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1 Bioresource Technology 101 (2010) Contents lists available at ScienceDirect Bioresource Technology journal homepage: Cellulose pretreatment with 1-n-butyl-3-methylimidazolium chloride for solid acid-catalyzed hydrolysis Soo-Jin Kim a,b, Adid Adep Dwiatmoko a,c, Jae Wook Choi a, Young-Woong Suh a, *, Dong Jin Suh a, Moonhyun Oh b a Clean Energy Center, Korea Institute of Science and Technology, Seoul , Republic of Korea b Department of Chemistry, Yonsei University, Seoul , Republic of Korea c Clean Energy and Chemical Engineering Department, University of Science and Technology, Daejeon , Republic of Korea article info abstract Article history: Received 5 March 2010 Received in revised form 27 May 2010 Accepted 7 June 2010 Available online 1 July 2010 Keywords: 1-n-Butyl-3-methylimidazolium chloride Pretreatment Hydrolysis Solid acid This study has been focused on developing a cellulose pretreatment process using 1-n-butyl-3-methylimidazolium chloride ([bmim]cl) for subsequent hydrolysis over Nafion Ò NR50. Thus, several pretreatment variables such as the pretreatment period and temperature, and the [bmim]cl amount were varied. Additionally, the [bmim]cl-treated cellulose samples were characterized by X-ray diffraction analysis, and their crystallinity index values including CI(XD), CI(XD-CI) and CI(XD-CII) were then calculated. When correlated with these values, the concentrations of total reducing sugars (TRS) obtained by the pretreatment of native cellulose (NC) and glucose produced by the hydrolysis reaction were found to show a distinct relationship with the [CI(NC) CI(XD)] and CI(XD-CII) values, respectively. Consequently, the cellulose pretreatment step with [bmim]cl is to loosen a crystalline cellulose through partial transformation of cellulose I to cellulose II and, furthermore, the TRS release, while the subsequent hydrolysis of [bmim]cl-treated cellulose over Nafion Ò NR50 is effective to convert cellulose II to glucose. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction A great deal of attention has been recently paid to development of biofuels and biochemicals technologies, which is associated with high dependency on fossil fuels and release of greenhouse gases that cause global climate change. Cellulose ((C 6 H 10 O 5 ) n ), the major component in lignocellulosic materials (35 50%), is a biodegradable polymer of simple carbohydrates (1000 < DP < 15,000) composed of b(1? 4)-linked D-glucose units, which has been recognized as a potential renewable source for biofuels and bio-based chemicals production (O Sullivan, 1997). However, due to the extensive network of inter- and intra-molecular hydrogen bonding between its fibrils, cellulose is insoluble in either water or most organic solvents. It is, thus, recalcitrant to process and hydrolyze in solution (Murakami et al., 2007). Therefore, a pretreatment process is highly required to make cellulose more susceptible for subsequent process, implying that the effective and economical pretreatment is the main key for the success of cellulose processing (Mosier et al., 2005; Hamelinck et al., 2005). To date, numerous methods have been applied for cellulose pretreatment prior to hydrolysis (Mosier et al., 2005; Hamelinck et al., 2005; Kumar et al., 2009; Sousa et al., 2009; Zheng et al., 2009). Although several processes including alkali and acid, oxidation and other chemical * Corresponding author. Tel.: ; fax: address: ywsuh@kist.re.kr (Y.-W. Suh). treatments have been reported to be a capable method in reducing the crystallinity of cellulose, they are high energy-demanding and often require severe conditions (Taherzadeh and Karimi, 2008). In recent years, a new type of non-volatile solvent, ionic liquids (ILs) have been thoroughly investigated to act as a powerful solvent for cellulose dissolution and hydrolysis owing to their solvent properties and process benefits (Kamiya et al., 2008; Zhao et al., 2009; Li et al., 2009). Swatloski et al. (2002) first reported that the ILs could act as the non-derivatizing solvent for cellulose and chloride-based ILs appear to be the most effective solvent solubilizing cellulose, where the greatest solubility of up to 25 wt.% was achieved under microwave irradiation with 1-n-butyl-3- methylimidazolium chloride ([bmim]cl). This resulted from formation of hydrogen bonding between hydroxyl functions of cellulose and chloride anions of ILs (Remsing et al., 2006). According to recent reports (Zhao et al., 2009), anions of chloride, formate, acetate or alkylphosphonate in ILs would be usually beneficial to dissolving cellulose due to such a strong bonding at elevated temperatures. Aside from their use for cellulose dissolution, ILs have been applied for cellulose pretreatment prior to enzymatic hydrolysis (Dadi et al., 2006, 2007; Liu and Chen, 2006; Li et al., 2009; Zhao et al., 2009; Lee et al., 2009; Samayam and Schall, 2010) or utilized as a medium for the hydrolysis reaction (Li and Zhao, 2007; Rinaldi et al., 2008, 2010; Kamiya et al., 2008; Li et al., 2008; Rinaldi et al., 2010). Particularly, Rinaldi et al. (2008, 2010) reported that the /$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi: /j.biortech

