Abstract
Download PDF
Full Article
Microstructure Properties and Cellulase Hydrolysis Efficiency of Hybrid Pennisetum with [Amim]Cl Pretreatment
Shengdan Wang,a Jiachuan Chen,b,* Guihua Yang,b Kefu Chen,a Rendang Yang,a and Jinsong Zeng a,*
The complex microstructure of lignocellulosic biomass restricts its conversion into bio-ethanol. In this study, the effects of an ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([Amim]Cl) pretreatment on the microstructure properties and cellulase hydrolysis efficiency of hybrid Pennisetum (P. americanum × P. purpureum, lignocellulosic biomass) were investigated. After the [Amim]Cl pretreatment, the bonds of lignin-carbohydrate complex (LCC) and C=O in xylan were destroyed and the content of inter-molecular H-bonds O(6)H…O(3’) decreased by 47.2%, while the content of intra-molecular H-bonds of O(2)H…O(6) and O(3)H…O(5) increased by 9.5% and 47.0%, respectively. The crystallinity and the crystallite size decreased by 20.8% and 42.22%, respectively, and the cellulose crystalline structure changed from cellulose crystalline I to cellulose crystalline II. The specific surface area increased from 0.15 to 10.11 m2/g after the [Amim]Cl pretreatment. The glucose recovery increased by 10.3 times after being pretreated with [Amim]Cl, compared with the unpretreated sample.
Keywords: Hybrid Pennisetum; Microstructure; Pretreatment; [Amim]Cl; Enzymatic hydrolysis
Contact information: a: State Key Laboratory of Pulp and Paper Engineering, Plant micro/nano fiber research center, School of Light Industry and Engineering, South China University of Technology, Guangzhou, 510640, China; b: Key Lab of Pulp & Paper Science and Technology of Education Ministry of China, Qi Lu University of Technology, Jinan, 250353, China;
* Corresponding authors: chenjc@qlu.edu.cn; fezengjs@scut.edu.cn
INTRODUCTION
Lignocellulosic biomass is a sustainable raw material used for the production of alternative fossil fuels, such as the conversion of cellulose to cellulosic ethanol, furfural, and other high value-added biopolymers (Rose and Palkovitz 2011). As a relatively new feedstock, the energy grass hybrid Pennisetum, a perennial herbaceous plant, has attracted growing research because of its advantages, such as its resistance to salinity and drought, suitability for marginal land quality, high productivity, low nutritional requirements, environmental benefits, and its versatility (Chen et al. 2014). The grass hybrid is cultivated in north China, the Middle-Lower Yangtze Plain, northeast China, and southwest China, and can grow up to more than 2 m tall in 5 to 6 months, with annual yields as high as 60 to 130 tons per hm2 (Liu 2009; Zhao and Zhou 2010). Using the cellulose in hybrid Pennisetum as a C6 carbon source to produce bio-ethanol would be an effective process for its utilization in China, instead of burning the plant. The hydrolysis of cellulose in the hybrid Pennisetum can be carried out to produce bio-ethanol (Tadesse and Luque 2011). However, the complex structure and recalcitrance of its plant cell wall makes it difficult to achieve bio-energy conversion efficiently (Jørgensen et al. 2007).
Therefore, a pretreatment is a necessary step to destroy the rigid protective sheath for the improved glucose recovery of cellulose hydrolysis during the production of bio-energy (Zheng et al. 2009; Xu et al. 2013). Quite a number of pre-processing operations have shown a tendency to improve the hydrolysis efficiency, including physical, biological, and chemical methods (Yang and Wyman 2008). Compared with the above pre-processing methods, ionic liquid (IL) pretreatments have attracted increasing research for the dissolution and subsequent hydrolysis of lignocellulosic biomass. This is due to their desirable properties, such as an environmentally benign nature, low melting point, low volatility and toxicity, and high thermal and chemical stability (Tadesse and Luque 2011). Cellulose samples could be dissolved by many ILs, such as 1-ethyl-3-methylimidazolium acetate ([Emim]Ac), 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), and 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Among the different ILs, [Amim]Cl possesses a high reactivity of allyl chloride and shows better solubility for cellulose samples than the others (Zhang et al. 2005; Liu et al. 2012).
