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Effects of combination of steam explosion and microwave irradiation (SE–MI) pretreatment on enzymatic hydrolysis, sugar yields and structural properties of corn stover
Industrial Crops and Products 42 (2013) 402–408
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Effects of combination of steam explosion and microwave irradiation (SE–MI) pretreatment on enzymatic hydrolysis, sugar yields and structural properties of corn stover Feng Pang, Shulin Xue, Shengshuan Yu, Chao Zhang, Bing Li, Yong Kang ∗ School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 29 March 2012 Received in revised form 2 June 2012 Accepted 9 June 2012 Keywords: Steam explosion (SE) Microwave irradiation (MI) Corn stover Enzymatic hydrolysis Crystallinity
a b s t r a c t A novel process named combination of steam explosion and microwave irradiation (SE–MI) method was investigated and compared with steam explosion (SE) method for pretreating corn stover under 540 W microwave power and 3 min microwave irradiation time. Both of the two pretreatment methods were operated at 170–210 ◦ C for 3–15 min. Results showed that compared to SE process, SE–MI process enhanced the enzymatic hydrolysis yields of both glucose and xylose, and slightly increased the total sugar yield. The maximum glucose yield, xylose yield and total sugar yield of the process were 57.4%, 17.8% and 75.2% (corresponding to 100% maximum total sugar in feedstock), respectively, obtained at 200 ◦ C for 5 min. SE–MI pretreatment showed attractive advantage in inhibiting the increase of biomass crystallinity. The crystallinity following SE–MI pretreatment was 19% lower than that following SE pretreatment at 190 ◦ C for 5 min. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The production of biofuels (e.g. bioethanol) and biobased products from renewable lignocellulosic biomass will promote rural economy, decrease greenhouse gas emissions, and enhance energy security (Sathitsuksanoh et al., 2009). The bioconversion process for bioethanol production from lignocellulosic biomass typically involves the three steps of pretreatment, hydrolysis and fermentation (Ewanick et al., 2007). A pretreatment step is essential to overcome the natural recalcitrance of lignocellulosic biomass to enzymatic hydrolysis to sugars through opening up the lignocellulosic complex and making high sugar yields possible (Mosier et al., 2005). A variety of physical, chemical and biological methods have been assessed for their technical and economical effectiveness at pretreating lignocellulosic residues. Among the different existing pretreatment methods, steam explosion (SE) is one of the most commonly used for fractionation of biomass components. In SE pretreatment, biomass is exposed to pressurized steam followed by rapid reduction in pressure (Ruiz et al., 2008). The process has been shown to be effective in providing a balance between
∗ Corresponding author. Tel.: +86 22 27408813; fax: +86 22 27408813. E-mail addresses: [email protected] (F. Pang), [email protected] (S. Xue), [email protected] (S. Yu), [email protected] (C. Zhang), [email protected] (B. Li), [email protected] (Y. Kang). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.06.016
the effective recovery of the water soluble hemicellulose sugars while increasing the enzyme digestibility of the water insoluble cellulosic fraction (Kumar et al., 2010). A variety of temperatures, retention times, initial moistures and particle sizes have been advocated by many researchers using SE method (Tucker et al., 2003; Cullis et al., 2004; Monavari et al., 2009; Wang et al., 2009). Compared with alternative pretreatment methods, the advantages of SE pretreatment include a significantly lower environmental impact, lower capital investment and less hazardous process chemicals (Li et al., 2001). Microwaves are electromagnetic waves spanning a frequency range from 300 MHz (3 × 108 cycles/s) to 300 GHz (3 × 1011 cycles/s), with most industrial and household microwave processing operating at a frequency of 2.45 GHz. Ooshima et al. (1984) and Azuma et al. (1985) first introduced microwave irradiation for processing cellulosic biomass and demonstrated enhanced cellulose saccharification following microwave treatment. The methods has been used to pretreat various lignocellulosic biomass, such as rice straw (Ooshima et al., 1984; Kitchaiya et al., 2003; Zhu et al., 2006), sugarcane bagasse (Ooshima et al., 1984; Kitchaiya et al., 2003), switchgrass (Keshwani and Cheng, 2010), barley husk (Palmarola-Adrados et al., 2005), softwood (Azuma et al., 1985; Liu et al., 2010) and beech wood (Verma et al., 2011). Shi et al. (2011) summarized literatures on microwave pretreatment of various biomass materials and concluded that sugar yields were enhanced following the pretreatments with just water or with added alkali or acid.
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Both SE pretreatment and microwave pretreatment could remove part of hemicellulose and lignin from biomass, and enhance the cellulose accessibility, therefore increase the enzymatic digestibility of materials at acidic conditions under temperature that higher than 160 ◦ C. Based on the hypothesis that steam and microwave irradiation could have synergistic effect on lignocellulosic biomass pretreatment in acidic conditions under high temperatures, a novel pretreatment process which combined steam explosion and microwave irradiation pretreatment (named SE–MI pretreatment) was proposed to separate lignocellulose components and improve sugar yield. The objective of this work was to evaluate the effectiveness and feasibility of combination of steam explosion and microwave irradiation (SE–MI) pretreatment as a new pretreatment method for lignocellulosic biomass in order to enhance the enzymatic hydrolysis yield and total sugar (glucan and xylan) yield. To accomplish this, corn stover was pretreated by SE–MI or SE method, and the effects of the two pretreatment processes were evaluated and compared on the basis of the sugar yields obtained from pretreatment and subsequent enzymatic hydrolysis step using cellulase and glucosidase. The effects of reaction temperature and reaction time on pretreatment and subsequent enzymatic hydrolysis were also evaluated. Various structural characteristics of raw and pretreated corn stover were also assessed by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analyses. 2. Materials and methods 2.1. Chemicals and materials Corn stover was locally harvested and dried in air, and then chopped into particles (approximately 6–20 mm) by ZC-1 chaffcutter (Qufu S&T Co., Ltd., China) and stored at room temperature in air-tight bags prior to use. The composition of raw corn stover was measured as described in Section 2.3. All chemicals were reagent-grade and purchased from Jiangtian Chemical Co. (Tianjin, China), unless otherwise noted. Celluclast 1.5L cellulase (65 FPU/ml, 12.0 IU/ml) and Novozyme 188 -glucosidase (350 CBU/ml, 32.4 IU/ml) were purchased from Sigma–Aldrich Chemical Co. (Shanghai, China). Enzymes activities were assayed according to the method recommended by Ghose (1987) before they were used. 2.2. Pretreatment SE–MI pretreatment was conducted according to the process flow chart shown in Fig. 1. The details of the process were described by Kang et al. (2010a,b,c). Prior to pretreatment, 100 g (dry weight basis, dwb) corn stover were weighed in clean plastic bags and impregnated by the addition of deionized water to adjust the moisture content of material to 25% (dwb) at room temperature. The bags were sealed and kept for more than 30 min in order to allow the penetration of water into the material. Then all of the material was added into a 15 l reaction vessel via the feed inlet valve. High pressure saturated steam generated by a steam generator (Jinan Sanheng Environmental Protection Heat Energy Equipment Co., Ltd., China) was injected into the vessel rapidly via the steam inlet valve until the target temperature was approached. Next, microwave with frequency of 2.45 GHz supplied by a microwave generator (Guangzhou Kewei Microwave Equipment Co., Ltd., China) was imported into the reaction vessel via two rectangular waveguides from both sides of the vessel. Ceramic material and PTFE (polytetrafluorethylene) sealing gasket were used in the microwave feed port of the vessel, which could prevent steam in the vessel from permeating into the
Fig. 1. The schematic flow chart of SE–MI process used in the present study.
waveguides (which might cause damage to the microwave generator), and ensure the good performance of waveguides under high pressure. Reaction temperature (◦ C), steam reaction time (min), microwave power (W) and microwave irradiation time (min) were monitored and controlled during pretreatment process. (In this study, the microwave irradiation power and microwave irradiation time were fixed at 540 W and 3 min, respectively, for all the SE–MI experiments.) Microwave irradiation treatment was terminated by interrupting running of the microwave generator. Steam treatment was then ended by opening the pneumatic ball valve rapidly and the treated slurry was immediately ejected into a 140 l collection vessel under differential pressure. The treated slurry in the collection vessel was discharged via the feed outlet valve and collected, and then vacuum filtered using a 500 mesh (25 m pore size) filter cloth to separate solids from the slurry. The solids were washed with 20 ◦ C tap water (1:20, w/v). The washing water and the hydrolyzate were uniform mixed, and the mixture was analyzed using HPLC. SE pretreatment process was similar to SE–MI pretreatment process, but microwave was not imported into the reaction vessel during the process. The pretreatment severity factor (Overend and Chornet, 1987) that incorporated reaction temperature T (◦ C) and reaction time t (min) was defined as lg R0 , where R0 = t × exp[(T − 100)/14.75]. For all the SE–MI experiments, microwave power and microwave irradiation time were the same, so the microwave effect was not accounted for when calculating the pretreatment severity factor.
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2.3. Chemical analysis The compositional analysis of the untreated corn stover and pretreated solids were performed according to the procedures of NREL LAPs (Sluiter et al., 2006, 2008). Absorbance data of acid soluble lignin was taken at 243 nm using UV-7500 UV–Vis spectrophotometer (Shanghai Tianmei Co., Ltd., China). Glucose and xylose concentrations of hydrolyzates after pretreatment and enzymatic hydrolysis were quantified by Waters 1525 HPLC system (Waters Co., USA) equipped with an Aminex HPX-87P column (300 mm × 7.8 mm; Bio-Rad, USA) and refractive index detection. Concentrations of furfural, 5-hydroxymethyl furfural (5-HMF), acetic acid and formic acid of hydrolyzates from pretreatment were quantified by Waters 1525 HPLC system equipped with an Aminex HPX-87H column (300 mm × 7.8 mm; Bio-Rad, USA) and refractive index detection.
