Elucidation of the effect of ionic liquid pretreatment on rice husk via structural analyses
© Ang et al.; licensee BioMed Central Ltd. 2012
Received: 26 March 2012
Accepted: 3 August 2012
Published: 7 September 2012
In the present study, three ionic liquids, namely 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), and 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM]DEP), were used to partially dissolve rice husk, after which the cellulose were regenerated by the addition of water. The aim of the investigation is to examine the implications of the ionic liquid pretreatments on rice husk composition and structure.
From the attenuated total reflectance Fourier transform-infrared (ATR FT-IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM) results, the regenerated cellulose were more amorphous, less crystalline, and possessed higher structural disruption compared with untreated rice husk. The major component of regenerated cellulose from [BMIM]Cl and [EMIM]DEP pretreatments was cellulose-rich material, while cellulose regenerated from [EMIM]OAc was a matrix of cellulose and lignin. Cellulose regenerated from ionic pretreatments could be saccharified via enzymatic hydrolysis, and resulted in relatively high reducing sugars yields, whereas enzymatic hydrolysis of untreated rice husk did not yield reducing sugars. Rice husk residues generated from the ionic liquid pretreatments had similar chemical composition and amorphousity to that of untreated rice husk, but with varying extent of surface disruption and swelling.
The structural architecture of the regenerated cellulose and rice husk residues showed that they could be used for subsequent fermentation or derivation of cellulosic compounds. Therefore, ionic liquid pretreatment is an alternative in the pretreatment of lignocellulosic biomass in addition to the conventional chemical pretreatments.
KeywordsRice husk Ionic liquid Dissolution Pretreatment Regenerated cellulose Structural analysis
However, enzymatic saccharification of these untreated crop residues leads to low reducing sugar yields. Hence, pretreatment that disrupts the recalcitrant lignocellulosic biomass is necessary to enhance the saccharification of cellulose/hemicellulose into reducing sugars. Physical, chemical and a combination of physical/chemical pretreatments are the commonly employed methods in pretreating lignocellulosic biomass. Some of these methods require long residence times, high energy consumption, and carry the risk of sugar degradation when pretreatment is conducted at high temperatures [8, 9]. In consideration of these shortcomings, continual efforts have been invested to explore alternative pretreatments, one of which is via the application of green solvent - ionic liquids that is reported in this study.
Ionic liquids with cellulose-dissolving ability offer a novel solution for pretreating lignocellulosic biomass . Various ionic liquids, such as 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) , 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]) , and 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM][DEP]) , have been applied as solvents in pretreatment step before enzymatic saccharification to enhance reducing sugars yield. Lignocellulosic biomass pretreated with ionic liquid is favorable for subsequent enzymatic hydrolysis due to their reduced cellulose crystallinity and decreased lignin content [11, 13–15]. Li et al.  reported a significant improvement of reducing sugars yield of 17-fold from enzymatic saccharification of ionic liquid-pretreated switchgrass. In another study, enzymatic hydrolysis of ionic liquid-pretreated forest residues too exhibited increment in reducing sugars yield . Besides the significant improvement of yield, ionic liquids are greener pretreatment media because they can be recycled and reused in the dissolution process .
Rice husk (Oryza sativa) is one of the lignocellulosic residues that have attracted much attention among researchers due to its relatively high cellulose content and its potential to be used in biofuel production. The effects of acid and alkaline pretreatments on rice husk have previously been reported by Ang et al. . However, the application of ionic liquid pretreatments on rice husk has not been reported elsewhere. To gain a greater insight into ionic liquid pretreatment, the effect of ionic liquids with different anionic groups on the structural changes of rice husk was investigated. The structural architecture studies were conducted on both the regenerated cellulose and the rice husk residues from ionic liquid pretreatments.
