Bioresource Technology 164 (2014) 198–202

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Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Combination of ultrasonic irradiation with ionic liquid pretreatmentfor enzymatic hydrolysis of rice straw

http://dx.doi.org/10.1016/j.biortech.2014.05.0040960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ 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: tjfang@nchu.edu.tw (T.J. Fang).

Chun-Yao Yang a, Tony J. Fang a,b,⇑a Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROCb 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 2014Received in revised form 30 April 2014Accepted 2 May 2014Available online 10 May 2014

Keywords:Rice strawUltrasoundIonic liquidCholine hydroxideEnzymatic 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 dif-ferent processes of pretreatment and enzymatic hydrolysis was investigated. Various substrates for enzy-matic 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) phospho-nium 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, theyield of total reducing sugar (TRS) converted from CHRS was up to 96.22% at 240 min and was greaterthan that from the other substrates; the TRS yield for CHRS with ultrasound was 19.5% greater than thatwithout irradiation. Lignocellulosic biomass pretreated with [Ch][OH] showed the highest efficiencyamong the tested ILs, and ultrasound can be applied effectively in rice-straw pretreatment and enzymatichydrolysis.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Rice straw (RS), the major residue of rice production, is acommon and abundant agricultural waste in Taiwan. It containsan 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 toserve as a raw material for the production of biofuel. However,RS contains around 11–15% of cuticle silica (Juliano, 1985), whichmakes it difficult for the lignocellulose to be utilized by enzymesand microorganisms. In Taiwan, the silica content of rice straw islow, generally less than 6% (Wang et al., 2007; Korndörfer et al.,

2001). In recent studies, various methods have been reported thatcan destroy the morphological structure of RS so that the lignocel-lulose can be used and converted to fermentable sugars moreeffectively; these methods include physical pretreatment (e.g.,steaming, electron beam irradiation, grinding, and milling), chem-ical pretreatment (e.g., alkali, acid, ammonia, oxidizing agent, andorgano-solvent), biological pretreatment, and enzymatic hydroly-sis (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 over20 kHz, has been applied to assist in the pretreatment of lignocel-lulosic biomass with different reaction solutions (Subhedar andGogate, 2013). For example, we have investigated the potential ofultrasound for treating rice hull as the fermentation substrate forthe production of xylooligosaccharides (Yang et al., 2012). In addi-tion, 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) 198–202 199

wheat straw (Sun and Tomkinson, 2002), kenaf powder (Ninomiyaet al., 2012), and rice hulls (Yu et al., 2009) also has been reported.Ultrasonic irradiation on liquid–solid interfaces showed some sur-face erosion or particle size reduction (Luche, 1998; Sasson andNeumann, 1997). The effect of ultrasonic irradiation is to producecavitation in the liquid to assist the progress of chemical reactionsfrom bubble creation and hot-spot generation. Cavitation, theproperty occurred by ultrasound, has the potential to destroy thesurface structure of lignocellulosic biomass.

Ionic liquids (ILs), regarded as a type of green solvent, areorganic salts that are comprised entirely of cations (usuallyorganic) and anions (usually inorganic). Ionic liquids have beenused extensively in many fields due to their properties of negligiblevapor pressure, high thermal stability, and non-flammability(Vancov et al., 2012; Quijano et al., 2010). The most common typesof ILs used in biotechnological processes are imidazolium, pyridini-um, pyrrolidinium, tetrafluoroborate, methylsulfate, quaternaryammonium, quaternary phosphonium, hexafluorophosphate, andbis[(trifluoromethyl) sulfonyl] amide, among which the ILs of imi-dazolium-based salts have been the most investigated in biotech-nology (Quijano et al., 2010).

Recently, ILs have been applied in cellulose dissolution or bio-mass pretreatment. Swatloski et al. (2002) found that cellulosecould be dissolved in the IL, 1-butyl-3-methylimidazolium chloride([Bmim]Cl), and many studies have focused on investigatingthe 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 havethe potential for removing lignin, reducing the crystallinity of cel-lulose, and enhancing the activities and stabilities of severalenzymes (Nguyen et al., 2010; Fu and Mazza, 2011; Lynam et al.,2012).