2.4. Enzymatic hydrolysis The washed pretreated residue was enzymatically hydrolyzed using a mixture of Celluclast 1.5L cellulase (60 FPU/g glucan) and Novozyme 188 -glucosidase (60 CBU/g glucan). The mixture contained 1% glucan concentrations of pretreated corn stover in 10 ml 0.05 M citrate buffer (pH 4.8) with 200 mg/l sodium azide. The reaction was performed at 50 ◦ C using a thermostatic shaker air bath (LRH-250-ZII; Guangzhou Medical Appliance Co., China) set at 150 rpm. Substrate blanks without enzyme and enzyme blanks without substrate were run in parallel. Samples were taken at 72 h, followed by centrifugation at 6000 rpm for 10 min, and the release of glucose and xylose was measured by HPLC as described in Section 2.3. All the experiments were performed in triplicate, with the average value being reported.
2.5. Crystallinity measurement X-ray powder diffraction patterns of both the untreated and pretreated corn stover were obtained using a X-ray diffractometer (Rigaku DMAX 2500 V/pc, Japan) in conjunction with a Cu K␣ radiation source ( = 0.154 nm) operated at 30 kV. Samples were scanned at a speed of 1◦ /min from 10◦ to 30◦ with a step size of 0.02◦ . Biomass crystallinity, as expressed by crystallinity index (CrI), was determined from XRD data and calculated using the formula CrI (%) = (I002 − Iam )/I002 × 100 (Segal et al., 1959), where I002 is the intensity for the crystalline portion of biomass (cellulose) at about 2 = 22.5◦ , and Iam is the peak for the amorphous portion (i.e. cellulose, hemicellulose and lignin) at about 2 = 15.5◦ . Crystallinity index (CrI) of biomass was also measured by Fourier transform infrared spectroscopy (FTIR), and expressed as the absorbance ratio of the bands at 1370 and 2900 cm−1 (Nelson and O’Connor, 2003).
2.6. FTIR spectroscopic analysis To investigate and quantify chemical changes in pretreated biomass, Fourier transform infrared spectroscopy (FTIR) was conducted with a Nicolet 560 ESP FTIR spectrometer (Thermal Nicolet Co., USA). Sample spectra were obtained using 32 scans over the range of 400–4000 cm−1 with a spectral resolution of 2 cm−1 .
2.7. Sugar yield calculations The enzymatic hydrolysis yields of glucose, xylose and total sugars were calculated by the following equations in this work:
Enzymatic hydrolysis yield of glucose (%) = [(glucose (g) released in enzymatic hydrolysis)/(total glucose (g) in the pretreated biomass)] × 100 Enzymatic hydrolysis yield of xylose (%) = [(xylose (g) released in enzymatic hydrolysis)/(total xylose (g) in the pretreated biomass)] × 100 Enzymatic hydrolysis yield of total sugars (%) = [(total sugar (g) released in enzymatic hydrolysis)/(total sugar (g) in the pretreated biomass)] × 100 The glucose, xylose and total sugar yields following pretreatment and enzymatic hydrolysis were calculated by the following equations: Glucose yield (%) = [(glucose (g) released in pretreatment and enzymatic hydrolysis)/(potential total sugar (g) in the feedstock)] × 100 Xylose yield (%) = [(xylose (g) released in pretreatment and enzymatic hydrolysis)/(potential total sugar (g) in the feedstock)] × 100 Total sugar yield (%) = [(total sugar (g) released in pretreatment and enzymatic hydrolysis)/(potential total sugar (g) in the feedstock)] × 100 3. Results and discussion 3.1. Compositional analysis of pretreated solids and hydrolyzate The raw corn stover used in this study contained about 30.3% glucan, 13.3% xylan, 0.4% arabinose, 17.2% Klason lignin, 3.0% acid-soluble lignin, 2.2% ash and 33.6% extractives. The maximum potential total sugar yield was calculated as 48.8 g per 100 g dry feedstock. The maximum yield of glucose was 69.0% and the maximum yield of xylose was 31.0%, for a sum total sugar of 100% in the feedstock. As shown in Table 1, composition of solid residues and hydrolyzate following pretreatment were reported. SE-1 and SE-4 represented SE experiments at 170 ◦ C and 190 ◦ C, respectively, for 5 min, and could be compared with SE–MI-1 and SE–MI-4 which represented SE–MI experiments at the same reaction temperature and reaction time, respectively. The solid recovery of corn stover was an important index for the effectiveness of its pretreatment. Solid recovery was the percentage content of pretreated and washed material in 100 g raw feedstock. The solid recovery of materials pretreated by SE–MI method was about 5% lower than that pretreated by SE method, which might be resulted by some components of biomass that converted to soluble chemicals under microwave irradiation. About 38.3–46.7 g solids could be recovered from 100 g feedstocks after SE–MI pretreatment. The solid recovery was gradually decreased with pretreatment severity increasing, which was resulted by glucan and xylan converted into soluble oligomeric and monomeric sugars and its degradation products. Compared to that of SE pretreatment, percent of glucan and xylan corresponding to pretreated solids were both lower following SE–MI pretreatment, while percent of lignin corresponding to pretreated solids was slightly higher. However, because the solid recovery for SE–MI pretreatment was lower than that for SE pretreatment, percent of glucan, xylan and lignin corresponding to feedstocks were all lower following SE–MI pretreatment than that following SE pretreatment. For SE–MI pretreatment, with reaction temperature and reaction time rising, percent of glucan corresponding to feedstocks was kept at about 22.8–23.9%. Meanwhile, percent of lignin corresponding to feedstocks was slightly increased from 9.4% to 10.8%, and percent of xylan corresponding to feedstocks was decreased from 7.7% to 2.1%. As SE–MI pretreatment was operated at acidic
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Table 1 Composition of solid residues and hydrolyzate following pretreatment at the conditions noted. No.a
Tb (◦ C)
Pb (MPa)
UT SE-1 SE-4 SE–MI-1 SE–MI-2 SE–MI-3 SE–MI-4 SE–MI-5 SE–MI-6 SE–MI-7 SE–MI-8
– 170 190 170 180 190 190 190 190 200 210
– 0.69 1.15 0.69 0.90 1.15 1.15 1.15 1.15 1.45 1.80
tb (min)
– 5 5 5 5 3 5 10 15 5 5
lg R0
Solid recovery (%)
– 2.76 3.35 2.76 3.05 3.13 3.35 3.65 3.83 3.64 3.94
100.0 51.6 45.8 46.7 42.2 42.9 41.0 39.6 38.7 39.7 38.3
Solid fractionc (%)
Liquid fractiond (g)
Glucan
Xylan
Lignin
Glucose
Xylose
30.3 28.0 (54.3) 27.5 (60.1) 23.9 (51.1) 22.8 (54.1) 23.8 (55.5) 23.5 (57.4) 23.7 (59.8) 23.4 (60.3) 23.8 (60.0) 23.2 (60.5)
13.3 9.1 (17.6) 5.7 (12.4) 7.7 (16.4) 5.3 (12.5) 5.3 (12.3) 4.4 (10.8) 3.3 (8.5) 2.4 (6.2) 3.2 (8.0) 2.1 (5.5)
17.2 10.0 (19.4) 10.5 (22.8) 9.4 (20.1) 9.5 (22.6) 9.7 (22.6) 9.8 (24.0) 10.0 (25.3) 10.5 (27.0) 10.1 (25.5) 10.8 (28.1)
– 3.05 (6.25) 4.69 (9.61) 3.16 (6.48) 4.02 (8.24) 4.15 (8.51) 5.17 (10.60) 5.95 (12.20) 6.89 (14.12) 6.21 (12.73) 6.52 (13.37)
– 3.53 (7.24) 5.37 (11.01) 3.83 (7.85) 5.18 (10.62) 5.28 (10.82) 5.83 (11.95) 6.82 (13.98) 5.61 (11.50) 6.84 (14.02) 4.77 (9.78)
Microwave power and microwave irradiation time were 540 W and 3 min, respectively, for all the SE–MI experiments. a UT, SE and SE–MI represent no pretreatment, steam explosion pretreatment, and combination of steam explosion and microwave irradiation pretreatment, respectively. b T, reaction temperature; P, pressure of saturated steam (gauge pressure); t, reaction time. c Dates before and in the parentheses represent percent contents of glucan, xylan and lignin in untreated corn stover and in pretreated solids, respectively. d Dates before and in the parentheses represent the amount of glucose and xylose (g) in 100 g feedstock and in 100 g total sugars of feedstock, respectively.
conditions, most xylan was dissolved or removed during the process. However, most of lignin could not be removed following the pretreatment, especially under high pretreatment severity, the reason might be that condensation reaction of lignin prevailed under the pretreatment conditions, and resulted in lignin content stabilized (Ramos, 2003). During SE or SE–MI pretreatment, many monosaccharides, oligosaccharides, and by-products such as organic acids and furans were dissolved in liquid. Compared to SE pretreatment, SE–MI pretreatment released more glucose, xylose and inhibitors into hydrolyzate (Table 1 and Fig. 2). Both the glucose and xylose yields increased with reaction time and reaction temperature rising, but a decrease of xylose was observed when the reaction temperature was higher than 200 ◦ C. The yields of furfural and HMF were an indication of the severity of pretreatment. As shown in Fig. 2, increased time and temperature resulted in furfural and HMF formation rising during SE–MI pretreatment. At the most severe condition (210 ◦ C for 5 min), the yields were 0.46 and 1.06 g per 100 g dry feedstock for HMF and furfural, respectively, while at the least severe condition (170 ◦ C for 5 min), the yields were only 0.07 and 0.23 g per 100 g dry feedstock, respectively. More degradation of pentose under severe conditions also increased the yield of acetic acid and formic acid. The results were in agreement with the findings of Linde et al. (2008) by steam pretreatment with dilute H2 SO4 -impregnation.
