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co-solvent for enzymatic hydrolysis enhancement into fermentable sugars
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Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars Ngoc Lan Mai a , Sung Ho Ha b , Yoon-Mo Koo a,b,∗ a b
Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea Department of Chemical Engineering and Nano-Bio Technology, Hannam University, Daejeon 305-811, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 January 2014 Received in revised form 21 February 2014 Accepted 26 March 2014 Available online xxx Keywords: Ionic liquid Lignocellulose Pretreatment Enzymatic hydrolysis Co-solvent
a b s t r a c t Ionic liquids (ILs) have been widely used as alternative solvents for biomass pretreatment, however, efficient methods that enable economically use of ILs at large scale have not been established. In this study, a new method in which ILs and polar organic solvents (ILs/co-solvent systems) was proposed for efficient pretreatment of lignocellulosic materials. The combination use of appropriate ILs and organic co-solvents can significantly enhance the solubility of lignocellulose due to the lower viscosity of ILs/cosolvent mixture as compared to those of pure ILs while the hydrogen bond basicity was maintained. In addition, the solubility of lignocellulosic materials in ILs/co-solvent system was found to be correlated with the Kamlet-Taft solvent parameters. Moreover, the use of microwave heating also enhances the efficiency of lignocellulose pretreatment. For example, the microwave-assisted [Emim][OAc]-DMSO (1:1 volume ratio) treated-rice straw could be hydrolyzed at least 22 times faster than that of untreated-rice straw by cellulase from Trichoderma reesei. This enhancement was attributed by several factors including more efficient lignin extraction, less crystalline cellulose and lower residual ILs in treated-rice straw. The produced sugars can be effectively fermented by Pichia stipitis for ethanol production. Moreover, [Emim][OAc]-DMSO mixture could be reused at least 5 times without significantly decrease in effectiveness demonstrated that the use of ILs/co-solvent was potential alternative method for large-scale biomass pretreatment. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Pretreatment is a vital step for the production of biofuels and platform chemicals from lignocellulosic biomass through enzymatic hydrolysis pathway. Pretreatment is required to reduce the crystallinity and increase the porosity of the lignocelluloses. A variety of pretreatment methods have been investigated, including biological, physical, chemical, and physiochemical processes [1]. These methods have been proved to provide hydrolysable sources of hemicelluloses and celluloses, however, none of these methods is able to sufficiently permit enzymatic hydrolysis at high solid loading, short residence time, and low enzyme loading concentrations
Abbreviations: [Emim]+ , 1-ethyl-3-methylimidazolium; [Bmim]+ , 1-butyl3-methylimidazolium; [Cl]− , chloride; [OAc]− , acetate; [DMP]− , dimethylphosphate; DMSO, dimethylsulfoxide; DMF, N,N-dimethylformamide; DMA, N,Ndimethylacetamide. ∗ Corresponding author at: Department of Biological Engineering, Inha University, 253 Younghyun-dong, Nam-gu, Incheon 402-751, Republic of Korea. Tel.: +82 32 860 7513; fax: +82 32 872 4046. E-mail address: [email protected] (Y.-M. Koo).
[2]. Moreover each of these methods has some drawbacks such as long treatment time (biological methods), high energy demand (physical methods), involved toxic and environmental unfriendly compounds (chemical methods), and high temperature/pressure (physicochemical methods). Recently, ionic liquids (ILs), consisting entirely ions and having low melting point, have been used as alternative solvents for cellulose and lignocelluloses pretreatment [3,4]. In comparison to traditional molecular solvent, ILs exhibit very interesting properties such as broad liquid temperature, high thermal stability and negligible vapor pressure [5]. It is common acknowledged that carbohydrates and lignin can be dissolved in ILs. As a result of dissolution in ILs, the intricate network of non-covalent interactions among cellulose, hemicellulose, and lignin were effectively disrupted while minimizing formation of degradation products [4]. The use of ILs for cellulosic biomass pretreatment has been effectively demonstrated on several lignocellulosic feedstocks such as straw [6] and wood [7]. Despite their efficacy, using ILs for biomass pretreatment suffers several drawbacks, such as slow dissolution rate, high viscosity of the obtained polymer/IL solution, and high cost of ILs [8–10]. Thus, advances toward improved processes for
http://dx.doi.org/10.1016/j.procbio.2014.03.024 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mai NL, et al. Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.03.024
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2 Table 1 Rice straw composition. Cellulose Hemicellulose Acid insoluble lignin Acid soluble lignin Ash Moisture Others
35.5% 20.7% 14.3% 1.6% 10.5% 8.9% 8.5%
dissolution of cellulose are still necessary. In our previous work, the use of microwave irradiation on the dissolution of cellulose with different degree of polymerization (DP) in ILs has shown promising result. Microwave heating not only enhanced the dissolution of cellulose in ILs but also significantly decreased the DP of cellulose dissolved in ILs. In addition, dissolution pretreatment of cellulose in ILs by microwave heating also reduced the residual amount of ILs retained in regenerated celluloses, thus enhancing enzymatic hydrolysis of treated cellulose and recovery of ILs [11]. Furthermore, major drawbacks associated with high viscosity of ILs/cellulose solution and high cost of ILs could be improved by using polar organic solvents as additives or cosolvents to minimize the use of ILs. Several aprotic organic solvents (e.g., dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-2-imidazolidinone (DMI), etc.) have been used as co-solvents on the dissolution of cellulose in ILs [12–16]. However, comprehensive data on the effect of co-solvent on the dissolution of lignocellulosic materials in ILs/cosolvent as well as the efficacy of ILs/co-solvent on the pretreatment of lignocelluloses are still limited [15–19]. In this study, the effect of ILs/co-solvents mixture on the dissolution of lignocellulosic materials as well as the synergetic effect of ILs/co-solvent and microwave heating on the pretreatment of lignocellulose was investigated. Additionally, the correlation between lignocellulose solubility and solvent properties was also examined. In other to evaluate the efficiency of the pretreatment method on lignocellulosic materials, rice straw (RS) was used as typical lignocellulose. RS is one of the abundant lignocellulosic waste materials in the world and has several characteristics that make it a potential feedstock for fuel production. For example, it has high cellulose and hemicelluloses content that can be readily hydrolyzed into fermentable sugars [20]. 2. Material and methods 2.1. Material [Emim][OAc] and [Bmim][Cl] were obtained from by C-Tri (Suwon, Korea). [Emim][DMP] was purchased from Sigma–Aldrich. All ILs were dried under vacuum at 90 ◦ C for 24 h before use. Avicel PH-101, xylan (as major component of hemicelluloses) from birchwood and lignin (alkali, low sulfonate content) were obtained from Sigma–Aldrich. Rice straw (RS) from urban area of Suwon, Korea was used as representative lignocelluloses. The chemistry composition of RS is shown in Table 1. RS was cut, ground into small pieces and sieved through disk with size of 0.125–0.250 mm. RS samples were dried at 50 ◦ C for 24 h before use. Cellulase from Trichoderma reesei, which activity of 421 FPU/g was obtained from Sigma-Aldrich. All other reagents were of analytical grade. 2.2. Lignocellulose dissolution and regeneration For determining the solubility of cellulose, hemicelluloses and lignin in ILs/co-solvent, every 10 mg of the materials were gradually added in 5 mL glass vial containing 1 mL of ILs/co-solvent at 80 ◦ C under nitrogen pressure and magnetic stirring at 500 rpm.