Results and discussion
Dissolution of rice husk
In this study, the rice husk sample contained 53.18 ± 0.44% (w/w) cellulose, 4.63 ± 0.58% (w/w) hemicellulose and 19.67 ± 0.28% (w/w) lignin . Rice husk is often used in “low value for money” applications; for instance, it is either disposed as waste or burnt as fuel [20, 21]. Sometimes it is used as a low-cost filler in animal feeds  or as fertilizer . By applying an appropriate pretreatment, rice husk with relatively high cellulose content could be an attractive source for saccharification or derivatization into other useful products.
The effects of three ionic liquids ([BMIM]Cl, [EMIM]DEP and [EMIM]OAc) on the dissolution of rice husk and subsequent regeneration of cellulose were investigated. Previous study showed that prolong pretreatment at high temperatures possesses the risk of degrading the dissolved cellulose . Thus, the current experimental conditions (heating at 100°C for 10 hours) were selected as a compromise to allow sufficient cellulose dissolution, while minimizing the possibility of cellulose degradation and also reducing energy for pretreatment.
On the other hand, rice husk residues separated from the reaction mixtures were swollen compared to the untreated rice husk. This is due to the diffusion of the ionic liquids into the rice husk matrix that subsequently facilitates the dissolution of the rice husk . Among the ionic liquids examined, rice husk residue from the [EMIM]OAc pretreatment were the most severely swollen, whereas [BMIM]Cl and [EMIM]DEP did not show extensive swelling.
ATR FT-IR analysis
Group frequency of absorption bands of rice husk and regenerated cellulose
Group frequency, wavenumber, cm-1
800 – 950
C-H deformation vibration in cellulose
C-O stretching vibration in cellulose/hemicellulose and aryl-OH group in lignin
Asymmetric bending of CH3 and methoxy (-OCH3) groups present in lignin
C = C-C a
Aromatic skeletal stretching in lignin
O-H bending vibration of adsorbed water molecules
C-H stretching in cellulose-rich material
2995 – 4000
Free and hydrogen-bonded OH stretching
[EMIM]OAc showed the highest dissolution of rice husk (Section ‘Dissolution of rice husk’), and its regenerated cellulose possessed all the absorption bands present in the untreated rice husk. This clearly suggests that [EMIM]OAc does not selectively dissolve cellulose, but both cellulose and lignin in the rice husk lignocellulosic matrix. This ionic liquid has been reported to be capable of dissolving cellulose and lignin , and various lignocellulosic biomass [24, 41, 42]. Furthermore, [EMIM]OAc-treated cellulose showed higher intensity at band 797 cm-1, indicating that the regenerated cellulose was more amorphous than the untreated rice husk. The band at about 800 cm-1 is sensitive to the amount of amorphous cellulose present in the regenerated material, where broadening of this band indicates higher amorphousity of the regenerated cellulose. The dissolution and subsequent regeneration of the hemicellulose fraction might contribute to higher degree of amorphousity of the regenerated cellulose.
In comparison, the spectra of regenerated cellulose from [BMIM]Cl and [EMIM]DEP dissolution were different from the spectrum of the untreated rice husk, where some absorption bands were absent. In the spectra of regenerated cellulose of these two ionic liquid pretreatments, the band in the region of 800 – 950 cm-1 is broader implying a higher amount of disordered cellulosic structure [37, 38]. The disorder of cellulosic structure is very likely caused by the deformation vibration of β-glycosidic linkages and hydrogen bond rearrangement [34, 37]. In addition, [BMIM]Cl- and [EMIM]DEP-regenerated cellulose exhibited reduced absorbance at 1035 cm-1, which might have resulted from the degradation of cellulose/hemicellulose during heating. The degradation shortens cellulose chains leading to the reduction in C-O-C pyranose ring skeletal stretching . Moreover, the degradation of cellulose also reduced C-H stretching at 2896 cm-1 and free/hydrogen-bonded OH stretching at 3312 cm-1 of these regenerated cellulose. The disappearance of absorption band at 1457 cm-1 suggests the removal of lignin in regenerated cellulose of [BMIM]Cl and [EMIM]DEP.