Fig. 2. Yields of inhibitors resulting from SE (SE-1 and SE-4) and SE–MI (from SE–MI1 to SE–MI-8) pretreatment process.
3.2. Enzymatic hydrolysis and sugar yields The water-insoluble pretreated residue was submitted to enzymatic hydrolysis by a cellulase complex (Celluclast 1.5L) supplemented with -glucosidase (Novozyme 188). For comparison purposes, enzymatic hydrolysis was also carried out on raw material not subjected to any pretreatment. As shown in Fig. 3, the enzymatic hydrolysis yields of glucose and xylose for raw material were both lower than 20%. Materials treated by SE–MI method gave higher enzymatic hydrolysis yields than that treated by SE method both at 170 ◦ C and 190 ◦ C for 5 min. For instance, when it was treated at 190 ◦ C for 5 min, the enzymatic hydrolysis yields of glucose and xylose by SE–MI method were 77.9% and 55.8%, which were 12% and 7.2% higher than that by SE method, respectively. For SE–MI pretreatment, when the pretreatment severity was lower than 3.64–3.65, enzymatic hydrolysis yields of glucose increased with reaction time and reaction temperature during SE–MI pretreatment, but a higher pretreatment severity of more than 3.64–3.65 could resulted in the yields decreasing. The maximum enzymatic hydrolysis yields of glucose were 82.4% and 82.8%, which were obtained at 190 ◦ C for 10 min and 200 ◦ C for 5 min,
Fig. 3. Enzymatic hydrolysis yields of glucose, xylose and total sugar for SE (SE-1 and SE-4) and SE–MI (from SE–MI-1 to SE–MI-8) pretreatment process after enzymatic hydrolysis for 72 h. UT represents untreated corn stover. Substrates were suspended in 50 mM Na–citrate buffer (pH 4.8) at 1% glucan concentrations and hydrolyzed by cellulases (Celluclast 1.5L) and -glucosidase (Novozymes 188) at a ratio of 60 FPU/g glucan to 60 CBU/g glucan at 50 ◦ C and 150 rpm for 72 h. All the experiments were performed in triplicate, with the average value being reported.
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Fig. 4. Glucose, xylose and total sugar yields for SE (SE-1 and SE-4) and SE–MI (from SE–MI-1 to SE–MI-8) pretreatment process after pretreatment (PT) and enzymatic hydrolysis (EH) from corn stover feedstock. UT represents untreated corn stover.
respectively. Enzymatic hydrolysis yields of xylose for all the samples were lower than that of glucose. The maximum enzymatic hydrolysis yield of xylose was 62.0%, which were achieved at 190 ◦ C for 3 min. When the reaction temperature was above 180 ◦ C, the xylose yield decreased with increasing reaction temperature and reaction time. As presented in Sections 3.1 and 3.2, more inhibitors were generated at elevated temperature, and the lignin contents in pretreated materials were also increased, which could play a negative role in xylose hydrolysis and thus reduced the xylose yields. The total sugar yield made by combining the total sugar conversion yield during pretreatment and enzymatic hydrolysis was an index of pretreatment efficiency for ethanol production (Chen et al., 2011). As shown in Fig. 4, the total sugar yields of materials following SE or SE–MI pretreatment were far higher than that of raw material. Compared to SE pretreatment, SE–MI pretreatment resulted in lower percent of glucan and xylan in pretreated solids, but caused higher enzymatic hydrolysis yields of glucose and xylose. As a result, the total sugar yields of SE–MI pretreatment were slightly higher (about more than 1.5%) than that of SE pretreatment. For SE–MI pretreatment, with reaction temperature and reaction time rising, the total sugar yields were increased, but then decreased when reaction time was more than 10 min or reaction temperature was higher than 200 ◦ C. When the material was treated at 200 ◦ C for 5 min (the pretreatment severity factor was 3.64), the maximum glucose yield, xylose yield and total sugar yield were 57.4%, 17.8% and 75.2%, respectively, corresponding to 28.0 g glucose, 8.7 g xylose and 36.7 g total sugar were generated from 100 g raw feedstock. This reaction condition could be regarded as the optimum SE–MI pretreatment conditions, when the microwave power and microwave irradiation time were 540 W and 3 min, respectively. The mass balance for corn stover at optimum SE–MI pretreatment conditions was shown in Fig. 5. When corn stover was pretreated by SE–MI method at 200 ◦ C for 5 min, 25.28 g glucan and 7.58 g xylan were released during pretreatment and enzymatic hydrolysis. Under this condition, in order to produce a liter of ethanol, 4.63 g dry material was required. During SE–MI pretreatment, about 0.4–0.6 l water was consumed corresponding to 0.1 kg dry material, then 18.52–27.78 l water was required to produce a liter of ethanol. 3.3. Crystallinity analysis The biomass crystallinity index (CrI) was believed to significantly affect enzymatic saccharification of glucan (Kumar et al.,
2009). Crystallinity analysis of the untreated and pretreated corn stover by XRD and FTIR method was done to investigate the crystalline behavior of cellulose (Table 2). The crystallinity of the biomass following SE or SE–MI pretreatment was much higher than that of untreated corn stover. The reason might be that non-cellulosic polysaccharides were removed and amorphous cellulose was dissolved, which resulted in the increase of cellulose crystallinity. The increase of crystallinity after low pH pretreatment has also been reported by Kumar et al. (2009). The crystallinity of biomasses pretreated by SE–MI method was much lower than that pretreated by SE method, especially at high reaction temperature. Compared to that of SE pretreatment at 170 ◦ C and 190 ◦ C for 5 min, after SE–MI pretreatment at the same reaction temperature and reaction time, the crystallinity measured by XRD method was decreased by about 5% and 19%, and the crystallinity measured by FTIR method was decreased by about 0.08 and 0.24, respectively. The reason could be interpreted by the structure of lignocellulose and the unique characterization of microwave irradiation. As cellulosic biomass was a highly heterogeneous complex of polymers, primarily lignin, cellulose, and hemicelluloses, some of the polymers might be coupling and reacting with carboxylic acid catalyst that released from water by auto-hydrolysis of hemicelluloses during pretreatment. The high water content of cellulosic biomass was also susceptible to microwave heating. Considering all these factors, application of microwave irradiation to lignocellulose might selectively heated the more polar part and created “hot spots” within heterogeneous materials. This unique heating feature could possibly resulted in an “explosion” effect among the particles and improved disruption of the recalcitrant structures of lignocellulose (Shi et al., 2011), therefore the crystal region of cellulose could be destroyed and resulted in the decline of crystallinity. For SE–MI pretreatment, the biomass crystallinity was significantly affected by reaction time and reaction temperature. Longer reaction time and higher reaction temperature resulted in higher crystallinity. When the reaction time was increased from 3 min to 15 min at 190 ◦ C, the biomass crystallinity measured by XRD method gradually increased from 36.0% to 43.6%, and the biomass crystallinity measured by FTIR method raised from 1.11 to 1.21. The biomass crystallinity was changed more significantly with reaction temperature rising. When the reaction temperature was increased from 170 ◦ C to 210 ◦ C for 5 min reaction time, the biomass crystallinity measured by XRD method gradually increased from 34.1% to 45.9%, and the biomass crystallinity measured by FTIR method raised from 1.07 to 1.25. However, the observation was inconsistent with the research results of Liu et al. (2009) by FeCl3 pretreatment, which showed that the crystallinity decreased slightly when pretreatment temperature increased from 140 ◦ C to 180 ◦ C. The inconsistency could be attributed to the different pretreatment methods and different pretreatment conditions the two groups used. 3.4. FTIR analysis FTIR spectroscopy was an analytical method that was frequently used to investigate the structure of constituents and chemical changes that occurred during the pretreatment of lignocellulosic biomass (Liu et al., 2009). The FTIR spectra of raw corn stover and solid residues following SE or SE–MI pretreatment were shown in Fig. 6. The peak at 900 cm−1 was characteristic of -glycosidic linkages. This peak existed in feedstock, as well as in corn stover pretreated by SE or SE–MI method, which demonstrated that most of -glycosidic linkages between the sugar units in cellulose and hemicellulose were preserved during pretreatment. The prominent bands at 1200–1000 cm−1 were typically related to the
F. Pang et al. / Industrial Crops and Products 42 (2013) 402–408
Reaction temperature : 200 Reaction time : 5 min Microwave power : 540 W Microwave irradiation time: 3 min
Corn stover 100 g dry weight
SE-MI Pretreatment
Enzymes 60 FPU/g glucan 60 CBU /g glucan
41.5 g dry solids
39.7 g dry solids
Washing Solids 23.8 g Glucan 3.2 g Xylan 12.7 g Others
30.3 g Glucan 13.3 g Xylan 0.4 g Arabinan 17.2 g Klason lignin 3.0 g Acid-soluble lignin 2.2 g ash 33.6 g extractives
407
Liquid
Enzymatic Hydrolysis
17.9 g dry solids
Liquid
Solids 4.01 g Glucan 1.54 g Xylan 12.35 g Others
21.88 g Glucose 1.77 g Xylose
6.21 g Glucose 6.84 g Xylose
Fig. 5. Mass balance for corn stover during SE–MI pretreatment (SE–MI-7) and 72 h enzymatic hydrolysis.
