primarily focus on the integrated conversion of the available polysaccharides found in lignocellulosic biomass into biofuels and bioproducts via hydrolysis and fermentation. Enzymatic hydrolysis has long been recognized as a potential low-cost means for extracting fermentable sugars from lignocellulosic biomass. Since lignocellulose is highly resistant to bio-degradation, largely due to the lignin-carbohydrate cross-linkages and the crystalline structure of cellulose, thermochemical pretreatment of feedstocks is required to achieve high saccharification efficiencies. Ionic liquids (ILs) are salts in the liquid state at temperatures below 100 °C. Many ILs are non-volatile, non-flammable, ⇑ Corresponding authors. Tel.: +82 33 250 8323 (S.-H. Lee), tel.: +81 82 420 8278 (T. Endo). E-mail addresses: [email protected] (S.-H. Lee), [email protected] (T. Endo). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.072
non-corrosive and thermally stable. The chemical and physical properties of ILs can be adjusted by using different combinations of anions and cations (Earle and Seddon, 2000). Furthermore, ILs can be recycled and reused with minimal loss of activity (Lozano et al., 2012). These features have made ILs a promising medium in many areas of chemistry (Olivier-Bourbigou et al., 2010). Some ILs can dissolve cellulose under relatively mild conditions supposedly by disrupting the intra- and inter-molecular hydrogen bonding network in crystalline cellulose (Michels and Kosan, 2005; Moulthrop et al., 2005; Remsing et al., 2006; Swatloski et al., 2002). Lately, the application of the cellulose-dissolving ILs composed of large organic cations (e.g., alkyl-substituted imidazolium cations) and organic/inorganic anions in biorefining processes has drawn increasing attention (Stark, 2011; Tadesse and Luque, 2011). IL pretreatment under certain conditions can change the composition and structure of the lignocellulosic biomass and make the carbohydrate polymers more susceptible to enzymatic hydrolysis (Bahcegul et al., 2012; Cheng et al., 2011; Li et al., 2010). A previous
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study showed that pretreatment with 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), among 6 ILs investigated, was most effective in improving the enzymatic digestibility of sugarcane bagasse (da Silva et al., 2011). Other recent studies have also suggested that [EMIM]OAc pretreatment can significantly increase the efficiency of enzymatic hydrolysis of various woody and herbaceous lignocellulosic biomasses (Labbé et al., 2012; Lee et al., 2009; Qiu et al., 2012). Although the effectiveness of ILs has been well documented, there are still technological and economic challenges that need to be addressed before the practical application of ILs in the biorefining industry can become a reality. For instance, [EMIM]OAc, like many other ILs, is relatively costly; it has very high viscosity in comparison to common aqueous thermochemical pretreatment reagents (e.g., acid and alkali); and it deactivates regular cellulolytic enzymes even at very low concentrations (Engel et al., 2010; Turner et al., 2003). Dimethyl sulfoxide (DMSO) is an important polar aprotic solvent, which can be obtained from a by-product (i.e., dimethyl sulfide) of kraft pulping. DMSO has been frequently used in the IL treatment of cellulose, in which context it reportedly played a wide range of roles, including co-solvent, cellulose swelling agent, viscosity reducer and antisolvent (Cuissinat et al., 2008; Fort et al., 2007; Kimona et al., 2011; Lv et al., 2012; Sun et al., 2012; Wang et al., 2011). Tian et al. (2011) found that treating microcrystalline cellulose with organic electrolyte solutions (OES, a mixture of ionic liquid (1-allyl-3-methylimidazolium chloride ([AMIM]Cl) and DMSO) with [AMIM]Cl molar fraction above a certain level effectively decreased the crystallinity of the cellulose and improved the efficiency of subsequent enzymatic hydrolysis. The authors also discussed the potential advantages of the OES, such as lowering pretreatment reagent cost, shortening pretreatment time, reducing energy requirements for stirring and transporting, and recyclability. So far, whether the addition of DMSO has any influence on the efficiency of [EMIM]OAc pretreatment of lignocellulosic biomass for enzymatic hydrolysis has not been fully investigated. The objective of this study is to elucidate the effect of DMSO on [EMIM]OAc pretreatment of eucalyptus wood in order to further improve the feasibility of the IL pretreatment for enzymatic production of fermentable sugars from lignocellulosic feedstock. 2. Methods 2.1. Materials Eucalyptus wood chips, kindly supplied by Oji Paper (Tokyo, Japan), were ground to an average particle size of less than 0.2 mm using a Fritsch P-14 rotor mill (Fritsch Japan, Yokohama, Japan) and stored at room temperature before use (referred to as untreated). The moisture content of the untreated sample was 3.8% (w.b.) according to oven drying method (i.e., drying 500 mg biomass at 105 °C for 24 h). [EMIM]OAc (>95% purity) was purchased from IoLiTec (Heilbronn, Germany). DMSO (>99% purity) was purchased from Wako Pure Chemical Industries (Tokyo, Japan). Acremonium-derived cellulase (Meiji Seika, Tokyo, Japan) and Optimash BG (Genencor Kyowa, Tokyo, Japan) were used for enzymatic hydrolysis. Other chemicals were obtained from commercial sources and used without any further purification. 2.2. [EMIM]OAc–DMSO pretreatment of biomass Pure [EMIM]OAc and DMSO were mixed at room temperature to obtain solutions with [EMIM]OAc-to-DMSO ratios of 4:1, 3:2, 2:3 and 1:4 (v/v, referred to as 4IL-1D, 3IL-2D, 2IL-3D and 1IL4D). The viscosity of each solution was measured at 25 °C using a Brookfield DV-III Ultra rheometer equipped with an UL adapter (Brookfield, MA, USA).