Recently, the optimization of [Amim]Cl pretreatment conditions for the maximum glucose recovery from hybrid Pennisetum has been obtained by response surface methodology (Wang et al. 2015). However, the microstructural changes and those effects on the cellulase hydrolysis efficiency of hybrid Pennisetum were not elucidated. This study aimed to investigate the microstructure properties of hybrid Pennisetum after [Amim]Cl pretreatment. A novel analytical approach of the Gaussian distribution function was used to analyze the H-bond energies (EH) and bond lengths (R) of different H-bond models, and the crystallite size of the cellulose in the hybrid Pennisetum. Furthermore, the effects of the change in microstructure properties of the hybridPennisetum on the cellulase hydrolysis efficiency were elucidated.
EXPERIMENTAL
Materials
The hybrid Pennisetum was collected from an experimental field of the Beijing Academy of Agricultural Sciences (China). It was milled with a FZ120 plant shredder (Truelab, Shanghai, China) and then sieved to 40- to 60-mesh. A comprehensive extraction process with toluene/ethanol (2:1, v/v) was performed in a Blst-250SQ Soxhlet apparatus (Bilon, Shanghai, China). The extractive-free samples were stored at 4 °C in a sealed bag. The ionic liquid ([Amim]Cl) was purchased from the Lanzhou Institute of Chemical Physics (Lanzhou, China). Commercial cellulase (Celluclast 1.5 L, activity 84.5 FPU/g) from Trichoderma reesei was purchased from Novozymes (Beijing, China).
Methods
Ionic liquid pretreatment
The pretreatment process was performed according to the authors’ previous work (Wang et al.2015). Briefly, 5 g of hybrid Pennisetum was placed in 49.95 g of [Amim]Cl in a 150-mL dried screw-capped Synthware tube (Synthware, Beijing, China). The mixture was heated to 139 °C in a preheated oil bath for 178 min. After cooling to 90 °C, deionized water (90 °C) was added to the slurry with an agitation rate of 250 rpm for 1 h. The mixture was centrifuged to separate the solid (pretreated samples). The precipitate (pretreated sample) was freeze-dried and collected for subsequent analyses.
Enzymatic hydrolysis
The enzymatic hydrolysis of pretreated and unpretreated samples was performed in 25-mL stoppered conical flasks at 50 °C in an air bath shaking incubator (ZWY-2102C, Shanghai, China) at 150 rpm for 72 h. Then, 0.2 g of the pretreated sample was suspended in 10 mL of a 50 mM citrate acid-sodium citrate buffer (pH 4.8) at an enzyme loading of 20 FPU/g substrate. After hydrolysis, the samples were kept in a boiling bath for 5 min to inactivate the enzyme.
The glucose concentrations from the enzymatic hydrolysates were measured by HPLC (Agilent 1200, CA, USA) with a Bio-Rad Aminex HPX-87H analytical column. The column temperature was set at 35 °C (Wang et al. 2015).
Component analysis
The chemical composition of the unpretreated and pretreated samples was analyzed using the methods of the Laboratory Analytical Procedure (LAP) provided by the National Renewable Energy Laboratory (NREL) (Sluiter et al. 2008).
FTIR analysis
The deconvolution of the spectra (3800 to 3000 cm-1) was carried out using Peak-Fit (Jinan, China) software (4.12.00) in conjunction with the Gaussian distribution function to analyze the H-bond energies (EH), bond strength, and bond lengths (R) of different H-bond models. After deconvolution, the H-bond characteristic parameters were calculated as follows (Struszczyk and Laine 1986),
where v0 is the standard free hydroxyl frequency (3650 cm-1), v is the sample hydroxyl frequency, and k is a constant (6.7 × 10-2 kJ-1).
In. Eq. 2, Δv = v0-v, v0 is the stretch vibrational frequencies of hydroxyl (3600 cm-1), v is the sample hydroxyl frequency (cm-1), and R is the bond length (Å).
X-ray diffraction (XRD) analysis
The crystallinity of cellulose I and cellulose II, and the average width of the crystal in the 002/101-lattice plane, were calculated as follows (Langford and Wilson 1978; Struszczyk and Laine 1986),
where I002 is the diffraction intensity at 2θ = 22.6° and Iam is the diffraction intensity at 2θ = 18°.
