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Lignin relocation contributed to the alkaline pretreatment efficiency of sweet sorghum bagasse
Fuel 158 (2015) 152–158
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Lignin relocation contributed to the alkaline pretreatment efficiency of sweet sorghum bagasse Zhipei Yan a,b, Jihong Li a,b,⇑, Sandra Chang a,b, Ting Cui a,b, Yan Jiang a,b, Menghui Yu a,b, Lei Zhang a,b, Gang Zhao b, Panlun Qi c, Shizhong Li a,b,⇑ a b c
Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, China Beijing Engineering Research Center of Biofuels, Tsinghua University, Beijing 100084, China Sinopec Research Institute of Petroleum Processing, Petro China Company Limited, Beijing 100083, China
h i g h l i g h t s The relocation of LCC occurred in Ca(OH)2 pretreatment. Complex formation of LCC and Ca
2+
resulted in the relocation.
Lignin did not adsorb cellulase during enzymatic hydrolysis.
a r t i c l e
i n f o
Article history: Received 15 January 2015 Received in revised form 23 April 2015 Accepted 15 May 2015 Available online 23 May 2015 Keywords: Sodium hydroxide pretreatment Calcium hydroxide pretreatment Lignin Enzymatic hydrolysis Relocation
a b s t r a c t Sodium hydroxide and calcium hydroxide are used to improve enzymatic hydrolysis of lignocellulose. Compared to NaOH, less carbohydrates was lost in Ca(OH)2 pretreatment. It was found that the substrates with similar lignin content prepared from NaOH and Ca(OH)2 pretreatments had distinct cellulose conversion rate. To understand the mechanism of action, two sweet sorghum bagasse (SSB) samples with similar lignin content were prepared. The enzymatic hydrolysis result showed that the cellulose conversion rate of SSB treated by Ca(OH)2 was about 1.62 times that of SSB treated by NaOH. SEM results showed tiny droplets on the SSB surface pretreated with Ca(OH)2. EDS analysis revealed that the droplets were a complex of lignin–carbohydrate and calcium ions. The results suggested that Ca(OH)2 not only increased the porosity of substrates, but also reduced carbohydrates loss by forming a complex with lignin and calcium ions. This demonstrated that calcium ions could be used to reduce the carbohydrates loss from the pretreatment procedure. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose is the most abundant renewable resources on the earth and a unique renewable feedstock to produce liquid fuels. Biofuel production from lignocellulose using bioconversion technology is low-energy and environmentally friendly [1]. However, the release of fermentable sugars from lignocellulose has become Abbreviations: ATR–FT/IR, attenuated total reflectance–fourier transform infrared spectroscopy; BET, Brunauer–Emmett–Teller; EDS, energy dispersive spectral analysis; G, guaiacyl propane; H, p-hydroxyl phenyl propane; LCC, lignin–carbohydrate complexes; S, syringyl propane; SEM, scanning electron microscope; SSB, sweet sorghum bagasse. ⇑ Corresponding authors at: Institute of New Energy Technology, Tsinghua University, Beijing 100084, China. Tel./fax: +86 10 80194050. E-mail addresses: [email protected] (J. Li), [email protected] (S. Li). http://dx.doi.org/10.1016/j.fuel.2015.05.029 0016-2361/Ó 2015 Elsevier Ltd. All rights reserved.
