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Combination of ultrasonic irradiation with ionic liquid pretreatment for enzymatic hydrolysis of rice straw
Bioresource Technology 164 (2014) 198–202
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Combination of ultrasonic irradiation with ionic liquid pretreatment for enzymatic hydrolysis of rice straw Chun-Yao Yang a, Tony J. Fang a,b,⇑ a b
Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROC Department of Nutrition, China Medical University, 91 Hsueh Shih Road, Taichung 40402, Taiwan, ROC
h i g h l i g h t s Ultrasound promotes the pretreatment and enzymatic hydrolysis of rice straw. Ionic liquids were used to pretreat rice straw under ultrasonic irradiation. Choline hydroxide pretreatment with ultrasound can erode the structure of rice straw. High total reducing sugar yield in enzymatic hydrolysis was obtained with ultrasound. The efficient process for treating rice straw with ultrasound and ILs was developed.
a r t i c l e
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Article history: Received 20 February 2014 Received in revised form 30 April 2014 Accepted 2 May 2014 Available online 10 May 2014 Keywords: Rice straw Ultrasound Ionic liquid Choline hydroxide Enzymatic hydrolysis
a b s t r a c t The application of ultrasonic irradiation and ionic liquids (ILs) in the degradation of rice straw under different processes of pretreatment and enzymatic hydrolysis was investigated. Various substrates for enzymatic hydrolysis by cellulase with and without ultrasound were as follows: untreated rice-straw powder (RS); RS treated by ILs of 1-ethyl-3-methylimidazolium ethylsulfate and trihexyl (tetradecyl) phosphonium decanoate with ultrasound at 300 W/(40 kHz, 28 kHz); RS treated by IL of choline hydroxide ([Ch][OH]) with ultrasound at 300 W/40 kHz (CHRS). In ultrasound-mediated enzymatic hydrolysis, the yield of total reducing sugar (TRS) converted from CHRS was up to 96.22% at 240 min and was greater than that from the other substrates; the TRS yield for CHRS with ultrasound was 19.5% greater than that without irradiation. Lignocellulosic biomass pretreated with [Ch][OH] showed the highest efficiency among the tested ILs, and ultrasound can be applied effectively in rice-straw pretreatment and enzymatic hydrolysis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Rice straw (RS), the major residue of rice production, is a common and abundant agricultural waste in Taiwan. It contains an abundant lignocellulosic biomass, including cellulose (24–34%), hemicellulose (19–29%), lignin (5–11%), and crude ash (10.4–21.8%) (Juliano, 1985); therefore, RS has the potential to serve as a raw material for the production of biofuel. However, RS contains around 11–15% of cuticle silica (Juliano, 1985), which makes it difficult for the lignocellulose to be utilized by enzymes and microorganisms. In Taiwan, the silica content of rice straw is low, generally less than 6% (Wang et al., 2007; Korndörfer et al., ⇑ Corresponding author at: Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROC. Tel.: +886 4 22861505; fax: +886 4 22876211. E-mail address: [email protected] (T.J. Fang). http://dx.doi.org/10.1016/j.biortech.2014.05.004 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
2001). In recent studies, various methods have been reported that can destroy the morphological structure of RS so that the lignocellulose can be used and converted to fermentable sugars more effectively; these methods include physical pretreatment (e.g., steaming, electron beam irradiation, grinding, and milling), chemical pretreatment (e.g., alkali, acid, ammonia, oxidizing agent, and organo-solvent), biological pretreatment, and enzymatic hydrolysis (Ranjan and Moholkar, 2013; Binod et al., 2010; Bak et al., 2009; Kim and Han, 2012; Sindhu et al., 2012; Nguyen et al., 2010). Ultrasound, the high frequency waves that are generally over 20 kHz, has been applied to assist in the pretreatment of lignocellulosic biomass with different reaction solutions (Subhedar and Gogate, 2013). For example, we have investigated the potential of ultrasound for treating rice hull as the fermentation substrate for the production of xylooligosaccharides (Yang et al., 2012). In addition, ultrasound pretreatment of sugarcane bagasse (Liu et al., 2006), buckwheat hulls (Hromádková and Ebringerová, 2003),
C.-Y. Yang, T.J. Fang / Bioresource Technology 164 (2014) 198–202
wheat straw (Sun and Tomkinson, 2002), kenaf powder (Ninomiya et al., 2012), and rice hulls (Yu et al., 2009) also has been reported. Ultrasonic irradiation on liquid–solid interfaces showed some surface erosion or particle size reduction (Luche, 1998; Sasson and Neumann, 1997). The effect of ultrasonic irradiation is to produce cavitation in the liquid to assist the progress of chemical reactions from bubble creation and hot-spot generation. Cavitation, the property occurred by ultrasound, has the potential to destroy the surface structure of lignocellulosic biomass. Ionic liquids (ILs), regarded as a type of green solvent, are organic salts that are comprised entirely of cations (usually organic) and anions (usually inorganic). Ionic liquids have been used extensively in many fields due to their properties of negligible vapor pressure, high thermal stability, and non-flammability (Vancov et al., 2012; Quijano et al., 2010). The most common types of ILs used in biotechnological processes are imidazolium, pyridinium, pyrrolidinium, tetrafluoroborate, methylsulfate, quaternary ammonium, quaternary phosphonium, hexafluorophosphate, and bis[(trifluoromethyl) sulfonyl] amide, among which the ILs of imidazolium-based salts have been the most investigated in biotechnology (Quijano et al., 2010). Recently, ILs have been applied in cellulose dissolution or biomass pretreatment. Swatloski et al. (2002) found that cellulose could be dissolved in the IL, 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), and many studies have focused on investigating the efficiencies of various ILs in treating lignocellulosic biomass, and diversifying their recovery, structures, enzymatic hydrolysis, and yields of fermentable sugar. In addition, ILs were found to have the potential for removing lignin, reducing the crystallinity of cellulose, and enhancing the activities and stabilities of several enzymes (Nguyen et al., 2010; Fu and Mazza, 2011; Lynam et al., 2012). The aim of this study was to investigate the application of ultrasound and ionic liquids in the degradation of rice straw under different processes of pretreatment and enzymatic hydrolysis. Three different types of ionic liquid including [EMIM][EtSO4], THTDPD, and [Ch][OH] were used to pretreat rice straw under ultrasound, the structural and elemental composition changes were verified by field emission scanning electron microscope (FE-SEM) and X-ray energy dispersive spectrometer (EDS). The enzymatic hydrolysis of substrates was conducted by cellulase from Trichoderma reesei ATCC 26921 with and without ultrasonic irradiation. Specifically, the new method of pretreatment with [Ch][OH] and ultrasound for the utilization of rice straw was developed in this study, due to the basic ionic liquid [Ch][OH] being able to replace the acid and alkali pretreatments for rice straw and effectively promoting the enzymatic hydrolysis under ultrasound. The pretreatment strategy by combing basic ionic liquid [Ch][OH] and ultrasound not only generates high availability of lignocellulosic biomass in high efficiency of bioconversion within limited processing time but also makes possible reduction of pretreatment cost by using [Ch][OH], which is cheaper than other types of ILs, demonstrating a rather preferred potential of commercial feasibility.
