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Reinforced alkali-pretreatment for enhancing enzymatic hydrolysis of sugarcane bagasse
Fuel Processing Technology 143 (2016) 1–6
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Fuel Processing Technology journal homepage: www.elsevier.com/locate/fuproc
Research article
Reinforced alkali-pretreatment for enhancing enzymatic hydrolysis of sugarcane bagasse Yun-Yun Liu a,b, Jing-Liang Xu a, Yu Zhang a, Cui-Yi Liang a, Min-Chao He a, Zhen-Hong Yuan a,⁎, Jun Xie b a b
Guangzhou Institute of Energy Conversion, Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, China Institute of New Energy and New Materials, South China Agriculture University, Guangzhou 510642, China
a r t i c l e
i n f o
Article history: Received 11 April 2015 Received in revised form 6 November 2015 Accepted 12 November 2015 Available online xxxx Keywords: Sugarcane bagasse Pretreatment Alkaline Microwave irradiation Ionic liquids Enzymatic hydrolysis
a b s t r a c t Microwave irradiation (MWI) and ionic liquid (IL) assisted methods were chosen to strengthen the alkali pretreatment process to improve saccharification efficiency of sugarcane bagasse (SCB). Four different pretreatment approaches namely alkali, IL, MWI-alkali and IL-alkali pretreatments were compared. The chemical composition, morphology and crystal performance of the substrates were analyzed by the standardized methods of the National Renewable Energy Laboratory, field emission scanning electron microscope (SEM) and X-ray diffraction (XRD), respectively. IL assisted treatment brought the highest lignin removal (78.51%), and the glucan and xylan conversion achieved 99.51% and 92.76%, respectively, after 72 h of enzymatic hydrolysis. The similar effect occurred for MWI assisted alkali-pretreatment approach. SEM and XRD analyses showed that the strengthened process obviously changed the structure and crystallinity of the substrates. Moreover, as a persuasive tool, the fractal-like theory was used to study the enzymatic hydrolysis kinetics. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lignocellulosic biomass mainly consists of hexose, pentose and lignin. Crystalline cellulose is partially surrounded by the amorphous hemicelluloses, which is aligned with cellulose fibers and densely packed by the layers of lignin. Lignin constructed of phenylpropane units is the most obstinate component of the plant cell wall, which is generally resistant to enzymatic hydrolysis [1]. The degree of polymerization, available surface area, lignin content and distribution are important factors that hinder the enzymes attack. Due to the inhomogeneous structure of lignocellulosic materials, efficient pretreatment technologies are required to disrupt the recalcitrance of lignocellulosic biomass for further downstream processing [2,3]. Among different pretreatment methods, alkali pretreatment has the advantage of simple device, convenient operation with high efficiency, which induces the swelling reactions and the removing of lignin, uronic acids and acetyl groups from polysaccharides, therefore, making the substrates more susceptible to enzymatic reaction [4,5]. Although alkali pretreatment can remove the majority of lignin, the residual lignin and hemicelluloses in the pretreated substrate can still hinder enzymatic hydrolysis. The digestibility improvement depended on the removal of lignin and parts of hemicelluloses, the reduction of acetyl content, and the disruption of lignin–carbohydrate matrix [6,7]. In order to achieve ⁎ Corresponding author at: No.2, Nengyuan Rd, Wushan, Tianhe District, Guangzhou, China. Tel: +8620-87057735. E-mail address: [email protected] (Z.-H. Yuan).
http://dx.doi.org/10.1016/j.fuproc.2015.11.004 0378-3820/© 2015 Elsevier B.V. All rights reserved.
efficient hydrolysis, using the synergistic benefits of the combined methods may be an integrated novel pretreatment approach. Microwave irradiation (MWI) with the advantages of high heating efficiency and easy operation has been widely used in many areas. It has been shown in some studies that MWI can change the ultrastructure of cellulose and degrade lignin and hemicelluloses. The pretreated particles were characterized by swelling and fragmentation, which was favorable for enzyme accessibility. This technique can be easily combined with chemical reaction and accelerate the reaction rate [8–10]. The recent discovery of ionic liquid (IL) can effectively dissolve cellulose and biomass and is suggested to be a potential alternative to established pretreatment techniques [11]. IL pretreatment can reduce the cellulose crystallinity and partially remove lignin and hemicelluloses, thus enhance the biomass digestibility. It has high degree of flexibility in process design with less energy costing, and is easier to handle than other pretreatment methods [12,13]. The enzymatic hydrolysis of cellulose can be thought as a heterogeneous reaction with less than three dimensions [14] along a cellulosic fiber chain. Before the reaction, enzyme must be absorbed on the substrate surface and then diffuse to a reactive site. These complex reactions and mechanisms can be understood using a mathematical model [15]. The fractal-like kinetic model has been applied to analyze the effect of pretreatment on the enzymatic saccharification kinetics. With two parameters (fractal exponent and rate coefficient), it fitted very well to the experimental data in the literatures [16–18]. In the present study, alkali pretreatment was enhanced by MWI or IL considering the synergistic benefits of them for improving the hydrolysis
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Y.-Y. Liu et al. / Fuel Processing Technology 143 (2016) 1–6
Table 1 Chemical compositions of untreated and pretreated SCBs. Pretreatment method
Untreated Alkali Alkali + MWI IL Alkali + IL
Composition (%) Glucan
Xylan
Klason lignin
38.51 63.43 65.62 44.82 69.24
20.65 26.11 26.54 21.49 21.68
25.03 8.26 7.13 27.16 5.47
efficiency. The comparison of single alkali and IL pretreatments was also applied on sugarcane bagasse (SCB). 1-Allyl-3-methylimidazolium chloride (AMIC) was used as a representative IL because of its enhanced hydrogen bond basicity and lower viscosity [19]. The structural characterization of SCB substrates was investigated by field emission scanning electron microscope (SEM) and X-ray diffraction (XRD), and the fractal-like model was used to theoretically analyze the effect of various pretreatments on enzymatic hydrolysis.