2 8274 S.-J. Kim et al. / Bioresource Technology 101 (2010) cellulose depolymerization could be carried out over solid catalysts in the solvent of [bmim]cl, where Amberlyst 15DRY converted a-cellulose to total reducing sugars (TRS) of about 12.5% (25% mono- and di-saccharides). However, that the cellulose pretreatment and hydrolysis in [bmim]cl over solid acids were conducted in one pot may cause the complexity in understanding the effects of [bmim]cl on cellulose pretreatment as well as the activity of solid acids in the hydrolysis of pretreated cellulose. In this report, microcrystalline cellulose has been pretreated with [bmim]cl and subsequently hydrolyzed over a solid acid. At this point, it should be noted that the utilization of acetate- or phosphonate-based ionic liquids is prohibited due to less activation of the glycosidic bonds by weakly basic anions-containing ILs (Rinaldi et al., 2008, 2010), thus choosing the chloride-containing IL in our work. In the pretreatment step, several experimental variables such as the pretreatment period and temperature, and the amount of [bmim]cl were varied. The resulting cellulose/ [bmim]cl mixture were then separated by adding water as an anti-solvent, where the precipitated cellulose solid samples were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD), while TRS in the supernatant liquid samples was quantitatively determined by 3,5-dinitrosalicylic acid (DNS) assay. The [bmim]cl-treated cellulose solid samples were then hydrolyzed over Nafion Ò NR50 in the solvent of water, where Nafion Ò NR50 has a very similar acidic character but good thermal stability, compared to Amberlyst 15DRY. Here, it is worth noting that the absence of [bmim]cl in the hydrolysis reaction mixture is necessary to evaluate the intrinsic activity of Nafion Ò NR50, according to our previous work. Consequently, it would be addressed through an optimization study to search for the best pretreatment condition using [bmim]cl that the crystallinity index (CI) values of [bmim]cl-treated cellulose has a distinct relationship to the TRS concentration in the cellulose pretreatment or the glucose concentration in the hydrolysis of pretreated cellulose. 2. Methods 2.1. Materials Microcrystalline cellulose and 1-n-butyl-3-methylimidazolium chloride ([bmim]cl, 98%), purchased from Sigma Aldrich Co., were used without further purification and stored in a glove box. Nafion Ò NR50 was supplied by Wako Chemicals and used as a solid acid catalyst for the hydrolysis reaction General procedure for cellulose pretreatment and hydrolysis The as-received microcrystalline cellulose (0.2 g; mmol calculated as anhydroglucose unit, C 5 H 10 O 5 ) was charged into a Teflon-lined autoclave equipped with a heating jacket, and mmol [bmim]cl in a solid form and a magnetic stirrer were placed onto the cellulose particles. Into this system, N 2 gas was purged to remove any reactive gas molecules including air and moisture. Then, the pretreatment temperature was raised to a desired temperature in the range of K and maintained for 1 4 h. While the temperature increased, a magnetic stirring of the reaction mixture was started at about 353 K which is little higher than the melting point of [bmim]cl (ca. 350 K). After cellulose pretreatment under a specified condition and cooling to about 308 K, the resulting reaction mixture was visually checked prior to precipitation using water as an anti-solvent. The purpose of this step is to confirm whether it is homogeneous or not. If not completely homogeneous, the pretreatment would be done again instead of conducting further steps. To the obtained homogeneous cellulose/[bmim]cl mixture was then added a certain amount of distilled water (x), leading to precipitation of [bmim]cl-treated cellulose solid parts. After centrifugation and subsequent separation of supernatant liquid portions, the regeneration step was repeated four times to completely take out the residual ionic liquid and the released total reducing sugars (TRS). The supernatant liquids collected in this