a bottleneck for bioconversion produced bioethanol due to its recalcitrance [1]. The natural recalcitrance of lignocellulosic materials to enzymatic hydrolysis is due to the structure of the plant cell wall, which is composed of cellulose, hemicellulose and lignin [2,3]. Lignin is relatively hydrophobic and covalently linked to hemicelluloses and fills the spaces in the cell wall between cellulose and hemicellulose. This structural arrangement protects the carbohydrates from degradation by micro-organisms or enzymes [1]. Thus, a pretreatment process is required to remove the recalcitrant physical and chemical barriers and increase the enzymatic digestibility of cellulose [4]. Among various pretreatments, numerous studies have focused on alkaline pretreatments because they have some practical operational advantages including lower reaction temperature and pressure, no need for complicated reactor, and allowing the reuse of residual alkali [1]. Alkali pretreatments weaken the recalcitrance of cell wall to cellulases by cleaving the
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hydrolyzable linkages between lignin and glycosidic bonds of polysaccharides, as well as disruption of the lignin structure [5]. In addition, alkaline saponification of acetyl and uronic ester bonds also improved the enzymatic accessibility of the polysaccharides [6,7]. Sodium hydroxide (NaOH) and calcium hydroxide (Ca(OH)2) are two common chemicals studied extensively in alkaline pretreatments. Mild NaOH pretreatment could effectively improve the enzymatic hydrolysis of herbaceous crops due to their lower lignin content, special lignin and lignin-carbohydrate complexes (LCC) structures which are more susceptible to alkaline pretreatment [8]. The glucan saccharification yield of sweet sorghum bagasse (SSB) pretreated with 2.5 M or 20% NaOH at room temperature for 120 min increased significantly compared to the control [9,10]. Ca(OH)2 could also successfully improve the enzymatic hydrolysis of lignocellulose. Corn stover pretreated with 0.5 g Ca(OH)2/g raw biomass at 55 °C for 4 weeks with aeration converted 91.3% of glucan and 51.8% of xylan to glucose and xylose, respectively [11]. The time of Ca(OH)2 pretreatment needed was longer than NaOH pretreatment due to its poor solubility in water (1.73 g/L at 20 °C). However, it is widely used because it is inexpensive (about 6% cost of NaOH), easier to handle and recover [1]. Most of the previous literatures were focused on process optimization of pretreatment. Although some papers discussed the changes to the substrates by NaOH or Ca(OH)2 pretreatment, the sensitivity of the substrates to these different alkalis was not studied. Moreover, due to most of the attention were focused on the enzymatic hydrolysis, comparison of the substrates with similar key features after pretreatment has not been investigated up to now. Lignin content is an important component influenced by alkaline pretreatment because lignin not only is the barrier to cellulose, but also can absorb cellulases [12]. However, other factors may play an important role in the bioconversion of cellulose. In previous studies the differences in lignin content of the substrate can obscure and make comparison of other factors difficult. In order to exclude the influence of the lignin content, two alkaline samples with similar lignin content were prepared by NaOH and Ca(OH)2 pretreatment. The changes to SSB after pretreatment, including chemical structure, porosity, surface topography, and surface composition of pretreated SSB were characterized in detail. The changes to the properties were correlated to the enzymatic digestibility of the samples. The differences between NaOH and Ca(OH)2 pretreatment on enzymatic hydrolysis and effect on SSB were discussed.
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2.4. Characterization of chemical structure The chemical structure of untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were characterized by attenuated total reflectance–fourier transform infrared spectroscopy (ATR–FT/IR). ATR–FT/IR was conducted using a Spectrum One FTIR system (Perkin Elmer) with a universal ATR (Attenuated Total reflection) accessory. Samples were washed completely with DI water and dried to constant mass at 45 °C in vacuum oven. Sample spectra were obtained using an average of 32 scans over the range between 600 cm 1 and 4500 cm 1 with a spectral resolution of 2 cm 1. Baseline and ATR corrections for penetration depth and frequency variations were applied using Spectrum software supplied with the equipment. 2.5. Thioacidolysis Thioacidolysis was used to estimate the relative abundance and proportion of S, G, and H monolignols according to Foster et al. [15]. 2.6. Preparation of lignins The lignin was isolated from SSB pretreated with NaOH or Ca(OH)2 according to the method reported by Hu et al. [16]. 2.7. Enzymatic hydrolysis of Avicel The hydrolysis of Avicel was carried out in 50 mM sodium acetate buffer (pH 4.8, 50 °C) at 2% consistency in the presence of the isolated lignin. The lignin loading was 1/4 of cellulose. The cellulase (CTec 3) loading for the Avicel was 10 FPU/g cellulose. The hydrolysis of Avicel, without the addition of isolated lignin, was performed as control. Aliquots were taken at the indicated time from the reaction mixture and centrifuged to remove the insoluble materials. The sugar content was measured according to the method reported by Li et al. [14]. All samples were measured in triplicates. 2.8. Porosity
2. Methods
Specific surface area determinations were performed using the Brunauer–Emmett–Teller (BET) method with an adsorptionmeter (Quadrasorb SI-MP, Quantachrome). Porosity was determined by BJH/DH method. Outgas Temp: 120.0 °C, Analysis gas: nitrogen.
