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dilute acid system for removal of non-cellulosic components and acceleration of enzymatic hydrolysis
Industrial Crops & Products 132 (2019) 21–28
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Pretreatment of Eucalyptus urophylla in γ-valerolactone/dilute acid system for removal of non-cellulosic components and acceleration of enzymatic hydrolysis
T
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Shao Ni Suna, Xue Chena, Ying Hua Taoa, Xue Fei Caoa, , Ming Fei Lia, Jia Long Wena, ⁎ Shuang Xi Nieb, Run Cang Suna, a
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, 100083, China Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control, College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China
b
A R T I C LE I N FO
A B S T R A C T
Keywords: γ-Valerolactone Pretreatment Structural characteristic Enzymatic hydrolysis Lignin
Enzymatic hydrolysis of biomass is highly dependent on the changes in chemical compositions and structural characteristics after pretreatment. γ-Valerolactone (GVL)/dilute acid pretreatment is an effective approach to remove hemicelluloses and lignin and disrupt the cell wall structure, leading to the improvement of enzymatic hydrolysis. This study examined the influence of chemical compositions and structural features on the enzymatic hydrolysis of Eucalyptus urophylla from GVL/dilute acid pretreatment. Results revealed that glucose yield showed a positive correlation with removal rate of hemicelluloses and lignin. Particularly, there was no direct correlation of cellulose crystallinity on enzymatic hydrolysis. The highest glucose yield of 89.1% was achieved when the pretreatment was performed with GVL/H2O solution (4:1, v/v) containing 100 mM H2SO4 at 120 °C for 60 min. Meanwhile, under the mentioned conditions, 16.5% of lignin with a high purity (only contained 0.09% sugars) and molecular weight (3450 g/mol) was fractionated, which can be served as feedstock for future utilization. In short, an extensive understanding of the pretreated biomass and the obtained lignin during the GVL/dilute acid pretreatment will be beneficial for value-added applications of biomass.
1. Introduction Lignocellulosic biomass is considered as an ideal feedstock for the production of bioethanol due to its abundance, renewability, high sugar content, and reasonable price. The use of biomass has benefits for addressing global warming, climate change, and energy demand. However, biomass recalcitrance limits its efficient utilization mainly owing to the complex and heterogeneous cell wall structure. The biomass recalcitrance is connected with physical and chemical features, including contents and distributions of hemicelluloses and lignin, accessible surface area, structure and crystallinity of cellulose, and other factors (Yoo et al., 2017). A pretreatment step is thus required to alter the chemical composition and structural features of biomass, simultaneously overcoming the recalcitrant barriers for increasing surface area and cellulose accessibility (Govumoni et al., 2013; Sun et al., 2016). Generally, an effective pretreatment method should be economical and effective on a diversity of biomass. Meanwhile, it should minimize the operating step, maximize the recovery of biomass components, and
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produce a cellulosic fraction that can be easily hydrolyzed with cellulase (Shuai et al., 2016). To date, many pretreatment methods have been developed to reduce the biomass recalcitrance and facilitate the accessibility of enzymes to cellulose, such as steam explosion, hydrothermal, ionic liquid, organosolv, acid and alkali pretreatment (Inalbon et al., 2017; Nie et al., 2018; Ninomiya et al., 2018; Sun and Xue, 2018; Yang et al., 2018). In the steam explosion or hydrothermal pretreatment process, a part of hemicelluloses was removed and lignin as a key barrier to enzymatic hydrolysis was still left in the pretreated substrates (Sun et al., 2014a). The acid pretreatment could dissolve hemicelluloses from biomass effectively; however, there are still some limitations, such as severe equipment corrosion, expensive equipment construction, and low removal rate of lignin. The biomass structure can be altered by alkaline pretreatment by swelling cellulose and removing lignin and hemicelluloses, whereas a significant limitation is that the alkali was converted into irrecoverable or incorporation salts (Chiaramonti et al., 2012; Hendriks and Zeeman, 2009). In the ionic liquid pretreatment,
Corresponding author. E-mail address: [email protected] (R.C. Sun).
https://doi.org/10.1016/j.indcrop.2019.02.004 Received 7 September 2018; Received in revised form 30 January 2019; Accepted 3 February 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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dilute HCl solution (pH 2.0) to precipitate lignin, and the solid fraction was washed completely with distilled water. The precipitated lignin and washed solid fraction were freeze-dried for further experiment. The solid fractions were labeled as R80-30, R80-60, R80-90, R100-30, R100-60, R100-90, R120-30, R120-60, R120-90, R140-30, R140-60, and R140-90, corresponding to the pretreatment temperature and time. The lignin fractions obtained were labeled as L80-30, L80-60, L80-90, L100-30, L100-60, L10090, L120-30, L120-60, L120-90, L140-30, L140-60, and L140-90, according to the corresponding solid fractions.
the cost of ionic liquid is high and its recovery is difficult, thus limiting its industrial application for biomass pretreatment. The above mentioned pretreatment, such as steam explosion, hydrothermal, or acid pretreatment alone, had limited for removal and recovery of lignin and enhancement of enzymatic hydrolysis. Thus a combined treatment, such as hydrothermal, dilute acid, steam explosion or ionic liquid pretreatment combined alkaline treatment, was studied to be a promising integrated technology (Li et al., 2016; Sun et al., 2014a, 2014c and 2016). However, before the alkaline treatment, a large amount of water and a long operation time were needed to separate the pretreatment liquid and solid, thus increasing the operating cost and decreasing the economic profit. Organosolv pretreatment is an effective lignin-targeting method by breaking the internal bonds of hemicelluloses and lignin using organic or aqueous organic solvents such as ethanol, methanol and acetone with or without the addition of an acid or base catalyst (Pan et al., 2006; Yoo et al., 2017; Zhao et al., 2009). It is a practical technology for fractionation of lignocellulosic biomass because it promotes the fractionation of high-quality lignin and cellulose. Recently, organic solvents such as tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-MeTHF), methyl isobutyl ketone (MIBK), and γ-valerolactone (GVL) have been successfully applied to pretreat biomass for increasing the enzymatic hydrolysis efficiency by dissolution of lignin (Katahira et al., 2014; Nguyen et al., 2015; Shuai et al., 2016; Zhang et al., 2016). Among these organic solvents, GVL has been paid great attention because it is a biomass-derived, green and non-toxic solvent and its structure is rather stable in water and oxygen (Wu et al., 2016). Recently, although the production of nonenzymatic sugar and its degradation products generated from biomass using GVL have been intensively investigated, only a small amount of research is being conducted on the enzymatic hydrolysis of biomass. Specifically, the effect of the structural features of biomass on the enzymatic hydrolysis has been generally ignored during the GVL pretreatment period, which is pivotal for understanding and overcoming biomass recalcitrance. In addition, fundamental characteristics of the soluble lignin are rarely reported. In this work, therefore, a GVL/dilute acid pretreatment process was performed at moderate temperatures (80–140 °C) for different periods (30–90 min) to explore its effect on the chemical compositions, structural characteristics and enzymatic hydrolysis of Eucalyptus urophylla. Meanwhile, the soluble lignin was recovered after the pretreatment, and its yield, purity and molecular weight have been thoroughly investigated.
2.3. Chemical characterization of the solid fractions The chemical compositions of the solid fractions were quantified by a two-step hydrolysis process (Sluiter et al., 2008). The hemicelluloses and cellulose contents were determined by a high-performance anion exchange chromatography (HPAEC) system (Sun et al., 2014d). The surface characteristics of RM and pretreated Eucalyptus were performed with Hitachi S-3400 N II (Hitachi, Japan) at 10 kV. FT-IR spectra were conducted on a Nicolet iN10 spectrophotometer. The Xray diffraction was obtained using an XRD-6000 apparatus (Shimadzu, Japan) with a Cu Ka radiation source (k = 1.54 Å) and recorded from 5 to 40° with a scanning speed of 2°/min. The crystallinity index (CrI) was calculated by height method, in which the CrI was calculated from the ratio of the height of the 002 peak (I002, 2θ ≈ 22.5°) and the height of the minimum (Iam, 2θ ≈ 18.5°) between the 002 and the 101 peaks (Sun et al., 2014c). CP/MAS 13C NMR spectra were detected by a Bruker AV-III 400 M spectrometer (Germany) at 100.6 MHz. TGA/DTG curves were obtained on a thermal analyzer (SDT Q600). The solid fractions (10 mg) were placed on an Al2O3 crucible and heated from room temperature to 700 °C at a constant heating rate of 20 °C/min under the purified nitrogen atmosphere. 2.4. Enzymatic hydrolysis The hydrolysis experiment was conducted at 50 °C for 72 h in a shaking incubator at 150 rpm. Typically, 0.2 g solid fraction, 10 mL sodium acetate buffer (50 mM, pH 4.8) and a cellulase activity (Cellic® CTec2, 100 FPU/mL) of 17 FPU/g substrate were added into a 25 mL Erlenmeyer flask. The glucose content in the hydrolysate was determined by a HPAEC system equipped with an integral amperometric detector and a CarboPac PA 100 (Dionex, U.S.) analytical column (Sun et al., 2014c).