2. Methods 2.1. Biomass and chemical reagent The biomass of rice straw was Taikeng 9, and it was obtained from Ershui Town, Changhua County, Taiwan. Prior to the experiments, the rice straw first was cut into short lengths and washed thoroughly with reverse osmosis (RO) water until it was clean. Then, it was dried, pulverized, and screened through 60-mesh sieves. The ILs used in the study were 1-ethyl-3-methylimidazolium
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ethylsulfate ([EMIM][EtSO4], 98%, from Strem Chemicals, Inc.), trihexyl(tetradecyl)phosphonium decanoate (THTDPD, 95%, from Strem Chemicals, Inc.), and choline hydroxide solution ([Ch][OH], 46 wt% in H2O, from Sigma–Aldrich). The commercial cellulase from T. reesei ATCC 26921 (aqueous solution, 1.2 g/mL of density at 25 °C, P700 endoglucanase units (EGU)/g, CAS Number 9012-54-8, EC Number 232-734-4, CelluclastÒ 1.5 L, C2730, Sigma–Aldrich Co., LLC.) was used in enzymatic hydrolysis. 2.2. Pretreatment of rice straw under ultrasonic irradiation The pretreatment of rice straw powder was conducted with treated solutions in a 150-mL, two-neck, round-bottom flask at 60 °C for 180 min in an ultrasonic bath (LEO-600, Ko Hsieh Instruments Co., Ltd., Taiwan). The ultrasonic bath was equipped with dual frequencies (28/40 kHz) and variable electric power (maximum = 300 W), and the power density was 0.0126 W/cm3 for 300 W (Yang et al., 2012; Yang and Chu, 2014). FE-SEM was used to observe the structural change of samples treated by the ILs and ultrasonic irradiation. 2.2.1. RS pretreated with the ionic liquids [EMIM][EtSO4] and THTDPD The solution of ILs was prepared with 10 g of [EMIM][EtSO4], 1 g of THTDPD, and 10 mL of RO water in a 150-mL, two-neck, roundbottom flask under ultrasound (600 W/40 kHz) for 10 min. Then, 1 g of RS was put into the solution of ILs to be pretreated by the ultrasonic system (300 W/40 kHz) at 60 °C for 180 min. The supernatant was removed by centrifugation at 2690g (5000 rpm) for 30 min at room temperature. The precipitate from the pretreatment was washed with deionized (DI) water and centrifuged at least five times. Finally, the precipitate was dried at 80 °C for 48 h, and the dried powder was called ILRS-A. The substrate of ILRS-B was prepared in the same pretreatment process, but with a different ultrasonic frequency of 28 kHz (300 W). In addition, the polysaccharide was extracted from the supernatant by precipitation with ethanol (Yang et al., 2012). The extracted polysaccharide was dried and milled to avoid aggregation. The extraction yield of polysaccharide from RS was calculated and total soluble sugar (TSS) was analyzed by phenol–sulfuric acid assay. 2.2.2. RS pretreated with ionic liquid [Ch][OH] The pretreatment solution was prepared with 5 g of [Ch][OH] and 45 g of DI water in a 150-mL, two-neck, round-bottom flask. The 2 g of RS were put into the prescribed solution to be pretreated by the ultrasonic system (300 W/40 kHz) at 60 °C for 180 min. The supernatant was removed by centrifugation at 2690g (5000 rpm) for 10 min. The precipitate was washed with DI water and centrifuged at least ten times to remove the [Ch][OH]. The precipitate was dried at 80 °C for 48 h and designated as CHRS. The procedure of polysaccharide extraction for the supernatant was the same with that described in Section 2.2.1. 2.3. Enzymatic hydrolysis and analysis The enzymatic hydrolysis reactions of untreated rice-straw powders (RS) and treated rice-straw powders of ILRS-A, ILRS-B, and CHRS were conducted in a glass tube at 50 °C under ultrasound (300 W/40 kHz) in the ultrasonic bath or without sonication. The reaction mixture contained 10 or 20 mg of RS, ILRS-A, ILRS-B, or CHRS using 7 mL of cellulase solution. The cellulase solution was prepared by using cellulase aqueous solution (10%, w/w) and acetate buffer (90%, w/w, pH 4.9 at room temperature). For each reaction time (0, 30, 60, 120, 180, and 240 min), the hydrolysis reaction was stopped by heating the sample in boiling water for 10 min, after which the sample was taken for subsequent analysis. After centrifuging the heated sample, the concentration of total reducing
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sugar (TRS) in the supernatant was determined using the 3,5-dinitrosalicylic acid (DNS) method (Yang et al., 2012; Miller, 1959) with a spectrophotometer (BioMate 3S UV–visible spectrophotometer, Thermo Fisher Scientific, Inc.). The hydrolysis product was assayed by using CARBOSep CHO-682 LEAD column (Transgenomic) in HPLC with refractive index (RI) detector. Different analytical techniques were applied on pretreated and untreated samples using FE-SEM and EDS. The ash contents of all samples were analyzed at 600 °C. The biomass recovery, TRS yield (Amarasekara and Shanbhag, 2013), and glucose content were calculated using Eqs. (1), (2), and (3), respectively:
Biomass recoveryð%Þ ¼
Weight of dry biomass after pretreatment Weight of raw materials 100 ð1Þ
TRS yieldð%Þ ¼
Weight of total reducing sugars 100 Weight of total dry substrate
Glucose contentð%Þ ¼
Weight of glucose 100 Weight of total dry substrate
ð2Þ
ð3Þ
effect of the ILs in treating RS; however, the effect of ultrasonic frequency on the pretreatment was not significant in this study. From ash analysis, the ash contents after pretreatments were 9.80%, 8.31%, 7.61%, and 3.63% in RS, ILRS-A, ILRS-B, and CHRS, respectively; the results indicated that the maximum carbohydrate contents after pretreatments were 90.20%, 91.69%, 92.39%, and 96.37% in RS, ILRS-A, ILRS-B, and CHRS, respectively. It shows that ILs and ultrasound can reduce ash content in pretreated RS to enhance enzymatic hydrolysis, especially the higher ash reduction for CHRS. TRS recoveries were 30.85%, 23.84%, and 83.87% for ILRSA, ILRS-B, and CHRS, respectively (Table 1). The supernatant after different pretreatments contained different ILs ([Ch][OH], or [EMIM][EtSO4] and THTDPD) and some soluble components; therefore, the polysaccharide in the supernatant was extracted by precipitation with ethanol. The extraction yield of polysaccharide from the supernatant after [Ch][OH] pretreatment was 14.98%, and the TSS was 33.8%. The extraction yields of polysaccharide from the supernatant after [EMIM][EtSO4] and THTDPD pretreatments were 2.68% (13.3% of TSS) with ultrasound of 300 W/40 kHz and 2.11% (10.0% of TSS) with 300 W/28 kHz. Thus, the results show that the supernatant after [Ch][OH] pretreatment can be extracted more polysaccharide than that after THTDPD and [EMIM][EtSO4] pretreatment.