For RS pretreatment, 50 mg RS was dissolved in 1 mL of ILs/cosolvent at 110 ◦ C (using oil bath) under nitrogen pressure and magnetic stirring at 500 rpm. For microwave-assisted RS dissolution, the ILs/co-solvent solution containing RS was placed under microwave irradiation using CEM discover microwave system (Matthews, USA) at constant power of 50 W for 1 min with magnetic stirring. The temperature of solution increased rapidly and the observed maximum temperature of RS/ILs/co-solvent solution was below 130 ◦ C during microwave irradiation. After dissolution, 3 mL of acetone/water (1:1 v/v) was added to the RS/ILs/cosolvent solution. The solution was vigorously mixing by vortexer. The precipitated cellulose-rich materials were separated from the supernatant by centrifugation (1500 rpm for 5 min). The regenerated RS was washed thoroughly at least 5 times with acetone/water to remove residual ILs/co-solvent. The regenerated RS was dried under vacuum condition at 50 ◦ C for 24 h before carrying out the enzymatic hydrolysis reaction. The lignin content in the combine supernatant was determined by the lignin precipitation with sulfuric acid and re-dissolution in 0.1 M NaOH. The absorbance of lignin in NaOH was measured at 280 nm with lignin as standard [21]. All experiments were duplicated. 2.3. Enzymatic hydrolysis of the regenerated rice straw A suspension of 30 mg (untreated and regenerated) cellulose (3% w/v) in 1.0 mL citrate buffer (5 mM, pH 4.8) was incubated at 50 ◦ C, 150 rpm. The reaction was started by adding 1.5 mg cellulase from Trichoderma reesei (20 FPU/g biomass). Twenty microliter of reaction aliquot was periodically withdrawn and the reducing sugars were determined by DNS method [22]. Glucose and xylose concentration were also determined by HPLC analysis. All experiments were duplicated. 2.4. Ethanol fermentation The enzymatic hydrolysate of [Emim][OAc]-DMSO treated-RS with glucose and xylose content of 10.2 and 5.0 g/l, respectively, was used as carbon source for ethanol production by Pichia stipitis KCTC 7222 (Biological Resource Center, Korea) under batch condition. Fermentation was carried out in 500 mL Erlenmeyer flask containing 100 mL of RS hydrolysate, supplemented with 3.0 g/L yeast extract, 3.0 g/L malt extract and 5.0 g/L peptone. Culture flasks were inoculated with 5% (v/v) P. stipitis KCTC 7222 and incubated at 30 ◦ C with shaking condition at 150 rpm. Sugar consumption and ethanol production were determined by HPLC and gas chromatography, respectively. All experiments were duplicated. 2.5. Analytical methods The Kamlet-Taft solvent parameter (˛: hydrogen bond donor/acidity, ˇ; hydrogen bond acceptor/basicity, and * dipolarity/polarizability) of ILs, solvents and IL/co-solvent mixture were determined by using set of dyes including 4-nitroanisole, 4-nitroaniline and Reichardt’s dye as described by KhodadadiMoghaddam et al. [23]. The correlations between the lignocellulose solubility in IL/cosolvent mixtures and their corresponding Kamlet-Taft solvent parameters were analyzed by Multivariate Adaptive Regression Splines (MARS) methods [24]. It is a non-parametric regression technique and can be considered as an extension of linear models that automatically models non-linearities and interaction between variables. The MARS modeling was carried out using “earth” package in R program [25]. The crystallinity of cellulose was determined by X-ray diffraction (XRD) using Rigaku DMAX 2500 X-ray diffractometer. Cellulose samples were scanned from 10◦ to 30◦ at scan speed of 2◦ /min and
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step size of 0.01◦ .The crystallinity index, Cr, which represents the percentage of crystalline materials was calculated from the XRD patterns by an empirical method described by Nelson and O’Connor [26] Cr =
Icr − Iam Icr
where Icr is the diffraction intensity at peak position 2 ≈ 22.6◦ for cellulose I, 21.7◦ for cellulose II and Iam is the intensity at suitable locations for the amorphous background (2 ≈ 19◦ for cellulose I, 16◦ for cellulose II) Residual [Emim][OAc] and DMSO in regenerated RS were determined by HPLC analysis. HPLC system composed of a LC-10AD pump (Shimadzu, Japan), two detectors: RI detector (for sugars analysis) and UV detector (for ionic liquid and DMSO detection) and a jacketed glass column (30 cm × 1.1 cm) packed with Dowex50WX4-400 cation exchanger resin. The functional group H+ of resin was exchanged with [Emim]+ of ILs by passing 10 column volumes (CV) of [Emim][OAc] in water through the column. Degassed and deionized water was used as mobile phase at flow rate of 1.5 mL/min. The analysis was performed at 65 ◦ C [27]. The structures of ILs/co-solvents before and after RS dissolution were examined by 400 MHz FT-NMR spectrometer (Varian Inova 400, USA) in acetone-D6 . Glucose and xylose concentration were measured by HPLC (Shimadzu Model LC-10A) equipped with a RI detector and a Bio-Rad HPX-87P column (300 mm × 7.8 mm) operated at 85 ◦ C. Deionized water was used as mobile phase at flow rate of 0.6 mL/min. Ethanol concentration from RS hydrolysate fermentation was measured by gas chromatography (CP 9001, Chrompack, Netherlands) using a DB23-capillary column (30 m × 0.25 mm ID; film thickness 0.25 mm; J&W Scientific, U.S.A.) and flame ionization detector (FID). 3. Results and discussion 3.1. Effect of organic co-solvents on dissolution of lignocellulose in ionic liquids Conventional aprotic polar organic solvents such as dimtheylsulfoxide (DMSO), N,N-dimethylformamide (DMF), and N,Ndimethylacetamide (DMA) themselves are not cellulose-dissolving solvents (Table 2) although they have been used as co-solvent to reduce the viscosity of cellulose/IL mixture for structure analysis of ILs-dissolved cellulose [28]. The addition of these solvent as viscous reducing co-solvent was carried out after the dissolution of cellulose in ILs. More recently, Rinaldi have reported that several electrolyte solution containing small fraction of ILs could instantaneously dissolve large amounts of cellulose [14]. These cellulose compatible solvents were known to reduce the viscosity of cellulose/ILs solution while maintaining the cellulose-dissolving capacity, or the hydrogen bond acceptor/basicity (ˇ) of ILs. In addition, the polar organic co-solvents promote the ILs dissociation into solvated cations and “free” anions to certain extent that enable the interaction of anions with cellulose, thus improving the cellulose dissolution [15]. As showed in Table 2, the use of organic co-solvent enhances the cellulose dissolution in terms of not only solubility but also dissolution rate. For instant, a mixture of [Emim][OAc] and DMSO, DMF or DMA with volume ratio of 1:1 could dissolve up to 28% cellulose in same order of time compared to 22% of pure ILs. The enhancement effect of these co-solvents on the dissolution of cellulose was also observed in [Emim][DMP]. Surprisingly, the cellulose dissolving capability of [Bmim][Cl], however, was deteriorated when these co-solvents were added (1:1 volume ratio). This may be explained by the reducing in the hydrogen-bond basicity of ILs/co-solvents at
3