Fig. 1. Effect of pretreatment method on enzymatic conversion of the substrates.
freeze-dried before subsequent enzymatic hydrolysis. The filtrate, containing water, IL, and some degraded carbohydrates as well as lignin, was separated under the reduced pressure.
2. Materials and methods 2.1. Materials
2.2.3. MWI assisted alkali pretreatment MWI was carried out in a MCR-3 microwave chemistry reactor. After the alkali pretreatment, the bottle with substrate mixtures was placed into the microwave reactor and the treatment program with the desired time of 5 min and a power of 600 W was started [22]. After the run, the bottle was removed from the reactor. The obtained solid was separated and dried according to Section 2.2.1.
SCB was obtained from Guangxi Fenghao Sugar Co., Ltd. (Chongzuo, China). It was pre-milled and screened, and the fraction between 18 and 40 meshes was used for alkali-pretreatment. AMIC was purchased from Shanghai Cheng Jie Chemical Co. Ltd. A cellulase mixture, CTec2, was kindly provided by Novozymes A/S (Bagsaevrd, Denmark), with an enzyme activity of 200 FPU/mL measured by the description of International Union of Pure and Applied Chemistry [20]. All other chemicals used were of analytical or reagent grade and directly used as purchased without further purification.
2.2.4. IL assisted alkali pretreatment The alkali-pretreated substrate was successively treated with IL, and the same pretreatment procedure was conducted according to Section 2.2.2. After the reaction, the wet regenerated SCB was freezedried for 24 h prior to enzymatic hydrolysis and further analysis.
2.2. Pretreatment procedure 2.2.1. Alkali pretreatment SCB was pretreated by 0.5 M NaOH in a round-bottom flask, at 80 °C for 2 h with agitation (solid–liquid ratio is 1:20). After the pretreatment, the solid fraction was separated by filtering and then washed with tap water until a neutral pH. The obtained residual fraction was dried at 65 °C and then used as substrates for the subsequent enzymatic hydrolysis. All experiments were carried out three times, and the given numbers were the mean values.
2.3. Enzymatic hydrolysis Enzymatic hydrolysis was carried out with 2% (w/v) substrate at pH 5.0, a temperature of 50 °C and a rotation speed of 150 rpm for 72 h in 250 mL Erlenmeyer flasks. The enzyme-loading was 9.6 FPU/g of biomass. Samples for sugar assay were taken at time points (specified in the text) and analyzed by high performance liquid chromatography (HPLC). Glucan and xylan conversion rates were estimated by the following equations:
2.2.2. IL pretreatment Details of IL pretreatment were described elsewhere [21]. 5 g of SCB was quickly mixed with 95 g of AMIC solution at a solid/liquid ratio of 1:20, in a 250 mL dried flask. The flask was sealed with a cork and placed in oil bath at 80 °C for 2 h with stirring. After the reaction, the obtained substrate was precipitated using deionized water (90 °C) with vigorous shaking for 10 s. The regenerated substrate was then transferred into a beaker and washed with 50 mL deionized water (75 °C) thoroughly, and
Glucan conversion rate ¼
Xylan conversion rate ¼
Glucose produced ðgÞ 0:9 Glucan amount in enzymatic substrate
Xylose produced ðgÞ 0:88 : Xylan amount in enzymatic substrate
ð1Þ
ð2Þ
Table 2 Glucose and xylose concentrations (g L−1) produced from enzymatic hydrolysis of different substrates. Time (h)
6 12 24 48 72
Raw
Alkali-treated
Alkali + MWI
IL-treated
Alkali + IL
Glucose
Xylose
Glucose
Xylose
Glucose
Xylose
Glucose
Xylose
Glucose
Xylose
0.96 1.12 1.25 1.28 1.35
0.44 0.48 0.55 0.60 0.62
8.79 10.12 11.33 11.95 12.02
3.79 4.19 4.59 4.61 4.55
10.09 11.16 12.04 12.47 12.90
3.91 4.20 4.54 4.62 4.73
1.27 1.38 1.69 1.69 1.73
0.49 0.54 0.64 0.66 0.66
12.06 12.72 13.39 13.61 13.78
3.69 3.88 4.04 4.00 4.02
Y.-Y. Liu et al. / Fuel Processing Technology 143 (2016) 1–6
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Fig. 2. SEM micrographs of the substrates. Raw material (A); pretreated SCB: alkali-pretreated (B), MWI assisted alkali-pretreated (C), AMIC pretreated (D), AMIC assisted alkali-pretreated (E) and enzyme hydrolyzed SCBs: raw material (a), alkali-pretreated (b), MWI assisted alkali-pretreated (c), AMIC pretreated (d) and AMIC assisted alkali-pretreated SCBs (e).
with a flow rate of 0.5 mL/min at 50 °C. Standard and hydrolyzed samples were filtrated by 0.22 μm filter before analysis.
2.4. Fractal-like kinetic modeling Fractal-like kinetic model considers reaction in materials that are prepared as fractals [23]. It assumes that cellulase and xylanase consist of several components to form a single combined effect on the substrate hydrolysis, and the reaction fits the fractal-like kinetic model, then the glucose and xylose expression can be deduced as follows: ½G ¼
C G ½S0 0:9kG 1−hG t 1− exp − 1−hG 0:9
ð3Þ
½X ¼
C X ½S0 0:88kX 1−hX t 1− exp − 1−hX 0:88
ð4Þ
where [G] and [X] are the concentrations of the generated glucose and xylose, CG and Cx are the glucan and xylan contents in the raw materials, KG and KX, hG and hX are the empirical constants representing rate constant and fractal dimension of glucan and xylan in substrates, respectively. 2.5. Analytical methods 2.5.1. Sugar assay Glucose and xylose concentrations were determined by HPLC using a Shodex sugar SH-1011 column coupled with a refractive index detector. The mobile phase of 5 mM H2SO4 in demineralized water was used
2.5.2. Compositional analysis Glucan, xylan, and Klason lignin contents in raw, pretreated, and enzyme hydrolyzed SCBs were analyzed in duplicate according to the standardized methods of the National Renewable Energy Laboratory, USA [24].