step (total volume = 4x) was subject to TRS analysis explained in section 2.4. Various pretreatment conditions investigated in this study are summarized in Table 1. Furthermore, the same pretreatment experiment was repeated at least 3 5 times for reproducibility. The regenerated cellulose samples, of which the amount was determined by subtracting the TRS from initial cellulose with respect to weight (cf. Table 1), were charged into an autoclave with distilled water (20 ml) and the catalyst (0.1 g), and then purged with N 2 flow. The suspension was heated to 433 K with stirring and maintained for 4 h, where the autogeneous pressure was roughly 0.5 MPa. The reaction mixture was centrifuged to separate the catalyst and unreacted cellulose from water-soluble products. The final solution was filtered and analyzed by high-performance liquid chromatography using a Bio-Rad HPX-87H column ( mm) at 308 K equipped with a refractive index detector (Young Lin YL 9170 RID), where the mobile phase was M sulfuric acid and the flow rate was 0.5 ml/min. Yield of glucose was calculated as follows: Glucose yieldð%þ glucose obtainedðmolþ ¼ regenerated cellulose; calculated as anhydroglucose ðmolþ 100 Aside from the desired product, glucose, other defined products such as fructose, cellobiose and HMF were detected in HPLC chromatograms. However, the concentration of these undesired products was very low in all cases investigated in this work, compared to the glucose concentration, where the difference in Table 1 Crystallinity index (CI) values and calculated amount of cellulose samples pretreated with [bmim]cl under different conditions and subsequently regenerated a. Run [bmim]cl amount (mmol) Pretreatment temperature (K) Pretreatment period (h) Regenerated cellulose b (g) CI(XD) CI(XD CI) CI(XD CII) n.d. c n.d. c n.d. c a The initial amount of cellulose for pretreatment with [bmim]cl in all runs was 0.2 g. b The amount of regenerated cellulose was determined by subtracting the TRS from initial cellulose with respect to weight. c After pretreatment at 418 K, the sample was not recovered to a sufficient amount for XRD analysis.

3 S.-J. Kim et al. / Bioresource Technology 101 (2010) an order of magnitude was about three. On the other hand, when hydrolysis product solutions were visually monitored with respect to humins formation, they appeared to be clear or very light yellow. Therefore, humins were not thought to be produced in a considerable amount, because it is well known that humins formation leads to a dark brown and turbid solution Characterization of cellulose samples pretreated with [bmim]cl For X-ray diffraction (XRD) and scanning electron microscopy (SEM) characterizations, the [bmim]cl-treated cellulose samples were obtained by washing the recovered cellulose solution with distilled water four times and subsequently with ethanol twice, and then dried in an oven. SEM images of these samples were obtained using a HITACHI S-4100 microscope. Also, their crystalline structures were characterized by XRD analysis performed with a Rigaku D/Max-2500 diffractometer with a step size of 0.02 and a scan speed of 4 /min from 3 to 60, using a Cu-Ka radiation X- ray generated at a voltage of 30 kv and a current of 100 ma. The determination of the crystallinity index by the resolution of wide-angle XRD patterns (CI(XD)) was taken from the literature method (Krässig, 1993). Amorphous halos were drawn by the Microcal ORIGIN program (Microcal Software Inc.) for the determination of h am. The values of CI(XD) were calculated by the following equation: CIðXDÞ ¼1 h am =h cr ¼ 1 h am =ðh tot h am Þ where the crystalline height (h cr ) represents the crystalline scatter of the (0 0 2) reflection at 2h of 22.5 for cellulose I or (1 0 1) reflection at 2h of 19.8 for cellulose II, and the amorphous height (h am ) indicates the height of the amorphous reflection at 2h of 18 for cellulose I or 16 for cellulose II (Krässig, 1993). With amorphous halo correction, deconvolution of the XRD patterns was conducted by using the Microcal ORIGIN program. After cellulose I and cellulose II components were resolved on the basis of the literature method (Oh et al., 2005), the values of CI(XD) were divided into those of CI(XD CI) for cellulose I and CI(XD CII) for cellulose II, calculated by the sum of peak areas of the same crystal system ( P A CI for cellulose I and P A CII for cellulose II), as follows: P ACI CIðXD CIÞ ¼P ðaci þ A CII Þ CIðXDÞ P ACII CIðXD CIIÞ ¼ PðACI þ A CII Þ CIðXDÞ 2.4. Measurement of total reducing sugars released during cellulose pretreatment with [bmim]cl 3,5-Dinitrosalicylic acid (DNS) assay was