The aim of this study was to investigate the application of ultra-sound and ionic liquids in the degradation of rice straw under dif-ferent processes of pretreatment and enzymatic hydrolysis. Threedifferent 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 verifiedby field emission scanning electron microscope (FE-SEM) andX-ray energy dispersive spectrometer (EDS). The enzymatic hydro-lysis of substrates was conducted by cellulase from Trichodermareesei 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 inthis study, due to the basic ionic liquid [Ch][OH] being able toreplace the acid and alkali pretreatments for rice straw and effec-tively promoting the enzymatic hydrolysis under ultrasound. Thepretreatment strategy by combing basic ionic liquid [Ch][OH]and ultrasound not only generates high availability of lignocellu-losic biomass in high efficiency of bioconversion within limitedprocessing time but also makes possible reduction of pretreatmentcost by using [Ch][OH], which is cheaper than other types of ILs,demonstrating a rather preferred potential of commercialfeasibility.

2. Methods

2.1. Biomass and chemical reagent

The biomass of rice straw was Taikeng 9, and it was obtainedfrom Ershui Town, Changhua County, Taiwan. Prior to the experi-ments, the rice straw first was cut into short lengths and washedthoroughly 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

ethylsulfate ([EMIM][EtSO4], 98%, from Strem Chemicals, Inc.), tri-hexyl(tetradecyl)phosphonium decanoate (THTDPD, 95%, fromStrem Chemicals, Inc.), and choline hydroxide solution ([Ch][OH],46 wt% in H2O, from Sigma–Aldrich). The commercial cellulase fromT. reesei ATCC 26921 (aqueous solution, 1.2 g/mL of density at 25 �C,P700 endoglucanase units (EGU)/g, CAS Number 9012-54-8, ECNumber 232-734-4, Celluclast� 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 withtreated solutions in a 150-mL, two-neck, round-bottom flask at60 �C for 180 min in an ultrasonic bath (LEO-600, Ko Hsieh Instru-ments Co., Ltd., Taiwan). The ultrasonic bath was equipped withdual frequencies (28/40 kHz) and variable electric power (maxi-mum = 300 W), and the power density was 0.0126 W/cm3 for300 W (Yang et al., 2012; Yang and Chu, 2014). FE-SEM was usedto observe the structural change of samples treated by the ILsand ultrasonic irradiation.

2.2.1. RS pretreated with the ionic liquids [EMIM][EtSO4] and THTDPDThe 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, round-bottom 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 theultrasonic system (300 W/40 kHz) at 60 �C for 180 min. The super-natant was removed by centrifugation at 2690�g (5000 rpm) for30 min at room temperature. The precipitate from the pretreat-ment was washed with deionized (DI) water and centrifuged atleast five times. Finally, the precipitate was dried at 80 �C for48 h, and the dried powder was called ILRS-A. The substrate ofILRS-B was prepared in the same pretreatment process, but witha different ultrasonic frequency of 28 kHz (300 W). In addition,the polysaccharide was extracted from the supernatant by precip-itation with ethanol (Yang et al., 2012). The extracted polysaccha-ride was dried and milled to avoid aggregation. The extractionyield of polysaccharide from RS was calculated and total solublesugar (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 pretreatedby the ultrasonic system (300 W/40 kHz) at 60 �C for 180 min. Thesupernatant was removed by centrifugation at 2690�g (5000 rpm)for 10 min. The precipitate was washed with DI water andcentrifuged at least ten times to remove the [Ch][OH]. The precip-itate was dried at 80 �C for 48 h and designated as CHRS. Theprocedure of polysaccharide extraction for the supernatant wasthe same with that described in Section 2.2.1.