In Eq. 4, I002 is the diffraction intensity at 2θ = 20.8° and Iam is the diffraction intensity at 2θ = 16°.
In Eq. 5, k is the Scherrer constant (0.89), λ is the wavelength of the X-ray source (0.15418 nm), βis the width of the middle height of the 002/101-lattice plane, in RAD, and θ is the maximum of the 002 reflection, in RAD.
The spectra were deconvolved using the Peak-Fit software in conjunction with the Gaussian distribution function to calculate the half-peak width and microcrystalline size.
Scanning electron microscopy (SEM) observation and BET surface area (BET) analysis
The surface characteristics of pretreated and unpretreated samples were observed using a scanning electron microscope (SEM, FEI Quanta-200 ESEM, EIN, Holland). Moreover, the specific surface area of the pretreated and unpretreated samples was analyzed by the nitrogen adsorption method (BET, V-Sorb 2800P, Beijing, China).
RESULTS AND DISCUSSION
Compositional Analysis
The chemical composition results show that the recovery yield of hybrid Pennisetum after [Amim]Cl pretreated was 69.6%. The cellulose, hemicelluloses, and lignin contents in the unpretreated sample were 36.64%, 18.05%, and 11.03%, respectively. The content of cellulose in the pretreated sample rose by 8.39%, to 45.03%, while the hemicelluloses and lignin contents fell by 5.64% and 1.41%, to 12.41% and 9.62%, respectively. This shows that part of the hemicelluloses and lignin in the hybrid Pennisetum were removed after the [Amim]Cl pretreatment, especially the hemicelluloses.
Changes in microstructure of cellulose of hybrid Pennisetum
A comparison of the FTIR spectra (Fig. 1a) of the unpretreated and pretreated hybrid Pennisetumwas used to detect the changes of the structural features. As shown in Fig. 1(a), the spectral intensity of characteristic function groups at 1730 cm-1 of C=O in xylan decreased (Kootstra et al.2009), while the spectral intensity at 1460 cm-1 of the CH2 in-plane bending vibration increased for the pretreated sample (Kumar et al. 2009). This indicates that the LCC and C=O bonds of xylan in hybrid Pennisetum were destroyed after the [Amim]Cl pretreatment (He et al. 2008).
The band at 3000 to 3800 cm-1 was attributed to the stretching vibration of the intermolecular and intra-molecular H-bonds (Sang et al. 2005; Popescu et al. 2007). The FTIR spectra in the region of 3000 to 38000 cm-1 were fitted into three peaks to analyze the intra-molecular and inter-molecular H-bonds of the pretreated and unpretreated samples. As shown in Fig. 1(b), the intra-molecular H-bonds for O(2)H…O(6) and O(3)H…O(5), and the inter-molecular H-bonds for O(6)H…O(3’) appeared at wave-numbers of 3455 to 3410 cm-1, 3375 to 3340cm-1, and 3310 to 3230 cm-1, respectively (Pimentel and Sederholm 1956). The peak-fitting parameters of different H-bonds are shown in Table 1.
As shown in Table 1, the content of inter-molecular H-bonds of O(6)H…O(3`) in the pretreated sample decreased by 47.25%, while the bond energy of inter-molecular H-bonds O(6)H…O(3’) was the maximum, although its bond length was the shortest. This result indicates that the inter-molecular H-bonds are difficult to break and had a more significant positive effect on cellulase hydrolysis efficiency than intra-molecular H-bonds. The decreasing of inter-molecular H-bonds resulted in the loosening of the cellulose chains and helped to expose the free hydroxyl groups of the hybrid Pennisetum after the [Amim]Cl pretreatment. Additionally, Table 1 shows that the content of intra-molecular H-bonds O(2)H…O(6) were more than the O(3)H…O(5) in hybrid Pennisetum. Moreover, the energy of intra-molecular H-bonds O(2)H…O(6) was higher than O(3)H…O(5). However, the increment of intra-molecular H-bonds O(3) H…O(5) (47.0%) was more than O(2)H…O(6) (9.5%) after the [Amim]Cl pretreatment. This indicated that the intra-molecular H-bonds O(3)H…O(5) were easier to form than O(2)H…O(6) during the [Amim]Cl pretreatment process. Similar results were reported for producing 5-hydroxymethyl furfural viawaste paper (Wan et al. 2015).