conducted to quantitatively analyze TRS released during cellulose pretreatment with [bmim]cl. DNS solution, which was prepared following the procedure of Miller (1959), was added to the supernatant liquid portions collected after cellulose pretreatment. A mixture with 0.5 ml of DNS reagent and 0.5 ml of a reaction sample was heated in a water bath of K for about 5 min, cooled to room temperature, and finally mixed with 4 ml of deionized water (Li and Zhao, 2007). The color intensity of the resulting mixture was measured using a VARIAN 5000 UV visible spectrometer at nm. The concentration of TRS was calculated based on a standard curve obtained with glucose. Yield of TRS was calculated as follows: TRS yieldð%þ TRS obtainedðmolþ ¼ initial cellulose; calculated as anhydroglucose ðmolþ Results and discussion 3.1. Characterization of cellulose samples pretreated with [bmim]cl From SEM images (not shown here), the cellulose samples pretreated by varying the amount of [bmim]cl appeared to have a relatively smooth surface, compared with a native microcrystalline cellulose. As the amount of [bmim]cl increased from 10 to 60 mmol, the surface of the [bmim]cl-treated cellulose samples becomes smooth. However, a difference between the cellulose samples pretreated with [bmim]cl of more than 20 mmol with respect to a surface roughness could not be clearly observed. This implies that the change in the morphology of [bmim]cl-treated cellulose samples rarely provides meaningful information into quantitatively determining the effect of pretreatment variables on cellulose pretreatment with [bmim]cl. In this respect, the cellulose samples pretreated under different conditions were characterized by X-ray diffraction (XRD) analysis, as shown in Fig. S1. The XRD pattern of native microcrystalline cellulose was also obtained for comparison, showing only two main peaks at 2h of 18 and 22.5 assigned to the amorphous and crystalline fraction of cellulose I, respectively (Fig. S1a, inset). After the [bmim]cl pretreatment, the reflection of cellulose crystal phase at 2h = 22.5 was diminished, which indicates that the [bmim]cl-treated cellulose samples have lower crystallinity than the native one. Besides, two additional peaks at 2h = 19.8 (crystalline fraction) and 16 (amorphous fraction), which was derived from cellulose II, were observed on the entire XRD patterns of cellulose samples pretreated by varying the [bmim]cl amount, pretreatment temperature and period. This result suggested that the structure of cellulose I in the native cellulose was partially transformed to cellulose II due to cellulose pretreatment with [bmim]cl (Schenzel et al., 2009; Sun et al., 2009). It is worth noting that various intensities of the peaks were noticed on the entire diffractograms of [bmim]cl-treated cellulose samples. As the [bmim]cl amount increased from 10 to 20 mmol, the decrease of the peak intensity at 2h of 22.5 was relatively large while the peak intensity at 2h of 19.8 was nearly same, and the [bmim]cl amount of higher than 20 mmol induced an insignificant change in the intensities (Fig. S1a). Similarly, as the pretreatment temperature and period increased, the peak at 2h of 19.8 appears to be more intense (Figs. S1b and S1c). Therefore, the extent of the transformation from cellulose I to cellulose II depends on the pretreatment conditions, that is, the amount of [bmim]cl, pretreatment temperature and period. In order to visualize changes of the structure, the CI values of pretreated cellulose samples were calculated from the XRD data, as summarized in Table 1. As the [bmim]cl amount increased from 10 to 60 mmol (Runs 1 4), the calculated CI(XD) values decreased from to This tendency was similarly observed in the case of the pretreatment period (Runs 2, 5 7) and the pretreatment temperature (Runs 2, 8 9). It could be, therefore, inferred that the crystalline phase of cellulose was partially altered to amorphous one. Furthermore, the values of CI(XD CI) and CI(XD CII) were varied with different pretreatment conditions. Among these, when microcrystalline cellulose was pretreated with 60 mmol [bmim]cl at 403 K for 2 h (Run 4), the highest value of CI(XD CII) was obtained, indicating that the amount of cellulose II component were largest in the [bmim]cl-treated samples. Only the aforementioned XRD information is, however, not sufficient to investigate the effect of pretreatment variables on cellulose pretreatment with [bmim]cl. Thus, the CI values summarized in Table 1 were afterwards correlated with the results obtained after the pretreatment step as well as the hydrolysis step (i.e., TRS and glucose concentrations).