2.3. Enzymatic hydrolysis and analysis

The enzymatic hydrolysis reactions of untreated rice-strawpowders (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. Thereaction mixture contained 10 or 20 mg of RS, ILRS-A, ILRS-B, orCHRS using 7 mL of cellulase solution. The cellulase solution wasprepared by using cellulase aqueous solution (10%, w/w) and ace-tate buffer (90%, w/w, pH 4.9 at room temperature). For each reac-tion time (0, 30, 60, 120, 180, and 240 min), the hydrolysis reactionwas stopped by heating the sample in boiling water for 10 min,after which the sample was taken for subsequent analysis. Aftercentrifuging the heated sample, the concentration of total reducing

200 C.-Y. Yang, T.J. Fang / Bioresource Technology 164 (2014) 198–202

sugar (TRS) in the supernatant was determined using the3,5-dinitrosalicylic acid (DNS) method (Yang et al., 2012; Miller,1959) with a spectrophotometer (BioMate 3S UV–visible spectro-photometer, Thermo Fisher Scientific, Inc.). The hydrolysis productwas assayed by using CARBOSep CHO-682 LEAD column (Transge-nomic) in HPLC with refractive index (RI) detector.

Different analytical techniques were applied on pretreated anduntreated samples using FE-SEM and EDS. The ash contents of allsamples were analyzed at 600 �C. The biomass recovery, TRS yield(Amarasekara and Shanbhag, 2013), and glucose content were cal-culated using Eqs. (1), (2), and (3), respectively:

Biomass recoveryð%Þ ¼Weight of dry biomass after pretreatmentWeight of raw materials

� 100

ð1Þ

TRS yieldð%Þ ¼Weight of total reducing sugarsWeight of total dry substrate

� 100 ð2Þ

Glucose contentð%Þ ¼ Weight of glucoseWeight of total dry substrate

� 100 ð3Þ

2.4. Statistical analysis

Statistical analysis was performed by one-way ANOVA followedby Turkey’s HSD post hoc tests using IBM SPSS Statistics 19.0, andstatistical 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, whichwere (1) mixture of THTDPD (phosphonium based IL) and[EMIM][EtSO4] (imidazolium based IL), and (2) [Ch][OH] (basicIL). The biomass was recovered as precipitate by the steps of cen-trifugation, water washing, and drying. Biomass recovery after pre-treatment and TRS yield of enzymatic hydrolysis for ILRS-A, ILRS-B,and CHRS are shown in Table 1. The ratios of the recovered biomasswere 92.72% for ILRS-A, 89.30% for ILRS-B, and 47.41% for CHRS,respectively. The results showed that the higher biomass recoveryratio revealed the lower extent of degradation for raw material bydifferent pretreatments. Using those treated-biomass samples assubstrates to conduct enzymatic hydrolysis, the TRS yields were28.29%, 22.03% and 80.83% for ILRS-A, ILRS-B, and CHRS, respec-tively; the order of overall TRS based on raw materials was CHRS(38.32%) > ILRS-A (26.23%) > ILRS-B (19.67%). Thus, the results indi-cated that different types of ILs affected the efficiencies of biomasspretreatment and enzymatic hydrolysis very differently, and thebest IL for treating RS was [Ch][OH]. Ultrasound can enhance the

Table 1Biomass recovery after pretreatment and TRS yield of enzymatic hydrolysisb of different s

Ultrasounda Ionic liquid Substrate designation Biom

40 kHz/300 W THTDPD and [EMIM][EtSO4] ILRS-A 92.728 kHz/300 W THTDPD and [EMIM][EtSO4] ILRS-B 89.340 kHz/300 W [Ch][OH] CHRS 47.4

a Biomass pretreated with ultrasound at 60 �C for 180 min.b Enzymatic hydrolysis of 20 mg substrates by cellulase under ultrasound (40 kHz/30c TRS yield based on raw materials, overall TRS (%) = (biomass recovery) � (TRS yieldd TRS yield based on total carbohydrate of pretreated biomass, TRS recovery ð%Þ ¼ We

* Values in the same column with different superscript letters indicate a significant diff

effect of the ILs in treating RS; however, the effect of ultrasonic fre-quency on the pretreatment was not significant in this study.