Fig. 1. The FTIR spectra of the pretreated and unpretreated samples (a), and the fitting of H-bonds stretching (b). (I) inter-molecular H-bonds O(6)H…O(3’); (II) intra-molecular H-bonds O(3)H…O(5); (III) intra-molecular H-bonds O(2)H…O(6); and (IV) raw spectra
The XRD spectra of the unpretreated and pretreated samples are shown in Fig. 2. The diffraction angles of the 002 and 101 lattice planes were changed after the [Amim]Cl pretreatment. This demonstrates that the cellulose crystal type of hybrid Pennisetum pretreated by [Amim]Cl changed from cellulose crystalline I to cellulose crystalline II.
Table 1. Characteristics of Different H-bond Models Obtained from FTIR Peak Fitting
Fig. 2. The XRD spectra of the pretreated and unpretreated samples
Table 2 shows the microcrystalline parameters of unpretreated samples and those pretreated with [Amim]Cl. The relative crystallinities of unpretreated and pretreated samples were 48.1% and 38.1%, respectively. The relative crystallinity of the pretreated sample decreased by 20.8% after the [Amim]Cl pretreatment. Moreover, the I002 lattice plane crystallite size of cellulose crystalline I in the unpretreated sample and the I101 lattice plane crystallite size of cellulose crystalline II in pretreated sample were 56.16 and 32.45 nm, respectively.
Compared with the unpretreated sample, the crystallite size of the characteristic lattice plane (cellulose crystalline II) in pretreated samples decreased by 42.22%. It is well known that inter-molecular H-bonds contribute to the formation of crystalline portions in cellulose molecules.
From a comparison of Tables 1 and 2, it can be found that the content of inter-molecular H-bonds and the microcrystalline size of cellulose in hybrid Pennisetum have similar variation tendencies after [Amim]Cl pretreatment. This result implies that when the content of inter-molecular H-bonds decreased, the adjacent cellulose chains loosened and led to a decrease in the amorphous portions (Vainio and Paulapuro 2007). Therefore, the related microcrystalline size in the crystalline portions decreased.
Table 2. Microcrystalline Parameters of Unpretreated and Pretreated Samples
Fig. 3. Scanning electron micrographs of (a) unpretreated and (b) pretreated samples
The SEM images of the unpretreated and pretreated samples are illustrated in Fig. 3. The pretreated samples show a different morphology compared with the unpretreated samples, with rough and porous surfaces because of the removal of lignin and hemicelluloses. Furthermore, the specific surface area of unpretreated samples (11.09 m2/g) increased by 73 times after the [Amim]Cl pretreatment (0.15 m2/g). This result implies that the surface structures of hybrid Pennisetum were severely damaged by [Amim]Cl pretreatment process.
Cellulase Hydrolysis Efficiency
A comparison of the cellulase hydrolysis efficiency of both the pretreated and unpretreated sample is shown in Fig. 4. Compared with the glucose concentration of the unpretreated sample (1.25 g/L), the glucose concentration of the pretreated sample was markedly increased (14.15 g/L) after 70 h of enzymatic hydrolysis.
Fig. 4. Comparison of enzymatic hydrolysis before and after [Amim]Cl pretreatment
It can be inferred that there is a direct relationship between the cellulase hydrolysis efficiency and hemicellulose/lignin removal. Similar results have been reported for a dilute sulfuric acid pretreatment of hybrid poplar (Sun et al. 2014). The high glucose recovery may be primarily a result of the decrease of inter-molecular H-bonds, resulting in the loosening of the cellulose chains, and the weaker hydrophobic interaction by transforming cellulose I to cellulose II (Wada et al. 2010). Moreover, the increasing specific surface area and roughness will accelerate the adsorption of cellulase enzymes and subsequently enhance the enzyme attack.