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Ground eucalyptus wood (500 mg) was evenly dispersed using a vortex mixer into the [EMIM]OAc–DMSO solutions at a solidto-liquid ratio of 15% (w/v) in a glass test tube with air-tight screw caps. The tubes containing the mixtures of the biomass and different solutions were placed in an autoclave and heated at a selected temperature (ranging from 80 to 140 °C) for a desired time period (120 or 240 min); the autoclave was then shut off and the samples were cooled down in situ to 50 °C, with heating up and cooling down taking approximately 0.5 and 1 h, respectively. Pretreatments with pure [EMIM]OAc or DMSO under the same conditions were also performed as controls. After pretreatment, the mixtures were transferred into plastic bottles and washed thoroughly with 250 mL of deionized water using a shaking incubator. The pretreated biomass was then collected by vacuum filtration using PTFE membrane filters with a pore size of 0.2 lm; the biomass that adhered to the membrane was manually recovered to minimize pretreatment loss. Preliminary experiments showed that, after the washing-filtration step under the present conditions, the reagents that remained in the biomass would not affect the subsequent enzymatic hydrolysis. 2.3. Enzymatic hydrolysis of pretreated biomass An enzyme cocktail comprising 1 mg/mL of Acremonium-derived cellulase and 0.2% (v/v) Optimash BG in 50 mM sodium citrate buffer (pH 4.8) was used for the enzymatic hydrolysis of the biomass. The impact that [EMIM]OAc and DMSO had on the FPase, CMCase and cellobiase activities of the enzyme cocktail over a range of concentrations was investigated according to the procedure proposed by Ghose (1987). Measurements of cellulase activities were performed with different amounts of [EMIM]OAc or DMSO taking place of the buffer in the reaction system (total volume remained unchanged, pure buffer as control showing original activities). Pretreated biomass, washed with deionized water and filtrated, was washed with 20 mL of 50 mM sodium citrate buffer (pH 4.8), centrifuged, and mixed with appropriate amounts of buffer and 10 mL of preheated (50 °C) enzyme solution (enzyme loading: 20 FPU/g glucan; solids loading: 2.5%, w/v), and incubated in a shaking incubator at 50 °C for 72 h. Hydrolysate samples (0.1 mL) were taken at the initial (4 h) and final (72 h) stages of the hydrolysis; then, the reaction was stopped by heating the samples at 100 °C for 15 min. Glucose and xylose concentrations in the samples were determined by HPLC following the method reported by da Silva et al. (2011).The hydrolysis yields of glucose and xylose were calculated according to the following equations:
Glucose yield ð%Þ ¼ 100 ð0:9 mass of glucose in hydrolysate= mass of glucan in 500 mg untreated biomassÞ ð1Þ Xylose yield ð%Þ ¼ 100 ð0:88 mass of xylose in hydrolysate= mass of xylan in 500 mg untreated biomassÞ
ð2Þ
All pretreatment-saccharification experiments were carried out in duplicate. Data are presented as mean ± standard deviation. 2.4. Compositional and structural analysis To preserve the structural features of the pretreated biomass, the biomass samples recovered by filtration (Section 2.2) were washed several times with t-butyl alcohol by centrifugation to remove residual water, and then transferred into tared 50-mL test tubes and freeze-dried for 72 h. The dried samples were weighed to determine the total solids recovery. Compositional analysis of the samples was performed according to the Laboratory Analytical Procedures provided by the National Renewable Energy Laboratory, USA (NREL). In brief, the structural polysaccharides (glucan
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and xylan) were broken down into monomers via two-step sulfuric acid hydrolysis for HPLC quantification, and the acid-insoluble lignin (AIL) found in the hydrolysis residues was determined by thermogravimetric analysis. The percent recoveries of the major components after pretreatment were calculated based on the results of compositional analysis according to the following equation:
of their mixture reduced exponentially with increasing DMSO amounts. It was observed that the lower viscosity facilitated the mixing of the biomass and pretreatment liquor, especially at the relatively high pretreatment solids loading tested in this study.
Recovery ð%Þ ¼ 100 ðmass of component in freeze-driedright
Wood cell wall primarily consists of cellulose, hemicellulose and lignin. The compositional analysis showed that glucan, xylan and AIL accounted for 40.6%, 11.4%, and 24.0% (dry weight) of the untreated eucalyptus wood, respectively. The composition of the biomass pretreated under certain conditions and the corresponding percent recoveries of the major components after pretreatment are presented in Fig. 2. Pretreatment at temperatures below 120 °C had a minor effect on the composition of the biomass, irrespective of the
biomass=mass of component in 500 mgright untreated biomassÞ
ð3Þ
The specific surface area (SSA) of the samples was estimated by the Brunauer–Emmett–Teller (BET) method (Brunauer et al., 1938) from nitrogen gas adsorption–desorption isotherms measured using a BELSORP-Max volumetric gas adsorption instrument (Bel Japan, Osaka, Japan). The measurements were performed in duplicate. Wide-angle X-ray diffraction (WAXRD) measurements were performed on a RINT-TTR III diffractometer (Rigaku, Tokyo, Japan) with Cu Ka radiation (k = 0.1542 nm, generated at 50 kV and 300 mA). The samples, pressed into 125-mg tablets each, were scanned at scattering angles varying from 2° to 60° in 0.02° increments at a scanning rate of 2°/min.
3.1. Effect of pretreatment on the composition of biomass
3. Results and discussion DMSO showed good miscibility with [EMIM]OAc under the experimental conditions tested. Since DMSO has a relatively high boiling point (189 °C), its presence does not cause any technical/ safety issue that jeopardizes the ‘‘green solvent’’ feature of the ionic liquid. The viscosities of [EMIM]OAc and DMSO at 25 °C were 128.9 and 1.96 cP, respectively. As shown in Fig. 1, the viscosity
Fig. 1. Effect of [EMIM]OAc-to-DMSO ratio on the viscosity of pretreatment solutions.
Fig. 2. Composition of pretreated biomass and percent recoveries of the major components after pretreatment (pretreatment temperature and time as follows: (a) 80 °C for 120 min; (b) 120 °C for 120 min; (c) 120 °C for 240 min; and (d) 140 °C for 120 min).
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composition of the pretreatment liquor. There were negligible compositional differences among the differently pretreated samples including the control groups; the recoveries of the major components steadily reached 90% (Fig. 2a–c). In comparison, pretreatment at 140 °C led to a remarkable change in the composition of the biomass (Fig. 2d). The pretreated biomass had higher glucan and AIL contents, but the xylan content was much lower than the biomass pretreated at the lower temperatures. Since the glucan and AIL recoveries still reached around 90%, the results suggested that hemicellulose depolymerization occurred during the pretreatment, which accounted for the significant loss of xylan (30–40%) observed in the 140 °C pretreatment group. Additionally, xylan loss appeared to be more severe for higher [EMIM]OAc-to-DMSO ratios under the present conditions. Some studies have indicated that the non-cellulose components, especially lignin, could be selectively extracted from lignocellulosic biomass by [EMIM]OAc treatment (Lee et al., 2009; Lynam et al., 2012; Qiu et al., 2012; Torr et al., 2012; Weerachanchai et al., 2012). However, under comparable conditions, in this study the delignification effect of the pretreatment was not as significant.