high ratio of organic co-solvents which was also observed in works of Rinaldi [14]. However, at lower ratio of organic solvent, mixture of [BmimCl]-DMSO (86:14 wt%) dissolves pine, eucalyptus and oak wood shaving after heating at 100 ◦ C for several hours [12]. Interestingly, the use of co-solvent could also enhance the solubility of hemicellulose and lignin in IL/co-solvent mixture. DMSO itself could dissolve large amount of hemicellulose and lignin at test condition, which are 70% and 37%, respectively, compared to 24% and 33% of pure [Emim][OAc]. As a result, the ILs/DMSO mixture could significantly enhance the dissolution of hemicelluloses and lignin. For example, the solubility of hemicelluloses and lignin in [Emim][OAc]-DMSO mixture (1:1 volume ratio) was increased 2.5and 1.1-fold compared to those of pure [Emim][OAc], respectively, and these solubility would further enhance as increasing the volume ratio of DMSO in [Emim][OAc]-DMSO mixture (Table 2). The higher solubility of lignocellulose components ILs/co-solvent might be explained by the lower viscosity of the IL/co-solvent mixture compared to that of pure ILs. The high viscosity of ILs was considered as limitation factor for dissolution of lignocellulosic materials. The viscosities of these tested co-solvents are far lower than those of pure ILs, therefore the use of less viscous ILs/co-solvent mixture will improve the lignocellulose dissolution (Table 2). To evaluate the effect of solvent properties on the lignocellulose dissolution, the Kamlet-Taft solvent parameters of IL/co-solvent mixture were correlated with the solubility of lignocellulosic components. A considerably correlation between cellulose solubility and Kamlet-Taft solvent parameters of IL/co-solvent mixture (R2 = 0.85) was found by MARS analysis (Fig. 1). In addition, the MARS models indicated non-linear relationship between solute solubility and Kamlet-Taft solvent parameters and showed the interaction of these parameters on the lignocellulose solubility in ILs/co-solvent. Because of these interactions, MARS model also showed better correlation than that of linear model (multiple linear regression models showed lower R2 value, data not showed). However, the solubility of hemicellulose and lignin in ILs/co-solvent mixture were less correlated to the Kamlet-Taft solvent parameters as represented by the lower correlation coefficient (R2 = 0.61 and 0.68, respectively). Among tested ILs and co-solvents, [Emim][OAc] and DMSO were shown as promising ILs/co-solvent system that could simultaneously enhance the solubility of cellulose, hemicellulose and lignin. Therefore, [Emim][OAc]-DMSO mixture (1:1 volume ratio) was used for pretreatment of RS. 3.2. Enzymatic hydrolysis of rice straw The enzymatic hydrolysis of untreated and ILs, ILs/DMSO treated-RS are showed in Fig. 2A and B. Significant increases in enzymatic hydrolytic rate as well as released sugars were observed in ILs and ILs/co-solvent treated-RS compared to those of untreated and DMSO treated-RS. For instance, the enzymatic hydrolytic rate of [Emim][OAc] treated-RS and [Emim][OAc]-DMSO treated-RS was 8 and 17 times higher than that of untreated-RS, respectively. In addition, the use of microwave heating in RS pretreatment in IL and ILs/co-solvent was significantly enhanced the enzymatic hydrolytic rate. For example, the initial hydrolytic rate of [Emim][OAc] and [Emim][OAc]-DMSO treated-RS using microwave heating was increased 19 and 22 folds as compared to that of the untreated-RS. However, there was only 1.7-fold improvement observed in DMSO treated-RS (Fig. 2B). Moreover, the maximum conversion yield of 95% was quickly achieved after 6 h in cases of microwave-assisted [Emim][OAc]-treated RS and both [Emim][OAc]-DMSO treated-RS sample with or without microwave heating. Whereas it takes 12 h for [Emim][OAc]-treated-RS with conventional heating to obtain the same conversion yield. On the contrary, only 22% and 43% conversion yield were obtained after 24 h in case of untreated
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Solvent
Cellulose
[Bmim][Cl] [Emim][OAc] [Emim][DMP] DMSO DMA DMF [Bmim][Cl]-DMSO (1:1) [Bmim][Cl]-DMSO (1:4) [Bmim][Cl]-DMA (1:1) [Bmim][Cl]-DMA (1:4) [Bmim][Cl]-DMF (1:1) [Bmim][Cl]-DMF (1:4) [Emim][OAc]-DMSO (1:1) [Emim][OAc]-DMSO (1:4) [Emim][OAc]-DMA (1:1) [Emim][OAc]-DMA (1:4) [Emim][OAc]-DMF (1:1) [Emim][OAc]-DMF (1:4) [Emim][DMP]-DMSO (1:1) [Emim][DMP]-DMSO (1:4) [Emim][DMP]-DMA (1:1) [Emim][DMP]-DMA (1:4) [Emim][DMP]-DMF (1:1) [Emim][DMP]-DMF (1:4)
14 22 18 0 0 0 0 0 0 0 0 0 28 12 28 4 28 8 24 5 25 2 25 2
Dis. rate (min) 3 1 1 – – – – – – – – – 2 1 2 1 2 1 3 1 3 30 3 30
Sol. (wt%) <1 24 4 70 0 0 60 70 2 0 2 0 60 70 20 10 30 14 60 70 6 2 10 4
(25 ◦ C)
Lignin
Dis. rate (min) – 25 240 10 – – 10 5 60 – 60 – 8 5 10 6 10 6 8 5 30 60 10 20
Sol. (wt%) 11 33 25 37 <1 3 22 32 8 4 12 6 36 40 11 7 18 14 32 34 8 5 15 11
Dis. rate (min) 60 5 10 2 – 120 2 2 4 5 3 5 2 2 3 3 3 2 2 2 3 5 2 3
a
15 – – 48.9 37.8 36.7 – – – – – – – – – – – – – – – – – –
Kamlet-Taft solvent parameters
Viscosity (cp) (20 ◦ C)
˛
ˇ
˘*
0.48 0.41 0.43 0.18 0.13 0.21 0.45 0.46 0.46 0.51 0.46 0.49 0.25 0.23 0.32 0.33 0.35 0.40 0.37 0.29 0.39 0.35 0.37 0.37
0.85 1.08 1.01 0.77 0.79 0.77 0.90 0.83 0.93 1.00 0.88 0.96 0.91 0.97 1.04 1.14 1.08 1.19 1.04 0.97 1.11 1.12 1.07 1.12
1.14 1.10 1.10 1.01 0.88 0.84 1.10 1.10 1.06 0.97 1.10 0.97 1.23 1.14 1.10 0.97 1.06 0.88 1.06 1.06 0.97 0.93 1.01 0.88
40,890b 162c 394d 2.14 1.96 (25 ◦ C) 0.92 – – – – – – – – – – – – – – – – – –
The solubility was determined at 80 ◦ C under nitrogen pressure. Solutes (10 mg) were gradually added until clear solution was obtained. Dis. rate – dissolution rate (min): time required for completely dissolving 2Ywt% lignocellulosic components in solvent. a: [35]; b: [36]; c: [37]; d:[38]; Solvent properties of organic co-solvent were taken from Ref. [39]
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Hemicellulose
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Table 2 Solubility of lignocellulosic components in ionic liquids/co-solvents mixture.
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Fig. 1. Experimental versus predicted lignocellulosic component solubilities in ionic liquids/co-solvents and correlation models between lignocellulosic component solubilites in ionic liquids/co-solvents and corresponding Kamlet-Taft solvent parameters.
and DMSO treated-RS, respectively (Fig. 2A). In comparison with previous report, this proposed method required shorter hydrolysis time to reach maximum conversion yield (6 h versus 24 h) [29]. Furthermore, the RS pretreatment in ILs and ILs/co-solvent also enhanced the amount of sugars released through enzymatic hydrolysis. For instance, the amount of sugars released from RS treated with [Emim][OAc] and [Emim][OAc]/DMSO was at least 4 times higher than that of untreated-RS. The highest released glucose and xylose of 10.4 and 5.1 mg/mL, respectively, were obtained in microwave-assisted [Emim][OAc]-DMSO treated-RS compared to those of 2.3 and 0.9 mg/mL in untreated-RS (Fig. 2A).
18 (A)
16
Reducing sugar (g/L)
14 Untreated-RS (2.3 - 0.9) [Emim][OAc] (8.4 - 3.8) DMSO (4.2 - 2.2) [Emim][OAc]-DMSO (9.1 - 3.9) [Emim][OAc]_MW (9.8 - 4.3) [Emim][OAc]-DMSO_MW (10.4 - 5.1)
12 10 8 6
3.3. Properties of treated-rice straw
4 2 0 0
5
10
15
20
25
Time (hrs) 10
The enzymatic hydrolysis enhancement of ILs, and ILs/cosolvent treated-RS might be explained by the synergetic effect of several factors. These include (i) the efficient lignin extraction of ILs and ILs/co-solvent (Fig. 3) (ii) a decrease in crystalline (CrI) and increase in amorphous (CrII) cellulose portion in ILs, ILs/co-solvent
(B)
40 6
Lignin Extraction (%)
Initial hydrolytic rate (mg/hrs)
50 8
4
2
30
20
10 0
U
e ntr
] ] l] S Ac [C MP dR m] [D ][O ate mi im m] i [B m m [E [E
O W W SO MS _M ]_M DM ]-D Ac SO c O M A [ [O ]-D m] m] mi Ac [E mi ][O [E m mi [E
Fig. 2. (A) Reducing sugars released during enzymatic hydrolysis of rice straw. (B) Initial hydrolytic rate of enzymatic hydrolysis of untreated and treated rice straw from ionic liquids and ionic liquids/co-solvent.
0 ] ] l] Ac [C MP [O m] ][D m] mi i m i B [ m m [E [E
O SO MW MW MS DM c]_ O_ ]-D A S c M A [O [O m] ]-D m] mi Ac [E mi [O ] [E m mi [E
Fig. 3. Lignin extraction from rice straw by ionic liquids and ionic liquids/co-solvent.