Table 2 Crystallinity index (CrI) changes of corn stover following SE (SE-1 and SE-4) and SE–MI (from SE–MI-1 to SE–MI-8) pretreatment process. No.a
Tb (◦ C)
Pb (MPa)
UT SE-1 SE-4 SE–MI-1 SE–MI-2 SE–MI-3 SE–MI-4 SE–MI-5 SE–MI-6 SE–MI-7 SE–MI-8
– 170 190 170 180 190 190 190 190 200 210
– 0.69 1.15 0.69 0.90 1.15 1.15 1.15 1.15 1.45 1.80
a b c d
tb (min) – 5 5 5 5 3 5 10 15 5 5
lg R0
CrIc (%)
CrId A1370/A2900
– 2.76 3.35 2.76 3.05 3.13 3.35 3.65 3.83 3.64 3.94
29.8 40.2 57.6 35.0 37.1 36.0 38.9 40.3 43.6 41.4 45.9
0.95 1.15 1.38 1.07 1.12 1.11 1.14 1.16 1.21 1.18 1.25
UT, SE and SE–MI represent no pretreatment, steam explosion pretreatment, and combination of steam explosion and microwave irradiation pretreatment, respectively. T, reaction temperature; P, pressure of saturated steam (gauge pressure); t, reaction time. Crystallinity index (CrI) measured by XRD method. Crystallinity index (CrI) measured by FTIR method.
that of untreated, suggested that SE–MI pretreatment increased the relative amount of lignin in the pretreated solid as a result of hemicellulose removal (Liu et al., 2009) and/or re-deposition of released lignin on the surface of solids.
3.5. Economic analysis of SE–MI process
Fig. 6. FTIR spectra of untreated (UT) corn stover and solid residues following SE (SE-1 and SE-4) and SE–MI pretreatment (SE–MI-1 and SE–MI-4).
structural features of cellulose and hemicelluloses. The vibrations of these bands overlapped the C–O–H stretching of primary and secondary alcohols at 1060 cm−1 , C–O–C glycosidic bond stretching at 1160 cm−1 and C–O–C ring skeletal vibration at 1100 cm−1 . After SE or SE–MI pretreatment, the adsorption peaks of these bands were appeared or enhanced, which suggested that the cellulose content in pretreated solid residue increased because the hemicelluloses fraction in raw materials was released by acid hydrolytic reaction (Hsu et al., 2010). Peak at 1510 cm−1 that related to aromatic ring stretching in lignin was stronger in spectra of pretreated solids than
To evaluate the economic performance of SE–MI process at optimum conditions, the cost of sugars was estimated by dividing the additional yield of sugars (grams of sugar per kg of biomass) obtained by SE–MI processing by microwave power cost (540 W microwave power multiplied by 3 min microwave irradiation time, and then multiplied by the cost of electricity during pretreatment) and electricity consumption of steam generator (0.42 kWh multiplied by the cost of electricity during pretreatment). At present, the cost of electricity in Tianjin was about $0.076 US/kWh. When corn stover was pretreated by SE–MI method at 200 ◦ C for 5 min, 0.367 kg sugars per kg of raw material was achieved, so the cost of sugars was calculated as follows.
(0.54 kW × 0.05 h + 0.42 kWh) × $0.076 US/kWh/0.367 kg of sugars = $0.093 US/kg of sugars.
As the cost of sugars ($0.093 US/kg of sugars) was much lower than $0.30 US/kg of sugars, so the process may be economically viable. However, further evaluation is needed to verify the economic feasibility of the new process.
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4. Conclusions Overall, compared with SE pretreatment, the SE–MI pretreatment process has attractive advantages in improving enzymatic hydrolysis yields and sugar yields. When the microwave power and microwave irradiation time were 540 W and 3 min, respectively, the maximum glucose yield, xylose yield and total sugar yield of the process were 57.4%, 17.8% and 75.2% (corresponding to 100% maximum total sugar in feedstock), respectively, obtained at 200 ◦ C for 5 min. SE–MI pretreatment also showed attractive advantage in inhibiting the increase of biomass crystallinity. The crystallinity following SE–MI pretreatment was 19% lower than that following SE pretreatment at 190 ◦ C for 5 min. However, further evaluation is needed to verify the economic feasibility of the new process. In addition, the effect of microwave power and microwave irradiation time on SE–MI pretreatment and subsequent enzymatic hydrolysis has been also investigated (Pang et al., 2012). As microwave pretreatment performed efficiently at acidic conditions, lower pH could be more favorable for SE–MI pretreatment. Further research could focus on evaluating the effectiveness and feasibility of SE–MI pretreatment with acid catalyst (such as dilute sulfuric acid, SO2 or other inorganic or organic acids) added at relatively low temperature, and that of SE–MI pretreatment of more recalcitrant biomass, such as softwood, at elevated temperature. Acknowledgement Financially support provided by the National Key Technology R&D Program of China during “the 11th Five-Year Plan” (Grant No. 2007BAD42B03) is greatly acknowledged. References Azuma, J., Higashino, J., Asai, T., Koshijima, T., 1985. Microwave irradiation of lignocellulosic materials. IV. Enhancement of enzymatic susceptibility of microwave-irradiated softwoods. Wood Res. 71, 13–24. Chen, W.H., Pen, B.L., Yu, C.T., Hwang, W.S., 2011. Pretreatment efficiency and structural characterization of rice straw by an integrated process of dilute-acid and steam explosion for bioethanol production. Bioresour. Technol. 102, 2916–2924. Cullis, I.F., Saddler, J.N., Mansfield, S.D., 2004. Effect of initial moisture content and chip size on the bioconversion efficiency of softwood lignocellulosics. Biotechnol. Bioeng. 85, 413–421. Ewanick, S.M., Bura, R., Saddler, J.N., 2007. Acid-catalyzed steam pretreatment of Lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol. Biotechnol. Bioeng. 98, 737–746. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Hsu, T.C., Guo, G.L., Chen, W.H., Hwang, W.S., 2010. Effect of dilute acid pretreatment of rice straw on structural properties and enzymatic hydrolysis. Bioresour. Technol. 101, 4907–4913. Kang, Y., Pang, F., Yu, S.S., Zhang, C., 2010a. A method by utilize combination of steam explosion and microwave irradiation pretreatment to realize cellulose decrystallization of plant stalk. Chinese Patent ZL 200910069710.9. Kang, Y., Yu, S.S., Pang, F., Zhang, C., 2010b. A set of equipment that utilize combination of steam explosion and microwave irradiation pretreatment to realize cellulose decrystallization of plant stalk. Chinese Patent ZL 200920097742.5. Kang, Y., Yu, S.S., Pang, F., Zhang, C., 2010c. A structure form of microwave inlet which is surround by saturation vapour with high temperature and high pressure. Chinese Patent ZL 200920098491.2. Keshwani, D.R., Cheng, J.J., 2010. Modeling changes in biomass composition during microwave-based alkali pretreatment of switchgrass. Biotechnol. Bioeng. 105, 88–97. Kitchaiya, P., Intanakul, P., Krairiksh, M., 2003. Enhancement of enzymatic hydrolysis of lignocellulosic wastes by microwave pretreatment under atmosphericpressure. J. Wood Chem. Technol. 23, 217–225.