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ment liquor reached a critical level, while pretreatment with excessive [EMIM]OAc would not induce any further improvement in the digestibility. In addition, care should be taken when selecting pretreatment temperature with regard to total hydrolysis yield, as xylan degradation during pretreatment at excessively high temperatures could have a significant effect on the final xylose yield. Under appropriate conditions, extending the pretreatment time had a positive effect on the hydrolysis yields, e.g., the biomass pretreated at 120 °C for 240 min displayed about 30% improvement in both initial hydrolysis rate and final sugar yields over the 120-min
3.2. Enzymatic hydrolysis of biomass Fig. 3 shows the influence of [EMIM]OAc and DMSO on the activities of the enzyme cocktail. [EMIM]OAc, as a salt, exhibited a strong inhibitive effect on the enzymes. The FPase, CMCase and cellobiase activities dropped to 52%, 67% and 48% of the original levels (marked with data labels in the figures), respectively, in the presence of merely 5% (v/v) of [EMIM]OAc in the reaction system, and rapidly decreased with further increase in the concentration of [EMIM]OAc. The effect of DMSO on the enzyme activity was relatively mild; the activities reached 58%, 76% and 88% of the original levels, respectively, even when 20% (v/v) of DMSO existed in the reaction system. Untreated eucalyptus wood was highly resistant to enzymatic hydrolysis. Less than 8 mg of glucose (4% of theoretical yield) and only trace amounts of xylose were released from 500 mg of the untreated biomass after 72 h of enzymatic hydrolysis under the conditions described in Section 2.3. Pretreatment with DMSO failed to improve the enzymatic digestibility of the biomass; there was little increase in the yields of the sugar monomers, even when the pretreatment was performed at 120 °C. Figs. 4 and 5 show the glucose and xylose yields from enzymatic hydrolysis of the biomass pretreated with [EMIM]OAc–DMSO solutions under certain conditions. Pretreatment at 80 °C showed a limited influence on the enzymatic digestibility of the biomass; 72 h glucose yield never exceeded 15% (xylose yield less than 10%) for all the [EMIM]OAc–DMSO groups (Figs. 4a and 5a). The hydrolysis yields were significantly enhanced with increased pretreatment temperature. In the case of the biomass pretreated with pure [EMIM]OAc at 120 °C for 120 min (IL groups in Figs. 4b and 5b), glucose yield reached 35% (4 h) and 67% (72 h), and xylose yield reached 27% (4 h) and 47% (72 h), respectively. The yields remained largely unaffected by increasing the DMSO content in the pretreatment liquor until a [EMIM]OAc-to-DMSO ratio of 3:2 (v/ v) (i.e., 4IL-1D and 3IL-2D groups in the figures), and then declined with further decrease of the ratio (i.e., 2IL-1D and 1IL-4D in the figures). Pretreatment at 140 °C led to even higher glucose yield. In fact, about 95% of theoretical yield could be reached after 72 h hydrolysis, provided that the [EMIM]OAc-to-DMSO ratio was above 2:3 (Fig. 4d). However, the xylose yield was not further increased for all the groups due to the massive loss of xylan after pretreatment (Fig. 5d). The above results indicated that the biomass pretreated at a sufficiently high temperature would be equally digestible as long as the [EMIM]OAc concentration in the pretreat-
Fig. 3. Effect of [EMIM]OAc and DMSO on the FPase, CMCase and cellobiase activities of enzyme cocktail (activities shown as percent of original levels marked with data labels).
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Fig. 4. Glucose yield from enzymatic hydrolysis of pretreated biomass (pretreatment temperature and time as follows: (a) 80 °C for 120 min; (b) 120 °C for 120 min; (c) 120 °C for 240 min; (d) 140 °C for 120 min).
Fig. 5. Xylose yield from enzymatic hydrolysis of pretreated biomass (pretreatment temperature and time as follows: (a) 80 °C for120 min; (b) 120 °C for 120 min; (c) 120 °C for 240 min; and (d) 140 °C for 120 min).
pretreatment groups across the range of [EMIM]OAc-to-DMSO ratio tested (Figs. 4c and 5c). Compared to the other thermochemical methods reported in the literature, such as acid (McIntosh et al., 2012), liquid hot water (Yu et al., 2010), steam explosion (Emmel et al., 2003) and alkali pretreatment (Park and Kim, 2012), the [EMIM]OAc–DMSO pretreatment yielded a great improvement in the enzymatic hydrolysis efficiency without drastically changing the composition of eucalyptus wood.
SSA was markedly increased even after pretreatment with pure DMSO at different temperatures. The increase in SSA was even more remarkable with the presence of EMIM]OAc. Moreover, the
Table 1 Specific surface area of the biomass pretreated with IL-DMSO solutions at different temperatures for 120 min. Pretreatment temperature (°C)
SSA (m2/g) IL
4IL1D
3IL2D
2IL3D
1IL4D
DMSO
80 120 140
8.57 23.18 23.54
11.01 19.46 33.09
9.47 18.60 35.70
10.10 17.28 47.74
10.34 14.35 40.37
3.28 5.32 15.83
3.3. Structural analysis of biomass The SSA of the untreated biomass determined by the BET method was 1.01 m2/g. According to the data presented in Table 1, the
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SSA of the pretreated biomass increased with increasing pretreatment temperature for a certain [EMIM]OAc-to-DMSO ratio. Nevertheless, the changes in SSA appeared to be irregular in terms of changing [EMIM]OAc-to-DMSO ratio, and largely inconsistent with the improvement in hydrolysis yields, particularly in the case of pretreatment at higher temperatures, which implied that other structural changes caused by the pretreatment decisively affected the digestibility of the biomass. X-ray diffraction patterns of the untreated and some of the pretreated biomass samples are presented in Fig. 6. The untreated and DMSO pretreated (120 °C for 120 min) samples had very similar diffraction patterns (Fig. 6a), showing the diffraction peaks from and 0 0 2 lattice planes characteristic of cellulose I found 101, 101 in higher plants. [EMIM]OAc–DMSO pretreatment at 80 °C for 120 min mildly changed the diffraction patterns (Fig. 6b); although a slight reduction in the diffraction intensity was observed, the diffraction profiles were generally similar to the untreated sample. The biomass pretreated with [EMIM]OAc at 120 °C for 120 min had a strikingly changed diffraction profile (IL spectrum in Fig. 6c), featuring remarkably decreased diffraction intensity with peaks almost unrecognizable, and slightly dethe 101 and 101 creased diffraction angle for the main peak. The drastic changes reflected a substantial disruption of the original crystal structure of the cellulose, due to the breakdown of the hydrogen bonding network by the ionic liquid. Similar diffraction patterns were also observed for certain [EMIM]OAc–DMSO groups (4IL-1D and 3IL-2D spectra in Fig. 6c), indicating that the pretreatment with the [EMIM]OAc–DMSO solutions also caused the disruption of the crystal structure. The results suggested that DMSO, an aprotic solvent, might not be involved in the interaction between the ionic liquid and crystalline cellulose. Pretreatment with lower amounts of [EMIM]OAc (i.e., [EMIM]OAc-to-DMSO ratio lower than 3:2) did not cause drastic structural changes under the present conditions, as evidenced by the diffraction patterns that were basically consistent with the patterns derived from the untreated biomass sample
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(2IL-3D and 1IL-4D spectra in Fig. 6c). In the case of the biomass pretreated at 140 °C (Fig. 6d), the diffraction patterns changed with the [EMIM]OAc-to-DMSO ratio in a similar manner except that the changes were even more drastic. Additionally, the higher pretreatment temperature seemed to help compensate the effect of low [EMIM]OAc concentration to some extent; similar diffraction patterns were observed even when the [EMIM]OAc-to-DMSO ratio was decreased to 2:3 (2IL-3D spectrum in Fig. 6d); however, the ratio of 1:4 was still too low to cause significant changes in the crystal structure. Although a longer pretreatment time led to a further reduction in the diffraction intensity over the temperature range tested, the overall diffraction profiles of the pretreated biomass were largely unaffected (data not shown). In association with the hydrolysis performance of differently pretreated biomass, the changes in cellulose crystal structure exhibited a crucial influence on the improvement of the enzymatic digestibility. Pretreatment at sufficiently high temperatures could substantially disrupt the crystal structure of cellulose and make the biomass easily digestible by the enzymes. The critical level of [EMIM]OAc concentration observed in the enzymatic hydrolysis experiment could be perfectly interpreted in terms of structural changes, because the disruption of the crystal structure occurred only when the amount of [EMIM]OAc in the pretreatment liquor reached that level; [EMIM]OAc above the critical level caused similar changes in the crystal structure, and led to the uniform hydrolysis performance of the pretreated biomass. The results were largely consistent with the conclusion drawn by Tian et al. (2011) based on the investigation into [AMIM]Cl-DMSO pretreatment of microcrystalline cellulose. According to the above results, the consumption of the costly ionic liquid for pretreatment could be minimized while ensuring the efficiency of the enzymatic hydrolysis of the pretreated biomass. By doing so, the risk of deactivation of the enzymes by residual ionic liquid after pretreatment could also be reduced. However, for a large-scale biorefinery, implementing pretreatment steps
Fig. 6. X-ray diffractograms of untreated and differently pretreated eucalyptus wood samples (pretreatment temperature and time as follows: (a) 120 °C for 120 min (DMSO pretreatment); (b) 80 °C for 120 min; (c) 120 °C for 120 min; and (d) 140 °C for 120 min).