2. Material and methods
2.5. Characterization of lignin
2.1. Materials
The associated carbohydrates and the molecular weights of the lignin fractions were analyzed by HPAEC and gel permeation chromatography (GPC), respectively (Sun et al., 2014b). 2D HSQC NMR spectrum and main substructures of L120-60 were acquired on a Bruker AVIII 400 MHz spectrometer according to the previous literature (Sun et al., 2014b).
Eucalyptus urophylla, which is a kind of popular Eucalyptus in Guangxi province (China), was ground to obtain fractions (40–60 mesh). The Eucalyptus was air-dried and dewaxed with toluene/ ethanol (2:1, v/v) for 8 h as RM, and then dried in an oven at 60 °C for 24 h for further treatment. Here the toluene/ethanol extraction was to avoid the possible effects of extractives on the chemical composition analysis and the lignin purity.
3. Results and discussion
2.2. Pretreatment procedure of Eucalyptus with GLV/dilute acid system
3.1. Effect of GVL/dilute acid pretreatment on the chemical compositions of Eucalyptus urophylla
The pretreatment of Eucalyptus was operated in a batch reactor made of Hastelloy C-276 (Sen Long Instruments Company, China). 3.0 g of oven-dried Eucalyptus and 30 mL of GVL/H2O solution (4:1, v/v) with 100 mM H2SO4 were mixed into the reactor and then reacted at the target temperatures of 80, 100, 120 and 140 °C for 30, 60 and 90 min, respectively. When the reaction was finished, the reactor was set in an ice bath to cool down. After releasing the gas and pressure, the mixture was filtered through Buchner funnels to separate the liquid and solid fractions. Then the liquid fraction was poured into 150 mL of
Due to the biomass recalcitrance, it is vital to pretreat biomass before its subsequent enzymatic hydrolysis. It has been reported that GVL is an attractive green solvent derived from biomass, and GVL/dilute acid system has been used as an excellent reaction medium for the fractionation of lignin from biomass at moderate reaction temperatures (Fang and Sixta, 2015). Meanwhile, the crystalline structure of cellulose was difficult to destroy with a low acid concentration at a moderate temperature, which could maximize its selective conversion by enzymes (Shuai et al., 2016). They reported that the use of a GVL-based 22
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Table 1 Solid yield and chemical composition (%, w/w) of the solid fractions. Sample
RM R80-30 R80-60 R80-90 R100-30 R100-60 R100-90 R120-30 R120-60 R120-90 R140-30 R140-60 R140-90
Solid yield (%)
86.9 74.5 67.5 63.0 51.3 46.4 43.0 45.3 38.4 35.1 23.4 21.2 18.7
Chemical compositiona
Pretreatment factor
Hemicelluloses
Cellulose
AILb
ASLc
15.6 ± 0.5 11.1 ± 0.3 9.7 ± 0.3 8.6 ± 0.2 3.2 ± 0.1 2.2 ± 0.1 1.6 ± 0.2 2.5 ± 0.1 0.6 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.1 ± 0.0 0.1 ± 0.0
42.3 39.4 37.3 36.2 39.4 36.6 35.9 34.6 32.6 30.9 19.5 18.7 15.1
23.7 ± 1.4 19.0 ± 1.4 16.4 ± 0.9 14.5 ± 0.8 7.0 ± 0.5 6.4 ± 0.4 4.5 ± 0.4 7.1 ± 0.5 4.3 ± 0.2 2.9 ± 0.1 3.1 ± 0.0 2.0 ± 0.0 3.1 ± 0.1
5.2 5.1 4.1 3.6 1.7 1.3 1.1 1.3 1.0 0.8 0.5 0.5 0.4
± ± ± ± ± ± ± ± ± ± ± ± ±
2.1 1.2 1.5 0.9 0.8 1.3 0.9 1.3 1.5 0.7 1.1 0.8 0.6
± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.5 0.2 0.2 0.1 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0
d
0.00 0.05 0.10 0.14 0.52 0.55 0.62 0.49 0.61 0.62 0.39 0.40 0.31
a
The contents of cellulose, hemicelluloses, and lignin were all calculated based on the initial weight of Eucalyptus. AIL, acid insoluble lignin. c ASL, acid soluble lignin. d Pretreatment factor = retention rate of cellulose × removal rate of hemicelluloses × total delignification rate, in which retention rate of cellulose = weight of cellulose in the pretreated samples / weight of cellulose in the raw material, removal rate of hemicelluloses = (weight of hemicelluloses in the raw material - weight of hemicelluloses in the pretreated samples) / weight of hemicelluloses in the raw material, and total delignification rate = (weight of total lignin in the raw material - weight of total lignin in the pretreated samples) / weight of total lignin in the raw material. b
pretreated Eucalyptus as compared with the initial 42.3% in RM. However, the relative content of cellulose (46.9–77.5%) in the pretreated Eucalyptus gradually increased with the pretreatment intensity raised owing to the significantly removal of hemicelluloses and lignin. It was reported that when the hydrothermal pretreatment alone was used to pretreat Eucalyptus, the hemicelluloses were degraded, while lignin was hardly changed, thus subsequent alkaline treatment was performed to remove lignin from the hydrothermal pretreated Eucalyptus (Sun et al., 2014c). The previous work using ionic liquids for Eucalyptus pretreatment also required the subsequent alkaline treatment to improve the removal rates of hemicelluloses and lignin, thus enhancing the enzymatic hydrolysis (Li et al., 2016). As compared to the two-step pretreatment methods mentioned above, the one-step pretreatment with GVL/dilute acid can effectively dissolve hemicelluloses and lignin, whereas the dissolved lignin can be recycled for further utilization. The effective dissolution of lignin was mainly because that GVL was completely miscible with aqueous solution and had a similar δ-value with lignin (Lê et al., 2016). Moreover, as compared with the pure GVL or aqueous solution, the property of GVL/water was changed, in which a strong hydrogen bond existed between GVL and aqueous solution (Xue et al., 2016). The changed property facilitated the dissolution of lignin. Unlike lignin, hemicelluloses were primarily released from biomass as xylose oligomers and monomers at mild temperature (e.g. 120 °C) during the GVL/dilute acid pretreatment (Motagamwala et al., 2016). As the temperature or time increased, the xylose oligomers were degraded to xylose monomers accompanied the formation of furfural. Moreover, the pretreatment factor was also calculated, as shown in Table 1. Here, the pretreatment factor was used as representative of the pretreatment effect on the main components of lignocellulosic biomass under the different pretreatment conditions, in which it was calculated based on the following formula: pretreatment factor = retention rate of cellulose × removal rate of hemicelluloses × total delignification rate. It was found that when the temperature was performed at 80 °C, the pretreatment factors of the pretreated Eucalyptus were only 0.05-0.14. As the temperature increased to 100–120 °C, the pretreatment factors increased significantly to 0.49-0.62 but decreased to 0.31-0.40 as the temperature reached to 140 °C. The result suggested that the GVL/dilute acid pretreatment at 100–120 °C was considered as the appropriate conditions for the comprehensive treatment of Eucalyptus in order to remove the hemicelluloses and lignin and simultaneously reserve the
pretreatment process at 120 °C can lead to high sugar yields after enzymatic hydrolysis (Shuai et al., 2016). When GVL/H2SO4 solvent at 120 °C with 75 mM H2SO4 was used to pretreat hardwood, up to 80% of original lignin was removed with 96–99% of original cellulose retained in the pretreated substrates. Wu et al. also pointed out that the treatment of cotton stalk with GVL/H2SO4 can release about 65% lignin of plant cell wall, whereas the enzymatic hydrolysis efficiency of the pretreated stalk increased by two-fold as compared to raw material (Wu et al., 2016). In addition, owning to the good solubility of lignin and hemicelluloses in GVL/H2SO4 solvent, a GVL/water solution with a relatively higher acid concentration liberated a higher amount of lignin and hemicelluloses. Under 100 mM H2SO4, the removal rates of lignin and hemicelluloses reached 86–92% and 97–98%, respectively (Li et al., 2019). Therefore, in this case, GVL/dilute acid pretreatment with 100 mM H2SO4 was performed at moderate temperatures (80–140 °C) for different periods (30–90 min) to explore its effect on the chemical compositions, structural characteristics and enzymatic hydrolysis of Eucalyptus urophylla. Table 1 summarizes the yield and chemical composition of Eucalyptus urophylla pretreated with GVL/dilute acid. To clarify the composition changes of the solid fractions after pretreatment, the contents of main components were calculated based on the initial weight of RM. As illustrated, the yields of solid fractions significantly decreased from 74.5 to 18.7% with the pretreatment intensity increased. The mass loss of the raw Eucalyptus was mainly contributed to the dissolution of lignin and hemicelluloses into the reaction medium. Results showed that when the pretreatment temperature was 80 °C, the hemicelluloses and cellulose contents in the pretreated Eucalyptus decreased slightly with the raise of the pretreatment time. However, the lignin content in the pretreated Eucalyptus, especially acid insoluble lignin (AIL), decreased obviously. The content of AIL decreased from 23.7 to 19.0-14.5% for different reaction times. It indicated that the GVL/dilute acid system was an excellent reaction medium for lignin removal even at a low pretreatment temperature. As the temperature rose to 100–120 °C, both lignin and hemicelluloses significantly removed from the biomass matrix, and only around 2.9–7.0% of AIL and 0.4–3.2% of hemicelluloses were remained in the pretreated Eucalyptus, respectively. Meanwhile, the degradation of cellulose became evident at 120 °C, which decreased from 42.3 to 30.9–34.6%. When the temperature rose to 140 °C, cellulose occurred significant degradation, and only 15.1–19.5% of original cellulose was reserved in the 23
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Fig. 1. SEM images of RM and the pretreated Eucalyptus at magnifications × 1000 and × 6000.