Kumar, L., Chandra, R., Chung, P.A., Saddler, J., 2010. Can the same steam pretreatment conditions be used for most softwoods to achieve good, enzymatic hydrolysis and sugar yields? Bioresour. Technol. 101, 7827–7833. Kumar, R., Mago, G., Balan, V., Wyman, C.E., 2009. Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies. Bioresour. Technol. 100, 3948–3962. Li, J., Lennholm, H., Henriksson, G., Gellerstedt, G., 2001. Bio-refinery of lignocellulosic materials for ethanol production. II. Fundaments and strategic design of steam explosion. In: Kyritsis, S., Beenackers, A.A.C.M., Helm, P., Grassi, A., Chiaramonti, D. (Eds.), Proceedings of the first World Conference on Biomass for Energy and Industry, vol. 1. , pp. 767–770. Linde, M., Jakobsson, E.L., Galbe, M., Zacchi, G., 2008. Steam pretreatment of dilute H2 SO4 -impregnated wheat straw and SSF with low yeast and enzyme loadings for bioethanol production. Biomass Bioenergy 32, 326–332. Liu, J., Takada, R., Karita, S., Watanabe, T., Honda, Y., Watanabe, T., 2010. Microwave assisted pretreatment of recalcitrant softwood in aqueous glycerol. Bioresour. Technol. 101, 9355–9360. Liu, L., Sun, J., Li, M., Wang, S., Pei, H., 2009. Enhanced enzymatic hydrolysis and structural features of corn stover by FeCl3 pretreatment. Bioresour. Technol. 100, 5853–5858. Monavari, S., Galbe, M., Zacchi, G., 2009. Impact of impregnation time and chip size on sugar yield in pretreatment of softwood for ethanol production. Bioresour. Technol. 100, 6312–6316. Mosier, N., Wyman, C.E., Dale, B.E., Elander, R.T., Lee, Y.Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686. Nelson, M.L., O’Connor, R.T., 2003. Relation of certain infrared bands to cellulose crystallinity and crystal lattice type. Part II. A new infrared ratio for estimation of crystallinity in celluloses I and II. J. Appl. Polym. Sci. 8, 1325–1341. Ooshima, H., Aso, K., Harano, Y., Yamamoto, T., 1984. Microwave treatment of cellulosic materials for their enzymatic-hydrolysis. Biotechnol. Lett. 6, 289–294. Overend, R.P., Chornet, E., 1987. Fractionation of lignocellulosics by steam aqueous pretreatments. Philos. Trans. R. Soc. Lond. A 321, 523–536. Palmarola-Adrados, B., Galbe, M., Zacchi, G., 2005. Pretreatment of barley husk for bioethanol production. J. Chem. Technol. Biotechnol. 80, 85–91. Pang, F., Xue, S.L., Yu, S.S., Zhang, C., Li, B., Kang, Y., 2012. Effects of microwave power and microwave irradiation time on pretreatment efficiency and characteristics of corn stover using combination of steam explosion and microwave irradiation (SE–MI) pretreatment. Bioresour. Technol. 118, 111–119. Ramos, L.P., 2003. The chemistry involved in the steam treatment of lignocellulosic materials. Quim. Nova 26, 863–871. 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Influence of steaming explosion time on the physic-chemical properties of cellulose from Lespedeza stalks (Lespedeza crytobotrya). Bioresour. Technol. 100, 5288–5294. Zhu, S.D., Wu, Y.X., Yu, Z.N., Zhang, X., Li, H., Gao, M., 2006. The effect of microwave irradiation on enzymatic hydrolysis of rice straw. Bioresour. Technol. 97, 1964–1968.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Effects of combination of steam explosion and microwave irradiation (SE–MI) pretreatment on enzymatic hydrolysis, sugar yields and structural properties of corn stover Feng Pang, Shulin Xue, Shengshuan Yu, Chao Zhang, Bing Li, Yong Kang ∗ School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
a r t i c l e
i n f o
Article history: Received 29 March 2012 Received in revised form 2 June 2012 Accepted 9 June 2012 Keywords: Steam explosion (SE) Microwave irradiation (MI) Corn stover Enzymatic hydrolysis Crystallinity
a b s t r a c t A novel process named combination of steam explosion and microwave irradiation (SE–MI) method was investigated and compared with steam explosion (SE) method for pretreating corn stover under 540 W microwave power and 3 min microwave irradiation time. Both of the two pretreatment methods were operated at 170–210 ◦ C for 3–15 min. Results showed that compared to SE process, SE–MI process enhanced the enzymatic hydrolysis yields of both glucose and xylose, and slightly increased the total sugar yield. The maximum glucose yield, xylose yield and total sugar yield of the process were 57.4%, 17.8% and 75.2% (corresponding to 100% maximum total sugar in feedstock), respectively, obtained at 200 ◦ C for 5 min. SE–MI pretreatment showed attractive advantage in inhibiting the increase of biomass crystallinity. The crystallinity following SE–MI pretreatment was 19% lower than that following SE pretreatment at 190 ◦ C for 5 min. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The production of biofuels (e.g. bioethanol) and biobased products from renewable lignocellulosic biomass will promote rural economy, decrease greenhouse gas emissions, and enhance energy security (Sathitsuksanoh et al., 2009). The bioconversion process for bioethanol production from lignocellulosic biomass typically involves the three steps of pretreatment, hydrolysis and fermentation (Ewanick et al., 2007). A pretreatment step is essential to overcome the natural recalcitrance of lignocellulosic biomass to enzymatic hydrolysis to sugars through opening up the lignocellulosic complex and making high sugar yields possible (Mosier et al., 2005). A variety of physical, chemical and biological methods have been assessed for their technical and economical effectiveness at pretreating lignocellulosic residues. Among the different existing pretreatment methods, steam explosion (SE) is one of the most commonly used for fractionation of biomass components. In SE pretreatment, biomass is exposed to pressurized steam followed by rapid reduction in pressure (Ruiz et al., 2008). The process has been shown to be effective in providing a balance between
∗ Corresponding author. Tel.: +86 22 27408813; fax: +86 22 27408813. E-mail addresses: [email protected] (F. Pang), [email protected] (S. Xue), [email protected] (S. Yu), [email protected] (C. Zhang), [email protected] (B. Li), [email protected] (Y. Kang). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.06.016
the effective recovery of the water soluble hemicellulose sugars while increasing the enzyme digestibility of the water insoluble cellulosic fraction (Kumar et al., 2010). A variety of temperatures, retention times, initial moistures and particle sizes have been advocated by many researchers using SE method (Tucker et al., 2003; Cullis et al., 2004; Monavari et al., 2009; Wang et al., 2009). Compared with alternative pretreatment methods, the advantages of SE pretreatment include a significantly lower environmental impact, lower capital investment and less hazardous process chemicals (Li et al., 2001). Microwaves are electromagnetic waves spanning a frequency range from 300 MHz (3 × 108 cycles/s) to 300 GHz (3 × 1011 cycles/s), with most industrial and household microwave processing operating at a frequency of 2.45 GHz. Ooshima et al. (1984) and Azuma et al. (1985) first introduced microwave irradiation for processing cellulosic biomass and demonstrated enhanced cellulose saccharification following microwave treatment. The methods has been used to pretreat various lignocellulosic biomass, such as rice straw (Ooshima et al., 1984; Kitchaiya et al., 2003; Zhu et al., 2006), sugarcane bagasse (Ooshima et al., 1984; Kitchaiya et al., 2003), switchgrass (Keshwani and Cheng, 2010), barley husk (Palmarola-Adrados et al., 2005), softwood (Azuma et al., 1985; Liu et al., 2010) and beech wood (Verma et al., 2011). Shi et al. (2011) summarized literatures on microwave pretreatment of various biomass materials and concluded that sugar yields were enhanced following the pretreatments with just water or with added alkali or acid.
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Both SE pretreatment and microwave pretreatment could remove part of hemicellulose and lignin from biomass, and enhance the cellulose accessibility, therefore increase the enzymatic digestibility of materials at acidic conditions under temperature that higher than 160 ◦ C. Based on the hypothesis that steam and microwave irradiation could have synergistic effect on lignocellulosic biomass pretreatment in acidic conditions under high temperatures, a novel pretreatment process which combined steam explosion and microwave irradiation pretreatment (named SE–MI pretreatment) was proposed to separate lignocellulose components and improve sugar yield. The objective of this work was to evaluate the effectiveness and feasibility of combination of steam explosion and microwave irradiation (SE–MI) pretreatment as a new pretreatment method for lignocellulosic biomass in order to enhance the enzymatic hydrolysis yield and total sugar (glucan and xylan) yield. To accomplish this, corn stover was pretreated by SE–MI or SE method, and the effects of the two pretreatment processes were evaluated and compared on the basis of the sugar yields obtained from pretreatment and subsequent enzymatic hydrolysis step using cellulase and glucosidase. The effects of reaction temperature and reaction time on pretreatment and subsequent enzymatic hydrolysis were also evaluated. Various structural characteristics of raw and pretreated corn stover were also assessed by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analyses. 2. Materials and methods 2.1. Chemicals and materials Corn stover was locally harvested and dried in air, and then chopped into particles (approximately 6–20 mm) by ZC-1 chaffcutter (Qufu S&T Co., Ltd., China) and stored at room temperature in air-tight bags prior to use. The composition of raw corn stover was measured as described in Section 2.3. All chemicals were reagent-grade and purchased from Jiangtian Chemical Co. (Tianjin, China), unless otherwise noted. Celluclast 1.5L cellulase (65 FPU/ml, 12.0 IU/ml) and Novozyme 188 -glucosidase (350 CBU/ml, 32.4 IU/ml) were purchased from Sigma–Aldrich Chemical Co. (Shanghai, China). Enzymes activities were assayed according to the method recommended by Ghose (1987) before they were used. 2.2. Pretreatment SE–MI pretreatment was conducted according to the process flow chart shown in Fig. 1. The details of the process were described by Kang et al. (2010a,b,c). Prior to pretreatment, 100 g (dry weight basis, dwb) corn stover were weighed in clean plastic bags and impregnated by the addition of deionized water to adjust the moisture content of material to 25% (dwb) at room temperature. The bags were sealed and kept for more than 30 min in order to allow the penetration of water into the material. Then all of the material was added into a 15 l reaction vessel via the feed inlet valve. High pressure saturated steam generated by a steam generator (Jinan Sanheng Environmental Protection Heat Energy Equipment Co., Ltd., China) was injected into the vessel rapidly via the steam inlet valve until the target temperature was approached. Next, microwave with frequency of 2.45 GHz supplied by a microwave generator (Guangzhou Kewei Microwave Equipment Co., Ltd., China) was imported into the reaction vessel via two rectangular waveguides from both sides of the vessel. Ceramic material and PTFE (polytetrafluorethylene) sealing gasket were used in the microwave feed port of the vessel, which could prevent steam in the vessel from permeating into the
Fig. 1. The schematic flow chart of SE–MI process used in the present study.
waveguides (which might cause damage to the microwave generator), and ensure the good performance of waveguides under high pressure. Reaction temperature (◦ C), steam reaction time (min), microwave power (W) and microwave irradiation time (min) were monitored and controlled during pretreatment process. (In this study, the microwave irradiation power and microwave irradiation time were fixed at 540 W and 3 min, respectively, for all the SE–MI experiments.) Microwave irradiation treatment was terminated by interrupting running of the microwave generator. Steam treatment was then ended by opening the pneumatic ball valve rapidly and the treated slurry was immediately ejected into a 140 l collection vessel under differential pressure. The treated slurry in the collection vessel was discharged via the feed outlet valve and collected, and then vacuum filtered using a 500 mesh (25 m pore size) filter cloth to separate solids from the slurry. The solids were washed with 20 ◦ C tap water (1:20, w/v). The washing water and the hydrolyzate were uniform mixed, and the mixture was analyzed using HPLC. SE pretreatment process was similar to SE–MI pretreatment process, but microwave was not imported into the reaction vessel during the process. The pretreatment severity factor (Overend and Chornet, 1987) that incorporated reaction temperature T (◦ C) and reaction time t (min) was defined as lg R0 , where R0 = t × exp[(T − 100)/14.75]. For all the SE–MI experiments, microwave power and microwave irradiation time were the same, so the microwave effect was not accounted for when calculating the pretreatment severity factor.