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with the use of viscous ionic liquids would be difficult. In this respect, considering the DMSO viscosity reduction effect on the pretreatment liquor, the addition of appropriate amounts of DMSO could contribute to the improvement in the efficiency of the pretreatment. 4. Conclusions [EMIM]OAc–DMSO pretreatment under appropriate conditions greatly improved the enzymatic hydrolysis performance of eucalyptus wood. At a sufficiently high temperature, the ionic liquid above a certain concentration could substantially disrupted the crystal structure of cellulose, making the biomass easily digestible. DMSO had little effect on the interaction between the ionic liquid and the cellulose, and its presence in the pretreatment liquor did not significantly affect the enzymatic hydrolysis performance of the pretreated biomass. Proper application of DMSO could contribute to improving the overall cost-effectiveness of the biorefining process without impairing the performance of the ionic liquid. Acknowledgements This work was part of the Japan–U.S. Cooperation Project for Research and Standardization of Clean Energy Technologies. The authors are grateful to Ms. Yuka Myouken, Mr. Ryouhei Shimodi and Mr. Takuya Tanioka for their technical assistance during this study. References Bahcegul, E., Apaydin, S., Haykir, N.I., Tatli, E., Bakir, U., 2012. Different ionic liquids favor different lignocellulosic biomass particle sizes during pretreatment to function efficiently. Green Chem. 14, 1896–1903. Brunauer, S., Emmet, P.H., Teller, E., 1938. Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 60, 309–319. Cheng, G., Varanasi, P., Li, C., Liu, H., Melnichenko, Y.B., Simmons, B.A., Kent, M.S., Singh, S., 2011. Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis. Biomacromolecules 12, 933–941. Cuissinat, C., Navard, P., Heinze, T., 2008. Swelling and dissolution of cellulose. Part IV: free floating cotton and wood fibres in ionic liquids. Carbohydr. Polym. 72, 590–596. Da Silva, A.S., Lee, S.-H., Endo, T., Bon, E.P.S., 2011. Major improvement in the rate and yield of enzymatic saccharification of sugarcane bagasse via pretreatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim] [Ac]). Bioresour. Technol. 102, 10505–10509. Earle, M.J., Seddon, K.R., 2000. Ionic liquids. Green solvents for the future. Pure Appl. Chem. 72, 1391–1398. Emmel, A., Mathias, A.L., Wypych, F., Ramos, L.P., 2003. Fractionation of Eucalyptus grandis chips by dilute acid-catalysed steam explosion. Bioresour. Technol. 86, 105–115. Engel, P., Mladenov, R., Wulfhorst, H., Jäger, G., Spiess, A.C., 2010. Point by point analysis: how ionic liquid affects the enzymatic hydrolysis of native and modified cellulose. Green Chem. 12, 1959–1966. Fort, D.A., Remsing, R.C., Swatloski, R.P., Moyna, P., Moyna, G., Rogers, R.D., 2007. Can ionic liquids dissolve wood? Processing and analysis of lignocellulosic materials with 1-n-butyl-3-methylimidazolium chloride. Green Chem. 9, 63– 69. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257– 268. Kimona, K.S., Alana, E.L., Sinclair, D.W.O., 2011. Enhanced saccharification kinetics of sugarcane bagasse pretreated in 1-butyl-3-methylimidazolium chloride at
high temperature and without complete dissolution. Bioresour. Technol. 102, 9325–9329. Labbé, N., Kline, L.M., Moens, L., Kim, K., Kim, P.C., Hayes, D.G., 2012. Activation of lignocellulosic biomass by ionic liquid for biorefinery fractionation. Bioresour. Technol. 104, 701–707. 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., 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. Lozano, P., Bernal, B., Recio, I., Belleville, M.P., 2012. A cyclic process for full enzymatic saccharification of pretreated cellulose with full recovery and reuse of the ionic liquid 1-butyl-3-methylimidazolium chloride. Green Chem. 14, 2631–2637. Lv, Y., Wu, J., Zhang, J., Niu, Y., Liu, C.Y., He, J., Zhang, J., 2012. Rheological properties of cellulose/ionic liquid/dimethylsulfoxide (DMSO) solutions. Polymer 53, 2524–2531. Lynam, J.G., Reza, M.T., Vasquez, V.R., Coronella, C.J., 2012. Pretreatment of rice hulls by ionic liquid dissolution. Bioresour. Technol. 114, 629–636. McIntosh, S., Vancov, T., Palmer, J., Spain, M., 2012. Ethanol production from Eucalyptus plantation thinnings. Bioresour. Technol. 110, 264–272. Michels, C., Kosan, B., 2005. Contribution to the dissolution state of cellulose and cellulose derivatives. Lenzinger Berichte 84, 62–70. Moulthrop, J.S., Swatloski, R.P., Moyna, G., Rogers, R.D., 2005. High-resolution 13C NMR studies of cellulose and cellulose oligomers in ionic liquid solutions. Chem. Commun., 1557–1559. National Renewable Energy Laboratory (NREL), Laboratory Analytical Procedure TP510-42618, TP-510-42619, TP-510-42620, TP-510-42621, TP-510-42622. http://www.nrel.gov/biomass/analytical_procedures.html. Olivier-Bourbigou, H., Magna, L., Morvan, D., 2010. Ionic liquids and catalysis: recent progress from knowledge to applications. Appl. Catal., A 373, 1–56. Park, Y.C., Kim, J.S., 2012. Comparison of various alkaline pretreatment methods of lignocellulosic biomass. Energy 47, 31–35. Qiu, Z., Aita, G.M., Walker, M.S., 2012. Effect of ionic liquid pretreatment on the chemical composition, structure and enzymatic hydrolysis of energy cane bagasse. Bioresour. Technol. 117, 251–256. Remsing, R.C., Swatloski, R.P., Rogers, R.D., Moyna, G., 2006. Mechanism of cellulose dissolution in the ionic liquid 1-n-butyl-3-methylimidazolium chloride: a 13C and 35/37Cl NMR relaxation study on model systems. Chem. Commun., 1271– 1273. Stark, A., 2011. Ionic liquids in the biorefinery: a critical assessment of their potential. Energy Environ. Sci. 4, 19–32. Sun, S.N., Li, M.F., Yuan, T.Q., Xu, F., Sun, R.C., 2012. Effect of ionic liquid pretreatment on the structure of hemicelluloses from Corncob. J. Agric. Food Chem. 60, 11120–11127. Swatloski, R.P., Spear, S.K., Holbrey, J.D., Rogers, R.D., 2002. Dissolution of cellulose with ionic liquids. J. Am. Chem. Soc. 124, 4974–4975. Tadesse, H., Luque, R., 2011. Advances on biomass pretreatment using ionic liquids: an overview. Energy Environ. Sci. 4, 3913–3929. Tian, X.F., Fang, Z., Jiang, D., Sun, X.Y., 2011. Pretreatment of microcrystalline cellulose in organic electrolyte solutions for enzymatic hydrolysis. Biotechnol. Biofuels 4, 53. Torr, K.M., Love, K.T., Çetinkol, Ö.P., Donaldson, L.A., George, A., Holmesb, B.M., Simmons, B.A., 2012. The impact of ionic liquid pretreatment on the chemistry and enzymatic digestibility of Pinus radiata compression wood. Green Chem. 14, 778–787. Turner, M.B., Spear, S.K., Huddleston, J.G., Holbrey, J.D., Rogers, R.D., 2003. Ionic liquid salt-induced inactivation and unfolding of cellulase from Trichoderma reesei. Green Chem. 5, 443–447. Wang, X., Li, H., Cao, Y., Tang, Q., 2011. Cellulose extraction from wood chip in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl). Bioresour. Technol. 102, 7959–7965. Weerachanchai, P., Leong, S.S.J., Chang, M.W., Ching, C.B., Lee, J.M., 2012. Improvement of biomass properties by pretreatment with ionic liquids for bioconversion process. Bioresour. Technol. 111, 453–459. Yu, Q., Zhuang, X., Yuan, Z., Wang, Q., Qi, W., Wang, W., Zhang, Y., Xu, J., Xu, H., 2010. Two-step liquid hot water pretreatment of Eucalyptus grandis to enhance sugar recovery and enzymatic digestibility of cellulose. Bioresour. Technol. 101, 4895–4899.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effect of dimethyl sulfoxide on ionic liquid 1-ethyl-3-methylimidazolium acetate pretreatment of eucalyptus wood for enzymatic hydrolysis Long Wu a, Seung-Hwan Lee b,⇑, Takashi Endo a,⇑ a b
Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