2.4. Statistical analysis Statistical analysis was performed by one-way ANOVA followed by Turkey’s HSD post hoc tests using IBM SPSS Statistics 19.0, and statistical significance was determined at the 0.05 level (P 6 0.05). 3. Results and discussion 3.1. RS treated with ultrasonic irradiation and various ILs RS was pretreated under ultrasound (300 W/40 kHz, 300 W/ 28 kHz) for 180 min at 60 °C in two different IL-solutions, which were (1) mixture of THTDPD (phosphonium based IL) and [EMIM][EtSO4] (imidazolium based IL), and (2) [Ch][OH] (basic IL). The biomass was recovered as precipitate by the steps of centrifugation, water washing, and drying. Biomass recovery after pretreatment and TRS yield of enzymatic hydrolysis for ILRS-A, ILRS-B, and CHRS are shown in Table 1. The ratios of the recovered biomass were 92.72% for ILRS-A, 89.30% for ILRS-B, and 47.41% for CHRS, respectively. The results showed that the higher biomass recovery ratio revealed the lower extent of degradation for raw material by different pretreatments. Using those treated-biomass samples as substrates to conduct enzymatic hydrolysis, the TRS yields were 28.29%, 22.03% and 80.83% for ILRS-A, ILRS-B, and CHRS, respectively; the order of overall TRS based on raw materials was CHRS (38.32%) > ILRS-A (26.23%) > ILRS-B (19.67%). Thus, the results indicated that different types of ILs affected the efficiencies of biomass pretreatment and enzymatic hydrolysis very differently, and the best IL for treating RS was [Ch][OH]. Ultrasound can enhance the
3.2. Morphological structure and elemental composition changes of RS by different ILs pretreatment with ultrasound The different pretreatments with ILs and ultrasound affected the micro-structure of the sample, the morphological structure changes of RS by various pretreatments were observed by FESEM. The micro-structure of RS was like an array of close, hollow bundles. The surface structure of ILRS-A after pretreatment with ILs under ultrasound revealed many regularly-arranged bumps and dumbbell-shaped cells when ILs of [EMIM][EtSO4] and THTDPD were used to treat RS under ultrasonic irradiation (300 W/40 kHz). Juliano (1985) also described the bumps, spikes, and dumbbell-shaped silica cells on the surface of rice straw that were revealed by removing the surface cuticular layer (possibly wax and hemicellulose) with hot acid detergent. There was significant corrosion and shrinkage of the surface of CHRS when it was pretreated by ultrasound and [Ch][OH]. In addition, several bumps and dumbbell-shaped cells were eroded severely on the surface; the morphological structure of CHRS was the most destroyed, making it the most advantageous for enzymatic hydrolysis. The elemental compositions (C, O and Si) of RS, ILRS-A and CHRS were observed by EDS. The contents of C and O are 46–51% and 48– 53% for RS, ILRS-A, and CHRS. The Si contents are 5.94% for RS, 0.94% for CHRS, and 0.12% for ILRS-A. The results show that the pretreatment using ILs under ultrasonic irradiation can effectively reduce silica content of rice straw to enhance enzymatic hydrolysis.
Table 1 Biomass recovery after pretreatment and TRS yield of enzymatic hydrolysisb of different substrates.* Ultrasounda 40 kHz/300 W 28 kHz/300 W 40 kHz/300 W a
THTDPD and [EMIM][EtSO4] THTDPD and [EMIM][EtSO4] [Ch][OH]
Substrate designation ILRS-A ILRS-B CHRS
Biomass recovery (%) 92.72 89.30 47.41
TRS yield (%) A
28.29 ± 1.20 22.03 ± 1.41A 80.83 ± 4.96B
Overall TRS (%)c
TRS recovery (%)d
26.23 19.67 38.32
30.85 ± 1.30A 23.84 ± 1.53A 83.87 ± 5.15B
Biomass pretreated with ultrasound at 60 °C for 180 min. Enzymatic hydrolysis of 20 mg substrates by cellulase under ultrasound (40 kHz/300 W) at 50 °C and 240 min. TRS yield based on raw materials, overall TRS (%) = (biomass recovery) (TRS yield (%)). Weight of total reducing sugar d TRS yield based on total carbohydrate of pretreated biomass, TRS recovery ð%Þ ¼ Weight of total 100. carbohydrate of pretreated biomass Values in the same column with different superscript letters indicate a significant difference at P 6 0.05. b
c
*
Ionic liquid
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C.-Y. Yang, T.J. Fang / Bioresource Technology 164 (2014) 198–202 Table 2 Enhancement of ultrasound (300 W/40 kHz) on enzymatic hydrolysis of different substrates.* Substrate
TRS yield (%) With ultrasound
a
RS ILRS-Aa ILRS-Ba CHRSa CHRSb
A
26.77 ± 2.10 28.29 ± 1.20A 22.03 ± 1.41A 80.83 ± 4.96B 96.22 ± 2.10C
Without ultrasound A
22.45 ± 0.48 16.55 ± 2.62A 18.38 ± 1.75A 73.39 ± 4.52B 80.52 ± 9.54B
Table 3 Effect of ultrasonic frequency on TRS yields of ILRS-A and ILRS-B in enzymatic hydrolysis.*
Enhancement ratioc (ER, %)
Enzymatic hydrolysis
19.24 70.94 19.86 10.14 19.50
Hydrolysis time (min)
ILRS-Aa
ILRS-Bb
With ultrasound (300 W/40 kHz)
60 120 180 240
15.16 ± 0.80 19.87 ± 0.05B 23.09 ± 0.60C 28.29 ± 1.20D
21.94 ± 0.68A 23.55 ± 1.27A 25.70 ± 2.58A 22.03 ± 1.41A
Without ultrasound
60 120 180 240
12.72 ± 0.83A 16.86 ± 0.21B 18.90 ± 0.87B 16.55 ± 2.62B
11.30 ± 0.78A 19.96 ± 0.46B 19.78 ± 0.14B 18.38 ± 1.75B
a
20 mg substrate was used for enzymatic hydrolysis at 50 °C and 240 min. b 10 mg substrate was used for enzymatic hydrolysis at 50 °C and 240 min. ultrasoundTRS yield without ultrasound c ER ð%Þ ¼ TRS yield withTRS 100. yield without ultrasound * Values in the same column with different superscript letters indicate a significant difference at P 6 0.05.
TRS yield (%) of substrate
A
a
Treated with ultrasound (300 W/40 kHz). Treated with ultrasound (300 W/28 kHz). * Values in the same column with different superscript letters indicate a significant difference at P 6 0.05. b
3.3. The effect of ultrasound and temperature on enzymatic hydrolysis To investigate the effects of ultrasound on enzymatic hydrolysis, TRS was derived from the enzymatic hydrolysis of various substrates, including RS, ILRS-A, ILRS-B, and CHRS, by cellulase with and without ultrasonic irradiation. The ratio of enhancement for TRS yield after being treated with ultrasound for 240 min is shown in Table 2. The enhancement ratios (ER,%) for 20-mg substrates of RS, ILRS-A, ILRS-B, CHRS were 19.24%, 70.94%, 19.86%, and 10.14%, respectively; but ER for 10 mg of CHRS was 19.50%. The ER for ILRSA was greater than that of other substrates, but the TRS yield of ILRS-A (28.29%) was much lower than that of CHRS (80.83%). The reason for those results can be explained by the images of FESEM. The surface pitting observed probably due to cavitation in the pretreatment using ultrasound. For ILRS-A, [EMIM][EtSO4] and THTDPD were forced to penetrate into the micro structure leading to the difficulty of washing off ILs. The surface still had bumps and dumbbell-shaped cells that impeded the penetration of cellulase into the networks of the substrates that caused the reduction of the hydrolysis reaction. However, for CHRS pretreated with hydrophilic [Ch][OH], greater TRS yield was obtained because the bumps and dumbbell-shaped cells on the surface were eroded severely, which enhanced the diffusion of cellulase into the substrate. The effect of temperature on enzymatic hydrolysis was explored using 10 mg of CHRS with and without ultrasound treatment for 30 min. The TRS yields at 50 °C were 65.05% with ultrasound and 56.38% without ultrasound; the yields at 40 °C were 60.71% with ultrasound and 42.79% without ultrasound. The TRS yield was higher at 50 °C than that at 40 °C with and without ultrasound, but the efficiency of ultrasonic irradiation was found to be more significant at the lower temperature due to the cavitation effect that enhanced the diffusion of the enzyme into the networks of substrates to a greater extent at the lower temperature.
