Research Note pubs.acs.org/IECR
Structure and Saccharification of Rice Straw Pretreated with Microwave-Assisted Dilute Lye Feng-he Li,* Hua-jia Hu, Ri-sheng Yao, Huai Wang, and Man-man Li Department of Pharmaceutical Engineering, School of Medical Engineering, Hefei University of Technology, Hefei 230009, China ABSTRACT: Surface characteristics of untreated and microwave-assisted dilute lye (MAL) treated rice straw have been investigated using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction (XRD). Rice straw composition was investigated by lignocelluloses determination. FTIR showed MAL treatment could effectively remove lipophilic extractives from the rice straw surface and break hydrogen bonds in the rice straw, and it was proven that more cellulose was exposed by MAL-treated with SEM. The composition analysis result indicated that the content of cellulose is 46.8% and content of lignin is 8.3% in rice straw with high-fire-power microwave-assisted 1% dilute lye for 1 h, and saccharification rate is 86% with high-fire-power microwave-assisted 1% dilute lye for 2 h.
2.2. Pretreatments. Microwave pretreatments were carried out in a domestic microwave oven (National/OF00243). The microwave oven had a maximal power of 800 W with six discrete settings. The mixtures (rice straw and lye) were placed in a sealed glass vessel and treated by the microwave. Different firepower microwave-treated rice straw was treated in 1% NaOH solution, about 1 g/15 mL substrate concentration for 5, 10, 20, 40, 60, 80, 100, and 120 min. After pretreatment, the slurry was filtered through eight-layer gauze to separate residues and liquid. The residues were dried at 50 °C and stored for enzymatic hydrolysis, while the liquid fraction was collected to determine the reducing sugar yields obtained in the process of microwave pretreatment by 3,5-dinitrosalicylic acid (DNS) colorimetry. 2.3. FTIR Spectroscopy. Perkin−Elmer infrared spectrophotometer was used for investigating the change in surface functional groups of the plant biomass after pretreatment. The dried original rice straw or pretreated rice straw samples were mixed with KBr of spectroscopic grade and made in the form of pellets at pressure of about 1 MPa. The pellets were about 10 mm in diameter and 1 mm thickness. The spectra were then subjected to baseline correction, and the bands were studied to quantify the changes in the chemical structure of lignocellulose matrix. 2.4. Crystalline Analyzed by XRD. The overall crystalline of sample were examined by XRD measurements performed on a Bruker D8 Advance diffractometer using Cu Kα radiation (λ = 0.1541 nm) at 30 kV and 30 mA. The sample was scanned and the recorded intensity in 2θ ranged from 5° to 50°.10 2.5. Surface Morphology Observation by SEM. A JEOL JSM-6490LV SEM instrument operated at 15 kV accelerated voltage was used to examine the morphology and size of different straw samples for comparison of the effect of pretreatment. There are four kinds of sample: (1) untreated
1. INTRODUCTION Rice straw is one of the most abundant agriculture residues, and mostly consisting of cellulose and hemicellulose, has been widely regarded as an important reproducible source for bioethanol, animal fodder, and organic chemicals. And cellulosic biomass for bioethanol production needs pretreatment. Thus, development of pretreatment methods that increase the material digestibility for the subsequent enzymatic hydrolysis becomes a focus in this research field. Hence, a myriad of different pretreatment methods have been reported to remove lignin and hemicellulose, reduce cellulose crystallinity, and change chemical groups, for instance, milling,1,2 dilute acid,3 steam explosion,4 liquid hot water, dilute lye, wet oxidation, and ammonia fiber explosion (AFEX), and so on.5−7 A microwave-assisted dilute lye (MAL) pretreatment that can effectively improve the enzymatic saccharification of rice straw has been studied,8,9 and the pretreatment conditions were optimized by response surface methodology and a Box− Benhnken Design. However, it is not clear how the rice straw structure was changed in the process of MAL pretreatment. The objective of this work is to study changes in the structure of rice straw with MAL by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). In this work, the changes of straw structure and enzymatic hydrolysis of treated-straw by low fire middle fire, and high-fire MAL were investigated. (High fire, medium fire, and low fire are 100%, 60%, and 20% output power of microwave, respectively.) 