CONCLUSIONS
- The highest glucose concentration of 14.15 g/L was obtained after 72 h of enzymatic hydrolysis from the pretreated sample. The glucose recovery increased by 10.3 times after the [Amim]Cl pretreatment compared with the unpretreated sample.
- A decrease in the relative content of inter-molecular H-bonds O(6)H…O(3’), hemicellulose, and lignin through [Amim]Cl pretreatment can greatly improve the cellulase hydrolysis efficiency of hybrid Pennisetum. Similar statements can be made for the decrease in the relative crystallinity and crystallite size.
- The increasing surface roughness and porosity by [Amim]Cl pretreatment can improve the cellulase hydrolysis efficiency of hybrid Pennisetum. Moreover, the cellulose crystalline II of hybrid Pennisetum is far easier to hydrolyze than that of cellulose crystalline I.
The authors are grateful for the support of the National Natural Science Foundation of China (Grant No. 31270627, 31370580, 31400511), the Major Science and Technology Projects of Shandong Province (No. 2014ZZCX09101, 2015ZDZX09002), the Taishan Scholars Project Special Funds, 111 plan, and the Guangdong provincial science and technology plan projects (Number: 2015B020241001, Name: Research and Application of Biomass Pretreatment and Ethanol Production Technology).
REFERENCES CITED
Chen, M., Liu, Y., Sui, N., Yuan, F., and Wang, B. S. (2014). “The bioenergy plant hybrid Pennisetum improves chilling resistance by enhancing the unsaturation of membrane lipid under low temperature,” Applied Mechanics and Materials 521, 81-87. DOI: 10.4028/www.scientific.net/AMM.521.81
He, J. X., Cui, S. Z., and Wang, S. Y. (2008). “Preparation and crystalline analysis of high-grade bamboo dissolving pulp for cellulose acetate,” Journal of Applied Polymer Science 107(2), 1029-1038. DOI: 10.1002/app.27061
Jørgensen, H., Kristensen, J. B., and Felby, C. (2007). “Enzymatic conversion of lignocellulose into fermentable sugars: Challenges and opportunities,” Biofuels Bioproducts and Biorefining 1(2), 119-134. DOI: 10.1002/bbb.4
Kootstra, A. M. J., Beeftink, H. H., Scott, E. L., and Sanders, J. P. M. (2009). “Comparison of dilute mineral and organic acid pretreatment for enzymatic hydrolysis of wheat straw,” Biochemical Engineering Journal 46(2), 126-131. DOI: 10.1016/j.bej. 2009.04.020
Kumar, R., Mago, G., Balan, V., and Wyman, C. E. (2009). “Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies,” Bioresource Technology 100(17), 3948-3962. DOI: 10.1016/j.biortech.2009.01.075
Liu, L. D. (2009). “Utilization of the high quality hybrid pennisetum,” Inner Mongolia Agricultural Science and Technology 5, 86-87. DOI: 10.3969/j.iissn.1007-0907.2009.05.045
Liu, D.-T., Xia, K.-F., Cai, W.-H., Yang, R.-D., Wang, L.-Q., and Wang, B. (2012). “Investigations about dissolution of cellulose in the 1-allyl-3-alkylimidazolium chloride ionic liquids,” Carbohydrate Polymers 87(2), 1058-1064. DOI: 10.1016/j.carbpol.2011.08.026
Langford, J. I., and Wilson, A. J. C. (1978). “Scherrer after sixty years: A survey and some new results in the determination of crystallite size,” Journal of Applied Crystallography 11(2), 102-113. DOI: 10.1063/1.4928401
Popescu, C. -M., Popescu, M. -C., Singurel, G., Vasile, C., Argyropoulos, D. S., and Willfor, S. (2007). “Spectral characterization of eucalyptus wood,” Society for Applied Spectroscopy 61(11), 1168-1177. DOI: 10.1366/000370207782597076
Pimentel, G. C., and Sederholm, C. H. (1956). “Correlation of infrared stretching frequencies and hydrogen bond distances in crystals,” The Journal of Chemical Physics 24(4), 639-641. DOI: 10.1063/1.1742588
Rose, M., and Palkovits, R. (2011). “Cellulose-based sustainable polymers: State of the art and future trends,” Macromolecular Rapid Communications 32(17), 1299-1311. DOI: 10.1002/marc.201100230
Sang, Y. O., Dong, I. Y., Shin, Y., Kim, H. C., and Kim, H. Y. (2005). “Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy,” Carbohydrate Research 340(15), 2376-2391. DOI: 10.1016/j.carres.2005.08.007
Sluiter, S., Hames, A., Ruiz, B., Scarlata, R., Sluiter, C., Templeton, J., and Crocker, D. (2008). Determination of Structural Carbohydrates and Lignin in Biomass (NREL/TP-510-42618), National Renewable Energy Laboratory, Golden, CO.