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2.3. Chemical analysis The compositional analysis of the untreated corn stover and pretreated solids were performed according to the procedures of NREL LAPs (Sluiter et al., 2006, 2008). Absorbance data of acid soluble lignin was taken at 243 nm using UV-7500 UV–Vis spectrophotometer (Shanghai Tianmei Co., Ltd., China). Glucose and xylose concentrations of hydrolyzates after pretreatment and enzymatic hydrolysis were quantified by Waters 1525 HPLC system (Waters Co., USA) equipped with an Aminex HPX-87P column (300 mm × 7.8 mm; Bio-Rad, USA) and refractive index detection. Concentrations of furfural, 5-hydroxymethyl furfural (5-HMF), acetic acid and formic acid of hydrolyzates from pretreatment were quantified by Waters 1525 HPLC system equipped with an Aminex HPX-87H column (300 mm × 7.8 mm; Bio-Rad, USA) and refractive index detection.
2.4. Enzymatic hydrolysis The washed pretreated residue was enzymatically hydrolyzed using a mixture of Celluclast 1.5L cellulase (60 FPU/g glucan) and Novozyme 188 -glucosidase (60 CBU/g glucan). The mixture contained 1% glucan concentrations of pretreated corn stover in 10 ml 0.05 M citrate buffer (pH 4.8) with 200 mg/l sodium azide. The reaction was performed at 50 ◦ C using a thermostatic shaker air bath (LRH-250-ZII; Guangzhou Medical Appliance Co., China) set at 150 rpm. Substrate blanks without enzyme and enzyme blanks without substrate were run in parallel. Samples were taken at 72 h, followed by centrifugation at 6000 rpm for 10 min, and the release of glucose and xylose was measured by HPLC as described in Section 2.3. All the experiments were performed in triplicate, with the average value being reported.
2.5. Crystallinity measurement X-ray powder diffraction patterns of both the untreated and pretreated corn stover were obtained using a X-ray diffractometer (Rigaku DMAX 2500 V/pc, Japan) in conjunction with a Cu K␣ radiation source ( = 0.154 nm) operated at 30 kV. Samples were scanned at a speed of 1◦ /min from 10◦ to 30◦ with a step size of 0.02◦ . Biomass crystallinity, as expressed by crystallinity index (CrI), was determined from XRD data and calculated using the formula CrI (%) = (I002 − Iam )/I002 × 100 (Segal et al., 1959), where I002 is the intensity for the crystalline portion of biomass (cellulose) at about 2 = 22.5◦ , and Iam is the peak for the amorphous portion (i.e. cellulose, hemicellulose and lignin) at about 2 = 15.5◦ . Crystallinity index (CrI) of biomass was also measured by Fourier transform infrared spectroscopy (FTIR), and expressed as the absorbance ratio of the bands at 1370 and 2900 cm−1 (Nelson and O’Connor, 2003).
2.6. FTIR spectroscopic analysis To investigate and quantify chemical changes in pretreated biomass, Fourier transform infrared spectroscopy (FTIR) was conducted with a Nicolet 560 ESP FTIR spectrometer (Thermal Nicolet Co., USA). Sample spectra were obtained using 32 scans over the range of 400–4000 cm−1 with a spectral resolution of 2 cm−1 .
2.7. Sugar yield calculations The enzymatic hydrolysis yields of glucose, xylose and total sugars were calculated by the following equations in this work:
Enzymatic hydrolysis yield of glucose (%) = [(glucose (g) released in enzymatic hydrolysis)/(total glucose (g) in the pretreated biomass)] × 100 Enzymatic hydrolysis yield of xylose (%) = [(xylose (g) released in enzymatic hydrolysis)/(total xylose (g) in the pretreated biomass)] × 100 Enzymatic hydrolysis yield of total sugars (%) = [(total sugar (g) released in enzymatic hydrolysis)/(total sugar (g) in the pretreated biomass)] × 100 The glucose, xylose and total sugar yields following pretreatment and enzymatic hydrolysis were calculated by the following equations: Glucose yield (%) = [(glucose (g) released in pretreatment and enzymatic hydrolysis)/(potential total sugar (g) in the feedstock)] × 100 Xylose yield (%) = [(xylose (g) released in pretreatment and enzymatic hydrolysis)/(potential total sugar (g) in the feedstock)] × 100 Total sugar yield (%) = [(total sugar (g) released in pretreatment and enzymatic hydrolysis)/(potential total sugar (g) in the feedstock)] × 100 3. Results and discussion 3.1. Compositional analysis of pretreated solids and hydrolyzate The raw corn stover used in this study contained about 30.3% glucan, 13.3% xylan, 0.4% arabinose, 17.2% Klason lignin, 3.0% acid-soluble lignin, 2.2% ash and 33.6% extractives. The maximum potential total sugar yield was calculated as 48.8 g per 100 g dry feedstock. The maximum yield of glucose was 69.0% and the maximum yield of xylose was 31.0%, for a sum total sugar of 100% in the feedstock. As shown in Table 1, composition of solid residues and hydrolyzate following pretreatment were reported. SE-1 and SE-4 represented SE experiments at 170 ◦ C and 190 ◦ C, respectively, for 5 min, and could be compared with SE–MI-1 and SE–MI-4 which represented SE–MI experiments at the same reaction temperature and reaction time, respectively. The solid recovery of corn stover was an important index for the effectiveness of its pretreatment. Solid recovery was the percentage content of pretreated and washed material in 100 g raw feedstock. The solid recovery of materials pretreated by SE–MI method was about 5% lower than that pretreated by SE method, which might be resulted by some components of biomass that converted to soluble chemicals under microwave irradiation. About 38.3–46.7 g solids could be recovered from 100 g feedstocks after SE–MI pretreatment. The solid recovery was gradually decreased with pretreatment severity increasing, which was resulted by glucan and xylan converted into soluble oligomeric and monomeric sugars and its degradation products. Compared to that of SE pretreatment, percent of glucan and xylan corresponding to pretreated solids were both lower following SE–MI pretreatment, while percent of lignin corresponding to pretreated solids was slightly higher. However, because the solid recovery for SE–MI pretreatment was lower than that for SE pretreatment, percent of glucan, xylan and lignin corresponding to feedstocks were all lower following SE–MI pretreatment than that following SE pretreatment. For SE–MI pretreatment, with reaction temperature and reaction time rising, percent of glucan corresponding to feedstocks was kept at about 22.8–23.9%. Meanwhile, percent of lignin corresponding to feedstocks was slightly increased from 9.4% to 10.8%, and percent of xylan corresponding to feedstocks was decreased from 7.7% to 2.1%. As SE–MI pretreatment was operated at acidic
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Table 1 Composition of solid residues and hydrolyzate following pretreatment at the conditions noted. No.a
Tb (◦ C)
Pb (MPa)
UT SE-1 SE-4 SE–MI-1 SE–MI-2 SE–MI-3 SE–MI-4 SE–MI-5 SE–MI-6 SE–MI-7 SE–MI-8
– 170 190 170 180 190 190 190 190 200 210
– 0.69 1.15 0.69 0.90 1.15 1.15 1.15 1.15 1.45 1.80
tb (min)
– 5 5 5 5 3 5 10 15 5 5
lg R0
Solid recovery (%)
– 2.76 3.35 2.76 3.05 3.13 3.35 3.65 3.83 3.64 3.94
100.0 51.6 45.8 46.7 42.2 42.9 41.0 39.6 38.7 39.7 38.3
Solid fractionc (%)
Liquid fractiond (g)
Glucan
Xylan
Lignin
Glucose
Xylose
30.3 28.0 (54.3) 27.5 (60.1) 23.9 (51.1) 22.8 (54.1) 23.8 (55.5) 23.5 (57.4) 23.7 (59.8) 23.4 (60.3) 23.8 (60.0) 23.2 (60.5)
13.3 9.1 (17.6) 5.7 (12.4) 7.7 (16.4) 5.3 (12.5) 5.3 (12.3) 4.4 (10.8) 3.3 (8.5) 2.4 (6.2) 3.2 (8.0) 2.1 (5.5)
17.2 10.0 (19.4) 10.5 (22.8) 9.4 (20.1) 9.5 (22.6) 9.7 (22.6) 9.8 (24.0) 10.0 (25.3) 10.5 (27.0) 10.1 (25.5) 10.8 (28.1)
– 3.05 (6.25) 4.69 (9.61) 3.16 (6.48) 4.02 (8.24) 4.15 (8.51) 5.17 (10.60) 5.95 (12.20) 6.89 (14.12) 6.21 (12.73) 6.52 (13.37)
– 3.53 (7.24) 5.37 (11.01) 3.83 (7.85) 5.18 (10.62) 5.28 (10.82) 5.83 (11.95) 6.82 (13.98) 5.61 (11.50) 6.84 (14.02) 4.77 (9.78)
Microwave power and microwave irradiation time were 540 W and 3 min, respectively, for all the SE–MI experiments. a UT, SE and SE–MI represent no pretreatment, steam explosion pretreatment, and combination of steam explosion and microwave irradiation pretreatment, respectively. b T, reaction temperature; P, pressure of saturated steam (gauge pressure); t, reaction time. c Dates before and in the parentheses represent percent contents of glucan, xylan and lignin in untreated corn stover and in pretreated solids, respectively. d Dates before and in the parentheses represent the amount of glucose and xylose (g) in 100 g feedstock and in 100 g total sugars of feedstock, respectively.