2. MATERIALS AND METHODS 2.1. Materials and Reagents. Rice straw, harvested in early October 2010 in rural Hefei, was obtained from raw materials and was collected and stored in a storehouse with adequate ventilation to dry under natural condition. All the rice straw was cut into 4−6 cm length. In the MAL treatment, the lye temperature would be increased to over the boiling point over time, the glass tube that held the sample was put into a beaker with the cold water, and the cold water was regularly replaced. © 2012 American Chemical Society
Received: Revised: Accepted: Published: 6270
November 7, 2011 March 30, 2012 April 10, 2012 April 10, 2012 dx.doi.org/10.1021/ie202547w | Ind. Eng. Chem. Res. 2012, 51, 6270−6274
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Research Note
rice straw; (2) low-fire MAL-treated rice straw treated for 1 h; (3) middle-fire MAL-treated rice straw treated for 1 h; (4) high-fire MAL-treated rice straw treated for 1 h. Specimens were prepared for SEM inspection by sticking the sample on carbon glue followed by Pt plating. 2.6. Enzymatic Hydrolysis. Crude cellulase from Trichoderma viride ZY-1, isolated from soil from the Hefei Dashu Mountain, with 3 IU/mL enzyme solution was used for hydrolysis experiments. The pretreated rice straws at 2% (w/v) solids loading in 0.1 M citrate buffer (pH 4.8) were incubated in flasks in a shaking water bath at 50 °C and 150 rpm for 48 h. The hydrolysis, conducted at a cellulase activity of 50 FPU/g substrate, was initiated by adding 5 mL of crude cellulase. The reaction was monitored by withdrawing samples from the supernatant periodically and measuring the release of soluble reducing sugars by the 3,5-dinitrosalicylic acid (DNS) assay using Dglucose as a standard.11 Total reducing sugars which can form some aldehyde or ketone (including disaccharide, monosaccharide) can be measured by the DNS method. Saccharification rate from pretreated rice straw was calculated as follows:
SR (%) =
RG × 100 PS
Figure 1. IR spectra of rice straw: (A) the original rice straw, (B) lowfire-power MAL-treated for 1 h, (C) medium-fire-power MAL-treated for 1 h, (D) high-fire-power MAL-treated for 1 h.
(1)
of the syringyl and guaiacyl units in lignin, respectively.15,17,18 The significant increase and decrease in their adsorption bands after the MAL treatment indicated that some chemical bonds and groups were broken by the heat effect of microwave, which led to the removal of more of the waxy layer and lignin and the exposure of more polysaccharides with dilute lye treatment. Therefore, the MAL treatment increased the amount of polysaccharides on the rice straw surface and cellulase hydrolysis sites. 3.2. X-ray Pattern. This article studied the effect of original, dilute lye-treated, MAL-treated samples on the crystallinity by X-ray diffraction (XRD) traces. I200 represents both crystalline and amorphous material while IAM represents amorphous material only.10 Therefore, the crystallinity material was the difference in value of I200 and IAM (I200 − IAM); hence the degree of crystallization of rice straw was the ratio of I200 − IAM and I200. Figure 2 shows that crystallinity was greater on the dilute lye-treated sample than original one. Thygesen et al.19
where RG is the dry-weight of reducing sugars in enzyme hydrolysis supernatant, PS is the dry-weight rice straw in pretreated solids 2.7. Yield of Pretreated Straw. Straw quality changes before and after pretreatment was calculated as follows: Y (%) =
W1 × 100 W2
(2)
where W1 is quality of straw before pretreatment and W2 is quality of straw after pretreatment.