Struszczyk, H., and Laine, J. E. (1986). “Modification of lignins. I. Reaction of lignins with chlorophosphazenes,” Journal of Macromolecular Science: Part A- Chemistry 23(8), 973-992. DOI: 10.1080/00222338608081105
Sun, Q. N., Foston, M., Meng, X. Z., Sawada, D., Pingali, S. V., O’Neill, H. M., Li, H. J., Wyman, C. E., Langan, P., Ragauskas, A. J., et al. (2014). “Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment,” Biotechnology for Biofuels 7, 150. DOI: 10.1186/s13068-014-0150-6
Tadesse, H., and Luque, R. (2011). “Advances on biomass pretreatment using ionic liquids: An overview,” Energy and Environmental Science 4(10), 3913-3929. DOI: 10.1039/c0ee00667j
Vainio, A., and Paulapuro, H. (2007). “The effect of wet pressing and drying on bonding and activation in paper,” Nordic Pulp and Paper Research Journal 22(4), 403-408. DOI: 10.3183/NPPRJ-2007-22-04-p403-408
Wan, J., Lian, J., Wang, Y., and Ma, Y. (2015). “Investigation of cellulose supramolecular structure changes during conversion of waste paper in near-critical water on producing 5-hydroxymethyl furfural,” Renewable Energy 80, 132-139. DOI: 10.1016/j.renene.2015.01.071
Wang, S. D., You, T. T., Xu, F., Chen, J. C., and Yang, G. H. (2015). “Optimization of [Amim]Cl pretreatment conditions for maximum glucose recovery from hybrid Pennisetum by response surface methodology,” BioResources 10(4), 7021-7037. DOI: 10.15376/biores.10.4.7021-7037
Wada, M., Ike, M., and Tokuyasu, K. (2010). “Enzymatic hydrolysis of cellulose I is greatly accelerated via its conversion to the cellulose II hydrate form,” Polymer Degradation and Stability 95 (4), 543-548. DOI: 10.1016/j.polymdegradstab.2009.12.014
Xu, F., Shi, Y. C., and Wang, D. (2013). “X-ray scattering studies of lignocellulosic biomass: A review,” Carbohydrate Polymers 94(2), 904-917. DOI: 10.1016/j.carbpol.2013.02.008
Yang, B., and Wyman, C. E. (2008). “Pretreatment: The key to unlocking low-cost cellulosic ethanol,” Biofuels Bioproducts and Biorefining 2(1), 26-40. DOI: 10.1002/bbb.49
Zhang, H., Wu, J., Jun Zhang, A., and He, J. (2005). “1-Allyl-3-methylimidazolium chloride room temperature ionic liquid: A new and powerful nonderivatizing solvent for cellulose,” Macromolecules 38(20), 8272-8277. DOI: 10.1021/ma0505676
Zhao, A., and Zhou, D. (2010). “Characteristics of Chinese Pennisetum as raw material for medium density fiberboard,” Journal of Nanjing Forestry University (Natural Sciences Edition) 4, 36. DOI: 10.3969/j.issn. 1000 -2006(2010)04 -0149 -04
Zheng, Y., Pan, Z., and Zhang, R. (2009). “Overview of biomass pretreatment for cellulosic ethanol production,” International Journal of Agricultural and Biological Engineering 2(3), 51-68. DOI: 10.3965/j.issn.1934-6344.2009.03.051-068
Article submitted: October 14, 2016; Peer review completed: November 25, 2016; Revised version received: December 1, 2016; Accepted: December 3, 2016; Published: December 13, 2016.
DOI: 10.15376/biores.12.1.1031-1040