2.5.3. SEM assay The surface morphology and characteristics of the different residual solids were observed using SEM. Images were taken by the model JEOL JSM-4800 LV SEM and performed at a beam accelerating voltage of 10 kV. Digital images were captured with 1280 × 960 resolution and 160 s dwell time.
2.5.4. XRD measurement X-ray powder diffraction pattern of samples was analyzed using an XRD instrument (PANalytical V.B., Netherlands). The determination was performed with Ni-filtered Cu Kα radiation (λ = 1.54 Å) at 40 kV and 40 mA, with a recorded range of 50 to 500. The sample crystallinity was quantified from XRD data using a crystallinity index and CrI for cellulose [25].
Fig. 3. XRD spectra of untreated, pretreated and enzyme hydrolyzed SCBs.
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Fig. 4. Fitted values from fractal-like model versus experimental values.
3. Results and discussion
The SCB used in this study consists of glucan, xylan, and lignin in the contents of 38.51%, 20.65%, and 25.03%, respectively. After the pretreatment, the composition of SCB was drastically changed, especially for the integrated alkali-pretreatment. Single-alkali-pretreatment removed approximately 67% of lignin, as shown in Table 1, and the percentage of glucan and xylan were all increased, which made cellulose partially exposed. When the assisted step of MWI pretreatment was added, more than 70% of the lignin was removed (71.51% removal), and the percentage of glucan and xylan (65.62% and 26.54%) were increased by 62.97% and 28.52%, respectively, compared to the raw material. When adding another step of IL pretreatment, the glucan content was increased to 69.24% with further delignification, and the largest Klason lignin removal of about 78.15% was found. However, for the sole IL pretreatment, the effect was not obvious in a short time. As shown in Table 1, the combined pretreatment could effectively remove much more lignin and keep the majority of cellulose in the residues than single alkali or IL pretreatment, especially for IL assisted alkali-pretreatment, which would facilitate the subsequent enzymatic hydrolysis.
respectively. This increase in sugar concentration can be attributed to the decrease in lignin/hemicellulose content, and the increase in the cellulose surface area. For the sole IL pretreatment, the relative lower sugar yield was caused by the insufficient cellulose dissolution in a relatively short period of time. When an assisted pretreatment step was added, the composition of the substrates had important changes as mentioned in Table 1. These changes could facilitate the subsequent hydrolysis and lead to the obvious increase of enzymatic digestibility. After the MWI assisted pretreatment, the glucan and xylan conversion reached 98.25% and 89.19%, respectively (Fig. 1). Under similar conditions, IL assisted pretreatment achieved better results. The glucan and xylan conversion (glucan, 99.51% and xylan, 92.76%) was significantly improved compared to the single alkali or IL treatment (Fig. 1). MWI based on intramolecular heat transfer can cause vibration and shock to polar bonds inside biomass and thus change the molecular activity [26]. After the alkalipretreatment, MWI could be easier to interact with hydrogen bonds and disrupt molecule structure in a short time. AMIC with strong hydrogen bond acceptor Cl−1 was capable to form hydrogen bonds with hydroxyl and ether oxygen of cellulose [27], and weaken the hydrogen bonds in the matrix. Meanwhile, [Amim]+ with its small bulk was more conducive to attack oxygen atoms on the hydroxides of cellulose, which made AMIC perform a nearly complete dissolution of the biomass (glucan conversion reached approximately 100%).
3.2. Enzymatic hydrolysis
3.3. SEM analysis
The pretreatment methods were evaluated by the produced sugars, and the enzymatic hydrolysis of the untreated and pretreated SCBs was comparatively studied (Table 2). In the low substrate loading, hydrolysis inhibition effect was not obvious, and enzymatic conversion worked in concert with the degree of the pretreatment. After 72 h of hydrolysis, the glucose and xylose concentrations only reached 1.35, 0.62 g/L and 1.73, 0.66 g/L, respectively, for untreated and sole IL pretreated SCBs. After the alkali-pretreatment, high cellulose digestibility was observed and the glucose and xylose concentrations reached 12.02 and 4.55 g/L,
The superficial physical structure changes of raw, alkali, MWI assisted alkali, IL, IL assisted alkali-pretreated, and enzyme hydrolyzed SCB substrates were analyzed with SEM, as shown in Fig. 2. The untreated SCB had a very compact and smooth morphology. Alkalitreated sample showed a rough structure and an obvious gully as a result of the lignin removal (Fig. 2B). After the MWI and IL assisted pretreatments, some smaller fragments were produced. For the IL assisted strategy, the regenerated cellulose morphology was largely changed into bundle fibers with relatively more porous grooves; this was
3.1. Effect of different pretreatments on SCB composition
Table 3 Values of rate constant and fractal dimension determined from the model fit of the data in Table 2. Sample
Raw Alkali Alkali + MWI IL Alkali + IL
Glucan hydrolysis
Xylan hydrolysis
Rate constant
Fractal dimension
Correlated coefficient
Rate constant
Fractal dimension
Correlated coefficient
0.01628 0.25468 0.29939 0.01868 0.38855
0.86468 0.60432 0.64820 0.86162 0.69257
0.99512 0.99905 0.99982 0.98913 0.99920
0.00128 0.14334 0.14280 0.01289 0.11431
0.85133 0.84554 0.84455 0.86760 0.91876
0.99900 0.99446 0.99890 0.98996 0.99863
Y.-Y. Liu et al. / Fuel Processing Technology 143 (2016) 1–6
probably due to the lignin and partial hemicellulose removal (Fig. 2E). These changes improved enzyme accessibility, which was in accordance with the obtained glucose yield. However, for the sole IL pretreatment, the morphologies were nearly untouched (Fig. 2D), indicating that single IL had no significant effect on SCB in a relatively short time. After 72 h of enzymatic hydrolysis, the pretreated samples became fractured with the obvious cracks, ravines and holes, particularly for MWI and IL assisted processes, suggesting that most of the substrate was sufficiently hydrolyzed.