24
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2017). Here the main effect of GVL/dilute acid pretreatment is the removal of amorphous substances. The X-ray diffraction testing is a better method to evaluate the effect of pretreatment on the CrI of biomass. Fig. S2a shows the X-ray diffraction curves and the CrI values of RM, R80-60, R100-60, R120-60, and R140-60. No remarkable differences were observed among the XRD patterns of the solid fractions, and the two characteristic peaks observed at around 16° and 22° suggested a typical pattern of cellulose I. It demonstrated that the crystalline structure of cellulose in biomass was not destructed after the GVL/dilute acid pretreatment. Furthermore, it was found that as compared to the CrI value (52.03%) of the untreated Eucalyptus (RM), the CrI values of the pretreated samples were gradually increased to 59.17–72.03% with the increment of the pretreatment severity. This could be the progressive solubilization and degradation of amorphous lignin and hemicelluloses, as illustrated in Table 1, which exposed the crystalline cellulose core and increased the cellulose proportion in the pretreated samples (Liu et al., 2009). The result was in accordance with many previous studies (Xiao et al., 2013; Zakaria et al., 2015). Zakaria et al. (2015) reported that the increased CrI in directly showed higher exposure of cellulose component amenable to cellulase attack. To further analyze the main components and structural variations of the pretreated Eucalyptus, solid state CP/MAS 13C NMR spectra (Fig. S2b) of RM, R80-60, R100-60, R120-60, and R140-60 were recorded. The spectral signals were interpreted according to the data from previous literatures (Jeong and Lee, 2016; Lü et al., 2017; Pereira et al., 2016). The signals of cellulose can be assigned as followed: C1 (103.5 ppm), C2/C3/C5 (71.0 ppm), C4 (82.5 and 86.7 ppm), and C6 (63.1 ppm). Since the signals in the 80–86 and 86–92 ppm were due to amorphous cellulose and crystalline cellulose, respectively, the CrI values were also calculated by taking the ratio of the integration in the region of 86–92 ppm to that of 80–92 ppm. It was found that the CrI values from CP/MAS 13C NMR method were relatively low as compared to those from XRD peak height method, which was because that the effect of the cellulose chains present on the surface of cellulose crystals was considered in the NMR method (Park et al., 2010). Note that a similar trend was detected, namely the CrI values increased with the pretreatment temperatures. In addition, the intensities of the signals at 19.4 and 170.5 ppm originated from acetyl groups of hemicelluloses cut down with the increasing pretreatment intensity. The decreasing trend was benefit for the enhancement of cellulose accessibility, which was because the decreased acetyl groups of hemicelluloses can reduce the steric obstacles of enzymes from hemicelluloses (Jiang et al., 2014). The signal intensities of methoxyl group (54.1 ppm) and aromatic group (151.3 ppm) from lignin also gradually reduced with the increment of the pretreatment severity, illustrating that the degree of biomass delignification increased as shown in Table 1. Additionally, the intensities of the signals at 86.7 and 63.1 ppm derived from the C4 and C6 of the crystalline cellulose were enhanced as the increased pretreatment temperatures. The thermal stabilities of the raw material and the pretreated Eucalyptus prepared with GVL/dilute acid were characterized by TGA and DTG (Fig. S3). TGA curves showed a significant decrease of char residues at 700 °C in R80-60 (17.35%) and R100-60 (16.17%) as compared to that in RM (20.69%), which could be due to the fact that the lignin with a high thermal stability was removed during the pretreatment process. However, the char percentages were increased to 19.16–19.58% when the pretreatment temperatures increased to 120–140 °C for a constant time of 60 min. The reason for this was that part of lignin was condensed at high pretreatment temperatures under the acid condition, resulting in an increment of thermal stabilities to some extent. In addition, a shoulder peak at 283 °C and a sharp peak at 367 °C, originated from the thermal degradation of hemicelluloses and cellulose, respectively, were observed in the DTG curves of RM and R8060 (Cao et al., 2014). However, because most hemicelluloses were removed from the Eucalyptus urophylla, almost no shoulder peak was found in the DTG curves of R100-60, R120-60, and R140-60.
cellulose. 3.2. SEM analysis of the solid fractions SEM is the powerful tool for studying the morphological information and fundamental physical properties of the biomass after pretreatment (Gabhane et al., 2015; Karimi and Taherzadeh, 2016). The morphology of RM, R80-60, R100-60, R120-60, and R140-60 was recorded by SEM. As shown in Fig. 1, the untreated Eucalyptus exhibited a rigid and compact surface structure preventing the accessibility of enzymes to cellulose. In contrast, the loosing fibrillar structure was observed in the pretreated Eucalyptus due to the removal of some non-cellulosic components. Furthermore, it is worth noting that the morphology of the pretreated sample was correlated to the pretreatment severity. Specifically, slight surface structural changes were observed when the Eucalyptus was pretreated at 80 °C, while some crevices appeared in the samples pretreated at mild temperatures (100–120 °C). It suggested that the GVL/dilute acid system was able to disrupt the plant cell wall of biomass effectively. When the temperature increased to 140 °C, a more disorganized morphology was detected in R140-60, in which the lignin and hemicelluloses were almost completely removed. The relatively severe pretreatment resulted in the exposure of more reactive area on the Eucalyptus surface, as well as the collapse of cellulose fibrils, which would be a negative factor on the enzymatic hydrolysis (Hou et al., 2013; Sun et al., 2011). Similar finding was reported when hulless oat straw was imposed at severe treatment condition which caused the biomass damage and led to the reduction of accessible surface area (Yang et al., 2013). In short, the disruption of the plant cell wall contributed to the improvement the enzymatic hydrolysis of the samples pretreated with GVL/dilute acid, which was confirmed by the results of the subsequent enzymatic hydrolysis experiment. (Fig. 1) 3.3. Characterization of the physicochemical properties of the solid fractions In order to further investigate the structural changes of Eucalyptus urophylla pretreated under different conditions, FTIR, XRD, CP/MAS 13 C NMR and TG were performed. FTIR spectra (Fig. S1) were used to probe the chemical composition changes among RM and the pretreated Eucalyptus. As compared with RM, the intensities of the absorption bands at 1740 and 1242 cm−1 originated from the C]O stretching vibration in acetyl groups and the C–O stretching of hemicelluloses (Kumar and Wyman, 2009), respectively, were sharply reduced in R10060 and even disappeared in R120-60 and R140-60. The result indicated that most of hemicelluloses were degraded in the samples pretreated at 100–140 °C for 60 min. This was in line with the changes observed in the chemical composition reported in Table 1. The bands at 1593, 1504 and 1458 cm−1 are attributed to the aromatic skeletal vibrations of lignin (Sun et al., 2014b). It was clearly observed that the intensities of these bands were declined dramatically in the pretreated Eucalyptus, especially in R100-60, R120-60 and R140-60, which was also in accordance with the results in Table 1. Moreover, the increasing intensity of the band at 1153 cm−1 for the characteristic assignment of cellulose was observed in R80-60-R140-60, which could owe to the relative high content of cellulose in the these samples. These results suggested that the pretreatment of Eucalyptus urophylla with GVL/dilute acid removed most non-cellulosic components and simultaneously reserved cellulose under the moderate pretreatment conditions. The cellulose structure and its CrI are regarded to affect the enzymatic hydrolysis (Inouye et al., 2014; Sathitsuksanoh et al., 2011). Generally, if the purpose of biomass pretreatment is to remove hemicelluloses and lignin, high CrI indicates good pretreatment effect (Wang et al., 2017; Zhang et al., 2018). However, if the purpose of biomass pretreatment is to destroy the crystallization zone of cellulose, low CrI indicates good pretreatment effect (Sahoo et al., 2018; Wang et al., 25