Please cite this article in press as: Mai NL, et al. Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.03.024
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6 0.5 CrI CrII
Crystallinity index
0.4
0.3
0.2
0.1
0.0
U
te ea ntr
dR
S [B
m] mi
] ] Ac MP [D ][O im m] i m m [E [E
l] [C
O W W SO MS _M ]_M DM ]-D Ac SO c O M A [ [O ]-D m] m] mi Ac mi [E [O ] [E m mi [E
Fig. 4. Cellulose crystallinity index of untreated and treated rice straw.
treated-RS (Fig. 4), and (iii) a decrease in residual ILs retained in regenerated-RS (Fig. 5). As presented in Fig. 3, 18.5% of RS lignin was efficiently extracted after dissolution and regeneration treatment in [Emim][OAc]. The relatively higher lignin extraction obtained in RS treated with [Emim][OAc] as compared to those with other ILs was in accordance with the high solubility of cellulose, hemicellulose and lignin in this ILs (Table 2). In addition, the use of this ILs in combination with DMSO and microwave heating also give rise to a higher lignin extraction. The highest lignin extraction (44.1%) was achieved by using microwave-assisted [Emim][OAc]-DMSO pretreatment, while the values for using [Emim][OAc]-DMSO and microwave-assisted [Emim][OAc] pretreatment were 27.2% and 33.7%, respectively. In all cases, the ILs/co-solvent system and system with microwave heating always showed higher lignin extraction efficiency compared to that pure ILs and system without microwave heating, respectively. This can be explained by the
ability of organic solvent to dissolve large amount of lignin, therefore facilitate the lignin extraction of the corresponding low viscous of ILs/co-solvent. In addition, the use of microwave heating also enhanced the lignin extraction. It is documented that polar and ionic solvents are efficiently and rapidly heated under microwave irradiation. ILs, which are ionic compounds and have moderate polarity (dielectric constant, ε, of 15), in mixture with DMSO, which is a relatively high polar solvent (ε of 48.9), can be efficiently heated under microwave irradiation. The combination of all the factors in microwave-assisted [Emim][OAc]-DMSO pretreatment, therefore, results in more efficient lignin extraction as compared to those of [Emim][OAc]-DMSO treatment and microwave-assisted [Emim][OAc] pretreatment. In addition, the enhanced enzymatic hydrolysis of ILs and ILs/co-solvent treated-RS were also attributed by the simultaneously decrease of CrI (corresponding to crystalline cellulose) and increase of CrII (corresponding to amorphous cellulose) in treatedRS (Fig. 4). It is recognized that, amorphous cellulose is easily hydrolyzed by enzyme than that of crystalline cellulose [11,30]. In rice straw, cellulose exists in native crystalline form, which is cellulose I [31]. However, in treated-RS the cellulose II of low crystallinity was recovered. The highest reduction in CrI was obtained in microwave-assisted [Emim][OAc]-DMSO treated-RS. These results indicate that [Emim][OAc]-DMSO cannot only efficiently extract lignin but also disrupt the crystalline structure of cellulose in RS. Furthermore, the pretreatment of RS with [Emim][OAc]-DMSO also efficiently reduced the residual ILs retained in regenerated RS as showed in Fig. 5A. It is reported that cellulase from T. reesei could be inactivated by high concentration of cellulose dissolving ILs such as [Bmim][Cl] and [Emim][OAc] [32,33]. During the regeneration of RS from ILs, some residual ILs might be trapped in the cellulose chains despite thoroughly washing. Thus, the efficient removal of ILs in treated RS is believed to not only facilitate the enzymatic hydrolysis but also improve economical uses of ILs in biomass pretreatment. The residual ILs retained in microwave-assisted [Emim][OAc]-DMSO treated-RS was around 0.4% and this amount was at least 25 times lower than those in [Emim][OAc] treated-RS. Interestingly, the residual DMSO was not detected under analysis condition indicates that the solvent was efficiently removed through washing steps.
12
120
(A)
(B) 100
Enzymatic conversion (%)
[Emim][OAc] content (wt%)
10
8
6
4
2
80
60
40
20
0 [O m] mi [E
0
] Ac m mi [E
A ][O
SO DM c]m [E
im
][O
W ]_M Ac
[E
[O m] mi
Ac
W _M SO M ]-D
1
2
3
4
5
Reuse of [Emim][OAc]\DMSO
Fig. 5. (A) Residual [Emim][OAc] content in treated-rice straw and (B) reuse of [Emim][OAc]-DMSO.
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Fig. 6. Fermentation profile of Pichia stipitis KCTC 7222.
3.4. Reuse of ILs/co-solvent
4. Conclusion
The economical feasible use of ILs in biomass processing could be improved by the reuse of ILs. In this study, [Emim][OAc]-DMSO could be reused at least 5 times for RS pretreatment without any reduction in enzymatic conversion of treated-RS (Fig. 5B). The structure of [Emim][OAc] and DMSO were not changed during repeated use for RS pretreatment (confirmed by 1 H NMR analysis, data not shown). In addition, the low viscosity of [Emim][OAc]DMSO mixture showed to be advantageous for regeneration of lignocellulose as well as recycling of IL/co-solvent. This would increase the number of recycled use of IL/co-solvent as minimizing the residual amount of lignocellulose in the solution [29].
The combination use of cellulose compatible organic solvents as co-solvent with ILs and microwave heating could significantly enhance the efficiency of lignocellulose pretreatment for enzymatic production of fermentable sugars from biomass. The use of organic co-solvents facilitates not only the dissolution of cellulose but also hemicelluloses and lignin, which gives rise to a higher lignin extraction, lower crystalline cellulose and lesser residual ILs amount retained in treated lignocellulosic biomass. In addition, the ILs/co-solvent could be recycled and reused at least 5 cycles without significant loss in pretreatment efficiency (on the pretreatment of RS) demonstrated that this method might be beneficial for the large-scale biomass pretreatment although the process optimization as well as the economical efficacy assessment are need to be proved. These are the objects of further study.
3.5. Ethanol fermentation The fermentability of sugars produced from the enzymatic hydrolysis of microwave-assisted [Emim][OAc]-DMSO treatd-RS were evaluated by ethanol production catalyzed by Pichia stipilis KCTC 7222. The microbial growth, sugar consumption and ethanol production were presented in Fig. 6. The results showed that the fermentation behavior of RS hydrolysate was identical to that of pure sugar mixtures in control experiments. Generally, glucose was preferably used by the microbial until they reached stationary state which represented by the plateau of OD value at 12 h. After 18 h, glucose was completely consumed and the xylose was up-taken by the cell. However, the xylose consumption not affected the microbial cell growth. The equilibrium ethanol concentration of 1.68 g/L was achieved after 24 h of fermentation which corresponded to yield of 0.14 g ethanol/g sugars. Although the ethanol yield in this study was lower than those reported in literature (0.45 g/g) [34] due to the low initial sugar concentration, the optimization of fermentation conditions would improve the ethanol yield of the present process. In conclusion, the fermentation profiles of hydrolysates from ILs/co-solvent treated-RS reinforced the efficiency of pretreatment method using ILs/co-solvent in production of fermentable sugars from lignocellulosic biomass
Acknowledgements This work was financially supported by the Ministry of Knowledge Economy, Korea through the Cleaner Production Technology Development Project (KC000619). This work was also supported by Inha University research Grant. This work is the outcome of a Manpower Development Program for Marine Energy by the Ministry of Land, Transport and Maritime Affairs (MLTM). References [1] Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 2010;101:4851–61. [2] Qureshi N, Maddox IS. Reduction in butanol inhibition by perstraction: utilization of concentrated lactose/whey permeate by Clostridium acetobutylicum to enhance butanol fermentation economics. Food Bioprod Process 2005;83:43–52. [3] Swatloski RP, Spear SK, Holbrey JD, Rogers RD. Dissolution of cellose with ionic liquids. J Am Chem Soc 2002;124:4974–5. [4] Maki-Arvela P, Anugwom I, Virtanen P, Sjoholm R, Mikkola JP. Dissolution of lignocellulosic materials and its constituents using ionic liquids – a review. Ind Crop Prod 2010;32:175–201.