ultrasound treatment in the early period at 30 min (ER: 40.80% for RS and 45.38% for CHRS) were greater than those obtained after treatment for 240 min (ER: 19.24% for RS and 10.14% for CHRS), indicating that the ultrasonic effect was more powerful in the early period of enzymatic hydrolysis by facilitating the diffusion of the enzyme. Several previous studies also indicated that ultrasonic treatment with a sonic horn promoted enzymatic hydrolysis by cellulase (Sulaiman et al., 2013) and that even low intensity ultrasound had a positive effect on the activities of free cellulase and immobilized cellulase (Wang et al., 2012). 3.5. Enzymatic hydrolysis from different amounts of substrate The hydrolysis reactions of 10- and 20-mg substrates (RS, ILRSA and CHRS) were compared under ultrasound (300 W/40 kHz) at 50 °C (Fig. 2). The TRS yields of 10 mg of RS and ILRS-A treated with ultrasonic irradiation at 240 min were only 35.99% and 37.79%, respectively. However, the yield of TRS for CHRS was as high as 96.22% with ultrasound for the same reaction time. As the amount of substrate increased to 20 mg with the same cellulase concentration, the yields of TRS for RS, ILRS-A and CHRS were reduced to 26.77%, 28.29% and 80.83%, respectively. Concerning the amount of TRS produced, 9.62 mg of TRS were converted from 10 mg of CHRS, and the larger amount of 16.16 mg of TRS would be obtained by using 20 mg of CHRS, indicating that more TRS can be acquired by increasing the amount of substrate under ultrasound even with the same cellulase concentration. The glucose contents of 10-mg
RS (With ultrasound) RS (Without ultrasound) CHRS (With ultrasound) CHRS (Without ultrasound)
100
3.4. Effect of hydrolysis time with and without ultrasound Table 3 shows the TRS yields from ILRS-A and ILRS-B for different enzymatic hydrolysis times with and without ultrasound. Table 3 shows that the yields of TRS from ILRS-A and ILRS-B with ultrasound were greater than the yields without sonication for all hydrolysis times. The TRS yield of ILRS-A was increased from 15.16% at 60 min to 28.29% at 240 min by using ultrasound. However, the TRS yield of ILRS-A was only 16.55% at 240 min without ultrasound. The results showed that ultrasonic irradiation was very effective at enhancing the effects of enzymatic hydrolysis. Fig. 1 shows the trend of enzymatic hydrolysis with respect to time for RS and CHRS with and without ultrasound. The TRS yields from RS and CHRS increased with increasing hydrolysis time. For a 4-h period of enzymatic hydrolysis, the enhancement ratios with
TRS yield (%)
80
C
C
C B
C
60
B
C
C B
40
B
20
A
B
B
A
A
A
A
A
A A
0 0
50
100
150
200
250
Time of enzymatic hydrolysis (min) Fig. 1. The trend of enzymatic hydrolysis with respect to time for 20-mg RS and 20mg CHRS with and without ultrasound (300 W/40 kHz). Each value is expressed as mean ± S.D. (n = 3). Means within the same group with different letters are significantly different at P 6 0.05.
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References
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TRS yield (%)
100 80
20 mg RS 10 mg RS 20 mg ILRS-A 10 mg ILRS-A 20 mg CHRS 10 mg CHRS
C
E
D D
B C C C
D D
60
C
40
BC
B B B AB BC C
20 A
AB
AB
A
A
A
A
AB
AB
A A
A
0 30
60
120
180
240
Time of enzymatic hydrolysis (min) Fig. 2. Enzymatic hydrolysis using different amounts of substrates of RS, ILRS-A, and CHRS. Each value is expressed as mean ± S.D. (n = 3). Means within the same group with different letters are significantly different at P 6 0.05.
RS, ILRS-A, and CHRS treated with ultrasonic irradiation at 240 min were 25.99%, 19.32%, and 55.45%, respectively; the results show that glucose is the most important component of the TRS released in the enzymatic hydrolysis for CHRS. In the same enzyme dose per unit mass of the substrate, the hydrolysis reactions of 10-, 20-, and 30-mg CHRS were compared under ultrasound (300 W/40 kHz) at 50 °C for 120 min, and the cellulase dose was kept at 0.15 g per mg of CHRS. The TRS yield for 10mg CHRS was 83.47%. As the amount of CHRS increased to 20 and 30 mg, TRS yields were 69.90% and 75.82%. TRS yields for 20 and 30 mg were lower than that from 10-mg CHRS, showing that TRS yield was not significant with increasing the amount of substrate in the same enzyme dose. 4. Conclusions In this study, different types of ILs were used to pretreat rice straw, and their use affected the efficiencies of biomass pretreatment and enzymatic hydrolysis under ultrasonic irradiation. After pretreatment, bumps and dumbbell-shaped cells were exposed on the surface of ILRS-A, and those bumps and cells on the surface of CHRS were eroded and shrunken significantly, thereby promoting enzymatic hydrolysis. The best IL to treat RS in this study is a basic ionic liquid [Ch][OH]. Higher temperatures increased TRS yields with and without ultrasound, and lower temperatures increased the efficiency of ultrasonic irradiation. Acknowledgement We thank National Science Council, ROC, project no. NSC1022313-B005-023-MY3 for financially supporting this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.05. 004.
Amarasekara, A.S., Shanbhag, P., 2013. Degradation of untreated switchgrass biomass into reducing sugars in 1-(alkylsulfonic)-3-methylimidazolium brönsted acidic ionic liquid medium under mild conditions. Bioenergy Res. 6, 719–724. Bak, J.S., Ko, J.K., Han, Y.H., Lee, B.C., Choi, I.G., Kim, K.H., 2009. Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment. Bioresour. Technol. 100, 1285–1290. Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshmi, S., Kurien, N., Sukumaran, R.K., Pandey, A., 2010. Bioethanol production from rice straw: an overview. Bioresour. Technol. 101, 4767–4774. Fu, D., Mazza, G., 2011. Aqueous ionic liquid pretreatment of straw. Bioresour. Technol. 102, 7008–7011. Hromádková, Z., Ebringerová, A., 2003. Ultrasonic extraction of plant materialsinvestigation of hemicellulose release from buckwheat hulls. Ultrason. Sonochem. 10, 127–133. Juliano, B.O., 1985. Rice: Chemistry and Technology. American Association of Cereal Chemists Inc., USA. Kim, I., Han, J.I., 2012. Optimization of alkaline pretreatment conditions for enhancing glucose yield of rice straw by response surface methodology. Biomass Bioenergy 46, 210–217. Korndörfer, G.H., Snyder, G.H., Ulloa, M., Powell, G., Datnoff, L.E., 2001. Calibration of soil and plant silicon analysis for rice production. J. Plant Nutr. 24, 1071–1084. Liu, C.F., Ren, J.L., Xu, F., Liu, J.J., Sun, J.X., Sun, R.C., 2006. Isolation and characterization of cellulose obtained from ultrasonic irradiated sugarcane bagasse. J. Agric. Food Chem. 54, 5742–5748. Luche, J.L., 1998. Synthetic Organic Sonochemistry. Plenum Press, New York. Lynam, J.G., Reza, M.T., Vasquez, V.R., Coronella, C.J., 2012. Pretreatment of rice hulls by ionic liquid dissolution. Bioresour. Technol. 114, 629–636. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Nguyen, T.A.D., Kim, K.R., Han, S.J., Cho, H.Y., Kim, J.W., Park, S.M., Park, J.C., Sim, S.J., 2010. Pretreatment of rice straw with ammonia and ionic liquid for lignocellulose conversion to fermentable sugars. Bioresour. Technol. 101, 7432–7438. Ninomiya, K., Kamide, K., Takahashi, K., Shimizu, N., 2012. Enhanced enzymatic saccharification of kenaf powder after ultrasonic pretreatment in ionic liquids at room temperature. Bioresour. Technol. 103, 259–265. Quijano, G., Couvert, A., Amrane, A., 2010. Ionic liquids: applications and future trends in bioreactor technology. Bioresour. Technol. 101, 8923–8930. Ranjan, A., Moholkar, V.S., 2013. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel 112, 567–571. Sasson, Y., Neumann, R., 1997. Handbook of Phase Transfer Catalysis. Blackie Academic & Professional, London. Sindhu, R., Binod, P., Janu, K.U., Sukumaran, R.K., Pandey, A., 2012. Organosolvent pretreatment and enzymatic hydrolysis of rice straw for the production of bioethanol. World J. Microbiol. Biotechnol. 