3.4. X-ray analysis Cellulose crystallinity is considered to be one of the major substrate properties that influence the hydrolysis kinetics and yields. To gain insight into the possible structure features affecting the cellulose hydrolysis, the untreated, pretreated and enzyme hydrolyzed SCBs were examined by XRD, as shown in Fig. 3. All samples exhibited typical diffraction patterns of the cellulose I. After the IL assisted approach, the main peak at 2θ = 22°–23° was obviously weakened but still presented as a broad peak, and the peak at 2θ = 34°–35° was nearly disappeared, which suggested an expansion of the cellulose I lattice. This was in line with the results obtained by Cheng et al. [28]. Moreover, the significantly reduced crystallinity indicates that IL might mainly break the cellulose crystalline zone, and make the treated cellulose to be more soluble. Alkali method might mostly disrupt the amorphous zone; the peak at 2θ = 22°–23° was higher than other samples, especially for MWI assisted approach. This was probably due to the degradation of lignin, hemicelluloses and the amorphous regions of the cellulose. After enzymatic hydrolysis, the crystallinity of all samples was slightly increased, and the peak intensity was lower compared to the pretreated samples, which suggested that the remaining residual was the unhydrolyzed crystalline regions of the cellulose.
3.5. Fractal-like kinetic modeling To further and deeply understand the mechanisms underlying the influence of various pretreatments on enzymatic hydrolysis, the experimental data was fitted into fractal-like kinetic Eqs. (1) and (2). Fig. 4 represented that the experimental points were very close to the simulated curves, and the correlation coefficients (R2) in Table 3 were all above 0.98. These evidenced that the fit was very perfect. The well fitted results indicate that the fractal-like theory is satisfactory for studying the enzymatic hydrolysis kinetics. The larger rate constant, the greater binding capacity between substrates and enzymes was. The fractal dimension was in the range of 0 to 1, the closer to 0, the more easily substrates hydrolyzed. For glucan hydrolysis, cellulase had higher binding capacity to the IL assisted treated sample, and weaker to the untreated substrate, compared to other samples. IL assisted strategy was able to efficiently remove lignin, reduce the non-productive adsorption of enzyme, and greatly improve the enzyme binding capacity. From the perspective of the fractal dimension, alkali-treated substrates were more likely to be hydrolyzed, and the subsequent step with MWI or IL increased the fractal dimension slightly. This may be because the further processing removed some of the smaller cellulose particles that were easier to be hydrolyzed than larger portions in alkalitreated residuals. For xylan hydrolysis, xylanase combined to the alkali treated sample with the strongest capacity. The fractal dimension had little changes for all substrates including unprocessed raw materials, suggesting that the behavior of the xylan hydrolysis differed from the cellulose hydrolysis. While, the IL assisted approach adversely accelerated the xylan hydrolysis, the probable reason was that the process removes its random, amorphous structure in the substrates.
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4. Conclusions MWI and IL are efficient heating and green methods for the pretreatment of SCB. The integration of MWI or IL method with alkalipretreatment increased the saccharification of the substrates by removing lignin in large quantity and disrupting the semi-crystalline supramolecular structure of the cellulose. AMIC has been proven to be effective in cellulose dissolution and regeneration. After enhancement based on alkali-pretreatment, the lignin removal and glucan yield reached approximately 78.15% and 69.24%, respectively, and the glucan and xylan achieved the highest saccharification of 99.51% and 92.76%, respectively, after 72 h of hydrolysis. The analysis of the fractal-like kinetic indicates that cellulase was more likely to be combined with the IL assisted treated samples for glucan hydrolysis. For xylan degradation, xylanase was strongly combined with the alkali-treated residual. Fractal dimension suggested that the effects of different pretreatment methods on xylan hydrolysis were not obvious. Nomenclature [G] concentration of the generated glucose in the raw materials [X] concentration of the generated xylan in the raw materials glucan rate constant in substrates KG xylan rate constant in substrates KX glucan fractal dimension hG xylan fractal dimension hX correlation coefficients R2 Abbreviation MWI IL SCB SEM XRD AMIC IUPAC HPLC
microwave irradiation ionic liquid sugarcane bagasse field emission scanning electron microscope X-ray diffraction 1-allyl-3-methylimidazolium chloride International Union of Pure and Applied Chemistry high performance liquid chromatography
Acknowledgments This work was supported by the Program of National Natural Science Foundation of China(21306196, 21206163, 21476233), Guangdong Science and Technology Research Program(2013B010403021), Science & Technology Project of Guangzhou (2013J4300026, 2014A010106023), and Cooperation Project between Chinese Academy of Sciences and Guangxi Academy of Sciences. References [1] M. Badiei, N. Asim, J.M. Jahim, K. Sopian, Comparison of chemical pretreatment methods for cellulosic biomass, APCBEE Procedia 9 (2014) 170–174. [2] P. Alvira, E. Tomás-Pejó, M. Ballesteros, M.J. Negro, Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review, Bioresour. Technol. 101 (2010) 4851–4861. [3] S. Behera, R. Arora, N. Nandhagopal, S. Kumar, Importance of chemical pretreatment for bioconversion of lignocellulosic biomass, Renew. Sust. Energ. Rev. 36 (2014) 91–106. [4] P. Kumar, D.M. Barrett, M.J. Delwiche, P. Stroeve, Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production, Ind. Eng. Chem. Res. 48 (8) (2009) 3713–3729. [5] J. Yang, J.C. Jiang, N. Zhang, C.C. Miao, M. Wei, J. Zhao, Enhanced enzyme saccharification of Sawtooth Oak shell using dilute alkali pretreatment, Fuel 139 (2015) 102–106. [6] S.J. Zhang, D.R. Keshwani, Y.X. Xu, M.A. Hanna, Alkali combined extrusion pretreatment of corn stover to enhance enzyme saccharification, Ind. Crop. Prod. 37 (1) (2012) 352–357. [7] M. Asgher, Z. Ahmad, H.M.N. Iqbal, Alkali and enzymatic delignification of sugarcane bagasse to expose cellulose polymers for saccharification and bio-ethanol production, Ind. Crop. Prod. 44 (2013) 488–495. [8] S.D. Zhu, Y.X. Wu, Z.N. Yu, J.T. Liao, Y. Zhang, Pretreatment by microwave/alkali of rice straw and its enzymatic hydrolysis, Process Biochem. 40 (9) (2005) 3082–3086.