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Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars Ngoc Lan Mai a , Sung Ho Ha b , Yoon-Mo Koo a,b,∗ a b
Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea Department of Chemical Engineering and Nano-Bio Technology, Hannam University, Daejeon 305-811, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 January 2014 Received in revised form 21 February 2014 Accepted 26 March 2014 Available online xxx Keywords: Ionic liquid Lignocellulose Pretreatment Enzymatic hydrolysis Co-solvent
a b s t r a c t Ionic liquids (ILs) have been widely used as alternative solvents for biomass pretreatment, however, efficient methods that enable economically use of ILs at large scale have not been established. In this study, a new method in which ILs and polar organic solvents (ILs/co-solvent systems) was proposed for efficient pretreatment of lignocellulosic materials. The combination use of appropriate ILs and organic co-solvents can significantly enhance the solubility of lignocellulose due to the lower viscosity of ILs/cosolvent mixture as compared to those of pure ILs while the hydrogen bond basicity was maintained. In addition, the solubility of lignocellulosic materials in ILs/co-solvent system was found to be correlated with the Kamlet-Taft solvent parameters. Moreover, the use of microwave heating also enhances the efficiency of lignocellulose pretreatment. For example, the microwave-assisted [Emim][OAc]-DMSO (1:1 volume ratio) treated-rice straw could be hydrolyzed at least 22 times faster than that of untreated-rice straw by cellulase from Trichoderma reesei. This enhancement was attributed by several factors including more efficient lignin extraction, less crystalline cellulose and lower residual ILs in treated-rice straw. The produced sugars can be effectively fermented by Pichia stipitis for ethanol production. Moreover, [Emim][OAc]-DMSO mixture could be reused at least 5 times without significantly decrease in effectiveness demonstrated that the use of ILs/co-solvent was potential alternative method for large-scale biomass pretreatment. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Pretreatment is a vital step for the production of biofuels and platform chemicals from lignocellulosic biomass through enzymatic hydrolysis pathway. Pretreatment is required to reduce the crystallinity and increase the porosity of the lignocelluloses. A variety of pretreatment methods have been investigated, including biological, physical, chemical, and physiochemical processes [1]. These methods have been proved to provide hydrolysable sources of hemicelluloses and celluloses, however, none of these methods is able to sufficiently permit enzymatic hydrolysis at high solid loading, short residence time, and low enzyme loading concentrations
Abbreviations: [Emim]+ , 1-ethyl-3-methylimidazolium; [Bmim]+ , 1-butyl3-methylimidazolium; [Cl]− , chloride; [OAc]− , acetate; [DMP]− , dimethylphosphate; DMSO, dimethylsulfoxide; DMF, N,N-dimethylformamide; DMA, N,Ndimethylacetamide. ∗ Corresponding author at: Department of Biological Engineering, Inha University, 253 Younghyun-dong, Nam-gu, Incheon 402-751, Republic of Korea. Tel.: +82 32 860 7513; fax: +82 32 872 4046. E-mail address: [email protected] (Y.-M. Koo).
[2]. Moreover each of these methods has some drawbacks such as long treatment time (biological methods), high energy demand (physical methods), involved toxic and environmental unfriendly compounds (chemical methods), and high temperature/pressure (physicochemical methods). Recently, ionic liquids (ILs), consisting entirely ions and having low melting point, have been used as alternative solvents for cellulose and lignocelluloses pretreatment [3,4]. In comparison to traditional molecular solvent, ILs exhibit very interesting properties such as broad liquid temperature, high thermal stability and negligible vapor pressure [5]. It is common acknowledged that carbohydrates and lignin can be dissolved in ILs. As a result of dissolution in ILs, the intricate network of non-covalent interactions among cellulose, hemicellulose, and lignin were effectively disrupted while minimizing formation of degradation products [4]. The use of ILs for cellulosic biomass pretreatment has been effectively demonstrated on several lignocellulosic feedstocks such as straw [6] and wood [7]. Despite their efficacy, using ILs for biomass pretreatment suffers several drawbacks, such as slow dissolution rate, high viscosity of the obtained polymer/IL solution, and high cost of ILs [8–10]. Thus, advances toward improved processes for
http://dx.doi.org/10.1016/j.procbio.2014.03.024 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
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2 Table 1 Rice straw composition. Cellulose Hemicellulose Acid insoluble lignin Acid soluble lignin Ash Moisture Others
35.5% 20.7% 14.3% 1.6% 10.5% 8.9% 8.5%
dissolution of cellulose are still necessary. In our previous work, the use of microwave irradiation on the dissolution of cellulose with different degree of polymerization (DP) in ILs has shown promising result. Microwave heating not only enhanced the dissolution of cellulose in ILs but also significantly decreased the DP of cellulose dissolved in ILs. In addition, dissolution pretreatment of cellulose in ILs by microwave heating also reduced the residual amount of ILs retained in regenerated celluloses, thus enhancing enzymatic hydrolysis of treated cellulose and recovery of ILs [11]. Furthermore, major drawbacks associated with high viscosity of ILs/cellulose solution and high cost of ILs could be improved by using polar organic solvents as additives or cosolvents to minimize the use of ILs. Several aprotic organic solvents (e.g., dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-2-imidazolidinone (DMI), etc.) have been used as co-solvents on the dissolution of cellulose in ILs [12–16]. However, comprehensive data on the effect of co-solvent on the dissolution of lignocellulosic materials in ILs/cosolvent as well as the efficacy of ILs/co-solvent on the pretreatment of lignocelluloses are still limited [15–19]. In this study, the effect of ILs/co-solvents mixture on the dissolution of lignocellulosic materials as well as the synergetic effect of ILs/co-solvent and microwave heating on the pretreatment of lignocellulose was investigated. Additionally, the correlation between lignocellulose solubility and solvent properties was also examined. In other to evaluate the efficiency of the pretreatment method on lignocellulosic materials, rice straw (RS) was used as typical lignocellulose. RS is one of the abundant lignocellulosic waste materials in the world and has several characteristics that make it a potential feedstock for fuel production. For example, it has high cellulose and hemicelluloses content that can be readily hydrolyzed into fermentable sugars [20]. 2. Material and methods 2.1. Material [Emim][OAc] and [Bmim][Cl] were obtained from by C-Tri (Suwon, Korea). [Emim][DMP] was purchased from Sigma–Aldrich. All ILs were dried under vacuum at 90 ◦ C for 24 h before use. Avicel PH-101, xylan (as major component of hemicelluloses) from birchwood and lignin (alkali, low sulfonate content) were obtained from Sigma–Aldrich. Rice straw (RS) from urban area of Suwon, Korea was used as representative lignocelluloses. The chemistry composition of RS is shown in Table 1. RS was cut, ground into small pieces and sieved through disk with size of 0.125–0.250 mm. RS samples were dried at 50 ◦ C for 24 h before use. Cellulase from Trichoderma reesei, which activity of 421 FPU/g was obtained from Sigma-Aldrich. All other reagents were of analytical grade. 2.2. Lignocellulose dissolution and regeneration For determining the solubility of cellulose, hemicelluloses and lignin in ILs/co-solvent, every 10 mg of the materials were gradually added in 5 mL glass vial containing 1 mL of ILs/co-solvent at 80 ◦ C under nitrogen pressure and magnetic stirring at 500 rpm.