Crystallinity indexes of untreated rice husk, regenerated cellulose and rice husk residue
Rice husk sample
Untreated rice husk
Regenerated cellulose ([BMIM]Cl)
Regenerated cellulose ([EMIM]OAc)
Regenerated cellulose ([EMIM]DEP)
Rice husk residue ([BMIM]Cl)
Rice husk residue ([EMIM]OAc)
Rice husk residue ([EMIM]DEP)
Rice husk residues of [BMIM]Cl and [EMIM]DEP pretreatments showed higher crystallinity index compared with the untreated rice husk; rice husk residue of [EMIM]OAc pretreatment showed slightly lower crystallinity index that is comparable to the untreated rice husk (Table 3). The dissolution of amorphous cellulose/hemicellulose of rice husk in ionic liquid, leaving the more crystalline lignocellulosic matrix in the residue, might be the main cause of the higher crystallinity index in rice husk residues of both [BMIM]Cl and [EMIM]DEP pretreatments. In contrast, the lower crystallinity of rice husk residue of [EMIM]OAc pretreatment might be due to the action of the ionic liquid that causes swelling to the structure of rice husk.
Comparison of ionic liquid pretreatments
The quantitative yield of regenerated cellulose reported in ‘Dissolution of rice husk’ indicates only the efficiency of the ionic liquids in dissolving rice husk. Information on structural characterization of the regenerated cellulose as well as the rice husk residues is helpful in the selection of a suitable ionic liquid pretreatment for lignocellulosic biomass. The ATR FT-IR and SEM analyses suggested that regenerated cellulose of the ionic liquid pretreatments comprise of cellulose-rich materials, which were more amorphous compared to the untreated rice husk. According to the XRD analysis, regenerated cellulose of the ionic liquid pretreatments had comparable crystallinity, with cellulose regenerated from [EMIM]DEP and [EMIM]OAc pretreatments showing a lower crystallinity.
Apart from the regenerated cellulose, rice husk residues from the ionic liquid pretreatments could be potential substrates for bioconversion into valuable compounds. The disrupted surface structure of the rice husk residues were favorable for solid-state fermentation, where it facilitates microbial growth by allowing access of microbes to the lignocellulosic matrix. SEM investigation demonstrated surface structure disruption of the rice husk residues after ionic liquid pretreatments, whereby [EMIM]OAc-treated rice husk residue showed the highest degree of structural disruption, followed by rice husk residues of [BMIM]Cl and [EMIM]DEP pretreatments. Besides, rice husk residue of [EMIM]OAc was found to have lower crystallinity after pretreatment compared to the other two ionic liquids. Nonetheless, chemical compositions of the rice husk residues remain relatively the same as those of the untreated rice husk.
The findings of structural characterization suggested that regenerated cellulose of [EMIM]OAc is amorphous and has low crystallinity, whereas its rice husk residue showed rigorously disrupted structure with reduced crystallinity. Therefore, [EMIM]OAc is a potential ionic liquid for the pretreatment of rice husk.
Enzymatic hydrolysis of regenerated cellulose and untreated rice husk
Besides acid and alkaline pretreatments, ionic liquid pretreatment can be used for pretreating lignocellulosic biomass. This study found that the chemical composition of the regenerated cellulose varies with the type of ionic liquid used. The ionic liquids [BMIM]Cl and [EMIM]DEP delignified the lignocellulosic rice husk, indicating their potential to be used in producing regenerated cellulose for enzymatic saccharification or cellulose derivatives. On the other hand, [EMIM]OAc dissolved the entire lignocelluloses and imparted surface structure disruption on the regenerated cellulose that is desired for subsequent fermentation or derivation of cellulosic compounds. The regenerated cellulose were more amorphous and had lower crystallinity compared with the untreated rice husk, whereas the rice husk residues showed a certain degree of structural disruption. The study also demonstrated that enzymatic hydrolysis of the regenerated cellulose resulted in higher yield compared to the untreated rice husk. Both the regenerated cellulose and rice husk residue revealed desirable structural changes in this study, which suggested that ionic liquid pretreatment is beneficial for conversion into value-added products. It is also essential to select a suitable ionic liquid pretreatment depending on the final application of the regenerated cellulose and rice husk residue.