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Contents lists available at ScienceDirect
Fuel Processing Technology journal homepage: www.elsevier.com/locate/fuproc
Research article
Reinforced alkali-pretreatment for enhancing enzymatic hydrolysis of sugarcane bagasse Yun-Yun Liu a,b, Jing-Liang Xu a, Yu Zhang a, Cui-Yi Liang a, Min-Chao He a, Zhen-Hong Yuan a,⁎, Jun Xie b a b
Guangzhou Institute of Energy Conversion, Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, China Institute of New Energy and New Materials, South China Agriculture University, Guangzhou 510642, China
a r t i c l e
i n f o
Article history: Received 11 April 2015 Received in revised form 6 November 2015 Accepted 12 November 2015 Available online xxxx Keywords: Sugarcane bagasse Pretreatment Alkaline Microwave irradiation Ionic liquids Enzymatic hydrolysis
a b s t r a c t Microwave irradiation (MWI) and ionic liquid (IL) assisted methods were chosen to strengthen the alkali pretreatment process to improve saccharification efficiency of sugarcane bagasse (SCB). Four different pretreatment approaches namely alkali, IL, MWI-alkali and IL-alkali pretreatments were compared. The chemical composition, morphology and crystal performance of the substrates were analyzed by the standardized methods of the National Renewable Energy Laboratory, field emission scanning electron microscope (SEM) and X-ray diffraction (XRD), respectively. IL assisted treatment brought the highest lignin removal (78.51%), and the glucan and xylan conversion achieved 99.51% and 92.76%, respectively, after 72 h of enzymatic hydrolysis. The similar effect occurred for MWI assisted alkali-pretreatment approach. SEM and XRD analyses showed that the strengthened process obviously changed the structure and crystallinity of the substrates. Moreover, as a persuasive tool, the fractal-like theory was used to study the enzymatic hydrolysis kinetics. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lignocellulosic biomass mainly consists of hexose, pentose and lignin. Crystalline cellulose is partially surrounded by the amorphous hemicelluloses, which is aligned with cellulose fibers and densely packed by the layers of lignin. Lignin constructed of phenylpropane units is the most obstinate component of the plant cell wall, which is generally resistant to enzymatic hydrolysis [1]. The degree of polymerization, available surface area, lignin content and distribution are important factors that hinder the enzymes attack. Due to the inhomogeneous structure of lignocellulosic materials, efficient pretreatment technologies are required to disrupt the recalcitrance of lignocellulosic biomass for further downstream processing [2,3]. Among different pretreatment methods, alkali pretreatment has the advantage of simple device, convenient operation with high efficiency, which induces the swelling reactions and the removing of lignin, uronic acids and acetyl groups from polysaccharides, therefore, making the substrates more susceptible to enzymatic reaction [4,5]. Although alkali pretreatment can remove the majority of lignin, the residual lignin and hemicelluloses in the pretreated substrate can still hinder enzymatic hydrolysis. The digestibility improvement depended on the removal of lignin and parts of hemicelluloses, the reduction of acetyl content, and the disruption of lignin–carbohydrate matrix [6,7]. In order to achieve ⁎ Corresponding author at: No.2, Nengyuan Rd, Wushan, Tianhe District, Guangzhou, China. Tel: +8620-87057735. E-mail address: [email protected] (Z.-H. Yuan).
http://dx.doi.org/10.1016/j.fuproc.2015.11.004 0378-3820/© 2015 Elsevier B.V. All rights reserved.