h i g h l i g h t s DMSO significantly reduces the viscosity of [EMIM]OAc. DMSO–[EMIM]OAc pretreatment has a mild effect on the composition of biomass. [EMIM]OAc improves biomass digestibility by disrupting cellulose crystal structures. DMSO has little influence on the interaction between cellulose and the ionic liquid. Proper use of DMSO facilitates the pretreatment without lowering hydrolysis yields.
a r t i c l e
i n f o
Article history: Received 22 January 2013 Received in revised form 17 April 2013 Accepted 18 April 2013 Available online 27 April 2013 Keywords: Lignocellulose Biorefinery Recalcitrance Crystallinity Saccharification
a b s t r a c t Ground eucalyptus wood was pretreated with 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc)– dimethyl sulfoxide (DMSO) solutions with different mixing ratios under various conditions. The changes in the composition and structure of the biomass were investigated; and the enzymatic hydrolysis performance of the pretreated biomass was evaluated. [EMIM]OAc–DMSO pretreatment had a relatively mild effect on the composition of the biomass, but excessively high pretreatment temperatures led to massive loss of xylan after pretreatment. The enzymatic digestibility of the biomass was significantly improved with increased pretreatment temperature. X-ray diffraction analysis revealed that the disruption of cellulose crystal structure by [EMIM]OAc at a sufficiently high temperature was primarily responsible for the remarkable improvement in the digestibility. Appropriate addition of DMSO could help minimize the consumption of [EMIM]OAc without impairing the performance of the ionic liquid, and contribute to the improvement in pretreatment efficiency due to the viscosity reduction effect on the pretreatment liquor. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Sugar-platform based biorefineries primarily focus on the integrated conversion of the available polysaccharides found in lignocellulosic biomass into biofuels and bioproducts via hydrolysis and fermentation. Enzymatic hydrolysis has long been recognized as a potential low-cost means for extracting fermentable sugars from lignocellulosic biomass. Since lignocellulose is highly resistant to bio-degradation, largely due to the lignin-carbohydrate cross-linkages and the crystalline structure of cellulose, thermochemical pretreatment of feedstocks is required to achieve high saccharification efficiencies. Ionic liquids (ILs) are salts in the liquid state at temperatures below 100 °C. Many ILs are non-volatile, non-flammable, ⇑ Corresponding authors. Tel.: +82 33 250 8323 (S.-H. Lee), tel.: +81 82 420 8278 (T. Endo). E-mail addresses: [email protected] (S.-H. Lee), [email protected] (T. Endo). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.072
non-corrosive and thermally stable. The chemical and physical properties of ILs can be adjusted by using different combinations of anions and cations (Earle and Seddon, 2000). Furthermore, ILs can be recycled and reused with minimal loss of activity (Lozano et al., 2012). These features have made ILs a promising medium in many areas of chemistry (Olivier-Bourbigou et al., 2010). Some ILs can dissolve cellulose under relatively mild conditions supposedly by disrupting the intra- and inter-molecular hydrogen bonding network in crystalline cellulose (Michels and Kosan, 2005; Moulthrop et al., 2005; Remsing et al., 2006; Swatloski et al., 2002). Lately, the application of the cellulose-dissolving ILs composed of large organic cations (e.g., alkyl-substituted imidazolium cations) and organic/inorganic anions in biorefining processes has drawn increasing attention (Stark, 2011; Tadesse and Luque, 2011). IL pretreatment under certain conditions can change the composition and structure of the lignocellulosic biomass and make the carbohydrate polymers more susceptible to enzymatic hydrolysis (Bahcegul et al., 2012; Cheng et al., 2011; Li et al., 2010). A previous
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study showed that pretreatment with 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), among 6 ILs investigated, was most effective in improving the enzymatic digestibility of sugarcane bagasse (da Silva et al., 2011). Other recent studies have also suggested that [EMIM]OAc pretreatment can significantly increase the efficiency of enzymatic hydrolysis of various woody and herbaceous lignocellulosic biomasses (Labbé et al., 2012; Lee et al., 2009; Qiu et al., 2012). Although the effectiveness of ILs has been well documented, there are still technological and economic challenges that need to be addressed before the practical application of ILs in the biorefining industry can become a reality. For instance, [EMIM]OAc, like many other ILs, is relatively costly; it has very high viscosity in comparison to common aqueous thermochemical pretreatment reagents (e.g., acid and alkali); and it deactivates regular cellulolytic enzymes even at very low concentrations (Engel et al., 2010; Turner et al., 2003). Dimethyl sulfoxide (DMSO) is an important polar aprotic solvent, which can be obtained from a by-product (i.e., dimethyl sulfide) of kraft pulping. DMSO has been frequently used in the IL treatment of cellulose, in which context it reportedly played a wide range of roles, including co-solvent, cellulose swelling agent, viscosity reducer and antisolvent (Cuissinat et al., 2008; Fort et al., 2007; Kimona et al., 2011; Lv et al., 2012; Sun et al., 2012; Wang et al., 2011). Tian et al. (2011) found that treating microcrystalline cellulose with organic electrolyte solutions (OES, a mixture of ionic liquid (1-allyl-3-methylimidazolium chloride ([AMIM]Cl) and DMSO) with [AMIM]Cl molar fraction above a certain level effectively decreased the crystallinity of the cellulose and improved the efficiency of subsequent enzymatic hydrolysis. The authors also discussed the potential advantages of the OES, such as lowering pretreatment reagent cost, shortening pretreatment time, reducing energy requirements for stirring and transporting, and recyclability. So far, whether the addition of DMSO has any influence on the efficiency of [EMIM]OAc pretreatment of lignocellulosic biomass for enzymatic hydrolysis has not been fully investigated. The objective of this study is to elucidate the effect of DMSO on [EMIM]OAc pretreatment of eucalyptus wood in order to further improve the feasibility of the IL pretreatment for enzymatic production of fermentable sugars from lignocellulosic feedstock. 2. Methods 2.1. Materials Eucalyptus wood chips, kindly supplied by Oji Paper (Tokyo, Japan), were ground to an average particle size of less than 0.2 mm using a Fritsch P-14 rotor mill (Fritsch Japan, Yokohama, Japan) and stored at room temperature before use (referred to as untreated). The moisture content of the untreated sample was 3.8% (w.b.) according to oven drying method (i.e., drying 500 mg biomass at 105 °C for 24 h). [EMIM]OAc (>95% purity) was purchased from IoLiTec (Heilbronn, Germany). DMSO (>99% purity) was purchased from Wako Pure Chemical Industries (Tokyo, Japan). Acremonium-derived cellulase (Meiji Seika, Tokyo, Japan) and Optimash BG (Genencor Kyowa, Tokyo, Japan) were used for enzymatic hydrolysis. Other chemicals were obtained from commercial sources and used without any further purification. 2.2. [EMIM]OAc–DMSO pretreatment of biomass Pure [EMIM]OAc and DMSO were mixed at room temperature to obtain solutions with [EMIM]OAc-to-DMSO ratios of 4:1, 3:2, 2:3 and 1:4 (v/v, referred to as 4IL-1D, 3IL-2D, 2IL-3D and 1IL4D). The viscosity of each solution was measured at 25 °C using a Brookfield DV-III Ultra rheometer equipped with an UL adapter (Brookfield, MA, USA).