conditions, most xylan was dissolved or removed during the process. However, most of lignin could not be removed following the pretreatment, especially under high pretreatment severity, the reason might be that condensation reaction of lignin prevailed under the pretreatment conditions, and resulted in lignin content stabilized (Ramos, 2003). During SE or SE–MI pretreatment, many monosaccharides, oligosaccharides, and by-products such as organic acids and furans were dissolved in liquid. Compared to SE pretreatment, SE–MI pretreatment released more glucose, xylose and inhibitors into hydrolyzate (Table 1 and Fig. 2). Both the glucose and xylose yields increased with reaction time and reaction temperature rising, but a decrease of xylose was observed when the reaction temperature was higher than 200 ◦ C. The yields of furfural and HMF were an indication of the severity of pretreatment. As shown in Fig. 2, increased time and temperature resulted in furfural and HMF formation rising during SE–MI pretreatment. At the most severe condition (210 ◦ C for 5 min), the yields were 0.46 and 1.06 g per 100 g dry feedstock for HMF and furfural, respectively, while at the least severe condition (170 ◦ C for 5 min), the yields were only 0.07 and 0.23 g per 100 g dry feedstock, respectively. More degradation of pentose under severe conditions also increased the yield of acetic acid and formic acid. The results were in agreement with the findings of Linde et al. (2008) by steam pretreatment with dilute H2 SO4 -impregnation.
Fig. 2. Yields of inhibitors resulting from SE (SE-1 and SE-4) and SE–MI (from SE–MI1 to SE–MI-8) pretreatment process.
3.2. Enzymatic hydrolysis and sugar yields The water-insoluble pretreated residue was submitted to enzymatic hydrolysis by a cellulase complex (Celluclast 1.5L) supplemented with -glucosidase (Novozyme 188). For comparison purposes, enzymatic hydrolysis was also carried out on raw material not subjected to any pretreatment. As shown in Fig. 3, the enzymatic hydrolysis yields of glucose and xylose for raw material were both lower than 20%. Materials treated by SE–MI method gave higher enzymatic hydrolysis yields than that treated by SE method both at 170 ◦ C and 190 ◦ C for 5 min. For instance, when it was treated at 190 ◦ C for 5 min, the enzymatic hydrolysis yields of glucose and xylose by SE–MI method were 77.9% and 55.8%, which were 12% and 7.2% higher than that by SE method, respectively. For SE–MI pretreatment, when the pretreatment severity was lower than 3.64–3.65, enzymatic hydrolysis yields of glucose increased with reaction time and reaction temperature during SE–MI pretreatment, but a higher pretreatment severity of more than 3.64–3.65 could resulted in the yields decreasing. The maximum enzymatic hydrolysis yields of glucose were 82.4% and 82.8%, which were obtained at 190 ◦ C for 10 min and 200 ◦ C for 5 min,
Fig. 3. Enzymatic hydrolysis yields of glucose, xylose and total sugar for SE (SE-1 and SE-4) and SE–MI (from SE–MI-1 to SE–MI-8) pretreatment process after enzymatic hydrolysis for 72 h. UT represents untreated corn stover. Substrates were suspended in 50 mM Na–citrate buffer (pH 4.8) at 1% glucan concentrations and hydrolyzed by cellulases (Celluclast 1.5L) and -glucosidase (Novozymes 188) at a ratio of 60 FPU/g glucan to 60 CBU/g glucan at 50 ◦ C and 150 rpm for 72 h. All the experiments were performed in triplicate, with the average value being reported.
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Fig. 4. Glucose, xylose and total sugar yields for SE (SE-1 and SE-4) and SE–MI (from SE–MI-1 to SE–MI-8) pretreatment process after pretreatment (PT) and enzymatic hydrolysis (EH) from corn stover feedstock. UT represents untreated corn stover.
respectively. Enzymatic hydrolysis yields of xylose for all the samples were lower than that of glucose. The maximum enzymatic hydrolysis yield of xylose was 62.0%, which were achieved at 190 ◦ C for 3 min. When the reaction temperature was above 180 ◦ C, the xylose yield decreased with increasing reaction temperature and reaction time. As presented in Sections 3.1 and 3.2, more inhibitors were generated at elevated temperature, and the lignin contents in pretreated materials were also increased, which could play a negative role in xylose hydrolysis and thus reduced the xylose yields. The total sugar yield made by combining the total sugar conversion yield during pretreatment and enzymatic hydrolysis was an index of pretreatment efficiency for ethanol production (Chen et al., 2011). As shown in Fig. 4, the total sugar yields of materials following SE or SE–MI pretreatment were far higher than that of raw material. Compared to SE pretreatment, SE–MI pretreatment resulted in lower percent of glucan and xylan in pretreated solids, but caused higher enzymatic hydrolysis yields of glucose and xylose. As a result, the total sugar yields of SE–MI pretreatment were slightly higher (about more than 1.5%) than that of SE pretreatment. For SE–MI pretreatment, with reaction temperature and reaction time rising, the total sugar yields were increased, but then decreased when reaction time was more than 10 min or reaction temperature was higher than 200 ◦ C. When the material was treated at 200 ◦ C for 5 min (the pretreatment severity factor was 3.64), the maximum glucose yield, xylose yield and total sugar yield were 57.4%, 17.8% and 75.2%, respectively, corresponding to 28.0 g glucose, 8.7 g xylose and 36.7 g total sugar were generated from 100 g raw feedstock. This reaction condition could be regarded as the optimum SE–MI pretreatment conditions, when the microwave power and microwave irradiation time were 540 W and 3 min, respectively. The mass balance for corn stover at optimum SE–MI pretreatment conditions was shown in Fig. 5. When corn stover was pretreated by SE–MI method at 200 ◦ C for 5 min, 25.28 g glucan and 7.58 g xylan were released during pretreatment and enzymatic hydrolysis. Under this condition, in order to produce a liter of ethanol, 4.63 g dry material was required. During SE–MI pretreatment, about 0.4–0.6 l water was consumed corresponding to 0.1 kg dry material, then 18.52–27.78 l water was required to produce a liter of ethanol. 3.3. Crystallinity analysis The biomass crystallinity index (CrI) was believed to significantly affect enzymatic saccharification of glucan (Kumar et al.,
2009). Crystallinity analysis of the untreated and pretreated corn stover by XRD and FTIR method was done to investigate the crystalline behavior of cellulose (Table 2). The crystallinity of the biomass following SE or SE–MI pretreatment was much higher than that of untreated corn stover. The reason might be that non-cellulosic polysaccharides were removed and amorphous cellulose was dissolved, which resulted in the increase of cellulose crystallinity. The increase of crystallinity after low pH pretreatment has also been reported by Kumar et al. (2009). The crystallinity of biomasses pretreated by SE–MI method was much lower than that pretreated by SE method, especially at high reaction temperature. Compared to that of SE pretreatment at 170 ◦ C and 190 ◦ C for 5 min, after SE–MI pretreatment at the same reaction temperature and reaction time, the crystallinity measured by XRD method was decreased by about 5% and 19%, and the crystallinity measured by FTIR method was decreased by about 0.08 and 0.24, respectively. The reason could be interpreted by the structure of lignocellulose and the unique characterization of microwave irradiation. As cellulosic biomass was a highly heterogeneous complex of polymers, primarily lignin, cellulose, and hemicelluloses, some of the polymers might be coupling and reacting with carboxylic acid catalyst that released from water by auto-hydrolysis of hemicelluloses during pretreatment. The high water content of cellulosic biomass was also susceptible to microwave heating. Considering all these factors, application of microwave irradiation to lignocellulose might selectively heated the more polar part and created “hot spots” within heterogeneous materials. This unique heating feature could possibly resulted in an “explosion” effect among the particles and improved disruption of the recalcitrant structures of lignocellulose (Shi et al., 2011), therefore the crystal region of cellulose could be destroyed and resulted in the decline of crystallinity. For SE–MI pretreatment, the biomass crystallinity was significantly affected by reaction time and reaction temperature. Longer reaction time and higher reaction temperature resulted in higher crystallinity. When the reaction time was increased from 3 min to 15 min at 190 ◦ C, the biomass crystallinity measured by XRD method gradually increased from 36.0% to 43.6%, and the biomass crystallinity measured by FTIR method raised from 1.11 to 1.21. The biomass crystallinity was changed more significantly with reaction temperature rising. When the reaction temperature was increased from 170 ◦ C to 210 ◦ C for 5 min reaction time, the biomass crystallinity measured by XRD method gradually increased from 34.1% to 45.9%, and the biomass crystallinity measured by FTIR method raised from 1.07 to 1.25. However, the observation was inconsistent with the research results of Liu et al. (2009) by FeCl3 pretreatment, which showed that the crystallinity decreased slightly when pretreatment temperature increased from 140 ◦ C to 180 ◦ C. The inconsistency could be attributed to the different pretreatment methods and different pretreatment conditions the two groups used. 3.4. FTIR analysis FTIR spectroscopy was an analytical method that was frequently used to investigate the structure of constituents and chemical changes that occurred during the pretreatment of lignocellulosic biomass (Liu et al., 2009). The FTIR spectra of raw corn stover and solid residues following SE or SE–MI pretreatment were shown in Fig. 6. The peak at 900 cm−1 was characteristic of -glycosidic linkages. This peak existed in feedstock, as well as in corn stover pretreated by SE or SE–MI method, which demonstrated that most of -glycosidic linkages between the sugar units in cellulose and hemicellulose were preserved during pretreatment. The prominent bands at 1200–1000 cm−1 were typically related to the
F. Pang et al. / Industrial Crops and Products 42 (2013) 402–408
Reaction temperature : 200 Reaction time : 5 min Microwave power : 540 W Microwave irradiation time: 3 min
Corn stover 100 g dry weight
SE-MI Pretreatment
Enzymes 60 FPU/g glucan 60 CBU /g glucan
41.5 g dry solids
39.7 g dry solids
Washing Solids 23.8 g Glucan 3.2 g Xylan 12.7 g Others
30.3 g Glucan 13.3 g Xylan 0.4 g Arabinan 17.2 g Klason lignin 3.0 g Acid-soluble lignin 2.2 g ash 33.6 g extractives
407
Liquid
Enzymatic Hydrolysis
17.9 g dry solids
Liquid
Solids 4.01 g Glucan 1.54 g Xylan 12.35 g Others
21.88 g Glucose 1.77 g Xylose
6.21 g Glucose 6.84 g Xylose
Fig. 5. Mass balance for corn stover during SE–MI pretreatment (SE–MI-7) and 72 h enzymatic hydrolysis.