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(%, oven dried RM) are given in Table 2. It was found that when the pretreatment temperature was performed at 80 °C, only 1.9–2.6% of lignin was extracted from RM. When the temperature rose to 100–140 °C, especially at 120 and 140 °C, the lignin yields were improved significantly to 10.8–21.0%, suggesting that the raise of the pretreatment severity had a dramatic effect on the removal of lignin, as in line with the component analysis of the solid fractions. Next, in order to verify the lignin purity, the associated carbohydrates were detected (Table 2). Results showed that the lignin extracted with GVL/dilute acid solution had low amounts of associated carbohydrates (0.08–1.48%), in which xylose was the main carbohydrates. Notably, when the temperature was increased to 120 and 140 °C, minor amounts of the associated carbohydrates (less than 0.2%) were detected. The low carbohydrates contents in these lignin fractions indicated that the linkages between lignin and hemicelluloses in biomass were broken obviously during the pretreatment process. Therefore, the GVL/dilute acid pretreatment could be used as a promising pretreatment technology for facilitating the extraction of lignin with a high purity from the plant cell walls, which was beneficial for its subsequent application. In addition, GPC analysis was used to explore the effect of the GVL/dilute acid pretreatment on the molecular weights and polydispersity index (Mw/ Mn) of the lignin fractions (Table 2). It was found that when the pretreatment temperature was 80 and 100 °C, prolonging pretreatment time from 30 to 90 min gave rise to the release of lignin with high molecular weights. As the temperature was 120 and 140 °C, the molecular weight of lignin was slightly reduced with the increasing time because of the degradation of lignin under the severe treatment conditions. The low Mw/Mn (1.36–1.83) of all the lignin fractions implied the relative homogeneity of the lignin macromolecule. Overall, during the GVL/dilute acid pretreatment process, especially at 120 and 140 °C, the lignin had high yields, purities, and Mw, as well as low Mw/Mn, which is a preferred feedstock for future utilization. Meanwhile, the 2D HSQC NMR spectrum and main substructures of L120-60 is shown in Fig. S5. Obviously, the main linkage of L120-60 was β-O-4′ (A), followed by ββ′ (B) and β-5′ (C), and syringyl (S) and guaiacyl (G) units were visible, which confirmed a primary structure of lignin. Therefore, GVL/dilute acid pretreatment was a promising technology to realize the efficient conversion of cellulose to glucose by enzymatic hydrolysis for bioethanol production, in which the obtained lignin can be converted into high value-added products (Dai et al., 2017, 2018).
Fig. 2. Enzymatic hydrolysis of RM and the pretreated Eucalyptus.
3.4. Enzymatic hydrolysis of the solid fractions To further explore the digestibility of the solid fractions, the raw material and the samples pretreated under various conditions were enzymatically hydrolyzed using cellulase at 50 °C for 72 h, as illustrated in Fig. 2. As compared to RM (21.1%), the pretreated Eucalyptus was more readily to be hydrolyzed by cellulase. Meanwhile, the enzymatic hydrolysis rates of the pretreated Eucalyptus increased with the increment of the temperature from 80 to 120 °C, especially at 100 and 120 °C, which was owing to the large release of the potential recalcitrance components (hemicelluloses and lignin) from biomass matrix. Meanwhile, the loosing fibrillar structure was visualized by SEM images, leading to the exposure of the internal surface of Eucalyptus and exhibition of adsorption sites of enzymes to more extent. As the temperature increased to 140 °C, the enzymatic hydrolysis rate of R14060 (88.2%) was similar to that of R120-60 (89.1%). The possible explanation may be that a large amount of amorphous cellulose was degraded under the relatively harsh pretreatment conditions and more crystalline cellulose was reserved in the pretreated sample, resulting in a relatively low enzymatic hydrolysis rate of R140-60. Another reason was that the excessive removal of lignin and hemicelluloses introduced the collapse of the cellulose multiple-layers, thus preventing the available surface area for cellulase (Ishizawa et al., 2009; Yang et al., 2013). Based on the above results, it is meaningful to establish the relationship of the glucose conversion ratios with the removal rates of hemicelluloses and lignin. As shown in Fig. S4a, a positive correlation between glucose conversion rate and removal rate of hemicelluloses and lignin with a relative high coefficient factor (R2 = 0.91) was observed in the solid fractions, which was consistent with the previous works (Ishizawa et al., 2007; Sun et al., 2011). Besides, the enzymatic hydrolysis efficiency is proportional increase to the raise of CrI value (Fig. S4b). Here note that the increment of CrI of the pretreated samples was mostly achieved by the release of hemicelluloses and lignin, thus indicating that the CrI did not significantly affect the glucose yield during the enzymatic hydrolysis (Hsu et al., 2010; Zakaria et al., 2015). That was the CrI had no direct correlation with hydrolysis efficiency. Overall, it can be concluded that both obvious removal of hemicelluloses and lignin during the GVL/dilute acid pretreatment had considerable influence on the structural changes of biomass matrix and expansion of available surface area, resulting in the enhancement of enzymatic hydrolysis.
4. Conclusions This study demonstrated that GVL/dilute acid pretreatment was an effective way to change the chemical composition and structural characteristic of biomass to improve enzymatic hydrolysis. Findings showed that the pretreatment successfully removed hemicelluloses and lignin and disrupted the cell wall structure, resulting in the increased cellulose crystallinity and enzyme accessibility. The highest glucose yield of 89.1% was obtained after the GVL/dilute acid pretreatment (80% GVL/20%H2O with100 mM H2SO4 at 120 °C for 60 min). Additionally, the by-product lignin with a high yield was obtained during the pretreatment had a high purity and molecular weight. Acknowledgments This work was supported by the National Natural Science Foundation of China (21706015, 31700518), Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control (KF201806-5) and the Foundation of State Key Laboratory of Biobased Material and Green Papermaking (No. KF201811).
3.5. Analysis of lignin fractions Appendix A. Supplementary data In this work, the dissolved lignin was recovered by precipitation of the liquid fraction obtained during the GVL/dilute acid pretreatment into aqueous dilute acid solution. The yields of lignin fractions obtained
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.02.004. 26
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Table 2 Yield of lignin (%, w/w), its associated carbohydrate content (%, w/w), weight-average (Mw) and number-average (Mn) molecular weights, and polydispersity (Mw/ Mn). Lignin fractions
Yield
Galactose
Glucose
L80-30 L80-60 L80-90 L100-30 L100-60 L100-90 L120-30 L120-60 L120-90 L140-30 L140-60 L140-90
1.9 2.3 2.6 10.8 12.0 14.1 11.6 16.5 18.3 19.9 21.0 20.2
0.18 0.20 0.14 0.10 0.12 0.15 0.10 0.00 0.15 0.00 0.00 0.00
0.28 0.18 0.17 0.17 0.16 0.20 0.12 0.02 0.05 0.10 0.11 0.09
± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.02 0.01 0.00 0.04 0.01 0.00 0.00 0.02 0.00 0.00 0.00
± ± ± ± ± ± ± ± ± ± ± ±
Xylose 0.05 0.04 0.04 0.03 0.01 0.04 0.02 0.00 0.00 0.01 0.00 0.00
0.78 1.10 0.85 0.34 0.76 1.01 0.99 0.07 0.09 0.00 0.10 0.05
± ± ± ± ± ± ± ± ± ± ± ±
0.10 0.04 0.03 0.00 0.07 0.09 0.07 0.00 0.00 0.00 0.01 0.00
Total sugars
Mw
1.24 1.48 1.16 0.61 1.04 1.36 1.21 0.09 0.29 0.10 0.21 0.14
3210 3760 3870 3070 3440 3590 3550 3450 2880 2610 2320 2270
± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.07 0.03 0.02 0.05 0.04 0.06 0.00 0.03 0.00 0.01 0.02
Mn ± ± ± ± ± ± ± ± ± ± ± ±
130 70 150 120 70 110 90 80 100 80 50 40
2360 2260 2290 1850 2010 2030 1990 1890 1690 1560 1480 1470
Mw/Mn ± ± ± ± ± ± ± ± ± ± ± ±
90 50 40 70 70 40 60 30 20 50 90 40
1.36 1.66 1.69 1.66 1.71 1.77 1.78 1.83 1.70 1.67 1.57 1.54
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Pretreatment of Eucalyptus urophylla in γ-valerolactone/dilute acid system for removal of non-cellulosic components and acceleration of enzymatic hydrolysis
T
⁎
Shao Ni Suna, Xue Chena, Ying Hua Taoa, Xue Fei Caoa, , Ming Fei Lia, Jia Long Wena, ⁎ Shuang Xi Nieb, Run Cang Suna, a
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, 100083, China Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control, College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China
b
A R T I C LE I N FO
A B S T R A C T
Keywords: γ-Valerolactone Pretreatment Structural characteristic Enzymatic hydrolysis Lignin
Enzymatic hydrolysis of biomass is highly dependent on the changes in chemical compositions and structural characteristics after pretreatment. γ-Valerolactone (GVL)/dilute acid pretreatment is an effective approach to remove hemicelluloses and lignin and disrupt the cell wall structure, leading to the improvement of enzymatic hydrolysis. This study examined the influence of chemical compositions and structural features on the enzymatic hydrolysis of Eucalyptus urophylla from GVL/dilute acid pretreatment. Results revealed that glucose yield showed a positive correlation with removal rate of hemicelluloses and lignin. Particularly, there was no direct correlation of cellulose crystallinity on enzymatic hydrolysis. The highest glucose yield of 89.1% was achieved when the pretreatment was performed with GVL/H2O solution (4:1, v/v) containing 100 mM H2SO4 at 120 °C for 60 min. Meanwhile, under the mentioned conditions, 16.5% of lignin with a high purity (only contained 0.09% sugars) and molecular weight (3450 g/mol) was fractionated, which can be served as feedstock for future utilization. In short, an extensive understanding of the pretreated biomass and the obtained lignin during the GVL/dilute acid pretreatment will be beneficial for value-added applications of biomass.