3. RESULTS AND DISCUSSION 3.1. FTIR Spectra. A comparison of the spectra in Figure 1 shows there were obvious decreases in the intensity of several typical peaks at 3320, 2916, 2859, 1733, 1648, and 1600 cm−1 of the MAL-treated samples than of those of original rice straw. The two peaks at 2916 and 2859 cm−1 were assigned, respectively, to the asymmetric and symmetric stretching of the CH2-group comprising the majority of the aliphatic fractions of waxes,5,12 and the broad band due to the hydrogen-bonded hydroxyl groups at 3320 cm−1 and the band at 1648 cm−1 were attributable to H−O−H bending of adsorption.13,14 This implied that MAL could effectively remove lipophilic extractives and break hydrogen bonded to the rice straw surface. Maybe one of reasons that more cellulose was exposed was because of the broken hydrogen bond and destroyed surface of rice straw, which was observed by SEM. The infrared bands at 1422 and 1600 cm−1, which were assigned to the methoxyl group in lignin and the aromatic skeletal vibrations and the aromatic skeletal vibrations coupled with C−H in plane deformations, respectively,15 were depressed. Moreover, the peak at 1245 cm−1 was decreased, which is more likely attributed to the C−O stretching of an acetyl group present in the lignin moiety as well as in the hemicellulose,16,17 and the peak at 1160 cm−1 was increased which is more likely assigned to C−O−C connection bridge in hemicelluloses and cellulose and to aromatic C−H deformation
Figure 2. Rice straw X-ray pattern: (a) dilute lye pretreatment on rice straw, (b) MAL pretreatment on rice straw, (c) the original rice straw. 6271
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Figure 3. Rice straw scanning electron microscopy (SEM) images: (A) untreated rice straw; (B) low-fire-power MAL-treated for 1 h, (C) mediumfire-power MAL-treated for 1 h, (D) high-fire-power MAL-treated for 1 h.
have reported that crystallite size can be increased because of the interfibrillar swelling with dilute lye-treatment. At the same, the dilute lye could remove lignin7 and expose more cellulose from the rice straw, and then enhance the relative ratio of cellulose in the dilute lye-treated straw. X-ray diffraction traces of MAL-treated rice straw displayed less crystallinity of the MAL-treated sample than of the dilute lye-treated one because the microwave-assisted treatment could destroy the crystalline of cellulose from rice straw. 3.3. Surface Morphology Observation by Scanning Electron Microscopy (SEM). Figure 3A shows a SEM image of a dried straw raw sample stored outdoors for 3 days. This dried specimen had some dent structure but also maintained plant cell wall composition such as epidermis and vascular bundles, and parenchyma sticking to the bundle surface. Furthermore, the dry MAL-treated sample with the wire of the cellulose and a distorted cell wall structure was shown in Figure 3 panels B−D. The surface morphology of the plant was significantly varied and during the processing of the MALtreated rice straw, dissolution caused the release of characteristic species such as silica, breaking of intramolecular Hbonding of microfibril, and liberation of amorphous pectin, hemicellulose and lignin. The appearance of two kinds of basic plant components including epidermis and vascular bundles was largely changed during low-fire-power MAL and medium-fire-power MAL treatment, respectively. It was clearly seen that concavity was increased on the rice straw (Figure 3B,C), while the wire of the cellulose was increased and even the surface rose up on the high-fire-power MAL-treated rice straw (Figure 3D). It indicated that the liposoluble constituent and lignin were shucked off for a short time from the rice straw, and more
cellulose was exposed with the MAL-treated method. This result explained that the inner space of the rice straw increased due to boil-off and liberation of the vaporization substance or small molecules by heat effect of the microwave, and then more dilute lye could enter into the rice straw and strip more substance. 3.4. Determination of Rice Straw Composition. The composition of the original rice straw sample and different firepower MAL-treated samples is showed in Table 1. The content Table 1. Rice Straw Composition pretreatmenta A B C D
cellulose (%) 30.5 35.4 40.5 46.8
± ± ± ±
0.3 0.5 0.2 0.3
hemicelluloses (%) 21.1 24.6 26.5 23.3
± ± ± ±
0.4 0.5 0.6 0.5
lignin (%) 19.7 11.9 9.7 8.3
± ± ± ±
0.3 0.4 0.6 0.4
a
Pretreatments: A, original rice straw; B, low-fire-power MAL-treated for 1 h; C, medium-fire-power MAL-treated for 1 h; D, high-fire-power MAL-treated for 1 h.