Table 2 Crystallinity index (CrI) changes of corn stover following SE (SE-1 and SE-4) and SE–MI (from SE–MI-1 to SE–MI-8) pretreatment process. No.a
Tb (◦ C)
Pb (MPa)
UT SE-1 SE-4 SE–MI-1 SE–MI-2 SE–MI-3 SE–MI-4 SE–MI-5 SE–MI-6 SE–MI-7 SE–MI-8
– 170 190 170 180 190 190 190 190 200 210
– 0.69 1.15 0.69 0.90 1.15 1.15 1.15 1.15 1.45 1.80
a b c d
tb (min) – 5 5 5 5 3 5 10 15 5 5
lg R0
CrIc (%)
CrId A1370/A2900
– 2.76 3.35 2.76 3.05 3.13 3.35 3.65 3.83 3.64 3.94
29.8 40.2 57.6 35.0 37.1 36.0 38.9 40.3 43.6 41.4 45.9
0.95 1.15 1.38 1.07 1.12 1.11 1.14 1.16 1.21 1.18 1.25
UT, SE and SE–MI represent no pretreatment, steam explosion pretreatment, and combination of steam explosion and microwave irradiation pretreatment, respectively. T, reaction temperature; P, pressure of saturated steam (gauge pressure); t, reaction time. Crystallinity index (CrI) measured by XRD method. Crystallinity index (CrI) measured by FTIR method.
that of untreated, suggested that SE–MI pretreatment increased the relative amount of lignin in the pretreated solid as a result of hemicellulose removal (Liu et al., 2009) and/or re-deposition of released lignin on the surface of solids.
3.5. Economic analysis of SE–MI process
Fig. 6. FTIR spectra of untreated (UT) corn stover and solid residues following SE (SE-1 and SE-4) and SE–MI pretreatment (SE–MI-1 and SE–MI-4).
structural features of cellulose and hemicelluloses. The vibrations of these bands overlapped the C–O–H stretching of primary and secondary alcohols at 1060 cm−1 , C–O–C glycosidic bond stretching at 1160 cm−1 and C–O–C ring skeletal vibration at 1100 cm−1 . After SE or SE–MI pretreatment, the adsorption peaks of these bands were appeared or enhanced, which suggested that the cellulose content in pretreated solid residue increased because the hemicelluloses fraction in raw materials was released by acid hydrolytic reaction (Hsu et al., 2010). Peak at 1510 cm−1 that related to aromatic ring stretching in lignin was stronger in spectra of pretreated solids than
To evaluate the economic performance of SE–MI process at optimum conditions, the cost of sugars was estimated by dividing the additional yield of sugars (grams of sugar per kg of biomass) obtained by SE–MI processing by microwave power cost (540 W microwave power multiplied by 3 min microwave irradiation time, and then multiplied by the cost of electricity during pretreatment) and electricity consumption of steam generator (0.42 kWh multiplied by the cost of electricity during pretreatment). At present, the cost of electricity in Tianjin was about $0.076 US/kWh. When corn stover was pretreated by SE–MI method at 200 ◦ C for 5 min, 0.367 kg sugars per kg of raw material was achieved, so the cost of sugars was calculated as follows.
(0.54 kW × 0.05 h + 0.42 kWh) × $0.076 US/kWh/0.367 kg of sugars = $0.093 US/kg of sugars.
As the cost of sugars ($0.093 US/kg of sugars) was much lower than $0.30 US/kg of sugars, so the process may be economically viable. However, further evaluation is needed to verify the economic feasibility of the new process.
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4. Conclusions Overall, compared with SE pretreatment, the SE–MI pretreatment process has attractive advantages in improving enzymatic hydrolysis yields and sugar yields. When the microwave power and microwave irradiation time were 540 W and 3 min, respectively, the maximum glucose yield, xylose yield and total sugar yield of the process were 57.4%, 17.8% and 75.2% (corresponding to 100% maximum total sugar in feedstock), respectively, obtained at 200 ◦ C for 5 min. SE–MI pretreatment also showed attractive advantage in inhibiting the increase of biomass crystallinity. The crystallinity following SE–MI pretreatment was 19% lower than that following SE pretreatment at 190 ◦ C for 5 min. However, further evaluation is needed to verify the economic feasibility of the new process. In addition, the effect of microwave power and microwave irradiation time on SE–MI pretreatment and subsequent enzymatic hydrolysis has been also investigated (Pang et al., 2012). As microwave pretreatment performed efficiently at acidic conditions, lower pH could be more favorable for SE–MI pretreatment. Further research could focus on evaluating the effectiveness and feasibility of SE–MI pretreatment with acid catalyst (such as dilute sulfuric acid, SO2 or other inorganic or organic acids) added at relatively low temperature, and that of SE–MI pretreatment of more recalcitrant biomass, such as softwood, at elevated temperature. Acknowledgement Financially support provided by the National Key Technology R&D Program of China during “the 11th Five-Year Plan” (Grant No. 2007BAD42B03) is greatly acknowledged. References Azuma, J., Higashino, J., Asai, T., Koshijima, T., 1985. Microwave irradiation of lignocellulosic materials. IV. Enhancement of enzymatic susceptibility of microwave-irradiated softwoods. Wood Res. 71, 13–24. Chen, W.H., Pen, B.L., Yu, C.T., Hwang, W.S., 2011. Pretreatment efficiency and structural characterization of rice straw by an integrated process of dilute-acid and steam explosion for bioethanol production. Bioresour. Technol. 102, 2916–2924. Cullis, I.F., Saddler, J.N., Mansfield, S.D., 2004. Effect of initial moisture content and chip size on the bioconversion efficiency of softwood lignocellulosics. Biotechnol. Bioeng. 85, 413–421. Ewanick, S.M., Bura, R., Saddler, J.N., 2007. Acid-catalyzed steam pretreatment of Lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol. Biotechnol. Bioeng. 98, 737–746. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Hsu, T.C., Guo, G.L., Chen, W.H., Hwang, W.S., 2010. Effect of dilute acid pretreatment of rice straw on structural properties and enzymatic hydrolysis. Bioresour. Technol. 101, 4907–4913. Kang, Y., Pang, F., Yu, S.S., Zhang, C., 2010a. A method by utilize combination of steam explosion and microwave irradiation pretreatment to realize cellulose decrystallization of plant stalk. Chinese Patent ZL 200910069710.9. Kang, Y., Yu, S.S., Pang, F., Zhang, C., 2010b. A set of equipment that utilize combination of steam explosion and microwave irradiation pretreatment to realize cellulose decrystallization of plant stalk. Chinese Patent ZL 200920097742.5. Kang, Y., Yu, S.S., Pang, F., Zhang, C., 2010c. A structure form of microwave inlet which is surround by saturation vapour with high temperature and high pressure. Chinese Patent ZL 200920098491.2. Keshwani, D.R., Cheng, J.J., 2010. Modeling changes in biomass composition during microwave-based alkali pretreatment of switchgrass. Biotechnol. Bioeng. 105, 88–97. Kitchaiya, P., Intanakul, P., Krairiksh, M., 2003. Enhancement of enzymatic hydrolysis of lignocellulosic wastes by microwave pretreatment under atmosphericpressure. J. Wood Chem. Technol. 23, 217–225.
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