1. Introduction Lignocellulosic biomass is considered as an ideal feedstock for the production of bioethanol due to its abundance, renewability, high sugar content, and reasonable price. The use of biomass has benefits for addressing global warming, climate change, and energy demand. However, biomass recalcitrance limits its efficient utilization mainly owing to the complex and heterogeneous cell wall structure. The biomass recalcitrance is connected with physical and chemical features, including contents and distributions of hemicelluloses and lignin, accessible surface area, structure and crystallinity of cellulose, and other factors (Yoo et al., 2017). A pretreatment step is thus required to alter the chemical composition and structural features of biomass, simultaneously overcoming the recalcitrant barriers for increasing surface area and cellulose accessibility (Govumoni et al., 2013; Sun et al., 2016). Generally, an effective pretreatment method should be economical and effective on a diversity of biomass. Meanwhile, it should minimize the operating step, maximize the recovery of biomass components, and
⁎
produce a cellulosic fraction that can be easily hydrolyzed with cellulase (Shuai et al., 2016). To date, many pretreatment methods have been developed to reduce the biomass recalcitrance and facilitate the accessibility of enzymes to cellulose, such as steam explosion, hydrothermal, ionic liquid, organosolv, acid and alkali pretreatment (Inalbon et al., 2017; Nie et al., 2018; Ninomiya et al., 2018; Sun and Xue, 2018; Yang et al., 2018). In the steam explosion or hydrothermal pretreatment process, a part of hemicelluloses was removed and lignin as a key barrier to enzymatic hydrolysis was still left in the pretreated substrates (Sun et al., 2014a). The acid pretreatment could dissolve hemicelluloses from biomass effectively; however, there are still some limitations, such as severe equipment corrosion, expensive equipment construction, and low removal rate of lignin. The biomass structure can be altered by alkaline pretreatment by swelling cellulose and removing lignin and hemicelluloses, whereas a significant limitation is that the alkali was converted into irrecoverable or incorporation salts (Chiaramonti et al., 2012; Hendriks and Zeeman, 2009). In the ionic liquid pretreatment,
Corresponding author. E-mail address: [email protected] (R.C. Sun).
https://doi.org/10.1016/j.indcrop.2019.02.004 Received 7 September 2018; Received in revised form 30 January 2019; Accepted 3 February 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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dilute HCl solution (pH 2.0) to precipitate lignin, and the solid fraction was washed completely with distilled water. The precipitated lignin and washed solid fraction were freeze-dried for further experiment. The solid fractions were labeled as R80-30, R80-60, R80-90, R100-30, R100-60, R100-90, R120-30, R120-60, R120-90, R140-30, R140-60, and R140-90, corresponding to the pretreatment temperature and time. The lignin fractions obtained were labeled as L80-30, L80-60, L80-90, L100-30, L100-60, L10090, L120-30, L120-60, L120-90, L140-30, L140-60, and L140-90, according to the corresponding solid fractions.
the cost of ionic liquid is high and its recovery is difficult, thus limiting its industrial application for biomass pretreatment. The above mentioned pretreatment, such as steam explosion, hydrothermal, or acid pretreatment alone, had limited for removal and recovery of lignin and enhancement of enzymatic hydrolysis. Thus a combined treatment, such as hydrothermal, dilute acid, steam explosion or ionic liquid pretreatment combined alkaline treatment, was studied to be a promising integrated technology (Li et al., 2016; Sun et al., 2014a, 2014c and 2016). However, before the alkaline treatment, a large amount of water and a long operation time were needed to separate the pretreatment liquid and solid, thus increasing the operating cost and decreasing the economic profit. Organosolv pretreatment is an effective lignin-targeting method by breaking the internal bonds of hemicelluloses and lignin using organic or aqueous organic solvents such as ethanol, methanol and acetone with or without the addition of an acid or base catalyst (Pan et al., 2006; Yoo et al., 2017; Zhao et al., 2009). It is a practical technology for fractionation of lignocellulosic biomass because it promotes the fractionation of high-quality lignin and cellulose. Recently, organic solvents such as tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-MeTHF), methyl isobutyl ketone (MIBK), and γ-valerolactone (GVL) have been successfully applied to pretreat biomass for increasing the enzymatic hydrolysis efficiency by dissolution of lignin (Katahira et al., 2014; Nguyen et al., 2015; Shuai et al., 2016; Zhang et al., 2016). Among these organic solvents, GVL has been paid great attention because it is a biomass-derived, green and non-toxic solvent and its structure is rather stable in water and oxygen (Wu et al., 2016). Recently, although the production of nonenzymatic sugar and its degradation products generated from biomass using GVL have been intensively investigated, only a small amount of research is being conducted on the enzymatic hydrolysis of biomass. Specifically, the effect of the structural features of biomass on the enzymatic hydrolysis has been generally ignored during the GVL pretreatment period, which is pivotal for understanding and overcoming biomass recalcitrance. In addition, fundamental characteristics of the soluble lignin are rarely reported. In this work, therefore, a GVL/dilute acid pretreatment process was performed at moderate temperatures (80–140 °C) for different periods (30–90 min) to explore its effect on the chemical compositions, structural characteristics and enzymatic hydrolysis of Eucalyptus urophylla. Meanwhile, the soluble lignin was recovered after the pretreatment, and its yield, purity and molecular weight have been thoroughly investigated.
2.3. Chemical characterization of the solid fractions The chemical compositions of the solid fractions were quantified by a two-step hydrolysis process (Sluiter et al., 2008). The hemicelluloses and cellulose contents were determined by a high-performance anion exchange chromatography (HPAEC) system (Sun et al., 2014d). The surface characteristics of RM and pretreated Eucalyptus were performed with Hitachi S-3400 N II (Hitachi, Japan) at 10 kV. FT-IR spectra were conducted on a Nicolet iN10 spectrophotometer. The Xray diffraction was obtained using an XRD-6000 apparatus (Shimadzu, Japan) with a Cu Ka radiation source (k = 1.54 Å) and recorded from 5 to 40° with a scanning speed of 2°/min. The crystallinity index (CrI) was calculated by height method, in which the CrI was calculated from the ratio of the height of the 002 peak (I002, 2θ ≈ 22.5°) and the height of the minimum (Iam, 2θ ≈ 18.5°) between the 002 and the 101 peaks (Sun et al., 2014c). CP/MAS 13C NMR spectra were detected by a Bruker AV-III 400 M spectrometer (Germany) at 100.6 MHz. TGA/DTG curves were obtained on a thermal analyzer (SDT Q600). The solid fractions (10 mg) were placed on an Al2O3 crucible and heated from room temperature to 700 °C at a constant heating rate of 20 °C/min under the purified nitrogen atmosphere. 2.4. Enzymatic hydrolysis The hydrolysis experiment was conducted at 50 °C for 72 h in a shaking incubator at 150 rpm. Typically, 0.2 g solid fraction, 10 mL sodium acetate buffer (50 mM, pH 4.8) and a cellulase activity (Cellic® CTec2, 100 FPU/mL) of 17 FPU/g substrate were added into a 25 mL Erlenmeyer flask. The glucose content in the hydrolysate was determined by a HPAEC system equipped with an integral amperometric detector and a CarboPac PA 100 (Dionex, U.S.) analytical column (Sun et al., 2014c).