From ash analysis, the ash contents after pretreatments were9.80%, 8.31%, 7.61%, and 3.63% in RS, ILRS-A, ILRS-B, and CHRS,respectively; the results indicated that the maximum carbohydratecontents after pretreatments were 90.20%, 91.69%, 92.39%, and96.37% in RS, ILRS-A, ILRS-B, and CHRS, respectively. It shows thatILs and ultrasound can reduce ash content in pretreated RS toenhance enzymatic hydrolysis, especially the higher ash reductionfor CHRS. TRS recoveries were 30.85%, 23.84%, and 83.87% for ILRS-A, ILRS-B, and CHRS, respectively (Table 1).

The supernatant after different pretreatments contained differ-ent ILs ([Ch][OH], or [EMIM][EtSO4] and THTDPD) and some solu-ble components; therefore, the polysaccharide in the supernatantwas extracted by precipitation with ethanol. The extraction yieldof polysaccharide from the supernatant after [Ch][OH] pretreat-ment was 14.98%, and the TSS was 33.8%. The extraction yieldsof polysaccharide from the supernatant after [EMIM][EtSO4] andTHTDPD pretreatments were 2.68% (13.3% of TSS) with ultrasoundof 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] pre-treatment can be extracted more polysaccharide than that afterTHTDPD and [EMIM][EtSO4] pretreatment.

3.2. Morphological structure and elemental composition changes of RSby different ILs pretreatment with ultrasound

The different pretreatments with ILs and ultrasound affectedthe micro-structure of the sample, the morphological structurechanges of RS by various pretreatments were observed by FE-SEM. The micro-structure of RS was like an array of close, hollowbundles. The surface structure of ILRS-A after pretreatment withILs under ultrasound revealed many regularly-arranged bumpsand dumbbell-shaped cells when ILs of [EMIM][EtSO4] andTHTDPD 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 thatwere revealed by removing the surface cuticular layer (possiblywax and hemicellulose) with hot acid detergent.

There was significant corrosion and shrinkage of the surface ofCHRS when it was pretreated by ultrasound and [Ch][OH]. In addi-tion, several bumps and dumbbell-shaped cells were erodedseverely on the surface; the morphological structure of CHRS wasthe most destroyed, making it the most advantageous for enzy-matic hydrolysis.

The elemental compositions (C, O and Si) of RS, ILRS-A and CHRSwere 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 thepretreatment using ILs under ultrasonic irradiation can effectivelyreduce silica content of rice straw to enhance enzymatichydrolysis.

ubstrates.*

ass recovery (%) TRS yield (%) Overall TRS (%)c TRS recovery (%)d

2 28.29 ± 1.20A 26.23 30.85 ± 1.30A

0 22.03 ± 1.41A 19.67 23.84 ± 1.53A

1 80.83 ± 4.96B 38.32 83.87 ± 5.15B

0 W) at 50 �C and 240 min.(%)).

Weight of total reducing sugaright of total carbohydrate of pretreated biomass � 100.erence at P 6 0.05.

Table 2Enhancement of ultrasound (300 W/40 kHz) on enzymatic hydrolysis of differentsubstrates.*

Substrate TRS yield (%) Enhancement ratioc

(ER, %)With ultrasound Without ultrasound

RSa 26.77 ± 2.10A 22.45 ± 0.48A 19.24ILRS-Aa 28.29 ± 1.20A 16.55 ± 2.62A 70.94ILRS-Ba 22.03 ± 1.41A 18.38 ± 1.75A 19.86CHRSa 80.83 ± 4.96B 73.39 ± 4.52B 10.14CHRSb 96.22 ± 2.10C 80.52 ± 9.54B 19.50

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.c ER ð%Þ ¼ TRS yield with ultrasound�TRS yield without ultrasound

TRS yield without ultrasound � 100.* Values in the same column with different superscript letters indicate a significantdifference at P 6 0.05.