efficient hydrolysis, using the synergistic benefits of the combined methods may be an integrated novel pretreatment approach. Microwave irradiation (MWI) with the advantages of high heating efficiency and easy operation has been widely used in many areas. It has been shown in some studies that MWI can change the ultrastructure of cellulose and degrade lignin and hemicelluloses. The pretreated particles were characterized by swelling and fragmentation, which was favorable for enzyme accessibility. This technique can be easily combined with chemical reaction and accelerate the reaction rate [8–10]. The recent discovery of ionic liquid (IL) can effectively dissolve cellulose and biomass and is suggested to be a potential alternative to established pretreatment techniques [11]. IL pretreatment can reduce the cellulose crystallinity and partially remove lignin and hemicelluloses, thus enhance the biomass digestibility. It has high degree of flexibility in process design with less energy costing, and is easier to handle than other pretreatment methods [12,13]. The enzymatic hydrolysis of cellulose can be thought as a heterogeneous reaction with less than three dimensions [14] along a cellulosic fiber chain. Before the reaction, enzyme must be absorbed on the substrate surface and then diffuse to a reactive site. These complex reactions and mechanisms can be understood using a mathematical model [15]. The fractal-like kinetic model has been applied to analyze the effect of pretreatment on the enzymatic saccharification kinetics. With two parameters (fractal exponent and rate coefficient), it fitted very well to the experimental data in the literatures [16–18]. In the present study, alkali pretreatment was enhanced by MWI or IL considering the synergistic benefits of them for improving the hydrolysis
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Table 1 Chemical compositions of untreated and pretreated SCBs. Pretreatment method
Untreated Alkali Alkali + MWI IL Alkali + IL
Composition (%) Glucan
Xylan
Klason lignin
38.51 63.43 65.62 44.82 69.24
20.65 26.11 26.54 21.49 21.68
25.03 8.26 7.13 27.16 5.47
efficiency. The comparison of single alkali and IL pretreatments was also applied on sugarcane bagasse (SCB). 1-Allyl-3-methylimidazolium chloride (AMIC) was used as a representative IL because of its enhanced hydrogen bond basicity and lower viscosity [19]. The structural characterization of SCB substrates was investigated by field emission scanning electron microscope (SEM) and X-ray diffraction (XRD), and the fractal-like model was used to theoretically analyze the effect of various pretreatments on enzymatic hydrolysis.
Fig. 1. Effect of pretreatment method on enzymatic conversion of the substrates.
freeze-dried before subsequent enzymatic hydrolysis. The filtrate, containing water, IL, and some degraded carbohydrates as well as lignin, was separated under the reduced pressure.
2. Materials and methods 2.1. Materials
2.2.3. MWI assisted alkali pretreatment MWI was carried out in a MCR-3 microwave chemistry reactor. After the alkali pretreatment, the bottle with substrate mixtures was placed into the microwave reactor and the treatment program with the desired time of 5 min and a power of 600 W was started [22]. After the run, the bottle was removed from the reactor. The obtained solid was separated and dried according to Section 2.2.1.
SCB was obtained from Guangxi Fenghao Sugar Co., Ltd. (Chongzuo, China). It was pre-milled and screened, and the fraction between 18 and 40 meshes was used for alkali-pretreatment. AMIC was purchased from Shanghai Cheng Jie Chemical Co. Ltd. A cellulase mixture, CTec2, was kindly provided by Novozymes A/S (Bagsaevrd, Denmark), with an enzyme activity of 200 FPU/mL measured by the description of International Union of Pure and Applied Chemistry [20]. All other chemicals used were of analytical or reagent grade and directly used as purchased without further purification.
2.2.4. IL assisted alkali pretreatment The alkali-pretreated substrate was successively treated with IL, and the same pretreatment procedure was conducted according to Section 2.2.2. After the reaction, the wet regenerated SCB was freezedried for 24 h prior to enzymatic hydrolysis and further analysis.
2.2. Pretreatment procedure 2.2.1. Alkali pretreatment SCB was pretreated by 0.5 M NaOH in a round-bottom flask, at 80 °C for 2 h with agitation (solid–liquid ratio is 1:20). After the pretreatment, the solid fraction was separated by filtering and then washed with tap water until a neutral pH. The obtained residual fraction was dried at 65 °C and then used as substrates for the subsequent enzymatic hydrolysis. All experiments were carried out three times, and the given numbers were the mean values.
2.3. Enzymatic hydrolysis Enzymatic hydrolysis was carried out with 2% (w/v) substrate at pH 5.0, a temperature of 50 °C and a rotation speed of 150 rpm for 72 h in 250 mL Erlenmeyer flasks. The enzyme-loading was 9.6 FPU/g of biomass. Samples for sugar assay were taken at time points (specified in the text) and analyzed by high performance liquid chromatography (HPLC). Glucan and xylan conversion rates were estimated by the following equations:
2.2.2. IL pretreatment Details of IL pretreatment were described elsewhere [21]. 5 g of SCB was quickly mixed with 95 g of AMIC solution at a solid/liquid ratio of 1:20, in a 250 mL dried flask. The flask was sealed with a cork and placed in oil bath at 80 °C for 2 h with stirring. After the reaction, the obtained substrate was precipitated using deionized water (90 °C) with vigorous shaking for 10 s. The regenerated substrate was then transferred into a beaker and washed with 50 mL deionized water (75 °C) thoroughly, and
Glucan conversion rate ¼
Xylan conversion rate ¼
Glucose produced ðgÞ 0:9 Glucan amount in enzymatic substrate
Xylose produced ðgÞ 0:88 : Xylan amount in enzymatic substrate
ð1Þ
ð2Þ
Table 2 Glucose and xylose concentrations (g L−1) produced from enzymatic hydrolysis of different substrates. Time (h)
6 12 24 48 72
Raw
Alkali-treated
Alkali + MWI
IL-treated
Alkali + IL
Glucose
Xylose
Glucose
Xylose
Glucose
Xylose
Glucose
Xylose
Glucose
Xylose
0.96 1.12 1.25 1.28 1.35
0.44 0.48 0.55 0.60 0.62
8.79 10.12 11.33 11.95 12.02
3.79 4.19 4.59 4.61 4.55
10.09 11.16 12.04 12.47 12.90
3.91 4.20 4.54 4.62 4.73
1.27 1.38 1.69 1.69 1.73
0.49 0.54 0.64 0.66 0.66
12.06 12.72 13.39 13.61 13.78
3.69 3.88 4.04 4.00 4.02
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Fig. 2. SEM micrographs of the substrates. Raw material (A); pretreated SCB: alkali-pretreated (B), MWI assisted alkali-pretreated (C), AMIC pretreated (D), AMIC assisted alkali-pretreated (E) and enzyme hydrolyzed SCBs: raw material (a), alkali-pretreated (b), MWI assisted alkali-pretreated (c), AMIC pretreated (d) and AMIC assisted alkali-pretreated SCBs (e).
with a flow rate of 0.5 mL/min at 50 °C. Standard and hydrolyzed samples were filtrated by 0.22 μm filter before analysis.