28, 473–483. Subhedar, P.B., Gogate, P.R., 2013. Intensification of enzymatic hydrolysis of lignocellulose using ultrasound for efficient bioethanol production: a review. Ind. Eng. Chem. Res. 52, 11816–11828. Sulaiman, A.Z., Ajit, A., Chisti, Y., 2013. Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose. Biotechnol. Prog. 29, 1448–1457. Sun, R.C., Tomkinson, J., 2002. Characterization of hemicelluloses obtained by classical and ultrasonically assisted extractions from wheat straw. Carbohydr. Polym. 50, 263–271. Swatloski, R.P., Spear, S.K., Holbrey, J.D., Rogers, R.D., 2002. Dissolution of cellulose with ionic liquids. J. Am. Chem. Soc. 124, 4974–4975. Vancov, T., Alston, A.S., Brown, T., McIntosh, S., 2012. Use of ionic liquids in converting lignocellulosic material to biofuels. Renew. Energy 45, 1–6. Wang, S.L., Tzoua, Y.M., Lua, Y.H., Sheng, G., 2007. Removal of 3-chlorophenol from water using rice-straw-based carbon. J. Hazard. Mater. 147, 313–318. Wang, Z., Lin, X., Li, P., Zhang, J., Wang, S., Ma, H., 2012. Effects of low intensity ultrasound on cellulase pretreatment. Bioresour. Technol. 117, 222–227. Yang, H.M., Chu, W.M., 2014. Esterification of sodium 4-hydroxybenzoate by ultrasound-assisted solid–liquid phase-transfer catalysis using dual-site phasetransfer catalyst. Ultrason. Sonochem. 21, 395–400. Yang, C.Y., Sheih, I.C., Fang, T.J., 2012. Fermentation of rice hull by Aspergillus japonicus under ultrasonic pretreatment. Ultrason. Sonochem. 19, 687–691. Yu, J., Zhang, J., He, J., Liu, Z., Yu, Z., 2009. Combinations of mild physical or chemical pretreatment with biological pretreatment for enzymatic hydrolysis of rice hull. Bioresour. Technol. 100, 903–908.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Combination of ultrasonic irradiation with ionic liquid pretreatment for enzymatic hydrolysis of rice straw Chun-Yao Yang a, Tony J. Fang a,b,⇑ a b
Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROC Department of Nutrition, China Medical University, 91 Hsueh Shih Road, Taichung 40402, Taiwan, ROC
h i g h l i g h t s Ultrasound promotes the pretreatment and enzymatic hydrolysis of rice straw. Ionic liquids were used to pretreat rice straw under ultrasonic irradiation. Choline hydroxide pretreatment with ultrasound can erode the structure of rice straw. High total reducing sugar yield in enzymatic hydrolysis was obtained with ultrasound. The efficient process for treating rice straw with ultrasound and ILs was developed.
a r t i c l e
i n f o
Article history: Received 20 February 2014 Received in revised form 30 April 2014 Accepted 2 May 2014 Available online 10 May 2014 Keywords: Rice straw Ultrasound Ionic liquid Choline hydroxide Enzymatic hydrolysis
a b s t r a c t The application of ultrasonic irradiation and ionic liquids (ILs) in the degradation of rice straw under different processes of pretreatment and enzymatic hydrolysis was investigated. Various substrates for enzymatic hydrolysis by cellulase with and without ultrasound were as follows: untreated rice-straw powder (RS); RS treated by ILs of 1-ethyl-3-methylimidazolium ethylsulfate and trihexyl (tetradecyl) phosphonium decanoate with ultrasound at 300 W/(40 kHz, 28 kHz); RS treated by IL of choline hydroxide ([Ch][OH]) with ultrasound at 300 W/40 kHz (CHRS). In ultrasound-mediated enzymatic hydrolysis, the yield of total reducing sugar (TRS) converted from CHRS was up to 96.22% at 240 min and was greater than that from the other substrates; the TRS yield for CHRS with ultrasound was 19.5% greater than that without irradiation. Lignocellulosic biomass pretreated with [Ch][OH] showed the highest efficiency among the tested ILs, and ultrasound can be applied effectively in rice-straw pretreatment and enzymatic hydrolysis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Rice straw (RS), the major residue of rice production, is a common and abundant agricultural waste in Taiwan. It contains an abundant lignocellulosic biomass, including cellulose (24–34%), hemicellulose (19–29%), lignin (5–11%), and crude ash (10.4–21.8%) (Juliano, 1985); therefore, RS has the potential to serve as a raw material for the production of biofuel. However, RS contains around 11–15% of cuticle silica (Juliano, 1985), which makes it difficult for the lignocellulose to be utilized by enzymes and microorganisms. In Taiwan, the silica content of rice straw is low, generally less than 6% (Wang et al., 2007; Korndörfer et al., ⇑ Corresponding author at: Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROC. Tel.: +886 4 22861505; fax: +886 4 22876211. E-mail address: [email protected] (T.J. Fang). http://dx.doi.org/10.1016/j.biortech.2014.05.004 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
2001). In recent studies, various methods have been reported that can destroy the morphological structure of RS so that the lignocellulose can be used and converted to fermentable sugars more effectively; these methods include physical pretreatment (e.g., steaming, electron beam irradiation, grinding, and milling), chemical pretreatment (e.g., alkali, acid, ammonia, oxidizing agent, and organo-solvent), biological pretreatment, and enzymatic hydrolysis (Ranjan and Moholkar, 2013; Binod et al., 2010; Bak et al., 2009; Kim and Han, 2012; Sindhu et al., 2012; Nguyen et al., 2010). Ultrasound, the high frequency waves that are generally over 20 kHz, has been applied to assist in the pretreatment of lignocellulosic biomass with different reaction solutions (Subhedar and Gogate, 2013). For example, we have investigated the potential of ultrasound for treating rice hull as the fermentation substrate for the production of xylooligosaccharides (Yang et al., 2012). In addition, ultrasound pretreatment of sugarcane bagasse (Liu et al., 2006), buckwheat hulls (Hromádková and Ebringerová, 2003),
C.-Y. Yang, T.J. Fang / Bioresource Technology 164 (2014) 198–202
wheat straw (Sun and Tomkinson, 2002), kenaf powder (Ninomiya et al., 2012), and rice hulls (Yu et al., 2009) also has been reported. Ultrasonic irradiation on liquid–solid interfaces showed some surface erosion or particle size reduction (Luche, 1998; Sasson and Neumann, 1997). The effect of ultrasonic irradiation is to produce cavitation in the liquid to assist the progress of chemical reactions from bubble creation and hot-spot generation. Cavitation, the property occurred by ultrasound, has the potential to destroy the surface structure of lignocellulosic biomass. Ionic liquids (ILs), regarded as a type of green solvent, are organic salts that are comprised entirely of cations (usually organic) and anions (usually inorganic). Ionic liquids have been used extensively in many fields due to their properties of negligible vapor pressure, high thermal stability, and non-flammability (Vancov et al., 2012; Quijano et al., 2010). The most common types of ILs used in biotechnological processes are imidazolium, pyridinium, pyrrolidinium, tetrafluoroborate, methylsulfate, quaternary ammonium, quaternary phosphonium, hexafluorophosphate, and bis[(trifluoromethyl) sulfonyl] amide, among which the ILs of imidazolium-based salts have been the most investigated in biotechnology (Quijano et al., 2010). Recently, ILs have been applied in cellulose dissolution or biomass pretreatment. Swatloski et al. (2002) found that cellulose could be dissolved in the IL, 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), and many studies have focused on investigating the efficiencies of various ILs in treating lignocellulosic biomass, and diversifying their recovery, structures, enzymatic hydrolysis, and yields of fermentable sugar. In addition, ILs were found to have the potential for removing lignin, reducing the crystallinity of cellulose, and enhancing the activities and stabilities of several enzymes (Nguyen et al., 