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Ground eucalyptus wood (500 mg) was evenly dispersed using a vortex mixer into the [EMIM]OAc–DMSO solutions at a solidto-liquid ratio of 15% (w/v) in a glass test tube with air-tight screw caps. The tubes containing the mixtures of the biomass and different solutions were placed in an autoclave and heated at a selected temperature (ranging from 80 to 140 °C) for a desired time period (120 or 240 min); the autoclave was then shut off and the samples were cooled down in situ to 50 °C, with heating up and cooling down taking approximately 0.5 and 1 h, respectively. Pretreatments with pure [EMIM]OAc or DMSO under the same conditions were also performed as controls. After pretreatment, the mixtures were transferred into plastic bottles and washed thoroughly with 250 mL of deionized water using a shaking incubator. The pretreated biomass was then collected by vacuum filtration using PTFE membrane filters with a pore size of 0.2 lm; the biomass that adhered to the membrane was manually recovered to minimize pretreatment loss. Preliminary experiments showed that, after the washing-filtration step under the present conditions, the reagents that remained in the biomass would not affect the subsequent enzymatic hydrolysis. 2.3. Enzymatic hydrolysis of pretreated biomass An enzyme cocktail comprising 1 mg/mL of Acremonium-derived cellulase and 0.2% (v/v) Optimash BG in 50 mM sodium citrate buffer (pH 4.8) was used for the enzymatic hydrolysis of the biomass. The impact that [EMIM]OAc and DMSO had on the FPase, CMCase and cellobiase activities of the enzyme cocktail over a range of concentrations was investigated according to the procedure proposed by Ghose (1987). Measurements of cellulase activities were performed with different amounts of [EMIM]OAc or DMSO taking place of the buffer in the reaction system (total volume remained unchanged, pure buffer as control showing original activities). Pretreated biomass, washed with deionized water and filtrated, was washed with 20 mL of 50 mM sodium citrate buffer (pH 4.8), centrifuged, and mixed with appropriate amounts of buffer and 10 mL of preheated (50 °C) enzyme solution (enzyme loading: 20 FPU/g glucan; solids loading: 2.5%, w/v), and incubated in a shaking incubator at 50 °C for 72 h. Hydrolysate samples (0.1 mL) were taken at the initial (4 h) and final (72 h) stages of the hydrolysis; then, the reaction was stopped by heating the samples at 100 °C for 15 min. Glucose and xylose concentrations in the samples were determined by HPLC following the method reported by da Silva et al. (2011).The hydrolysis yields of glucose and xylose were calculated according to the following equations:
Glucose yield ð%Þ ¼ 100 ð0:9 mass of glucose in hydrolysate= mass of glucan in 500 mg untreated biomassÞ ð1Þ Xylose yield ð%Þ ¼ 100 ð0:88 mass of xylose in hydrolysate= mass of xylan in 500 mg untreated biomassÞ
ð2Þ
All pretreatment-saccharification experiments were carried out in duplicate. Data are presented as mean ± standard deviation. 2.4. Compositional and structural analysis To preserve the structural features of the pretreated biomass, the biomass samples recovered by filtration (Section 2.2) were washed several times with t-butyl alcohol by centrifugation to remove residual water, and then transferred into tared 50-mL test tubes and freeze-dried for 72 h. The dried samples were weighed to determine the total solids recovery. Compositional analysis of the samples was performed according to the Laboratory Analytical Procedures provided by the National Renewable Energy Laboratory, USA (NREL). In brief, the structural polysaccharides (glucan
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and xylan) were broken down into monomers via two-step sulfuric acid hydrolysis for HPLC quantification, and the acid-insoluble lignin (AIL) found in the hydrolysis residues was determined by thermogravimetric analysis. The percent recoveries of the major components after pretreatment were calculated based on the results of compositional analysis according to the following equation:
of their mixture reduced exponentially with increasing DMSO amounts. It was observed that the lower viscosity facilitated the mixing of the biomass and pretreatment liquor, especially at the relatively high pretreatment solids loading tested in this study.
Recovery ð%Þ ¼ 100 ðmass of component in freeze-driedright
Wood cell wall primarily consists of cellulose, hemicellulose and lignin. The compositional analysis showed that glucan, xylan and AIL accounted for 40.6%, 11.4%, and 24.0% (dry weight) of the untreated eucalyptus wood, respectively. The composition of the biomass pretreated under certain conditions and the corresponding percent recoveries of the major components after pretreatment are presented in Fig. 2. Pretreatment at temperatures below 120 °C had a minor effect on the composition of the biomass, irrespective of the
biomass=mass of component in 500 mgright untreated biomassÞ
ð3Þ
The specific surface area (SSA) of the samples was estimated by the Brunauer–Emmett–Teller (BET) method (Brunauer et al., 1938) from nitrogen gas adsorption–desorption isotherms measured using a BELSORP-Max volumetric gas adsorption instrument (Bel Japan, Osaka, Japan). The measurements were performed in duplicate. Wide-angle X-ray diffraction (WAXRD) measurements were performed on a RINT-TTR III diffractometer (Rigaku, Tokyo, Japan) with Cu Ka radiation (k = 0.1542 nm, generated at 50 kV and 300 mA). The samples, pressed into 125-mg tablets each, were scanned at scattering angles varying from 2° to 60° in 0.02° increments at a scanning rate of 2°/min.