4 8276 S.-J. Kim et al. / Bioresource Technology 101 (2010) Fig. 1. Effect of [bmim]cl amount in the cellulose pretreatment step: (a) the concentration of total reducing sugars (TRS) released after cellulose pretreatment with [bmim]cl at 403 K for 2 h; and (b) the concentration of glucose obtained by the hydrolysis of the corresponding [bmim]cl-treated cellulose over Nafion Ò NR50 at 433 K for 4 h. Fig. 2. Effect of pretreatment temperature in the cellulose pretreatment step: (a) the concentration of total reducing sugars (TRS) released after cellulose pretreatment with 20 mmol [bmim]cl for 2 h; and (b) the concentration of glucose obtained by the hydrolysis of the corresponding [bmim]cl-treated cellulose over Nafion Ò NR50 at 433 K for 4 h Effects of pretreatment variables on cellulose pretreatment and hydrolysis Effect of [bmim]cl amount in the pretreatment step The native microcrystalline cellulose was pretreated with different amounts of [bmim]cl from 10 to 60 mmol at 403 K for 2 h and subsequently hydrolyzed over Nafion Ò NR50 at the same reaction condition. From the measurement of the concentration of TRS released during the pretreatment, TRS was not detected in the solution with the [bmim]cl amount of not more than 10 mmol, but the higher [bmim]cl amount similarly yielded TRS at the concentration of about 2.5 mm (Fig. 1a). As shown in Fig. 1b, the yield of glucose produced without the [bmim]cl pretreatment was only 2.1 mol%. However, when cellulose was pretreated with mmol [bmim]cl, the glucose yield was increased up to 35 mol%. This result suggests that the cellulose pretreatment with [bmim]cl is necessary and effective for the solid acid catalyst to hydrolyze the regenerated cellulose Effect of pretreatment temperature The temperature was varied from 388 to 418 K in pretreating the microcrystalline cellulose with 20 mmol [bmim]cl for 2 h, followed by the hydrolysis reaction over Nafion Ò NR50. As a result, the TRS concentration increased up to 8.6 mm with the pretreatment temperature (Fig. 2a), whereas the glucose yield reached at 21 mol% in the pretreatment temperature of K and then decreased to a lower value of ca. 9 mol% at higher temperatures (Fig. 2b). This implies that a higher pretreatment temperature leads to a release of more TRS, hence resulting in a lower glucose concentration by the hydrolysis reaction Effect of pretreatment period Finally, the pretreatment period in the step of cellulose pretreatment with [bmim]cl was varied from 1 to 4 h. As shown in Fig. 3, the trends of TRS and glucose concentrations were very similar to the ones observed in varying the pretreatment temperature: the TRS concentration increased up to 6.6 mm with the pretreatment period, whereas the glucose yield obtained from the cellulose samples pretreated for up to 2 h reached at 21.4 mol% and then declined to a lower value of 9.5 mol% at longer pretreatment periods. Therefore, as explained above, the decrease of glucose concentration was attributed to the higher conversion of microcrystalline cellulose into the TRS, due to excessive depolymerization during the prolonged pretreatment period with [bmim]cl Correlation of CI values of [bmim]cl-treated cellulose with TRS and glucose concentrations As shown in Fig. 4, the concentrations of TRS released during cellulose pretreatment and glucose obtained after the hydrolysis reaction were plotted against the CI values summarized in Table 1. With respect to the TRS concentration, the differences between the CI value of native cellulose (CI(NC)) and those of [bmim]cl-treated cellulose samples were calculated in order to investigate the effect

5 S.-J. Kim et al. / Bioresource Technology 101 (2010) Fig. 3. Effect of pretreatment period in the cellulose pretreatment step: (a) the concentration of total reducing sugars (TRS) released after cellulose pretreatment with 20 mmol [bmim]cl at 403 K; and (b) the concentration of glucose obtained by the hydrolysis of the corresponding [bmim]cl-treated cellulose over Nafion Ò NR50 at 433 K for 4 h. of pretreatment conditions, since TRS would be produced from the starting cellulose material. As a result, the TRS concentration was found to have a linear relationship with the [CI(NC) CI(XD)] value, as shown by a fitted line in Fig. 4a. This indicates that the overall decrease of cellulose crystallinity leads to the formation of TRS during the pretreatment. Furthermore, Fig. 4b exhibits a much better fit between the glucose yields and the CI(XD-CII) values, rather than the CI(XD) and CI(XD-CI) values, of [bmim]cl-treated cellulose samples. In other words, the glucose yield were lower when a [bmim]cl-treated cellulose sample contained a smaller CI(XD- CII) value, that is, a lower fraction of cellulose II component. From the above results, the depolymerization behavior of microcrystalline cellulose using [bmim]cl and Nafion Ò NR50 could be addressed. First, the pretreatment with [bmim]cl made possible the transformation of cellulose I to cellulose II (Schenzel et al., 2009; Sun et al., 2009). Moreover, more TRS was released in more severe pretreatment conditions, as observed in the previous sections. However, it is very difficult to determine whether TRS results from cellulose I or II, since TRS concentrations appeared to be randomly distributed with respect to the [CI(NC) (CI(XD-CI) or CI(XD- CII))] values (Fig. 4a). Thus, a linear relationship between the TRS concentration and the [CI(NC) CI(XD)] value suggests that TRS can be produced from both parts of cellulose I and cellulose II. The next investigation was focused on understanding the role of Nafion Ò NR50 catalyst for the hydrolysis of [bmim]cl-treated cellulose. As shown in Fig. 4b, the glucose yield was closely associated with the CI(XD-CII) value. This implies that Nafion Ò NR50 was active to attack and depolymerize cellulose II into glucose rather Fig. 4. Correlation between (a) the concentration of total reducing sugars released after cellulose pretreatment with [bmim]cl, and the difference of the CI values of [bmim]cl-treated cellulose (CI(XD), CI(XD-CI) or CI(XD-CII) from the crystallinity index of native cellulose (CI(NC)); (b) the concentration of glucose obtained by the hydrolysis of [bmim]cl-treated cellulose over Nafion Ò NR50, and the CI values of [bmim]cl-treated cellulose. than cellulose I, because the former is more susceptible for hydrolysis. From this finding, the solid-type Nafion Ò NR50 catalyst is not considered to be as strong as mineral acids commonly used for the hydrolysis reaction. Taking into account the aforementioned assessment, we can straightforwardly describe a couple of pretreatment and hydrolysis reactions investigated in this study. The first step for cellulose pretreatment with [bmim]cl is to loosen a crystalline cellulose through the partial transformation of cellulose I to cellulose II and, furthermore, the release of total reducing sugars. The subsequent step for the hydrolysis of [bmim]cl-treated cellulose over Nafion Ò NR50 is effective to convert cellulose II to the desired product, glucose Process strategy for cellulose depolymerization using ILs and solid acids The main objective of the present study was to clearly understand the effects of the cellulose pretreatment followed by hydrolysis over solid acids by decoupling these steps into a selfsupported single one, as elucidated in the previous section. It is, nevertheless, necessary to compare the data presented herein with the result reported by Rinaldi et al. (2010) in which the glucose yield obtained with [bmim]cl (572.5 mmol) and Amberlyst 15DRY (equivalent to 6.9 mmol of H 3 O + ) from a-cellulose was about 28% at 100 C for 3 h. In this study, both the amount of TRS released through the treatment step (1st step) and that of

6 8278 S.-J. Kim et al. / Bioresource Technology 101 (2010) Fig. 5. Schematic of (a) decoupled steps of cellulose pretreatment with [bmim]cl and solid acid-catalyzed hydrolysis conducted in this study and (b) proposed process of lignocellulose conversion into fermentable sugars on the basis of a combination of ILs, solid acids and enzymes. glucose produced by solid acid-catalyzed hydrolysis (2nd step), as shown in Fig. 5a, were dependent on the conditions applied for cellulose pretreatment. When cellulose was pretreated with mmol of [bmim]cl at 130 C for 2 h (cf. Fig. 1), the TRS of 4.6 mol% was released, and the hydrolysis of the corresponding regenerated cellulose containing both cellulose I (40%) and cellulose II (60%) over Nafion Ò NR50 (equivalent to 0.08 mmol H 3 O + ), resulted in the maximum glucose yield of ca. 35 mol% based on the amount of regenerated cellulose (ca. 16 mol% based on the initial amount of microcrystalline cellulose). Therefore, the glucose yield presented in this report was considered to be comparable to the result in the report of Rinaldi et al. (2010). These values obtained over solid acids were, however, evaluated to be relatively low, compared to those reported using H 2 SO 4 (Li and Zhao, 2007) and enzyme (Dadi et al., 2007; Lee et al., 2009) as a catalyst, due to mass transfer