2.1. Alkali treatment
2.9. Surface topography
NaOH pretreatment of SSB was carried out by the following conditions other than stated specially: 10 g NaOH/100 g SSB, 95 °C, 0.5 h, 1 g SSB/20 mL water; The condition for Ca(OH)2 pretreatment of SSB was: 10 g Ca(OH)2/100 g SSB, 25 °C, 24 h, 1 g SSB/10 mL water. The pretreated solids were washing to pH 7.0 by deionized water.
SEM images were taken at 250 and 10,000 times using JSM-6460/LV SEM (Hitachi, Tokyo, Japan) at 10 kV; the samples were sputter-coated with a thin layer of gold. 3. Results and discussion 3.1. Effect of lignin on the enzymatic hydrolysis
2.2. Composition analysis Compositions of samples were determined according to the NREL method [13]. All samples were measured in triplicates. 2.3. Enzymatic hydrolysis of the pretreated samples Enzymatic hydrolysis of the SSB pretreated with NaOH or Ca(OH)2 were carried out according to the method reported by Li et al. [14]. All samples were measured in triplicates.
NaOH and Ca(OH)2 are two important alkalis in alkaline pretreatment protocols. Although their basicity is different, each reacts with the biomass to open the natural recalcitrance of lignocellulose by solvation and saponification of lignin structures [1]. After alkaline pretreatment, the structure of lignocelluloses changes in many aspects. Among them, the lignin content changed significantly. Since the effect of lignin content is well established and studied [17–20], other changes influencing enzymatic hydrolysis caused by alkaline pretreatment were investigated. In order to
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exclude the effect of lignin content, SSB samples with about 17.3–17.4% lignin content resulting from NaOH and Ca(OH)2 pretreatment were prepared. The pretreatment conditions for NaOH was 10% NaOH at 95 °C for 30 min, and for Ca(OH)2 was 10% Ca(OH)2 at 25 °C for 24 h. The composition of these two samples was listed in Table 1. The enzymatic hydrolysis result showed that the cellulose conversion rate of SSB treated with Ca(OH)2 (61.11%) was 1.62 times of that for SSB treated with NaOH (37.73%) (Fig. 1). The result revealed that the enzymatic digestibility of substrates pretreated with the two different chemicals had no correlation with the lignin content. A similar result was reported by Yu et al. [20], that the lignin content of loblolly pine delignified by ozone and sodium chlorite was 12.8% and 18.0%, respectively. However, the carbohydrate conversion was 58% and 80%, respectively. It was believed that the different delignification mechanism between sodium chlorite and ozone caused the difference in enzymatic hydrolysis efficiency. Therefore, the pretreatment mechanism of NaOH and Ca(OH)2 was likely different as well. The properties of lignocellulosic substrates including chemical structure, porosity, surface topography, and surface composition of SSB pretreated with NaOH or Ca(OH)2 were systematically studied. 3.2. Comparison of chemical structure of SSB pretreated with NaOH or Ca(OH)2 In addition to lignin content, the structure of the substrate contributed to the efficiency of enzymatic hydrolysis of the lignocellulose. The acetyl groups of hemicellulose can interfere with enzyme recognition [1], inhibit hydrogen bonds between cellulose and the catalytic domain of cellulases, and increase the diameter of the cellulose chain [12]. Lignin is a phenolic polymer composed of three major types of building blocks: p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units. In general, S/G ratios and/or S content are indicative of efficiency of pretreatment [18]. The chemical structure of SSB pretreated with NaOH or Ca(OH)2 was characterized by attenuated total reflectance–fourier transform infrared spectroscopy (ATR–FT/IR). Normalized ATR–FT/IR spectra at a band position of 1510 cm 1 [21] of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were