3.1. Effect of pretreatment on the composition of biomass
3. Results and discussion DMSO showed good miscibility with [EMIM]OAc under the experimental conditions tested. Since DMSO has a relatively high boiling point (189 °C), its presence does not cause any technical/ safety issue that jeopardizes the ‘‘green solvent’’ feature of the ionic liquid. The viscosities of [EMIM]OAc and DMSO at 25 °C were 128.9 and 1.96 cP, respectively. As shown in Fig. 1, the viscosity
Fig. 1. Effect of [EMIM]OAc-to-DMSO ratio on the viscosity of pretreatment solutions.
Fig. 2. Composition of pretreated biomass and percent recoveries of the major components after pretreatment (pretreatment temperature and time as follows: (a) 80 °C for 120 min; (b) 120 °C for 120 min; (c) 120 °C for 240 min; and (d) 140 °C for 120 min).
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composition of the pretreatment liquor. There were negligible compositional differences among the differently pretreated samples including the control groups; the recoveries of the major components steadily reached 90% (Fig. 2a–c). In comparison, pretreatment at 140 °C led to a remarkable change in the composition of the biomass (Fig. 2d). The pretreated biomass had higher glucan and AIL contents, but the xylan content was much lower than the biomass pretreated at the lower temperatures. Since the glucan and AIL recoveries still reached around 90%, the results suggested that hemicellulose depolymerization occurred during the pretreatment, which accounted for the significant loss of xylan (30–40%) observed in the 140 °C pretreatment group. Additionally, xylan loss appeared to be more severe for higher [EMIM]OAc-to-DMSO ratios under the present conditions. Some studies have indicated that the non-cellulose components, especially lignin, could be selectively extracted from lignocellulosic biomass by [EMIM]OAc treatment (Lee et al., 2009; Lynam et al., 2012; Qiu et al., 2012; Torr et al., 2012; Weerachanchai et al., 2012). However, under comparable conditions, in this study the delignification effect of the pretreatment was not as significant.
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ment liquor reached a critical level, while pretreatment with excessive [EMIM]OAc would not induce any further improvement in the digestibility. In addition, care should be taken when selecting pretreatment temperature with regard to total hydrolysis yield, as xylan degradation during pretreatment at excessively high temperatures could have a significant effect on the final xylose yield. Under appropriate conditions, extending the pretreatment time had a positive effect on the hydrolysis yields, e.g., the biomass pretreated at 120 °C for 240 min displayed about 30% improvement in both initial hydrolysis rate and final sugar yields over the 120-min
3.2. Enzymatic hydrolysis of biomass Fig. 3 shows the influence of [EMIM]OAc and DMSO on the activities of the enzyme cocktail. [EMIM]OAc, as a salt, exhibited a strong inhibitive effect on the enzymes. The FPase, CMCase and cellobiase activities dropped to 52%, 67% and 48% of the original levels (marked with data labels in the figures), respectively, in the presence of merely 5% (v/v) of [EMIM]OAc in the reaction system, and rapidly decreased with further increase in the concentration of [EMIM]OAc. The effect of DMSO on the enzyme activity was relatively mild; the activities reached 58%, 76% and 88% of the original levels, respectively, even when 20% (v/v) of DMSO existed in the reaction system. Untreated eucalyptus wood was highly resistant to enzymatic hydrolysis. Less than 8 mg of glucose (4% of theoretical yield) and only trace amounts of xylose were released from 500 mg of the untreated biomass after 72 h of enzymatic hydrolysis under the conditions described in Section 2.3. Pretreatment with DMSO failed to improve the enzymatic digestibility of the biomass; there was little increase in the yields of the sugar monomers, even when the pretreatment was performed at 120 °C. Figs. 4 and 5 show the glucose and xylose yields from enzymatic hydrolysis of the biomass pretreated with [EMIM]OAc–DMSO solutions under certain conditions. Pretreatment at 80 °C showed a limited influence on the enzymatic digestibility of the biomass; 72 h glucose yield never exceeded 15% (xylose yield less than 10%) for all the [EMIM]OAc–DMSO groups (Figs. 4a and 5a). The hydrolysis yields were significantly enhanced with increased pretreatment temperature. In the case of the biomass pretreated with pure [EMIM]OAc at 120 °C for 120 min (IL groups in Figs. 4b and 5b), glucose yield reached 35% (4 h) and 67% (72 h), and xylose yield reached 27% (4 h) and 47% (72 h), respectively. The yields remained largely unaffected by increasing the DMSO content in the pretreatment liquor until a [EMIM]OAc-to-DMSO ratio of 3:2 (v/ v) (i.e., 4IL-1D and 3IL-2D groups in the figures), and then declined with further decrease of the ratio (i.e., 2IL-1D and 1IL-4D in the figures). Pretreatment at 140 °C led to even higher glucose yield. In fact, about 95% of theoretical yield could be reached after 72 h hydrolysis, provided that the [EMIM]OAc-to-DMSO ratio was above 2:3 (Fig. 4d). However, the xylose yield was not further increased for all the groups due to the massive loss of xylan after pretreatment (Fig. 5d). The above results indicated that the biomass pretreated at a sufficiently high temperature would be equally digestible as long as the [EMIM]OAc concentration in the pretreat-
Fig. 3. Effect of [EMIM]OAc and DMSO on the FPase, CMCase and cellobiase activities of enzyme cocktail (activities shown as percent of original levels marked with data labels).
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Fig. 4. Glucose yield from enzymatic hydrolysis of pretreated biomass (pretreatment temperature and time as follows: (a) 80 °C for 120 min; (b) 120 °C for 120 min; (c) 120 °C for 240 min; (d) 140 °C for 120 min).
Fig. 5. Xylose yield from enzymatic hydrolysis of pretreated biomass (pretreatment temperature and time as follows: (a) 80 °C for120 min; (b) 120 °C for 120 min; (c) 120 °C for 240 min; and (d) 140 °C for 120 min).
pretreatment groups across the range of [EMIM]OAc-to-DMSO ratio tested (Figs. 4c and 5c). Compared to the other thermochemical methods reported in the literature, such as acid (McIntosh et al., 2012), liquid hot water (Yu et al., 2010), steam explosion (Emmel et al., 2003) and alkali pretreatment (Park and Kim, 2012), the [EMIM]OAc–DMSO pretreatment yielded a great improvement in the enzymatic hydrolysis efficiency without drastically changing the composition of eucalyptus wood.