2. Material and methods
2.5. Characterization of lignin
2.1. Materials
The associated carbohydrates and the molecular weights of the lignin fractions were analyzed by HPAEC and gel permeation chromatography (GPC), respectively (Sun et al., 2014b). 2D HSQC NMR spectrum and main substructures of L120-60 were acquired on a Bruker AVIII 400 MHz spectrometer according to the previous literature (Sun et al., 2014b).
Eucalyptus urophylla, which is a kind of popular Eucalyptus in Guangxi province (China), was ground to obtain fractions (40–60 mesh). The Eucalyptus was air-dried and dewaxed with toluene/ ethanol (2:1, v/v) for 8 h as RM, and then dried in an oven at 60 °C for 24 h for further treatment. Here the toluene/ethanol extraction was to avoid the possible effects of extractives on the chemical composition analysis and the lignin purity.
3. Results and discussion
2.2. Pretreatment procedure of Eucalyptus with GLV/dilute acid system
3.1. Effect of GVL/dilute acid pretreatment on the chemical compositions of Eucalyptus urophylla
The pretreatment of Eucalyptus was operated in a batch reactor made of Hastelloy C-276 (Sen Long Instruments Company, China). 3.0 g of oven-dried Eucalyptus and 30 mL of GVL/H2O solution (4:1, v/v) with 100 mM H2SO4 were mixed into the reactor and then reacted at the target temperatures of 80, 100, 120 and 140 °C for 30, 60 and 90 min, respectively. When the reaction was finished, the reactor was set in an ice bath to cool down. After releasing the gas and pressure, the mixture was filtered through Buchner funnels to separate the liquid and solid fractions. Then the liquid fraction was poured into 150 mL of
Due to the biomass recalcitrance, it is vital to pretreat biomass before its subsequent enzymatic hydrolysis. It has been reported that GVL is an attractive green solvent derived from biomass, and GVL/dilute acid system has been used as an excellent reaction medium for the fractionation of lignin from biomass at moderate reaction temperatures (Fang and Sixta, 2015). Meanwhile, the crystalline structure of cellulose was difficult to destroy with a low acid concentration at a moderate temperature, which could maximize its selective conversion by enzymes (Shuai et al., 2016). They reported that the use of a GVL-based 22
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Table 1 Solid yield and chemical composition (%, w/w) of the solid fractions. Sample
RM R80-30 R80-60 R80-90 R100-30 R100-60 R100-90 R120-30 R120-60 R120-90 R140-30 R140-60 R140-90
Solid yield (%)
86.9 74.5 67.5 63.0 51.3 46.4 43.0 45.3 38.4 35.1 23.4 21.2 18.7
Chemical compositiona
Pretreatment factor
Hemicelluloses
Cellulose
AILb
ASLc
15.6 ± 0.5 11.1 ± 0.3 9.7 ± 0.3 8.6 ± 0.2 3.2 ± 0.1 2.2 ± 0.1 1.6 ± 0.2 2.5 ± 0.1 0.6 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.1 ± 0.0 0.1 ± 0.0
42.3 39.4 37.3 36.2 39.4 36.6 35.9 34.6 32.6 30.9 19.5 18.7 15.1
23.7 ± 1.4 19.0 ± 1.4 16.4 ± 0.9 14.5 ± 0.8 7.0 ± 0.5 6.4 ± 0.4 4.5 ± 0.4 7.1 ± 0.5 4.3 ± 0.2 2.9 ± 0.1 3.1 ± 0.0 2.0 ± 0.0 3.1 ± 0.1
5.2 5.1 4.1 3.6 1.7 1.3 1.1 1.3 1.0 0.8 0.5 0.5 0.4
± ± ± ± ± ± ± ± ± ± ± ± ±
2.1 1.2 1.5 0.9 0.8 1.3 0.9 1.3 1.5 0.7 1.1 0.8 0.6
± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.5 0.2 0.2 0.1 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0
d
0.00 0.05 0.10 0.14 0.52 0.55 0.62 0.49 0.61 0.62 0.39 0.40 0.31
a
The contents of cellulose, hemicelluloses, and lignin were all calculated based on the initial weight of Eucalyptus. AIL, acid insoluble lignin. c ASL, acid soluble lignin. d Pretreatment factor = retention rate of cellulose × removal rate of hemicelluloses × total delignification rate, in which retention rate of cellulose = weight of cellulose in the pretreated samples / weight of cellulose in the raw material, removal rate of hemicelluloses = (weight of hemicelluloses in the raw material - weight of hemicelluloses in the pretreated samples) / weight of hemicelluloses in the raw material, and total delignification rate = (weight of total lignin in the raw material - weight of total lignin in the pretreated samples) / weight of total lignin in the raw material. b
pretreated Eucalyptus as compared with the initial 42.3% in RM. However, the relative content of cellulose (46.9–77.5%) in the pretreated Eucalyptus gradually increased with the pretreatment intensity raised owing to the significantly removal of hemicelluloses and lignin. It was reported that when the hydrothermal pretreatment alone was used to pretreat Eucalyptus, the hemicelluloses were degraded, while lignin was hardly changed, thus subsequent alkaline treatment was performed to remove lignin from the hydrothermal pretreated Eucalyptus (Sun et al., 2014c). The previous work using ionic liquids for Eucalyptus pretreatment also required the subsequent alkaline treatment to improve the removal rates of hemicelluloses and lignin, thus enhancing the enzymatic hydrolysis (Li et al., 2016). As compared to the two-step pretreatment methods mentioned above, the one-step pretreatment with GVL/dilute acid can effectively dissolve hemicelluloses and lignin, whereas the dissolved lignin can be recycled for further utilization. The effective dissolution of lignin was mainly because that GVL was completely miscible with aqueous solution and had a similar δ-value with lignin (Lê et al., 2016). Moreover, as compared with the pure GVL or aqueous solution, the property of GVL/water was changed, in which a strong hydrogen bond existed between GVL and aqueous solution (Xue et al., 2016). The changed property facilitated the dissolution of lignin. Unlike lignin, hemicelluloses were primarily released from biomass as xylose oligomers and monomers at mild temperature (e.g. 120 °C) during the GVL/dilute acid pretreatment (Motagamwala et al., 2016). As the temperature or time increased, the xylose oligomers were degraded to xylose monomers accompanied the formation of furfural. Moreover, the pretreatment factor was also calculated, as shown in Table 1. Here, the pretreatment factor was used as representative of the pretreatment effect on the main components of lignocellulosic biomass under the different pretreatment conditions, in which it was calculated based on the following formula: pretreatment factor = retention rate of cellulose × removal rate of hemicelluloses × total delignification rate. It was found that when the temperature was performed at 80 °C, the pretreatment factors of the pretreated Eucalyptus were only 0.05-0.14. As the temperature increased to 100–120 °C, the pretreatment factors increased significantly to 0.49-0.62 but decreased to 0.31-0.40 as the temperature reached to 140 °C. The result suggested that the GVL/dilute acid pretreatment at 100–120 °C was considered as the appropriate conditions for the comprehensive treatment of Eucalyptus in order to remove the hemicelluloses and lignin and simultaneously reserve the
pretreatment process at 120 °C can lead to high sugar yields after enzymatic hydrolysis (Shuai et al., 2016). When GVL/H2SO4 solvent at 120 °C with 75 mM H2SO4 was used to pretreat hardwood, up to 80% of original lignin was removed with 96–99% of original cellulose retained in the pretreated substrates. Wu et al. also pointed out that the treatment of cotton stalk with GVL/H2SO4 can release about 65% lignin of plant cell wall, whereas the enzymatic hydrolysis efficiency of the pretreated stalk increased by two-fold as compared to raw material (Wu et al., 2016). In addition, owning to the good solubility of lignin and hemicelluloses in GVL/H2SO4 solvent, a GVL/water solution with a relatively higher acid concentration liberated a higher amount of lignin and hemicelluloses. Under 100 mM H2SO4, the removal rates of lignin and hemicelluloses reached 86–92% and 97–98%, respectively (Li et al., 2019). Therefore, in this case, GVL/dilute acid pretreatment with 100 mM H2SO4 was performed at moderate temperatures (80–140 °C) for different periods (30–90 min) to explore its effect on the chemical compositions, structural characteristics and enzymatic hydrolysis of Eucalyptus urophylla. Table 1 summarizes the yield and chemical composition of Eucalyptus urophylla pretreated with GVL/dilute acid. To clarify the composition changes of the solid fractions after pretreatment, the contents of main components were calculated based on the initial weight of RM. As illustrated, the yields of solid fractions significantly decreased from 74.5 to 18.7% with the pretreatment intensity increased. The mass loss of the raw Eucalyptus was mainly contributed to the dissolution of lignin and hemicelluloses into the reaction medium. Results showed that when the pretreatment temperature was 80 °C, the hemicelluloses and cellulose contents in the pretreated Eucalyptus decreased slightly with the raise of the pretreatment time. However, the lignin content in the pretreated Eucalyptus, especially acid insoluble lignin (AIL), decreased obviously. The content of AIL decreased from 23.7 to 19.0-14.5% for different reaction times. It indicated that the GVL/dilute acid system was an excellent reaction medium for lignin removal even at a low pretreatment temperature. As the temperature rose to 100–120 °C, both lignin and hemicelluloses significantly removed from the biomass matrix, and only around 2.9–7.0% of AIL and 0.4–3.2% of hemicelluloses were remained in the pretreated Eucalyptus, respectively. Meanwhile, the degradation of cellulose became evident at 120 °C, which decreased from 42.3 to 30.9–34.6%. When the temperature rose to 140 °C, cellulose occurred significant degradation, and only 15.1–19.5% of original cellulose was reserved in the 23
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Fig. 1. SEM images of RM and the pretreated Eucalyptus at magnifications × 1000 and × 6000.