Table 3Effect of ultrasonic frequency on TRS yields of ILRS-A and ILRS-B in enzymatichydrolysis.*

Enzymatichydrolysis

Hydrolysis time(min)

TRS yield (%) of substrate

ILRS-Aa ILRS-Bb

With ultrasound(300 W/40 kHz)

60 15.16 ± 0.80A 21.94 ± 0.68A

120 19.87 ± 0.05B 23.55 ± 1.27A

180 23.09 ± 0.60C 25.70 ± 2.58A

240 28.29 ± 1.20D 22.03 ± 1.41A

Without ultrasound 60 12.72 ± 0.83A 11.30 ± 0.78A

120 16.86 ± 0.21B 19.96 ± 0.46B

180 18.90 ± 0.87B 19.78 ± 0.14B

240 16.55 ± 2.62B 18.38 ± 1.75B

a Treated with ultrasound (300 W/40 kHz).b Treated with ultrasound (300 W/28 kHz).

* Values in the same column with different superscript letters indicate a significantdifference at P 6 0.05.

Time of enzymatic hydrolysis (min)

0 50 100 150 200 250

TR

S yi

eld

(%)

0

20

40

60

80

100 RS (With ultrasound) RS (Without ultrasound) CHRS (With ultrasound) CHRS (Without ultrasound)

A

A

A

A

B

C

B

C

B

C

B

B

A

C

C

A

A

A

B

C

Fig. 1. The trend of enzymatic hydrolysis with respect to time for 20-mg RS and 20-mg CHRS with and without ultrasound (300 W/40 kHz). Each value is expressed asmean ± S.D. (n = 3). Means within the same group with different letters aresignificantly different at P 6 0.05.

C.-Y. Yang, T.J. Fang / Bioresource Technology 164 (2014) 198–202 201

3.3. The effect of ultrasound and temperature on enzymatic hydrolysis

To investigate the effects of ultrasound on enzymatic hydroly-sis, TRS was derived from the enzymatic hydrolysis of various sub-strates, including RS, ILRS-A, ILRS-B, and CHRS, by cellulase withand without ultrasonic irradiation. The ratio of enhancement forTRS yield after being treated with ultrasound for 240 min is shownin Table 2. The enhancement ratios (ER,%) for 20-mg substrates ofRS, 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 ILRS-A was greater than that of other substrates, but the TRS yield ofILRS-A (28.29%) was much lower than that of CHRS (80.83%). Thereason for those results can be explained by the images of FE-SEM. The surface pitting observed probably due to cavitation inthe pretreatment using ultrasound. For ILRS-A, [EMIM][EtSO4]and THTDPD were forced to penetrate into the micro structureleading to the difficulty of washing off ILs. The surface still hadbumps and dumbbell-shaped cells that impeded the penetrationof cellulase into the networks of the substrates that caused thereduction of the hydrolysis reaction. However, for CHRS pretreatedwith hydrophilic [Ch][OH], greater TRS yield was obtained becausethe bumps and dumbbell-shaped cells on the surface were erodedseverely, which enhanced the diffusion of cellulase into thesubstrate.

The effect of temperature on enzymatic hydrolysis wasexplored using 10 mg of CHRS with and without ultrasound treat-ment for 30 min. The TRS yields at 50 �C were 65.05% with ultra-sound and 56.38% without ultrasound; the yields at 40 �C were60.71% with ultrasound and 42.79% without ultrasound. The TRSyield was higher at 50 �C than that at 40 �C with and without ultra-sound, but the efficiency of ultrasonic irradiation was found to bemore significant at the lower temperature due to the cavitationeffect that enhanced the diffusion of the enzyme into the networksof substrates to a greater extent at the lower temperature.

3.4. Effect of hydrolysis time with and without ultrasound

Table 3 shows the TRS yields from ILRS-A and ILRS-B for differ-ent enzymatic hydrolysis times with and without ultrasound. Table3 shows that the yields of TRS from ILRS-A and ILRS-B with ultra-sound were greater than the yields without sonication for allhydrolysis times. The TRS yield of ILRS-A was increased from15.16% at 60 min to 28.29% at 240 min by using ultrasound. How-ever, the TRS yield of ILRS-A was only 16.55% at 240 min withoutultrasound. The results showed that ultrasonic irradiation was veryeffective at enhancing the effects of enzymatic hydrolysis.