limitation induced by solid solid contact. Thus, a deliberate strategy would be required to enhance the overall process efficiency by a combination of ILs and solid acids, and to be viable for practical application of such a process. The first consideration in this regard is to select an appropriate IL for biomass dissolution. Although the major role of [bmim]cl used in cellulose pretreatment was clearly investigated, its inherent disadvantages, such as high viscosity, hygroscopic property, relatively high melting point and corrosive nature, make biomass processing expensive and inefficient. Recent research efforts on efficient cellulose dissolution have been, therefore, devoted to designing a new class of ILs including acetate- and alkylphosphonate-based ILs (Ohno and Fukaya, 2009; Vitz et al., 2009) to show process-friendly features different from chloride-based ILs. However, utilization of these ILs may cause consideration of two additional aspects; choice of a solid acid and judgment of right moment for separating and recovering TRS and/or glucose from a reaction medium containing an IL and a solid acid. Prior to assessment of the former aspect, it should be noted that ion-exchange resins such as Amberlyst 15DRY and Nafion Ò NR50 are inappropriate for ILs containing a weakly basic anion such as acetate or alkylphosphonate, because of their easy protonation by available H 3 O + species supplied from these solid acids (Rinaldi et al., 2010). Therefore, a suitable solid acid should be developed to exhibit both strong acid strength and non proton-donating power, which is now under our investigation. On the other hand, the other aspect would be intimately associated with a whole process scheme based on use of ILs and solid acids. As shown in Fig. 5b, a new process scheme would be proposed utilizing ILs and solid acids to convert lignocellulosic biomass to fermentable sugars, which can replace environmentally unfriendly concentrated or dilute H 2 SO 4 process. After preliminary treatment such as cleaning, size reduction and drying in order to achieve substrates of uniform particle size and be efficient for further steps, the first-stage hydrolysis in this scheme would be conducted using a solid acid catalyst in an IL medium for extracting a certain amount of cellulose and hemicellulose, followed by depolymerization to sugar monomers and oligomers (TRS). Here, it is worth noting that the amount of TRS obtained is highly dependent on the hydrolysis condition. Afterwards, a catalyst is recycled and the residual substrate is then separated by adding an anti-solvent, resulting into regenerated (hemi)cellulose solid which will be hydrolyzed into fermentable sugars by enzymes (one part of 2nd-stage hydrolysis). The supernatant liquid portion containing an IL, an anti-solvent and dissolved lignin compounds is subjected to a multi-purposed fractionation to regenerate lignin, recycle IL and recover TRS. The resulting TRS may be either utilized directly as a source for fermentation to alcohol or hydrolyzed over solid acids into fermentable sugars under a mild condition (the other part of 2nd-stage hydrolysis), where the latter conversion is conceptually designed because some of sugar oligomers may not be prone to fermentation and sugar degradation products formed in the 1st-stage hydrolysis, even if trivial, may cause undesirable effects on enzymes. Consequently, if the proposed process scheme based on a combined utilization of ILs, solid acids and enzymes becomes viable for depolymerization of lignocellulosic biomass into fermentable sugars, ILs and solid acids could potentially be applied in part for biomass conversion. 4. Conclusions The present study demonstrated that a solid acid-catalyzed hydrolysis of cellulose was facilitated by the pretreatment with [bmim]cl. Through the optimization study to search for the best pretreatment condition using [bmim]cl, the TRS concentration in cellulose pretreatment with [bmim]cl and the glucose concentration in the hydrolysis reaction over Nafion Ò NR50 were found to show a distinct relationship with the [CI(NC) CI(XD)] and CI(XD- CII) values, respectively. Consequently, the deliberate process scheme would be proposed on the basis of our results, hence being