SSA was markedly increased even after pretreatment with pure DMSO at different temperatures. The increase in SSA was even more remarkable with the presence of EMIM]OAc. Moreover, the
Table 1 Specific surface area of the biomass pretreated with IL-DMSO solutions at different temperatures for 120 min. Pretreatment temperature (°C)
SSA (m2/g) IL
4IL1D
3IL2D
2IL3D
1IL4D
DMSO
80 120 140
8.57 23.18 23.54
11.01 19.46 33.09
9.47 18.60 35.70
10.10 17.28 47.74
10.34 14.35 40.37
3.28 5.32 15.83
3.3. Structural analysis of biomass The SSA of the untreated biomass determined by the BET method was 1.01 m2/g. According to the data presented in Table 1, the
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SSA of the pretreated biomass increased with increasing pretreatment temperature for a certain [EMIM]OAc-to-DMSO ratio. Nevertheless, the changes in SSA appeared to be irregular in terms of changing [EMIM]OAc-to-DMSO ratio, and largely inconsistent with the improvement in hydrolysis yields, particularly in the case of pretreatment at higher temperatures, which implied that other structural changes caused by the pretreatment decisively affected the digestibility of the biomass. X-ray diffraction patterns of the untreated and some of the pretreated biomass samples are presented in Fig. 6. The untreated and DMSO pretreated (120 °C for 120 min) samples had very similar diffraction patterns (Fig. 6a), showing the diffraction peaks from and 0 0 2 lattice planes characteristic of cellulose I found 101, 101 in higher plants. [EMIM]OAc–DMSO pretreatment at 80 °C for 120 min mildly changed the diffraction patterns (Fig. 6b); although a slight reduction in the diffraction intensity was observed, the diffraction profiles were generally similar to the untreated sample. The biomass pretreated with [EMIM]OAc at 120 °C for 120 min had a strikingly changed diffraction profile (IL spectrum in Fig. 6c), featuring remarkably decreased diffraction intensity with peaks almost unrecognizable, and slightly dethe 101 and 101 creased diffraction angle for the main peak. The drastic changes reflected a substantial disruption of the original crystal structure of the cellulose, due to the breakdown of the hydrogen bonding network by the ionic liquid. Similar diffraction patterns were also observed for certain [EMIM]OAc–DMSO groups (4IL-1D and 3IL-2D spectra in Fig. 6c), indicating that the pretreatment with the [EMIM]OAc–DMSO solutions also caused the disruption of the crystal structure. The results suggested that DMSO, an aprotic solvent, might not be involved in the interaction between the ionic liquid and crystalline cellulose. Pretreatment with lower amounts of [EMIM]OAc (i.e., [EMIM]OAc-to-DMSO ratio lower than 3:2) did not cause drastic structural changes under the present conditions, as evidenced by the diffraction patterns that were basically consistent with the patterns derived from the untreated biomass sample
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(2IL-3D and 1IL-4D spectra in Fig. 6c). In the case of the biomass pretreated at 140 °C (Fig. 6d), the diffraction patterns changed with the [EMIM]OAc-to-DMSO ratio in a similar manner except that the changes were even more drastic. Additionally, the higher pretreatment temperature seemed to help compensate the effect of low [EMIM]OAc concentration to some extent; similar diffraction patterns were observed even when the [EMIM]OAc-to-DMSO ratio was decreased to 2:3 (2IL-3D spectrum in Fig. 6d); however, the ratio of 1:4 was still too low to cause significant changes in the crystal structure. Although a longer pretreatment time led to a further reduction in the diffraction intensity over the temperature range tested, the overall diffraction profiles of the pretreated biomass were largely unaffected (data not shown). In association with the hydrolysis performance of differently pretreated biomass, the changes in cellulose crystal structure exhibited a crucial influence on the improvement of the enzymatic digestibility. Pretreatment at sufficiently high temperatures could substantially disrupt the crystal structure of cellulose and make the biomass easily digestible by the enzymes. The critical level of [EMIM]OAc concentration observed in the enzymatic hydrolysis experiment could be perfectly interpreted in terms of structural changes, because the disruption of the crystal structure occurred only when the amount of [EMIM]OAc in the pretreatment liquor reached that level; [EMIM]OAc above the critical level caused similar changes in the crystal structure, and led to the uniform hydrolysis performance of the pretreated biomass. The results were largely consistent with the conclusion drawn by Tian et al. (2011) based on the investigation into [AMIM]Cl-DMSO pretreatment of microcrystalline cellulose. According to the above results, the consumption of the costly ionic liquid for pretreatment could be minimized while ensuring the efficiency of the enzymatic hydrolysis of the pretreated biomass. By doing so, the risk of deactivation of the enzymes by residual ionic liquid after pretreatment could also be reduced. However, for a large-scale biorefinery, implementing pretreatment steps
Fig. 6. X-ray diffractograms of untreated and differently pretreated eucalyptus wood samples (pretreatment temperature and time as follows: (a) 120 °C for 120 min (DMSO pretreatment); (b) 80 °C for 120 min; (c) 120 °C for 120 min; and (d) 140 °C for 120 min).
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with the use of viscous ionic liquids would be difficult. In this respect, considering the DMSO viscosity reduction effect on the pretreatment liquor, the addition of appropriate amounts of DMSO could contribute to the improvement in the efficiency of the pretreatment. 4. Conclusions [EMIM]OAc–DMSO pretreatment under appropriate conditions greatly improved the enzymatic hydrolysis performance of eucalyptus wood. At a sufficiently high temperature, the ionic liquid above a certain concentration could substantially disrupted the crystal structure of cellulose, making the biomass easily digestible. DMSO had little effect on the interaction between the ionic liquid and the cellulose, and its presence in the pretreatment liquor did not significantly affect the enzymatic hydrolysis performance of the pretreated biomass. Proper application of DMSO could contribute to improving the overall cost-effectiveness of the biorefining process without impairing the performance of the ionic liquid. Acknowledgements This work was part of the Japan–U.S. Cooperation Project for Research and Standardization of Clean Energy Technologies. The authors are grateful to Ms. Yuka Myouken, Mr. Ryouhei Shimodi and Mr. Takuya Tanioka for their technical assistance during this study. References Bahcegul, E., Apaydin, S., Haykir, N.I., Tatli, E., Bakir, U., 2012. Different ionic liquids favor different lignocellulosic biomass particle sizes during pretreatment to function efficiently. Green Chem. 14, 1896–1903. Brunauer, S., Emmet, P.H., Teller, E., 1938. Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 60, 309–319. Cheng, G., Varanasi, P., Li, C., Liu, H., Melnichenko, Y.B., Simmons, B.A., Kent, M.S., Singh, S., 2011. Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis. Biomacromolecules 12, 933–941. Cuissinat, C., Navard, P., Heinze, T., 2008. Swelling and dissolution of cellulose. Part IV: free floating cotton and wood fibres in ionic liquids. Carbohydr. Polym. 72, 590–596. Da Silva, A.S., Lee, S.-H., Endo, T., Bon, E.P.S., 2011. Major improvement in the rate and yield of enzymatic saccharification of sugarcane bagasse via pretreatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim] [Ac]). Bioresour. Technol. 102, 10505–10509. Earle, M.J., Seddon, K.R., 2000. Ionic liquids. Green solvents for the future. Pure Appl. Chem. 72, 1391–1398. Emmel, A., Mathias, A.L., Wypych, F., Ramos, L.P., 2003. Fractionation of Eucalyptus grandis chips by dilute acid-catalysed steam explosion. Bioresour. Technol. 86, 105–115. Engel, P., Mladenov, R., Wulfhorst, H., Jäger, G., Spiess, A.C., 2010. Point by point analysis: how ionic liquid affects the enzymatic hydrolysis of native and modified cellulose. Green Chem. 12, 1959–1966. Fort, D.A., Remsing, R.C., Swatloski, R.P., Moyna, P., Moyna, G., Rogers, R.D., 2007. Can ionic liquids dissolve wood? Processing and analysis of lignocellulosic materials with 1-n-butyl-3-methylimidazolium chloride. Green Chem. 9, 63– 69. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257– 268. Kimona, K.S., Alana, E.L., Sinclair, D.W.O., 2011. Enhanced saccharification kinetics of sugarcane bagasse pretreated in 1-butyl-3-methylimidazolium chloride at
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