Fig. 1 shows the trend of enzymatic hydrolysis with respect totime for RS and CHRS with and without ultrasound. The TRS yieldsfrom RS and CHRS increased with increasing hydrolysis time. For a4-h period of enzymatic hydrolysis, the enhancement ratios with

ultrasound treatment in the early period at 30 min (ER: 40.80%for RS and 45.38% for CHRS) were greater than those obtained aftertreatment for 240 min (ER: 19.24% for RS and 10.14% for CHRS),indicating that the ultrasonic effect was more powerful in the earlyperiod of enzymatic hydrolysis by facilitating the diffusion of theenzyme. Several previous studies also indicated that ultrasonictreatment with a sonic horn promoted enzymatic hydrolysis bycellulase (Sulaiman et al., 2013) and that even low intensity ultra-sound had a positive effect on the activities of free cellulase andimmobilized cellulase (Wang et al., 2012).

3.5. Enzymatic hydrolysis from different amounts of substrate

The hydrolysis reactions of 10- and 20-mg substrates (RS, ILRS-A and CHRS) were compared under ultrasound (300 W/40 kHz) at50 �C (Fig. 2). The TRS yields of 10 mg of RS and ILRS-A treated withultrasonic irradiation at 240 min were only 35.99% and 37.79%,respectively. However, the yield of TRS for CHRS was as high as96.22% with ultrasound for the same reaction time. As the amountof substrate increased to 20 mg with the same cellulase concentra-tion, the yields of TRS for RS, ILRS-A and CHRS were reduced to26.77%, 28.29% and 80.83%, respectively. Concerning the amountof TRS produced, 9.62 mg of TRS were converted from 10 mg ofCHRS, and the larger amount of 16.16 mg of TRS would be obtainedby using 20 mg of CHRS, indicating that more TRS can be acquiredby increasing the amount of substrate under ultrasound even withthe same cellulase concentration. The glucose contents of 10-mg

Time of enzymatic hydrolysis (min)

30 60 120 180 240

TR

S yi

eld

(%)

0

20

40

60

80

100

12020 mg RS 10 mg RS 20 mg ILRS-A 10 mg ILRS-A 20 mg CHRS 10 mg CHRS

A AB

A

BC

AB

C

D

E

A

A

AA

B

C

B

AB

A

B

C

D

AB AB

A

B

C C

C

D

BC

D

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 samegroup with different letters are significantly different at P 6 0.05.

202 C.-Y. Yang, T.J. Fang / Bioresource Technology 164 (2014) 198–202

RS, ILRS-A, and CHRS treated with ultrasonic irradiation at 240 minwere 25.99%, 19.32%, and 55.45%, respectively; the results showthat glucose is the most important component of the TRS releasedin the enzymatic hydrolysis for CHRS.

In the same enzyme dose per unit mass of the substrate, thehydrolysis reactions of 10-, 20-, and 30-mg CHRS were comparedunder ultrasound (300 W/40 kHz) at 50 �C for 120 min, and the cel-lulase dose was kept at 0.15 g per mg of CHRS. The TRS yield for 10-mg CHRS was 83.47%. As the amount of CHRS increased to 20 and30 mg, TRS yields were 69.90% and 75.82%. TRS yields for 20 and30 mg were lower than that from 10-mg CHRS, showing that TRSyield was not significant with increasing the amount of substratein the same enzyme dose.

4. Conclusions

In this study, different types of ILs were used to pretreat ricestraw, and their use affected the efficiencies of biomass pretreat-ment and enzymatic hydrolysis under ultrasonic irradiation. Afterpretreatment, bumps and dumbbell-shaped cells were exposedon the surface of ILRS-A, and those bumps and cells on the surfaceof CHRS were eroded and shrunken significantly, thereby promot-ing enzymatic hydrolysis. The best IL to treat RS in this study is abasic ionic liquid [Ch][OH]. Higher temperatures increased TRSyields with and without ultrasound, and lower temperaturesincreased the efficiency of ultrasonic irradiation.

Acknowledgement

We thank National Science Council, ROC, project no. NSC102-2313-B005-023-MY3 for financially supporting this research.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2014.05.004.

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