For RS pretreatment, 50 mg RS was dissolved in 1 mL of ILs/cosolvent at 110 ◦ C (using oil bath) under nitrogen pressure and magnetic stirring at 500 rpm. For microwave-assisted RS dissolution, the ILs/co-solvent solution containing RS was placed under microwave irradiation using CEM discover microwave system (Matthews, USA) at constant power of 50 W for 1 min with magnetic stirring. The temperature of solution increased rapidly and the observed maximum temperature of RS/ILs/co-solvent solution was below 130 ◦ C during microwave irradiation. After dissolution, 3 mL of acetone/water (1:1 v/v) was added to the RS/ILs/cosolvent solution. The solution was vigorously mixing by vortexer. The precipitated cellulose-rich materials were separated from the supernatant by centrifugation (1500 rpm for 5 min). The regenerated RS was washed thoroughly at least 5 times with acetone/water to remove residual ILs/co-solvent. The regenerated RS was dried under vacuum condition at 50 ◦ C for 24 h before carrying out the enzymatic hydrolysis reaction. The lignin content in the combine supernatant was determined by the lignin precipitation with sulfuric acid and re-dissolution in 0.1 M NaOH. The absorbance of lignin in NaOH was measured at 280 nm with lignin as standard [21]. All experiments were duplicated. 2.3. Enzymatic hydrolysis of the regenerated rice straw A suspension of 30 mg (untreated and regenerated) cellulose (3% w/v) in 1.0 mL citrate buffer (5 mM, pH 4.8) was incubated at 50 ◦ C, 150 rpm. The reaction was started by adding 1.5 mg cellulase from Trichoderma reesei (20 FPU/g biomass). Twenty microliter of reaction aliquot was periodically withdrawn and the reducing sugars were determined by DNS method [22]. Glucose and xylose concentration were also determined by HPLC analysis. All experiments were duplicated. 2.4. Ethanol fermentation The enzymatic hydrolysate of [Emim][OAc]-DMSO treated-RS with glucose and xylose content of 10.2 and 5.0 g/l, respectively, was used as carbon source for ethanol production by Pichia stipitis KCTC 7222 (Biological Resource Center, Korea) under batch condition. Fermentation was carried out in 500 mL Erlenmeyer flask containing 100 mL of RS hydrolysate, supplemented with 3.0 g/L yeast extract, 3.0 g/L malt extract and 5.0 g/L peptone. Culture flasks were inoculated with 5% (v/v) P. stipitis KCTC 7222 and incubated at 30 ◦ C with shaking condition at 150 rpm. Sugar consumption and ethanol production were determined by HPLC and gas chromatography, respectively. All experiments were duplicated. 2.5. Analytical methods The Kamlet-Taft solvent parameter (˛: hydrogen bond donor/acidity, ˇ; hydrogen bond acceptor/basicity, and * dipolarity/polarizability) of ILs, solvents and IL/co-solvent mixture were determined by using set of dyes including 4-nitroanisole, 4-nitroaniline and Reichardt’s dye as described by KhodadadiMoghaddam et al. [23]. The correlations between the lignocellulose solubility in IL/cosolvent mixtures and their corresponding Kamlet-Taft solvent parameters were analyzed by Multivariate Adaptive Regression Splines (MARS) methods [24]. It is a non-parametric regression technique and can be considered as an extension of linear models that automatically models non-linearities and interaction between variables. The MARS modeling was carried out using “earth” package in R program [25]. The crystallinity of cellulose was determined by X-ray diffraction (XRD) using Rigaku DMAX 2500 X-ray diffractometer. Cellulose samples were scanned from 10◦ to 30◦ at scan speed of 2◦ /min and
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step size of 0.01◦ .The crystallinity index, Cr, which represents the percentage of crystalline materials was calculated from the XRD patterns by an empirical method described by Nelson and O’Connor [26] Cr =
Icr − Iam Icr
where Icr is the diffraction intensity at peak position 2 ≈ 22.6◦ for cellulose I, 21.7◦ for cellulose II and Iam is the intensity at suitable locations for the amorphous background (2 ≈ 19◦ for cellulose I, 16◦ for cellulose II) Residual [Emim][OAc] and DMSO in regenerated RS were determined by HPLC analysis. HPLC system composed of a LC-10AD pump (Shimadzu, Japan), two detectors: RI detector (for sugars analysis) and UV detector (for ionic liquid and DMSO detection) and a jacketed glass column (30 cm × 1.1 cm) packed with Dowex50WX4-400 cation exchanger resin. The functional group H+ of resin was exchanged with [Emim]+ of ILs by passing 10 column volumes (CV) of [Emim][OAc] in water through the column. Degassed and deionized water was used as mobile phase at flow rate of 1.5 mL/min. The analysis was performed at 65 ◦ C [27]. The structures of ILs/co-solvents before and after RS dissolution were examined by 400 MHz FT-NMR spectrometer (Varian Inova 400, USA) in acetone-D6 . Glucose and xylose concentration were measured by HPLC (Shimadzu Model LC-10A) equipped with a RI detector and a Bio-Rad HPX-87P column (300 mm × 7.8 mm) operated at 85 ◦ C. Deionized water was used as mobile phase at flow rate of 0.6 mL/min. Ethanol concentration from RS hydrolysate fermentation was measured by gas chromatography (CP 9001, Chrompack, Netherlands) using a DB23-capillary column (30 m × 0.25 mm ID; film thickness 0.25 mm; J&W Scientific, U.S.A.) and flame ionization detector (FID). 3. Results and discussion 3.1. Effect of organic co-solvents on dissolution of lignocellulose in ionic liquids Conventional aprotic polar organic solvents such as dimtheylsulfoxide (DMSO), N,N-dimethylformamide (DMF), and N,Ndimethylacetamide (DMA) themselves are not cellulose-dissolving solvents (Table 2) although they have been used as co-solvent to reduce the viscosity of cellulose/IL mixture for structure analysis of ILs-dissolved cellulose [28]. The addition of these solvent as viscous reducing co-solvent was carried out after the dissolution of cellulose in ILs. More recently, Rinaldi have reported that several electrolyte solution containing small fraction of ILs could instantaneously dissolve large amounts of cellulose [14]. These cellulose compatible solvents were known to reduce the viscosity of cellulose/ILs solution while maintaining the cellulose-dissolving capacity, or the hydrogen bond acceptor/basicity (ˇ) of ILs. In addition, the polar organic co-solvents promote the ILs dissociation into solvated cations and “free” anions to certain extent that enable the interaction of anions with cellulose, thus improving the cellulose dissolution [15]. As showed in Table 2, the use of organic co-solvent enhances the cellulose dissolution in terms of not only solubility but also dissolution rate. For instant, a mixture of [Emim][OAc] and DMSO, DMF or DMA with volume ratio of 1:1 could dissolve up to 28% cellulose in same order of time compared to 22% of pure ILs. The enhancement effect of these co-solvents on the dissolution of cellulose was also observed in [Emim][DMP]. Surprisingly, the cellulose dissolving capability of [Bmim][Cl], however, was deteriorated when these co-solvents were added (1:1 volume ratio). This may be explained by the reducing in the hydrogen-bond basicity of ILs/co-solvents at
3