Material and reagents
Rice husk samples were obtained from Selangor, Malaysia. The rice husk was first washed and dried before being ground to approximately 30 mesh sizes (500 μm). Ground rice husk samples were stored in a dry cabinet prior to use.
Cellulase from Trichoderma viride (Cellulase Onozuka R-10, catalogue # 102321) was purchased from Merck (Germany). The CMC activity of the Cellulase Onozuka R-10 was reported to be ≥ 1 U/mg. The IUPAC Filter Paper Assay was determined according to the procedure outlined by Ghose . All the chemicals and reagents used were of analytical grade.
Dissolution of rice husk
In ionic liquid pretreatment, a rice husk-ionic liquid mixture in a ratio of 1.5% (w/v) was heated to 100°C and pretreated for 10 hours in a block heater (HACH DRB200, USA). At the end of the pretreatment, the reaction mixture consisted of ionic liquid-dissolved cellulose and undissolved rice husk (hereafter called rice husk residue). The dissolution of rice husk was carried out in triplicate.
Cellulose regeneration and residue separation
After the dissolution, an equal volume of deionised water (Sartorius, arium® 611UF, Germany) was added to the clear reaction mixture to precipitate regenerated cellulose before the rice husk residue was filtered according to the procedure as outlined by Ang et al. . The cellulose-rich material (henceforth called regenerated cellulose) precipitated from the mixture was filtered. Both the regenerated cellulose and rice husk residue were washed with deionised water to remove the ionic liquid completely, and dried in an oven at 60°C prior to analyses.
ATR FT-IR analysis
The ATR FT-IR spectra of the samples between 600 and 4000 cm-1 at 4 cm-1 nominal resolution at room temperature were recorded using a FT-IR/FT-FIR spectrometer (Perkin Elmer, Spectrum 400, USA). The spectra are presented as relative transmittance percentage (%) of wave number (cm-1) and their background was recorded with an empty cell.
where I002 = maximum intensity of crystalline portion in rice husk sample at about 2θ = 22.6°, Iam = intensity attributed to the amorphous portion of rice husk sample at 2θ = ~18.7°.
SEM images were obtained with a Quanta 200 FESEM (FEI, USA) scanning electron microscope operated at 10 kV accelerating voltage. The samples obtained in the dissolution of rice husk and regeneration of cellulose were affixed onto aluminum stubs with double sided adhesive carbon tapes and examined without metal-coating under low vacuum mode.
Regenerated cellulose from the ionic liquid pretreatments was hydrolyzed using Cellulase Onozuka R-10 with a loading of 50 FPU/g substrate. Enzymatic hydrolysis of rice husk samples was carried out in 50 mM acetate buffer solution (pH 4.8) at 50°C for 48 h . After the reaction, the samples were centrifuged at 10,000 g for 3 minutes. The concentration of total reducing sugars in supernatant was determined via DNS method [46, 48]. The total reducing sugars yield obtained from enzymatic hydrolysis was computed according to Li et al. . All the experiments were conducted in duplicate.
1-ethyl-3-methylimidazolium diethyl phosphate
- ATR FT-IR:
Attenuated total reflectance Fourier transform-infrared
Scanning electron microscopy
This work was financially supported by the University of Malaya Research Grant (RG006/09AET) and the University of Malaya Postgraduate Research Grant (PS059/2009A and PV076/2011B). The authors are grateful to Ng Trading Company, Selangor, Malaysia for providing the rice husk samples.
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