24
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2017). Here the main effect of GVL/dilute acid pretreatment is the removal of amorphous substances. The X-ray diffraction testing is a better method to evaluate the effect of pretreatment on the CrI of biomass. Fig. S2a shows the X-ray diffraction curves and the CrI values of RM, R80-60, R100-60, R120-60, and R140-60. No remarkable differences were observed among the XRD patterns of the solid fractions, and the two characteristic peaks observed at around 16° and 22° suggested a typical pattern of cellulose I. It demonstrated that the crystalline structure of cellulose in biomass was not destructed after the GVL/dilute acid pretreatment. Furthermore, it was found that as compared to the CrI value (52.03%) of the untreated Eucalyptus (RM), the CrI values of the pretreated samples were gradually increased to 59.17–72.03% with the increment of the pretreatment severity. This could be the progressive solubilization and degradation of amorphous lignin and hemicelluloses, as illustrated in Table 1, which exposed the crystalline cellulose core and increased the cellulose proportion in the pretreated samples (Liu et al., 2009). The result was in accordance with many previous studies (Xiao et al., 2013; Zakaria et al., 2015). Zakaria et al. (2015) reported that the increased CrI in directly showed higher exposure of cellulose component amenable to cellulase attack. To further analyze the main components and structural variations of the pretreated Eucalyptus, solid state CP/MAS 13C NMR spectra (Fig. S2b) of RM, R80-60, R100-60, R120-60, and R140-60 were recorded. The spectral signals were interpreted according to the data from previous literatures (Jeong and Lee, 2016; Lü et al., 2017; Pereira et al., 2016). The signals of cellulose can be assigned as followed: C1 (103.5 ppm), C2/C3/C5 (71.0 ppm), C4 (82.5 and 86.7 ppm), and C6 (63.1 ppm). Since the signals in the 80–86 and 86–92 ppm were due to amorphous cellulose and crystalline cellulose, respectively, the CrI values were also calculated by taking the ratio of the integration in the region of 86–92 ppm to that of 80–92 ppm. It was found that the CrI values from CP/MAS 13C NMR method were relatively low as compared to those from XRD peak height method, which was because that the effect of the cellulose chains present on the surface of cellulose crystals was considered in the NMR method (Park et al., 2010). Note that a similar trend was detected, namely the CrI values increased with the pretreatment temperatures. In addition, the intensities of the signals at 19.4 and 170.5 ppm originated from acetyl groups of hemicelluloses cut down with the increasing pretreatment intensity. The decreasing trend was benefit for the enhancement of cellulose accessibility, which was because the decreased acetyl groups of hemicelluloses can reduce the steric obstacles of enzymes from hemicelluloses (Jiang et al., 2014). The signal intensities of methoxyl group (54.1 ppm) and aromatic group (151.3 ppm) from lignin also gradually reduced with the increment of the pretreatment severity, illustrating that the degree of biomass delignification increased as shown in Table 1. Additionally, the intensities of the signals at 86.7 and 63.1 ppm derived from the C4 and C6 of the crystalline cellulose were enhanced as the increased pretreatment temperatures. The thermal stabilities of the raw material and the pretreated Eucalyptus prepared with GVL/dilute acid were characterized by TGA and DTG (Fig. S3). TGA curves showed a significant decrease of char residues at 700 °C in R80-60 (17.35%) and R100-60 (16.17%) as compared to that in RM (20.69%), which could be due to the fact that the lignin with a high thermal stability was removed during the pretreatment process. However, the char percentages were increased to 19.16–19.58% when the pretreatment temperatures increased to 120–140 °C for a constant time of 60 min. The reason for this was that part of lignin was condensed at high pretreatment temperatures under the acid condition, resulting in an increment of thermal stabilities to some extent. In addition, a shoulder peak at 283 °C and a sharp peak at 367 °C, originated from the thermal degradation of hemicelluloses and cellulose, respectively, were observed in the DTG curves of RM and R8060 (Cao et al., 2014). However, because most hemicelluloses were removed from the Eucalyptus urophylla, almost no shoulder peak was found in the DTG curves of R100-60, R120-60, and R140-60.
cellulose. 3.2. SEM analysis of the solid fractions SEM is the powerful tool for studying the morphological information and fundamental physical properties of the biomass after pretreatment (Gabhane et al., 2015; Karimi and Taherzadeh, 2016). The morphology of RM, R80-60, R100-60, R120-60, and R140-60 was recorded by SEM. As shown in Fig. 1, the untreated Eucalyptus exhibited a rigid and compact surface structure preventing the accessibility of enzymes to cellulose. In contrast, the loosing fibrillar structure was observed in the pretreated Eucalyptus due to the removal of some non-cellulosic components. Furthermore, it is worth noting that the morphology of the pretreated sample was correlated to the pretreatment severity. Specifically, slight surface structural changes were observed when the Eucalyptus was pretreated at 80 °C, while some crevices appeared in the samples pretreated at mild temperatures (100–120 °C). It suggested that the GVL/dilute acid system was able to disrupt the plant cell wall of biomass effectively. When the temperature increased to 140 °C, a more disorganized morphology was detected in R140-60, in which the lignin and hemicelluloses were almost completely removed. The relatively severe pretreatment resulted in the exposure of more reactive area on the Eucalyptus surface, as well as the collapse of cellulose fibrils, which would be a negative factor on the enzymatic hydrolysis (Hou et al., 2013; Sun et al., 2011). Similar finding was reported when hulless oat straw was imposed at severe treatment condition which caused the biomass damage and led to the reduction of accessible surface area (Yang et al., 2013). In short, the disruption of the plant cell wall contributed to the improvement the enzymatic hydrolysis of the samples pretreated with GVL/dilute acid, which was confirmed by the results of the subsequent enzymatic hydrolysis experiment. (Fig. 1) 3.3. Characterization of the physicochemical properties of the solid fractions In order to further investigate the structural changes of Eucalyptus urophylla pretreated under different conditions, FTIR, XRD, CP/MAS 13 C NMR and TG were performed. FTIR spectra (Fig. S1) were used to probe the chemical composition changes among RM and the pretreated Eucalyptus. As compared with RM, the intensities of the absorption bands at 1740 and 1242 cm−1 originated from the C]O stretching vibration in acetyl groups and the C–O stretching of hemicelluloses (Kumar and Wyman, 2009), respectively, were sharply reduced in R10060 and even disappeared in R120-60 and R140-60. The result indicated that most of hemicelluloses were degraded in the samples pretreated at 100–140 °C for 60 min. This was in line with the changes observed in the chemical composition reported in Table 1. The bands at 1593, 1504 and 1458 cm−1 are attributed to the aromatic skeletal vibrations of lignin (Sun et al., 2014b). It was clearly observed that the intensities of these bands were declined dramatically in the pretreated Eucalyptus, especially in R100-60, R120-60 and R140-60, which was also in accordance with the results in Table 1. Moreover, the increasing intensity of the band at 1153 cm−1 for the characteristic assignment of cellulose was observed in R80-60-R140-60, which could owe to the relative high content of cellulose in the these samples. These results suggested that the pretreatment of Eucalyptus urophylla with GVL/dilute acid removed most non-cellulosic components and simultaneously reserved cellulose under the moderate pretreatment conditions. The cellulose structure and its CrI are regarded to affect the enzymatic hydrolysis (Inouye et al., 2014; Sathitsuksanoh et al., 2011). Generally, if the purpose of biomass pretreatment is to remove hemicelluloses and lignin, high CrI indicates good pretreatment effect (Wang et al., 2017; Zhang et al., 2018). However, if the purpose of biomass pretreatment is to destroy the crystallization zone of cellulose, low CrI indicates good pretreatment effect (Sahoo et al., 2018; Wang et al., 25