2010; Fu and Mazza, 2011; Lynam et al., 2012). The aim of this study was to investigate the application of ultrasound and ionic liquids in the degradation of rice straw under different processes of pretreatment and enzymatic hydrolysis. Three different types of ionic liquid including [EMIM][EtSO4], THTDPD, and [Ch][OH] were used to pretreat rice straw under ultrasound, the structural and elemental composition changes were verified by field emission scanning electron microscope (FE-SEM) and X-ray energy dispersive spectrometer (EDS). The enzymatic hydrolysis of substrates was conducted by cellulase from Trichoderma reesei ATCC 26921 with and without ultrasonic irradiation. Specifically, the new method of pretreatment with [Ch][OH] and ultrasound for the utilization of rice straw was developed in this study, due to the basic ionic liquid [Ch][OH] being able to replace the acid and alkali pretreatments for rice straw and effectively promoting the enzymatic hydrolysis under ultrasound. The pretreatment strategy by combing basic ionic liquid [Ch][OH] and ultrasound not only generates high availability of lignocellulosic biomass in high efficiency of bioconversion within limited processing time but also makes possible reduction of pretreatment cost by using [Ch][OH], which is cheaper than other types of ILs, demonstrating a rather preferred potential of commercial feasibility.
2. Methods 2.1. Biomass and chemical reagent The biomass of rice straw was Taikeng 9, and it was obtained from Ershui Town, Changhua County, Taiwan. Prior to the experiments, the rice straw first was cut into short lengths and washed thoroughly with reverse osmosis (RO) water until it was clean. Then, it was dried, pulverized, and screened through 60-mesh sieves. The ILs used in the study were 1-ethyl-3-methylimidazolium
199
ethylsulfate ([EMIM][EtSO4], 98%, from Strem Chemicals, Inc.), trihexyl(tetradecyl)phosphonium decanoate (THTDPD, 95%, from Strem Chemicals, Inc.), and choline hydroxide solution ([Ch][OH], 46 wt% in H2O, from Sigma–Aldrich). The commercial cellulase from T. reesei ATCC 26921 (aqueous solution, 1.2 g/mL of density at 25 °C, P700 endoglucanase units (EGU)/g, CAS Number 9012-54-8, EC Number 232-734-4, CelluclastÒ 1.5 L, C2730, Sigma–Aldrich Co., LLC.) was used in enzymatic hydrolysis. 2.2. Pretreatment of rice straw under ultrasonic irradiation The pretreatment of rice straw powder was conducted with treated solutions in a 150-mL, two-neck, round-bottom flask at 60 °C for 180 min in an ultrasonic bath (LEO-600, Ko Hsieh Instruments Co., Ltd., Taiwan). The ultrasonic bath was equipped with dual frequencies (28/40 kHz) and variable electric power (maximum = 300 W), and the power density was 0.0126 W/cm3 for 300 W (Yang et al., 2012; Yang and Chu, 2014). FE-SEM was used to observe the structural change of samples treated by the ILs and ultrasonic irradiation. 2.2.1. RS pretreated with the ionic liquids [EMIM][EtSO4] and THTDPD The solution of ILs was prepared with 10 g of [EMIM][EtSO4], 1 g of THTDPD, and 10 mL of RO water in a 150-mL, two-neck, roundbottom flask under ultrasound (600 W/40 kHz) for 10 min. Then, 1 g of RS was put into the solution of ILs to be pretreated by the ultrasonic system (300 W/40 kHz) at 60 °C for 180 min. The supernatant was removed by centrifugation at 2690g (5000 rpm) for 30 min at room temperature. The precipitate from the pretreatment was washed with deionized (DI) water and centrifuged at least five times. Finally, the precipitate was dried at 80 °C for 48 h, and the dried powder was called ILRS-A. The substrate of ILRS-B was prepared in the same pretreatment process, but with a different ultrasonic frequency of 28 kHz (300 W). In addition, the polysaccharide was extracted from the supernatant by precipitation with ethanol (Yang et al., 2012). The extracted polysaccharide was dried and milled to avoid aggregation. The extraction yield of polysaccharide from RS was calculated and total soluble sugar (TSS) was analyzed by phenol–sulfuric acid assay. 2.2.2. RS pretreated with ionic liquid [Ch][OH] The pretreatment solution was prepared with 5 g of [Ch][OH] and 45 g of DI water in a 150-mL, two-neck, round-bottom flask. The 2 g of RS were put into the prescribed solution to be pretreated by the ultrasonic system (300 W/40 kHz) at 60 °C for 180 min. The supernatant was removed by centrifugation at 2690g (5000 rpm) for 10 min. The precipitate was washed with DI water and centrifuged at least ten times to remove the [Ch][OH]. The precipitate was dried at 80 °C for 48 h and designated as CHRS. The procedure of polysaccharide extraction for the supernatant was the same with that described in Section 2.2.1. 2.3. Enzymatic hydrolysis and analysis The enzymatic hydrolysis reactions of untreated rice-straw powders (RS) and treated rice-straw powders of ILRS-A, ILRS-B, and CHRS were conducted in a glass tube at 50 °C under ultrasound (300 W/40 kHz) in the ultrasonic bath or without sonication. The reaction mixture contained 10 or 20 mg of RS, ILRS-A, ILRS-B, or CHRS using 7 mL of cellulase solution. The cellulase solution was prepared by using cellulase aqueous solution (10%, w/w) and acetate buffer (90%, w/w, pH 4.9 at room temperature). For each reaction time (0, 30, 60, 120, 180, and 240 min), the hydrolysis reaction was stopped by heating the sample in boiling water for 10 min, after which the sample was taken for subsequent analysis. After centrifuging the heated sample, the concentration of total reducing
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sugar (TRS) in the supernatant was determined using the 3,5-dinitrosalicylic acid (DNS) method (Yang et al., 2012; Miller, 1959) with a spectrophotometer (BioMate 3S UV–visible spectrophotometer, Thermo Fisher Scientific, Inc.). The hydrolysis product was assayed by using CARBOSep CHO-682 LEAD column (Transgenomic) in HPLC with refractive index (RI) detector. Different analytical techniques were applied on pretreated and untreated samples using FE-SEM and EDS. The ash contents of all samples were analyzed at 600 °C. The biomass recovery, TRS yield (Amarasekara and Shanbhag, 2013), and glucose content were calculated using Eqs. (1), (2), and (3), respectively:
Biomass recoveryð%Þ ¼
Weight of dry biomass after pretreatment Weight of raw materials 100 ð1Þ
TRS yieldð%Þ ¼
Weight of total reducing sugars 100 Weight of total dry substrate
Glucose contentð%Þ ¼
Weight of glucose 100 Weight of total dry substrate
ð2Þ
ð3Þ
effect of the ILs in treating RS; however, the effect of ultrasonic frequency on the pretreatment was not significant in this study. From ash analysis, the ash contents after pretreatments were 9.80%, 8.31%, 7.61%, and 3.63% in RS, ILRS-A, ILRS-B, and CHRS, respectively; the results indicated that the maximum carbohydrate contents after pretreatments were 90.20%, 91.69%, 92.39%, and 96.37% in RS, ILRS-A, ILRS-B, and CHRS, respectively. It shows that ILs and ultrasound can reduce ash content in pretreated RS to enhance enzymatic hydrolysis, especially the higher ash reduction for CHRS. TRS recoveries were 30.85%, 23.84%, and 83.87% for ILRSA, ILRS-B, and CHRS, respectively (Table 1). The supernatant after different pretreatments contained different ILs ([Ch][OH], or [EMIM][EtSO4] and THTDPD) and some soluble components; therefore, the polysaccharide in the supernatant was extracted by precipitation with ethanol. The extraction yield of polysaccharide from the supernatant after [Ch][OH] pretreatment was 14.98%, and the TSS was 33.8%. The extraction yields of polysaccharide from the supernatant after [EMIM][EtSO4] and THTDPD pretreatments were 2.68% (13.3% of TSS) with ultrasound of 300 W/40 kHz and 2.11% (10.0% of TSS) with 300 W/28 kHz. Thus, the results show that the supernatant after [Ch][OH] pretreatment can be extracted more polysaccharide than that after THTDPD and [EMIM][EtSO4] pretreatment.