2.4. Fractal-like kinetic modeling Fractal-like kinetic model considers reaction in materials that are prepared as fractals [23]. It assumes that cellulase and xylanase consist of several components to form a single combined effect on the substrate hydrolysis, and the reaction fits the fractal-like kinetic model, then the glucose and xylose expression can be deduced as follows: ½G ¼
C G ½S0 0:9kG 1−hG t 1− exp − 1−hG 0:9
ð3Þ
½X ¼
C X ½S0 0:88kX 1−hX t 1− exp − 1−hX 0:88
ð4Þ
where [G] and [X] are the concentrations of the generated glucose and xylose, CG and Cx are the glucan and xylan contents in the raw materials, KG and KX, hG and hX are the empirical constants representing rate constant and fractal dimension of glucan and xylan in substrates, respectively. 2.5. Analytical methods 2.5.1. Sugar assay Glucose and xylose concentrations were determined by HPLC using a Shodex sugar SH-1011 column coupled with a refractive index detector. The mobile phase of 5 mM H2SO4 in demineralized water was used
2.5.2. Compositional analysis Glucan, xylan, and Klason lignin contents in raw, pretreated, and enzyme hydrolyzed SCBs were analyzed in duplicate according to the standardized methods of the National Renewable Energy Laboratory, USA [24].
2.5.3. SEM assay The surface morphology and characteristics of the different residual solids were observed using SEM. Images were taken by the model JEOL JSM-4800 LV SEM and performed at a beam accelerating voltage of 10 kV. Digital images were captured with 1280 × 960 resolution and 160 s dwell time.
2.5.4. XRD measurement X-ray powder diffraction pattern of samples was analyzed using an XRD instrument (PANalytical V.B., Netherlands). The determination was performed with Ni-filtered Cu Kα radiation (λ = 1.54 Å) at 40 kV and 40 mA, with a recorded range of 50 to 500. The sample crystallinity was quantified from XRD data using a crystallinity index and CrI for cellulose [25].
Fig. 3. XRD spectra of untreated, pretreated and enzyme hydrolyzed SCBs.
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Fig. 4. Fitted values from fractal-like model versus experimental values.
3. Results and discussion
The SCB used in this study consists of glucan, xylan, and lignin in the contents of 38.51%, 20.65%, and 25.03%, respectively. After the pretreatment, the composition of SCB was drastically changed, especially for the integrated alkali-pretreatment. Single-alkali-pretreatment removed approximately 67% of lignin, as shown in Table 1, and the percentage of glucan and xylan were all increased, which made cellulose partially exposed. When the assisted step of MWI pretreatment was added, more than 70% of the lignin was removed (71.51% removal), and the percentage of glucan and xylan (65.62% and 26.54%) were increased by 62.97% and 28.52%, respectively, compared to the raw material. When adding another step of IL pretreatment, the glucan content was increased to 69.24% with further delignification, and the largest Klason lignin removal of about 78.15% was found. However, for the sole IL pretreatment, the effect was not obvious in a short time. As shown in Table 1, the combined pretreatment could effectively remove much more lignin and keep the majority of cellulose in the residues than single alkali or IL pretreatment, especially for IL assisted alkali-pretreatment, which would facilitate the subsequent enzymatic hydrolysis.
respectively. This increase in sugar concentration can be attributed to the decrease in lignin/hemicellulose content, and the increase in the cellulose surface area. For the sole IL pretreatment, the relative lower sugar yield was caused by the insufficient cellulose dissolution in a relatively short period of time. When an assisted pretreatment step was added, the composition of the substrates had important changes as mentioned in Table 1. These changes could facilitate the subsequent hydrolysis and lead to the obvious increase of enzymatic digestibility. After the MWI assisted pretreatment, the glucan and xylan conversion reached 98.25% and 89.19%, respectively (Fig. 1). Under similar conditions, IL assisted pretreatment achieved better results. The glucan and xylan conversion (glucan, 99.51% and xylan, 92.76%) was significantly improved compared to the single alkali or IL treatment (Fig. 1). MWI based on intramolecular heat transfer can cause vibration and shock to polar bonds inside biomass and thus change the molecular activity [26]. After the alkalipretreatment, MWI could be easier to interact with hydrogen bonds and disrupt molecule structure in a short time. AMIC with strong hydrogen bond acceptor Cl−1 was capable to form hydrogen bonds with hydroxyl and ether oxygen of cellulose [27], and weaken the hydrogen bonds in the matrix. Meanwhile, [Amim]+ with its small bulk was more conducive to attack oxygen atoms on the hydroxides of cellulose, which made AMIC perform a nearly complete dissolution of the biomass (glucan conversion reached approximately 100%).