high ratio of organic co-solvents which was also observed in works of Rinaldi [14]. However, at lower ratio of organic solvent, mixture of [BmimCl]-DMSO (86:14 wt%) dissolves pine, eucalyptus and oak wood shaving after heating at 100 ◦ C for several hours [12]. Interestingly, the use of co-solvent could also enhance the solubility of hemicellulose and lignin in IL/co-solvent mixture. DMSO itself could dissolve large amount of hemicellulose and lignin at test condition, which are 70% and 37%, respectively, compared to 24% and 33% of pure [Emim][OAc]. As a result, the ILs/DMSO mixture could significantly enhance the dissolution of hemicelluloses and lignin. For example, the solubility of hemicelluloses and lignin in [Emim][OAc]-DMSO mixture (1:1 volume ratio) was increased 2.5and 1.1-fold compared to those of pure [Emim][OAc], respectively, and these solubility would further enhance as increasing the volume ratio of DMSO in [Emim][OAc]-DMSO mixture (Table 2). The higher solubility of lignocellulose components ILs/co-solvent might be explained by the lower viscosity of the IL/co-solvent mixture compared to that of pure ILs. The high viscosity of ILs was considered as limitation factor for dissolution of lignocellulosic materials. The viscosities of these tested co-solvents are far lower than those of pure ILs, therefore the use of less viscous ILs/co-solvent mixture will improve the lignocellulose dissolution (Table 2). To evaluate the effect of solvent properties on the lignocellulose dissolution, the Kamlet-Taft solvent parameters of IL/co-solvent mixture were correlated with the solubility of lignocellulosic components. A considerably correlation between cellulose solubility and Kamlet-Taft solvent parameters of IL/co-solvent mixture (R2 = 0.85) was found by MARS analysis (Fig. 1). In addition, the MARS models indicated non-linear relationship between solute solubility and Kamlet-Taft solvent parameters and showed the interaction of these parameters on the lignocellulose solubility in ILs/co-solvent. Because of these interactions, MARS model also showed better correlation than that of linear model (multiple linear regression models showed lower R2 value, data not showed). However, the solubility of hemicellulose and lignin in ILs/co-solvent mixture were less correlated to the Kamlet-Taft solvent parameters as represented by the lower correlation coefficient (R2 = 0.61 and 0.68, respectively). Among tested ILs and co-solvents, [Emim][OAc] and DMSO were shown as promising ILs/co-solvent system that could simultaneously enhance the solubility of cellulose, hemicellulose and lignin. Therefore, [Emim][OAc]-DMSO mixture (1:1 volume ratio) was used for pretreatment of RS. 3.2. Enzymatic hydrolysis of rice straw The enzymatic hydrolysis of untreated and ILs, ILs/DMSO treated-RS are showed in Fig. 2A and B. Significant increases in enzymatic hydrolytic rate as well as released sugars were observed in ILs and ILs/co-solvent treated-RS compared to those of untreated and DMSO treated-RS. For instance, the enzymatic hydrolytic rate of [Emim][OAc] treated-RS and [Emim][OAc]-DMSO treated-RS was 8 and 17 times higher than that of untreated-RS, respectively. In addition, the use of microwave heating in RS pretreatment in IL and ILs/co-solvent was significantly enhanced the enzymatic hydrolytic rate. For example, the initial hydrolytic rate of [Emim][OAc] and [Emim][OAc]-DMSO treated-RS using microwave heating was increased 19 and 22 folds as compared to that of the untreated-RS. However, there was only 1.7-fold improvement observed in DMSO treated-RS (Fig. 2B). Moreover, the maximum conversion yield of 95% was quickly achieved after 6 h in cases of microwave-assisted [Emim][OAc]-treated RS and both [Emim][OAc]-DMSO treated-RS sample with or without microwave heating. Whereas it takes 12 h for [Emim][OAc]-treated-RS with conventional heating to obtain the same conversion yield. On the contrary, only 22% and 43% conversion yield were obtained after 24 h in case of untreated
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Solvent
Cellulose
[Bmim][Cl] [Emim][OAc] [Emim][DMP] DMSO DMA DMF [Bmim][Cl]-DMSO (1:1) [Bmim][Cl]-DMSO (1:4) [Bmim][Cl]-DMA (1:1) [Bmim][Cl]-DMA (1:4) [Bmim][Cl]-DMF (1:1) [Bmim][Cl]-DMF (1:4) [Emim][OAc]-DMSO (1:1) [Emim][OAc]-DMSO (1:4) [Emim][OAc]-DMA (1:1) [Emim][OAc]-DMA (1:4) [Emim][OAc]-DMF (1:1) [Emim][OAc]-DMF (1:4) [Emim][DMP]-DMSO (1:1) [Emim][DMP]-DMSO (1:4) [Emim][DMP]-DMA (1:1) [Emim][DMP]-DMA (1:4) [Emim][DMP]-DMF (1:1) [Emim][DMP]-DMF (1:4)
14 22 18 0 0 0 0 0 0 0 0 0 28 12 28 4 28 8 24 5 25 2 25 2
Dis. rate (min) 3 1 1 – – – – – – – – – 2 1 2 1 2 1 3 1 3 30 3 30
Sol. (wt%) <1 24 4 70 0 0 60 70 2 0 2 0 60 70 20 10 30 14 60 70 6 2 10 4
(25 ◦ C)
Lignin
Dis. rate (min) – 25 240 10 – – 10 5 60 – 60 – 8 5 10 6 10 6 8 5 30 60 10 20
Sol. (wt%) 11 33 25 37 <1 3 22 32 8 4 12 6 36 40 11 7 18 14 32 34 8 5 15 11
Dis. rate (min) 60 5 10 2 – 120 2 2 4 5 3 5 2 2 3 3 3 2 2 2 3 5 2 3
a
15 – – 48.9 37.8 36.7 – – – – – – – – – – – – – – – – – –
Kamlet-Taft solvent parameters
Viscosity (cp) (20 ◦ C)
˛
ˇ
˘*
0.48 0.41 0.43 0.18 0.13 0.21 0.45 0.46 0.46 0.51 0.46 0.49 0.25 0.23 0.32 0.33 0.35 0.40 0.37 0.29 0.39 0.35 0.37 0.37
0.85 1.08 1.01 0.77 0.79 0.77 0.90 0.83 0.93 1.00 0.88 0.96 0.91 0.97 1.04 1.14 1.08 1.19 1.04 0.97 1.11 1.12 1.07 1.12
1.14 1.10 1.10 1.01 0.88 0.84 1.10 1.10 1.06 0.97 1.10 0.97 1.23 1.14 1.10 0.97 1.06 0.88 1.06 1.06 0.97 0.93 1.01 0.88
40,890b 162c 394d 2.14 1.96 (25 ◦ C) 0.92 – – – – – – – – – – – – – – – – – –
The solubility was determined at 80 ◦ C under nitrogen pressure. Solutes (10 mg) were gradually added until clear solution was obtained. Dis. rate – dissolution rate (min): time required for completely dissolving 2Ywt% lignocellulosic components in solvent. a: [35]; b: [36]; c: [37]; d:[38]; Solvent properties of organic co-solvent were taken from Ref. [39]
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Sol. (wt%)
Hemicellulose
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Table 2 Solubility of lignocellulosic components in ionic liquids/co-solvents mixture.
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5
Fig. 1. Experimental versus predicted lignocellulosic component solubilities in ionic liquids/co-solvents and correlation models between lignocellulosic component solubilites in ionic liquids/co-solvents and corresponding Kamlet-Taft solvent parameters.
and DMSO treated-RS, respectively (Fig. 2A). In comparison with previous report, this proposed method required shorter hydrolysis time to reach maximum conversion yield (6 h versus 24 h) [29]. Furthermore, the RS pretreatment in ILs and ILs/co-solvent also enhanced the amount of sugars released through enzymatic hydrolysis. For instance, the amount of sugars released from RS treated with [Emim][OAc] and [Emim][OAc]/DMSO was at least 4 times higher than that of untreated-RS. The highest released glucose and xylose of 10.4 and 5.1 mg/mL, respectively, were obtained in microwave-assisted [Emim][OAc]-DMSO treated-RS compared to those of 2.3 and 0.9 mg/mL in untreated-RS (Fig. 2A).
18 (A)
16
Reducing sugar (g/L)
14 Untreated-RS (2.3 - 0.9) [Emim][OAc] (8.4 - 3.8) DMSO (4.2 - 2.2) [Emim][OAc]-DMSO (9.1 - 3.9) [Emim][OAc]_MW (9.8 - 4.3) [Emim][OAc]-DMSO_MW (10.4 - 5.1)
12 10 8 6
3.3. Properties of treated-rice straw
4 2 0 0
5
10
15
20
25
Time (hrs) 10
The enzymatic hydrolysis enhancement of ILs, and ILs/cosolvent treated-RS might be explained by the synergetic effect of several factors. These include (i) the efficient lignin extraction of ILs and ILs/co-solvent (Fig. 3) (ii) a decrease in crystalline (CrI) and increase in amorphous (CrII) cellulose portion in ILs, ILs/co-solvent
(B)
40 6
Lignin Extraction (%)
Initial hydrolytic rate (mg/hrs)
50 8
4
2
30
20
10 0
U
e ntr
] ] l] S Ac [C MP dR m] [D ][O ate mi im m] i [B m m [E [E
O W W SO MS _M ]_M DM ]-D Ac SO c O M A [ [O ]-D m] m] mi Ac [E mi ][O [E m mi [E
Fig. 2. (A) Reducing sugars released during enzymatic hydrolysis of rice straw. (B) Initial hydrolytic rate of enzymatic hydrolysis of untreated and treated rice straw from ionic liquids and ionic liquids/co-solvent.
0 ] ] l] Ac [C MP [O m] ][D m] mi i m i B [ m m [E [E
O SO MW MW MS DM c]_ O_ ]-D A S c M A [O [O m] ]-D m] mi Ac [E mi [O ] [E m mi [E
Fig. 3. Lignin extraction from rice straw by ionic liquids and ionic liquids/co-solvent.