2.4. Statistical analysis Statistical analysis was performed by one-way ANOVA followed by Turkey’s HSD post hoc tests using IBM SPSS Statistics 19.0, and statistical significance was determined at the 0.05 level (P 6 0.05). 3. Results and discussion 3.1. RS treated with ultrasonic irradiation and various ILs RS was pretreated under ultrasound (300 W/40 kHz, 300 W/ 28 kHz) for 180 min at 60 °C in two different IL-solutions, which were (1) mixture of THTDPD (phosphonium based IL) and [EMIM][EtSO4] (imidazolium based IL), and (2) [Ch][OH] (basic IL). The biomass was recovered as precipitate by the steps of centrifugation, water washing, and drying. Biomass recovery after pretreatment and TRS yield of enzymatic hydrolysis for ILRS-A, ILRS-B, and CHRS are shown in Table 1. The ratios of the recovered biomass were 92.72% for ILRS-A, 89.30% for ILRS-B, and 47.41% for CHRS, respectively. The results showed that the higher biomass recovery ratio revealed the lower extent of degradation for raw material by different pretreatments. Using those treated-biomass samples as substrates to conduct enzymatic hydrolysis, the TRS yields were 28.29%, 22.03% and 80.83% for ILRS-A, ILRS-B, and CHRS, respectively; the order of overall TRS based on raw materials was CHRS (38.32%) > ILRS-A (26.23%) > ILRS-B (19.67%). Thus, the results indicated that different types of ILs affected the efficiencies of biomass pretreatment and enzymatic hydrolysis very differently, and the best IL for treating RS was [Ch][OH]. Ultrasound can enhance the
3.2. Morphological structure and elemental composition changes of RS by different ILs pretreatment with ultrasound The different pretreatments with ILs and ultrasound affected the micro-structure of the sample, the morphological structure changes of RS by various pretreatments were observed by FESEM. The micro-structure of RS was like an array of close, hollow bundles. The surface structure of ILRS-A after pretreatment with ILs under ultrasound revealed many regularly-arranged bumps and dumbbell-shaped cells when ILs of [EMIM][EtSO4] and THTDPD were used to treat RS under ultrasonic irradiation (300 W/40 kHz). Juliano (1985) also described the bumps, spikes, and dumbbell-shaped silica cells on the surface of rice straw that were revealed by removing the surface cuticular layer (possibly wax and hemicellulose) with hot acid detergent. There was significant corrosion and shrinkage of the surface of CHRS when it was pretreated by ultrasound and [Ch][OH]. In addition, several bumps and dumbbell-shaped cells were eroded severely on the surface; the morphological structure of CHRS was the most destroyed, making it the most advantageous for enzymatic hydrolysis. The elemental compositions (C, O and Si) of RS, ILRS-A and CHRS were observed by EDS. The contents of C and O are 46–51% and 48– 53% for RS, ILRS-A, and CHRS. The Si contents are 5.94% for RS, 0.94% for CHRS, and 0.12% for ILRS-A. The results show that the pretreatment using ILs under ultrasonic irradiation can effectively reduce silica content of rice straw to enhance enzymatic hydrolysis.
Table 1 Biomass recovery after pretreatment and TRS yield of enzymatic hydrolysisb of different substrates.* Ultrasounda 40 kHz/300 W 28 kHz/300 W 40 kHz/300 W a
THTDPD and [EMIM][EtSO4] THTDPD and [EMIM][EtSO4] [Ch][OH]
Substrate designation ILRS-A ILRS-B CHRS
Biomass recovery (%) 92.72 89.30 47.41
TRS yield (%) A
28.29 ± 1.20 22.03 ± 1.41A 80.83 ± 4.96B
Overall TRS (%)c
TRS recovery (%)d
26.23 19.67 38.32
30.85 ± 1.30A 23.84 ± 1.53A 83.87 ± 5.15B
Biomass pretreated with ultrasound at 60 °C for 180 min. Enzymatic hydrolysis of 20 mg substrates by cellulase under ultrasound (40 kHz/300 W) at 50 °C and 240 min. TRS yield based on raw materials, overall TRS (%) = (biomass recovery) (TRS yield (%)). Weight of total reducing sugar d TRS yield based on total carbohydrate of pretreated biomass, TRS recovery ð%Þ ¼ Weight of total 100. carbohydrate of pretreated biomass Values in the same column with different superscript letters indicate a significant difference at P 6 0.05. b
c
*
Ionic liquid
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C.-Y. Yang, T.J. Fang / Bioresource Technology 164 (2014) 198–202 Table 2 Enhancement of ultrasound (300 W/40 kHz) on enzymatic hydrolysis of different substrates.* Substrate
TRS yield (%) With ultrasound
a
RS ILRS-Aa ILRS-Ba CHRSa CHRSb
A
26.77 ± 2.10 28.29 ± 1.20A 22.03 ± 1.41A 80.83 ± 4.96B 96.22 ± 2.10C
Without ultrasound A
22.45 ± 0.48 16.55 ± 2.62A 18.38 ± 1.75A 73.39 ± 4.52B 80.52 ± 9.54B
Table 3 Effect of ultrasonic frequency on TRS yields of ILRS-A and ILRS-B in enzymatic hydrolysis.*
Enhancement ratioc (ER, %)
Enzymatic hydrolysis
19.24 70.94 19.86 10.14 19.50
Hydrolysis time (min)
ILRS-Aa
ILRS-Bb
With ultrasound (300 W/40 kHz)
60 120 180 240
15.16 ± 0.80 19.87 ± 0.05B 23.09 ± 0.60C 28.29 ± 1.20D
21.94 ± 0.68A 23.55 ± 1.27A 25.70 ± 2.58A 22.03 ± 1.41A
Without ultrasound
60 120 180 240
12.72 ± 0.83A 16.86 ± 0.21B 18.90 ± 0.87B 16.55 ± 2.62B
11.30 ± 0.78A 19.96 ± 0.46B 19.78 ± 0.14B 18.38 ± 1.75B
a
20 mg substrate was used for enzymatic hydrolysis at 50 °C and 240 min. b 10 mg substrate was used for enzymatic hydrolysis at 50 °C and 240 min. ultrasoundTRS yield without ultrasound c ER ð%Þ ¼ TRS yield withTRS 100. yield without ultrasound * Values in the same column with different superscript letters indicate a significant difference at P 6 0.05.