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(%, oven dried RM) are given in Table 2. It was found that when the pretreatment temperature was performed at 80 °C, only 1.9–2.6% of lignin was extracted from RM. When the temperature rose to 100–140 °C, especially at 120 and 140 °C, the lignin yields were improved significantly to 10.8–21.0%, suggesting that the raise of the pretreatment severity had a dramatic effect on the removal of lignin, as in line with the component analysis of the solid fractions. Next, in order to verify the lignin purity, the associated carbohydrates were detected (Table 2). Results showed that the lignin extracted with GVL/dilute acid solution had low amounts of associated carbohydrates (0.08–1.48%), in which xylose was the main carbohydrates. Notably, when the temperature was increased to 120 and 140 °C, minor amounts of the associated carbohydrates (less than 0.2%) were detected. The low carbohydrates contents in these lignin fractions indicated that the linkages between lignin and hemicelluloses in biomass were broken obviously during the pretreatment process. Therefore, the GVL/dilute acid pretreatment could be used as a promising pretreatment technology for facilitating the extraction of lignin with a high purity from the plant cell walls, which was beneficial for its subsequent application. In addition, GPC analysis was used to explore the effect of the GVL/dilute acid pretreatment on the molecular weights and polydispersity index (Mw/ Mn) of the lignin fractions (Table 2). It was found that when the pretreatment temperature was 80 and 100 °C, prolonging pretreatment time from 30 to 90 min gave rise to the release of lignin with high molecular weights. As the temperature was 120 and 140 °C, the molecular weight of lignin was slightly reduced with the increasing time because of the degradation of lignin under the severe treatment conditions. The low Mw/Mn (1.36–1.83) of all the lignin fractions implied the relative homogeneity of the lignin macromolecule. Overall, during the GVL/dilute acid pretreatment process, especially at 120 and 140 °C, the lignin had high yields, purities, and Mw, as well as low Mw/Mn, which is a preferred feedstock for future utilization. Meanwhile, the 2D HSQC NMR spectrum and main substructures of L120-60 is shown in Fig. S5. Obviously, the main linkage of L120-60 was β-O-4′ (A), followed by ββ′ (B) and β-5′ (C), and syringyl (S) and guaiacyl (G) units were visible, which confirmed a primary structure of lignin. Therefore, GVL/dilute acid pretreatment was a promising technology to realize the efficient conversion of cellulose to glucose by enzymatic hydrolysis for bioethanol production, in which the obtained lignin can be converted into high value-added products (Dai et al., 2017, 2018).
Fig. 2. Enzymatic hydrolysis of RM and the pretreated Eucalyptus.
3.4. Enzymatic hydrolysis of the solid fractions To further explore the digestibility of the solid fractions, the raw material and the samples pretreated under various conditions were enzymatically hydrolyzed using cellulase at 50 °C for 72 h, as illustrated in Fig. 2. As compared to RM (21.1%), the pretreated Eucalyptus was more readily to be hydrolyzed by cellulase. Meanwhile, the enzymatic hydrolysis rates of the pretreated Eucalyptus increased with the increment of the temperature from 80 to 120 °C, especially at 100 and 120 °C, which was owing to the large release of the potential recalcitrance components (hemicelluloses and lignin) from biomass matrix. Meanwhile, the loosing fibrillar structure was visualized by SEM images, leading to the exposure of the internal surface of Eucalyptus and exhibition of adsorption sites of enzymes to more extent. As the temperature increased to 140 °C, the enzymatic hydrolysis rate of R14060 (88.2%) was similar to that of R120-60 (89.1%). The possible explanation may be that a large amount of amorphous cellulose was degraded under the relatively harsh pretreatment conditions and more crystalline cellulose was reserved in the pretreated sample, resulting in a relatively low enzymatic hydrolysis rate of R140-60. Another reason was that the excessive removal of lignin and hemicelluloses introduced the collapse of the cellulose multiple-layers, thus preventing the available surface area for cellulase (Ishizawa et al., 2009; Yang et al., 2013). Based on the above results, it is meaningful to establish the relationship of the glucose conversion ratios with the removal rates of hemicelluloses and lignin. As shown in Fig. S4a, a positive correlation between glucose conversion rate and removal rate of hemicelluloses and lignin with a relative high coefficient factor (R2 = 0.91) was observed in the solid fractions, which was consistent with the previous works (Ishizawa et al., 2007; Sun et al., 2011). Besides, the enzymatic hydrolysis efficiency is proportional increase to the raise of CrI value (Fig. S4b). Here note that the increment of CrI of the pretreated samples was mostly achieved by the release of hemicelluloses and lignin, thus indicating that the CrI did not significantly affect the glucose yield during the enzymatic hydrolysis (Hsu et al., 2010; Zakaria et al., 2015). That was the CrI had no direct correlation with hydrolysis efficiency. Overall, it can be concluded that both obvious removal of hemicelluloses and lignin during the GVL/dilute acid pretreatment had considerable influence on the structural changes of biomass matrix and expansion of available surface area, resulting in the enhancement of enzymatic hydrolysis.
4. Conclusions This study demonstrated that GVL/dilute acid pretreatment was an effective way to change the chemical composition and structural characteristic of biomass to improve enzymatic hydrolysis. Findings showed that the pretreatment successfully removed hemicelluloses and lignin and disrupted the cell wall structure, resulting in the increased cellulose crystallinity and enzyme accessibility. The highest glucose yield of 89.1% was obtained after the GVL/dilute acid pretreatment (80% GVL/20%H2O with100 mM H2SO4 at 120 °C for 60 min). Additionally, the by-product lignin with a high yield was obtained during the pretreatment had a high purity and molecular weight. Acknowledgments This work was supported by the National Natural Science Foundation of China (21706015, 31700518), Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control (KF201806-5) and the Foundation of State Key Laboratory of Biobased Material and Green Papermaking (No. KF201811).
3.5. Analysis of lignin fractions Appendix A. Supplementary data In this work, the dissolved lignin was recovered by precipitation of the liquid fraction obtained during the GVL/dilute acid pretreatment into aqueous dilute acid solution. The yields of lignin fractions obtained
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.02.004. 26
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Table 2 Yield of lignin (%, w/w), its associated carbohydrate content (%, w/w), weight-average (Mw) and number-average (Mn) molecular weights, and polydispersity (Mw/ Mn). Lignin fractions
Yield
Galactose
Glucose
L80-30 L80-60 L80-90 L100-30 L100-60 L100-90 L120-30 L120-60 L120-90 L140-30 L140-60 L140-90
1.9 2.3 2.6 10.8 12.0 14.1 11.6 16.5 18.3 19.9 21.0 20.2
0.18 0.20 0.14 0.10 0.12 0.15 0.10 0.00 0.15 0.00 0.00 0.00
0.28 0.18 0.17 0.17 0.16 0.20 0.12 0.02 0.05 0.10 0.11 0.09
± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.02 0.01 0.00 0.04 0.01 0.00 0.00 0.02 0.00 0.00 0.00
± ± ± ± ± ± ± ± ± ± ± ±
Xylose 0.05 0.04 0.04 0.03 0.01 0.04 0.02 0.00 0.00 0.01 0.00 0.00
0.78 1.10 0.85 0.34 0.76 1.01 0.99 0.07 0.09 0.00 0.10 0.05
± ± ± ± ± ± ± ± ± ± ± ±
0.10 0.04 0.03 0.00 0.07 0.09 0.07 0.00 0.00 0.00 0.01 0.00
Total sugars
Mw
1.24 1.48 1.16 0.61 1.04 1.36 1.21 0.09 0.29 0.10 0.21 0.14
3210 3760 3870 3070 3440 3590 3550 3450 2880 2610 2320 2270
± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.07 0.03 0.02 0.05 0.04 0.06 0.00 0.03 0.00 0.01 0.02
Mn ± ± ± ± ± ± ± ± ± ± ± ±
130 70 150 120 70 110 90 80 100 80 50 40
2360 2260 2290 1850 2010 2030 1990 1890 1690 1560 1480 1470
Mw/Mn ± ± ± ± ± ± ± ± ± ± ± ±
90 50 40 70 70 40 60 30 20 50 90 40
1.36 1.66 1.69 1.66 1.71 1.77 1.78 1.83 1.70 1.67 1.57 1.54
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