7 S.-J. Kim et al. / Bioresource Technology 101 (2010) potentially applied for biomass conversion if a combined utilization of ILs, solid acids and enzymes becomes viable. Acknowledgement The financial support by the Korea Ministry of Knowledge Economy (Project No E ) is greatly acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.biortech References Dadi, A.P., Varanasi, S., Schall, C.A., Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95 (5), Dadi, A.P., Schall, C.A., Varanasi, S., Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. In: Mielenz, J.R., Klasson, K.T., Adney, W.S., McMillan, J.D. (Eds.), Applied Biochemistry and Biotechnology: The Twenty-Eighth Symposium Proceedings of the Twenty- Eight Symposium on Biotechnology for Fuels and Chemicals. Humana Press Inc., New Jersey, pp Hamelinck, C.N., Hooijdonk, G.V., Faaij, A.P.C., Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle- and long-term. Biomass Bioenergy 28 (4), Kamiya, N., Matsushita, Y., Hanaki, M., Nakashima, K., Narita, M., Goto, M., Takahashi, H., Enzymatic in situ saccharification of cellulose in aqueous-ionic liquid media. Biotechnol. Lett. 30 (6), Krässig, H.A., Cellulose: Structure, Accessibility and Reactivity, first ed. Gordon and Breach Science Publishers, Yverdon, Switzerland. Kumar, P., Barrett, D.M., Delwiche, M.J., Stroeve, P., Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 48 (8), Lee, S.H., Doherty, T.V., Linhardt, R.J., Dordick, J.S., Ionic liquid-mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis. Biotechnol. Bioeng. 102 (5), Li, C., Zhao, Z.K., Efficient acid-catalyzed hydrolysis of cellulose in ionic liquid. Adv. Synth. Catal. 349 (11 12), Li, C., Wang, Q., Zhao, Z.K., Acid in ionic liquid: an efficient system for hydrolysis of lignocellulose. Green Chem. 10, Li, Q., He, Y.C., Xian, M., Jun, G., Xu, X., Yang, J.M., Li, L.Z., Improving enzymatic hydrolysis of wheat straw using ionic liquid 1-ethyl-3-methyl imidazolium diethyl phosphate pretreatment. Bioresour. Technol. 100 (14), Liu, L., Chen, H., Enzymatic hydrolysis of cellulose materials treated with ionic liquid [bmim]cl. Chin. Sci. Bull. 51 (20), Miller, G.L., Use of dynitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31 (3), Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M., Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96 (6), Murakami, M., Kaneko, Y., Kadokawa, J., Preparation of cellulose-polymerized ionic liquid composite by in-situ polymerization of polymerizable ionic liquid in cellulose-dissolving solution. Carbohydr. Polym. 69 (2), Oh, S.Y., Yoo, D.I., Shin, Y., Kim, H.C., Kim, H.Y., Chung, Y.S., Park, W.H., Youk, J.H., Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy. Carbohydr. Res. 340 (15), Ohno, H., Fukaya, Y., Task specific ionic liquids for cellulose technology. Chem. Lett. 38 (1), 2 7. O Sullivan, A.C., Cellulose: the structure slowly unravels. Cellulose 4 (3), Remsing, R.C., Swatloski, R.P., Rogers, R.D., Moyna, G., Mechanism of cellulose dissolution in the ionic liquid 1-n-butyl-3-methylimidazolium chloride: a 13 C and 35/37 Cl NMR relaxation study on model systems. Chem. Commun., Rinaldi, R., Palkovits, R., Schüth, F., Depolymerization of cellulose using solid catalysts in ionic liquids. Angew. Chem. Int. Edit. 120 (47), Rinaldi, R., Meine, N., vom Stein, J., Palkovits, R., Schüth, F., Which controls the depolymerization of cellulose in ionic liquids: the solid acid catalyst or cellulose? ChemSusChem 3 (2), Samayam, I.P., Schall, C.A., Saccharification of ionic liquid pretreated biomass with commercial enzyme mixtures. Bioresour. Technol. 101 (10), Schenzel, K., Almlöf, H., Germgård, U., Quantitative analysis of the transformation process of cellulose I? cellulose II using NIR FT Raman spectroscopy and chemometric methods. Cellulose 16 (3), Sousa, L.C., Chundawat, S.P.S., Balan, V., Dale, B.E., Cradle-to-grave assessment of existing lignocellulose pretreatment technologies. Curr. Opin. Biotechnol. 20 (3), Sun, N., Rahman, M., Qin, Y., Maxim, M.L., Rodriguez, H., Rogers, R.D., Complete dissolution and partial delignification of wood in the ionic liquid 1- ethyl-3-methylimidazolium acetate. Green Chem. 11, Swatloski, R.P., Spear, S.K., Holbrey, J.D., Rogers, R.D., Dissolution of cellulose with ionic liquids. J. Am. Chem. Soc. 124 (18), Taherzadeh, M.J., Karimi, K., Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int. J. Mol. Sci. 9 (9), Vitz, J., Erdmenger, T., Haensch, C., Schubert, U.S., Extended dissolution studies of cellulose in imidazolium based ionic liquids. Green Chem. 11, Zhao, H., Jones, C.L., Baker, G.A., Xia, S., Olubajo, O., Person, V.N., Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis. J. Biotechnol. 139 (1), Zheng, Y., Pan, Z., Zhang, R., Overview of biomass pretreatment for cellulosic ethanol production. Int. J. Agri. Biol. Eng. 2 (3),