of lignin was removed significantly with the MAL treatment; 56% of the lignin was moved by high-fire-power MAL treatment as compared to that of the original rice straw. This result was consistent with the above discussion. The content of hemicellulose increased in low- and medium-fire power MALtreated rice straw because some lignin and soluble substances were stripped leading to the increase of the relative ratio of hemicelluloses in these treatments, while it reduced in high-firepower MAL-treated rice straw because the structure and connection bonds of hemicellulose were broken, and then hemicellulose was removed. The content of cellulose increased 6272
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with the fire-power MAL-treatment. Our results showed that lignin and partial hemicellulose could be removed effectively and more cellulose was exposed. 3.5. Pretreatment Yield and Saccharification Rate. It was found that the pretreatment yield was decreased while the saccharification rate was increased during the process of highfire-power MAL for 5−120 min in the high-fire-power MALtreated rice straw compared to the original straw (Figure 4).
Research Note
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 0551 2901771. Fax: +86 2904675. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to thank the Hefei University of Technology Center of Forecasting and Analysis for the technology support of the scanning electron microscope and X-ray diffraction meter.
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REFERENCES
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Figure 4. Pretreatment yield and saccharification rate. -▲-, pretreatment yield; -●-, saccharification rate; high-fire-power microwaveassisted 1% dilute lye.
The reason for loss of pretreatment yield was that the waxes and lignin were shucked off, while the MAL-treated straw saccharification rate was increased. This could be explained that it contributes to an increase in the enzyme-action area for cellulose,20 where the rice straw structure was open and more cellulose was exposed in the MAL-treated. Finally, the straw saccharification rate was 86% with high-fire-power MAL-treated for 120 min. Pretreatment yield dramatically declined in the beginning of the MAL treatment, but the increase of saccharification rate was not high. The cause of this phenomenon was that the small molecule or soluble substances were vaporized and liberated initially by the heat effect of the microwave in the MAL treatment; however, the general surface structure of the rice straw changed little, therefore the alteration of the saccharification rate was not highlighted before 20 min of treatment. With the increase of MAL treatment time, dilute lye entered into the rice straw and stripped the lignin and liposoluble constituent, so the pretreatment yield decreased. Simultaneously, MAL treatment exposed more cellulose and increased more enzyme action areas, resulting in the increase of saccharification over time.
4. CONCLUSIONS IR and X-ray showed that lipophilic substances and lignin were shucked off and more cellulose was exposed in a short time with dilute lye in a microwave field. A smooth structure and filamentous cellulose were seen by scanning electron microscopy (SEM) photographs. Key factors for the enzymatic saccharification rate of rice straw was increased with the MALtreated method. However future research on a reasonable microwave reactor design and applications to the pretreatment is still required. 6273
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(18) Fang, J.; Fowler, P.; Tomkinson, J.; Hill, C. Preparation and characterisation of methylated hemicelluloses from wheat straw. Carbohydr. Polym. 2002, 47 (3), 285−293. (19) Thygesen, A.; Oddershede, J.; Lilholt, H.; Thomsen, A. B.; Stahl, K. On the determination of crystallinity and cellulose content in plant fibres. Cellulose 2005, 12 (6), 563−576. (20) McMillan, J. D. Pretreatment of Lignocellulosic Biomass. In Enzymatic Conversion of Biomass for Fuels Production; American Chemical Society: Washington, DC, 1994; Vol. 566, pp 292−324.
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