Please cite this article in press as: Mai NL, et al. Efficient pretreatment of lignocellulose in ionic liquids/co-solvent for enzymatic hydrolysis enhancement into fermentable sugars. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.03.024
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6 0.5 CrI CrII
Crystallinity index
0.4
0.3
0.2
0.1
0.0
U
te ea ntr
dR
S [B
m] mi
] ] Ac MP [D ][O im m] i m m [E [E
l] [C
O W W SO MS _M ]_M DM ]-D Ac SO c O M A [ [O ]-D m] m] mi Ac mi [E [O ] [E m mi [E
Fig. 4. Cellulose crystallinity index of untreated and treated rice straw.
treated-RS (Fig. 4), and (iii) a decrease in residual ILs retained in regenerated-RS (Fig. 5). As presented in Fig. 3, 18.5% of RS lignin was efficiently extracted after dissolution and regeneration treatment in [Emim][OAc]. The relatively higher lignin extraction obtained in RS treated with [Emim][OAc] as compared to those with other ILs was in accordance with the high solubility of cellulose, hemicellulose and lignin in this ILs (Table 2). In addition, the use of this ILs in combination with DMSO and microwave heating also give rise to a higher lignin extraction. The highest lignin extraction (44.1%) was achieved by using microwave-assisted [Emim][OAc]-DMSO pretreatment, while the values for using [Emim][OAc]-DMSO and microwave-assisted [Emim][OAc] pretreatment were 27.2% and 33.7%, respectively. In all cases, the ILs/co-solvent system and system with microwave heating always showed higher lignin extraction efficiency compared to that pure ILs and system without microwave heating, respectively. This can be explained by the
ability of organic solvent to dissolve large amount of lignin, therefore facilitate the lignin extraction of the corresponding low viscous of ILs/co-solvent. In addition, the use of microwave heating also enhanced the lignin extraction. It is documented that polar and ionic solvents are efficiently and rapidly heated under microwave irradiation. ILs, which are ionic compounds and have moderate polarity (dielectric constant, ε, of 15), in mixture with DMSO, which is a relatively high polar solvent (ε of 48.9), can be efficiently heated under microwave irradiation. The combination of all the factors in microwave-assisted [Emim][OAc]-DMSO pretreatment, therefore, results in more efficient lignin extraction as compared to those of [Emim][OAc]-DMSO treatment and microwave-assisted [Emim][OAc] pretreatment. In addition, the enhanced enzymatic hydrolysis of ILs and ILs/co-solvent treated-RS were also attributed by the simultaneously decrease of CrI (corresponding to crystalline cellulose) and increase of CrII (corresponding to amorphous cellulose) in treatedRS (Fig. 4). It is recognized that, amorphous cellulose is easily hydrolyzed by enzyme than that of crystalline cellulose [11,30]. In rice straw, cellulose exists in native crystalline form, which is cellulose I [31]. However, in treated-RS the cellulose II of low crystallinity was recovered. The highest reduction in CrI was obtained in microwave-assisted [Emim][OAc]-DMSO treated-RS. These results indicate that [Emim][OAc]-DMSO cannot only efficiently extract lignin but also disrupt the crystalline structure of cellulose in RS. Furthermore, the pretreatment of RS with [Emim][OAc]-DMSO also efficiently reduced the residual ILs retained in regenerated RS as showed in Fig. 5A. It is reported that cellulase from T. reesei could be inactivated by high concentration of cellulose dissolving ILs such as [Bmim][Cl] and [Emim][OAc] [32,33]. During the regeneration of RS from ILs, some residual ILs might be trapped in the cellulose chains despite thoroughly washing. Thus, the efficient removal of ILs in treated RS is believed to not only facilitate the enzymatic hydrolysis but also improve economical uses of ILs in biomass pretreatment. The residual ILs retained in microwave-assisted [Emim][OAc]-DMSO treated-RS was around 0.4% and this amount was at least 25 times lower than those in [Emim][OAc] treated-RS. Interestingly, the residual DMSO was not detected under analysis condition indicates that the solvent was efficiently removed through washing steps.
12
120
(A)
(B) 100
Enzymatic conversion (%)
[Emim][OAc] content (wt%)
10
8
6
4
2
80
60
40
20
0 [O m] mi [E
0
] Ac m mi [E
A ][O
SO DM c]m [E
im
][O
W ]_M Ac
[E
[O m] mi
Ac
W _M SO M ]-D
1
2
3
4
5
Reuse of [Emim][OAc]\DMSO
Fig. 5. (A) Residual [Emim][OAc] content in treated-rice straw and (B) reuse of [Emim][OAc]-DMSO.
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7
1.6
4
12
1.4
2
OD 600nm
Ethanol (g/L)
8 1.0 0.8
6
0.6 4
1
Glucose - Xylose (g/L)
10 1.2
3
0.4 2 0.2
0
0.0
0 0
10
20
30
40
Time (hrs) OD-Control OD - RS hydrolysate Ethanol - Control Ethanol - RS hydrolysate
Glucose - Control Glucose - RS hydrolysate Xylose- Control Xylose- RS hydrolysate
Fig. 6. Fermentation profile of Pichia stipitis KCTC 7222.
3.4. Reuse of ILs/co-solvent
4. Conclusion
The economical feasible use of ILs in biomass processing could be improved by the reuse of ILs. In this study, [Emim][OAc]-DMSO could be reused at least 5 times for RS pretreatment without any reduction in enzymatic conversion of treated-RS (Fig. 5B). The structure of [Emim][OAc] and DMSO were not changed during repeated use for RS pretreatment (confirmed by 1 H NMR analysis, data not shown). In addition, the low viscosity of [Emim][OAc]DMSO mixture showed to be advantageous for regeneration of lignocellulose as well as recycling of IL/co-solvent. This would increase the number of recycled use of IL/co-solvent as minimizing the residual amount of lignocellulose in the solution [29].
The combination use of cellulose compatible organic solvents as co-solvent with ILs and microwave heating could significantly enhance the efficiency of lignocellulose pretreatment for enzymatic production of fermentable sugars from biomass. The use of organic co-solvents facilitates not only the dissolution of cellulose but also hemicelluloses and lignin, which gives rise to a higher lignin extraction, lower crystalline cellulose and lesser residual ILs amount retained in treated lignocellulosic biomass. In addition, the ILs/co-solvent could be recycled and reused at least 5 cycles without significant loss in pretreatment efficiency (on the pretreatment of RS) demonstrated that this method might be beneficial for the large-scale biomass pretreatment although the process optimization as well as the economical efficacy assessment are need to be proved. These are the objects of further study.
3.5. Ethanol fermentation The fermentability of sugars produced from the enzymatic hydrolysis of microwave-assisted [Emim][OAc]-DMSO treatd-RS were evaluated by ethanol production catalyzed by Pichia stipilis KCTC 7222. The microbial growth, sugar consumption and ethanol production were presented in Fig. 6. The results showed that the fermentation behavior of RS hydrolysate was identical to that of pure sugar mixtures in control experiments. Generally, glucose was preferably used by the microbial until they reached stationary state which represented by the plateau of OD value at 12 h. After 18 h, glucose was completely consumed and the xylose was up-taken by the cell. However, the xylose consumption not affected the microbial cell growth. The equilibrium ethanol concentration of 1.68 g/L was achieved after 24 h of fermentation which corresponded to yield of 0.14 g ethanol/g sugars. Although the ethanol yield in this study was lower than those reported in literature (0.45 g/g) [34] due to the low initial sugar concentration, the optimization of fermentation conditions would improve the ethanol yield of the present process. In conclusion, the fermentation profiles of hydrolysates from ILs/co-solvent treated-RS reinforced the efficiency of pretreatment method using ILs/co-solvent in production of fermentable sugars from lignocellulosic biomass
Acknowledgements This work was financially supported by the Ministry of Knowledge Economy, Korea through the Cleaner Production Technology Development Project (KC000619). This work was also supported by Inha University research Grant. This work is the outcome of a Manpower Development Program for Marine Energy by the Ministry of Land, Transport and Maritime Affairs (MLTM). References [1] Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 2010;101:4851–61. [2] Qureshi N, Maddox IS. Reduction in butanol inhibition by perstraction: utilization of concentrated lactose/whey permeate by Clostridium acetobutylicum to enhance butanol fermentation economics. Food Bioprod Process 2005;83:43–52. [3] Swatloski RP, Spear SK, Holbrey JD, Rogers RD. Dissolution of cellose with ionic liquids. J Am Chem Soc 2002;124:4974–5. [4] Maki-Arvela P, Anugwom I, Virtanen P, Sjoholm R, Mikkola JP. Dissolution of lignocellulosic materials and its constituents using ionic liquids – a review. Ind Crop Prod 2010;32:175–201.
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