3.2. Enzymatic hydrolysis
3.3. SEM analysis
The pretreatment methods were evaluated by the produced sugars, and the enzymatic hydrolysis of the untreated and pretreated SCBs was comparatively studied (Table 2). In the low substrate loading, hydrolysis inhibition effect was not obvious, and enzymatic conversion worked in concert with the degree of the pretreatment. After 72 h of hydrolysis, the glucose and xylose concentrations only reached 1.35, 0.62 g/L and 1.73, 0.66 g/L, respectively, for untreated and sole IL pretreated SCBs. After the alkali-pretreatment, high cellulose digestibility was observed and the glucose and xylose concentrations reached 12.02 and 4.55 g/L,
The superficial physical structure changes of raw, alkali, MWI assisted alkali, IL, IL assisted alkali-pretreated, and enzyme hydrolyzed SCB substrates were analyzed with SEM, as shown in Fig. 2. The untreated SCB had a very compact and smooth morphology. Alkalitreated sample showed a rough structure and an obvious gully as a result of the lignin removal (Fig. 2B). After the MWI and IL assisted pretreatments, some smaller fragments were produced. For the IL assisted strategy, the regenerated cellulose morphology was largely changed into bundle fibers with relatively more porous grooves; this was
3.1. Effect of different pretreatments on SCB composition
Table 3 Values of rate constant and fractal dimension determined from the model fit of the data in Table 2. Sample
Raw Alkali Alkali + MWI IL Alkali + IL
Glucan hydrolysis
Xylan hydrolysis
Rate constant
Fractal dimension
Correlated coefficient
Rate constant
Fractal dimension
Correlated coefficient
0.01628 0.25468 0.29939 0.01868 0.38855
0.86468 0.60432 0.64820 0.86162 0.69257
0.99512 0.99905 0.99982 0.98913 0.99920
0.00128 0.14334 0.14280 0.01289 0.11431
0.85133 0.84554 0.84455 0.86760 0.91876
0.99900 0.99446 0.99890 0.98996 0.99863
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probably due to the lignin and partial hemicellulose removal (Fig. 2E). These changes improved enzyme accessibility, which was in accordance with the obtained glucose yield. However, for the sole IL pretreatment, the morphologies were nearly untouched (Fig. 2D), indicating that single IL had no significant effect on SCB in a relatively short time. After 72 h of enzymatic hydrolysis, the pretreated samples became fractured with the obvious cracks, ravines and holes, particularly for MWI and IL assisted processes, suggesting that most of the substrate was sufficiently hydrolyzed.
3.4. X-ray analysis Cellulose crystallinity is considered to be one of the major substrate properties that influence the hydrolysis kinetics and yields. To gain insight into the possible structure features affecting the cellulose hydrolysis, the untreated, pretreated and enzyme hydrolyzed SCBs were examined by XRD, as shown in Fig. 3. All samples exhibited typical diffraction patterns of the cellulose I. After the IL assisted approach, the main peak at 2θ = 22°–23° was obviously weakened but still presented as a broad peak, and the peak at 2θ = 34°–35° was nearly disappeared, which suggested an expansion of the cellulose I lattice. This was in line with the results obtained by Cheng et al. [28]. Moreover, the significantly reduced crystallinity indicates that IL might mainly break the cellulose crystalline zone, and make the treated cellulose to be more soluble. Alkali method might mostly disrupt the amorphous zone; the peak at 2θ = 22°–23° was higher than other samples, especially for MWI assisted approach. This was probably due to the degradation of lignin, hemicelluloses and the amorphous regions of the cellulose. After enzymatic hydrolysis, the crystallinity of all samples was slightly increased, and the peak intensity was lower compared to the pretreated samples, which suggested that the remaining residual was the unhydrolyzed crystalline regions of the cellulose.
3.5. Fractal-like kinetic modeling To further and deeply understand the mechanisms underlying the influence of various pretreatments on enzymatic hydrolysis, the experimental data was fitted into fractal-like kinetic Eqs. (1) and (2). Fig. 4 represented that the experimental points were very close to the simulated curves, and the correlation coefficients (R2) in Table 3 were all above 0.98. These evidenced that the fit was very perfect. The well fitted results indicate that the fractal-like theory is satisfactory for studying the enzymatic hydrolysis kinetics. The larger rate constant, the greater binding capacity between substrates and enzymes was. The fractal dimension was in the range of 0 to 1, the closer to 0, the more easily substrates hydrolyzed. For glucan hydrolysis, cellulase had higher binding capacity to the IL assisted treated sample, and weaker to the untreated substrate, compared to other samples. IL assisted strategy was able to efficiently remove lignin, reduce the non-productive adsorption of enzyme, and greatly improve the enzyme binding capacity. From the perspective of the fractal dimension, alkali-treated substrates were more likely to be hydrolyzed, and the subsequent step with MWI or IL increased the fractal dimension slightly. This may be because the further processing removed some of the smaller cellulose particles that were easier to be hydrolyzed than larger portions in alkalitreated residuals. For xylan hydrolysis, xylanase combined to the alkali treated sample with the strongest capacity. The fractal dimension had little changes for all substrates including unprocessed raw materials, suggesting that the behavior of the xylan hydrolysis differed from the cellulose hydrolysis. While, the IL assisted approach adversely accelerated the xylan hydrolysis, the probable reason was that the process removes its random, amorphous structure in the substrates.
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4. Conclusions MWI and IL are efficient heating and green methods for the pretreatment of SCB. The integration of MWI or IL method with alkalipretreatment increased the saccharification of the substrates by removing lignin in large quantity and disrupting the semi-crystalline supramolecular structure of the cellulose. AMIC has been proven to be effective in cellulose dissolution and regeneration. After enhancement based on alkali-pretreatment, the lignin removal and glucan yield reached approximately 78.15% and 69.24%, respectively, and the glucan and xylan achieved the highest saccharification of 99.51% and 92.76%, respectively, after 72 h of hydrolysis. The analysis of the fractal-like kinetic indicates that cellulase was more likely to be combined with the IL assisted treated samples for glucan hydrolysis. For xylan degradation, xylanase was strongly combined with the alkali-treated residual. Fractal dimension suggested that the effects of different pretreatment methods on xylan hydrolysis were not obvious. Nomenclature [G] concentration of the generated glucose in the raw materials [X] concentration of the generated xylan in the raw materials glucan rate constant in substrates KG xylan rate constant in substrates KX glucan fractal dimension hG xylan fractal dimension hX correlation coefficients R2 Abbreviation MWI IL SCB SEM XRD AMIC IUPAC HPLC
microwave irradiation ionic liquid sugarcane bagasse field emission scanning electron microscope X-ray diffraction 1-allyl-3-methylimidazolium chloride International Union of Pure and Applied Chemistry high performance liquid chromatography
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