TRS yield (%) of substrate
A
a
Treated with ultrasound (300 W/40 kHz). Treated with ultrasound (300 W/28 kHz). * Values in the same column with different superscript letters indicate a significant difference at P 6 0.05. b
3.3. The effect of ultrasound and temperature on enzymatic hydrolysis To investigate the effects of ultrasound on enzymatic hydrolysis, TRS was derived from the enzymatic hydrolysis of various substrates, including RS, ILRS-A, ILRS-B, and CHRS, by cellulase with and without ultrasonic irradiation. The ratio of enhancement for TRS yield after being treated with ultrasound for 240 min is shown in Table 2. The enhancement ratios (ER,%) for 20-mg substrates of RS, ILRS-A, ILRS-B, CHRS were 19.24%, 70.94%, 19.86%, and 10.14%, respectively; but ER for 10 mg of CHRS was 19.50%. The ER for ILRSA was greater than that of other substrates, but the TRS yield of ILRS-A (28.29%) was much lower than that of CHRS (80.83%). The reason for those results can be explained by the images of FESEM. The surface pitting observed probably due to cavitation in the pretreatment using ultrasound. For ILRS-A, [EMIM][EtSO4] and THTDPD were forced to penetrate into the micro structure leading to the difficulty of washing off ILs. The surface still had bumps and dumbbell-shaped cells that impeded the penetration of cellulase into the networks of the substrates that caused the reduction of the hydrolysis reaction. However, for CHRS pretreated with hydrophilic [Ch][OH], greater TRS yield was obtained because the bumps and dumbbell-shaped cells on the surface were eroded severely, which enhanced the diffusion of cellulase into the substrate. The effect of temperature on enzymatic hydrolysis was explored using 10 mg of CHRS with and without ultrasound treatment for 30 min. The TRS yields at 50 °C were 65.05% with ultrasound and 56.38% without ultrasound; the yields at 40 °C were 60.71% with ultrasound and 42.79% without ultrasound. The TRS yield was higher at 50 °C than that at 40 °C with and without ultrasound, but the efficiency of ultrasonic irradiation was found to be more significant at the lower temperature due to the cavitation effect that enhanced the diffusion of the enzyme into the networks of substrates to a greater extent at the lower temperature.
ultrasound treatment in the early period at 30 min (ER: 40.80% for RS and 45.38% for CHRS) were greater than those obtained after treatment for 240 min (ER: 19.24% for RS and 10.14% for CHRS), indicating that the ultrasonic effect was more powerful in the early period of enzymatic hydrolysis by facilitating the diffusion of the enzyme. Several previous studies also indicated that ultrasonic treatment with a sonic horn promoted enzymatic hydrolysis by cellulase (Sulaiman et al., 2013) and that even low intensity ultrasound had a positive effect on the activities of free cellulase and immobilized cellulase (Wang et al., 2012). 3.5. Enzymatic hydrolysis from different amounts of substrate The hydrolysis reactions of 10- and 20-mg substrates (RS, ILRSA and CHRS) were compared under ultrasound (300 W/40 kHz) at 50 °C (Fig. 2). The TRS yields of 10 mg of RS and ILRS-A treated with ultrasonic irradiation at 240 min were only 35.99% and 37.79%, respectively. However, the yield of TRS for CHRS was as high as 96.22% with ultrasound for the same reaction time. As the amount of substrate increased to 20 mg with the same cellulase concentration, the yields of TRS for RS, ILRS-A and CHRS were reduced to 26.77%, 28.29% and 80.83%, respectively. Concerning the amount of TRS produced, 9.62 mg of TRS were converted from 10 mg of CHRS, and the larger amount of 16.16 mg of TRS would be obtained by using 20 mg of CHRS, indicating that more TRS can be acquired by increasing the amount of substrate under ultrasound even with the same cellulase concentration. The glucose contents of 10-mg
RS (With ultrasound) RS (Without ultrasound) CHRS (With ultrasound) CHRS (Without ultrasound)
100
3.4. Effect of hydrolysis time with and without ultrasound Table 3 shows the TRS yields from ILRS-A and ILRS-B for different enzymatic hydrolysis times with and without ultrasound. Table 3 shows that the yields of TRS from ILRS-A and ILRS-B with ultrasound were greater than the yields without sonication for all hydrolysis times. The TRS yield of ILRS-A was increased from 15.16% at 60 min to 28.29% at 240 min by using ultrasound. However, the TRS yield of ILRS-A was only 16.55% at 240 min without ultrasound. The results showed that ultrasonic irradiation was very effective at enhancing the effects of enzymatic hydrolysis. Fig. 1 shows the trend of enzymatic hydrolysis with respect to time for RS and CHRS with and without ultrasound. The TRS yields from RS and CHRS increased with increasing hydrolysis time. For a 4-h period of enzymatic hydrolysis, the enhancement ratios with
TRS yield (%)
80
C
C
C B
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Time of enzymatic hydrolysis (min) Fig. 1. The trend of enzymatic hydrolysis with respect to time for 20-mg RS and 20mg CHRS with and without ultrasound (300 W/40 kHz). Each value is expressed as mean ± S.D. (n = 3). Means within the same group with different letters are significantly different at P 6 0.05.
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TRS yield (%)
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Time of enzymatic hydrolysis (min) Fig. 2. Enzymatic hydrolysis using different amounts of substrates of RS, ILRS-A, and CHRS. Each value is expressed as mean ± S.D. (n = 3). Means within the same group with different letters are significantly different at P 6 0.05.
RS, ILRS-A, and CHRS treated with ultrasonic irradiation at 240 min were 25.99%, 19.32%, and 55.45%, respectively; the results show that glucose is the most important component of the TRS released in the enzymatic hydrolysis for CHRS. In the same enzyme dose per unit mass of the substrate, the hydrolysis reactions of 10-, 20-, and 30-mg CHRS were compared under ultrasound (300 W/40 kHz) at 50 °C for 120 min, and the cellulase dose was kept at 0.15 g per mg of CHRS. The TRS yield for 10mg CHRS was 83.47%. As the amount of CHRS increased to 20 and 30 mg, TRS yields were 69.90% and 75.82%. TRS yields for 20 and 30 mg were lower than that from 10-mg CHRS, showing that TRS yield was not significant with increasing the amount of substrate in the same enzyme dose. 4. Conclusions In this study, different types of ILs were used to pretreat rice straw, and their use affected the efficiencies of biomass pretreatment and enzymatic hydrolysis under ultrasonic irradiation. After pretreatment, bumps and dumbbell-shaped cells were exposed on the surface of ILRS-A, and those bumps and cells on the surface of CHRS were eroded and shrunken significantly, thereby promoting enzymatic hydrolysis. The best IL to treat RS in this study is a basic ionic liquid [Ch][OH]. Higher temperatures increased TRS yields with and without ultrasound, and lower temperatures increased the efficiency of ultrasonic irradiation. Acknowledgement We thank National Science Council, ROC, project no. NSC1022313-B005-023-MY3 for financially supporting this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.05. 004.
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