presented in Fig. 2. NaOH and Ca(OH)2 pretreatments did not change the cellulose structure significantly, but altered certain functional groups and linkages. The peak near 3348 cm 1 represented –OH stretching. The intensity of the peak of SSB pretreated with NaOH increased, suggested the increase of hydrogen bonds in the cellulose. The peak for SSB pretreated with Ca(OH)2 reduced in the intensity of this peak, indicated that hydrogen bonds of cellulose were disrupted [2,22,23]. The band position at 2900 cm 1 is attributed to C–H stretching, and both NaOH and Ca(OH)2 pretreatments reduced the intensity, indicated that the methyl and methylene portions of cellulose were reduced. Both pretreatments caused significant changes in the 1735 cm 1 band which is attributed to ester carbonyl vibration in acetyl, feruloyl, and p-coumaroyl groups in lignin and hemicellulose, indicated that the ester bonds between hydroxycinnamic acids and lignin were ruptured [24]. The intensity of SSB pretreated with NaOH was reduced significantly while the peak for the SSB pretreated with Ca(OH)2 disappeared. The disappearance of the Table 1 The composition for SSB pretreated with NaOH and Ca(OH)2.
Cellulose (%) Hemicellulose (%) Lignin (%)
Untreated
NaOH
Ca(OH)2
39.42 ± 0.73 25.25 ± 0.26 18.93 ± 0.31
44.66 ± 0.70 28.23 ± 0.54 17.32 ± 0.19
48.17 ± 0.88 25.72 ± 0.00 17.45 ± 0.40
Fig. 1. Comparison of enzymatic hydrolysis of SSB pretreated with NaOH and Ca(OH)2. The white column was the cellulose conversion; the black column was the hemicellulose conversion; the gray column was the utilization of substrate.Comparison of enzymatic hydrolysis of SSB pretreated with NaOH and Ca(OH)2. The black column was the cellulose conversion; the red column was the hemicellulose conversion; the blue column was the utilization of substrate.
peak indicated that the ester bonds between hydroxycinnamic acids and lignin were cleaved during the pretreatment or the content of these groups were so low that they were below the detection limit for the spectroscopical methods employed. Both pretreatments reduced the intensity of the 1245 cm 1 band, which is attributed to the cleavage and/or alterations of acetyl groups [24]. The acetyl group was likely removed from the hemicellulose in both NaOH and Ca(OH)2 pretreatments. These changes likely contributed to the enzymatic hydrolysis but did not explain the difference in enzymatic hydrolysis efficiency. In both pretreatments the peak at the 1600 cm 1 band which is associated with aromatic skeletal vibration [24] did not change much. It suggested that skeleton of the benzene ring of lignin did not change during NaOH and Ca(OH)2 pretreatments. The band at 1462 cm 1 is associated with C–H deformation by methyl and methylene. The band at 1425 cm 1 is associated with C–H in-plane deformation by aromatic ring stretching [25]. With both peaks the SSB pretreated with NaOH did not change as compared to the untreated SSB, while the sample pretreated with Ca(OH)2 were significantly higher. This suggested that the proportion of G or H unit increased in lignin of the sample pretreated with Ca(OH)2 [25]. Furthermore, the C–H asymmetric deformation occurs at 1370 cm 1 is associated with the G unit. The band at 1323 cm 1 is attributed to the C–C and C–O skeletal vibrations, which is associated with S unit and G unit condensation. The peak at 1164 cm 1 is related to HGS lignin [26]. The relative reductions of all the above peaks by the Ca(OH)2 pretreatment pointed to the removal of the aromatic ring on lignin and solubilization of lignin polymers, especially of the G unit. Through ATR–FT/IR analysis, it was suggested that Ca(OH)2 pretreatment removed the G unit and increased the H unit. In order to quantify the changes, the lignin monomer of untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were analyzed (Table 2). Table 2 shows that the H unit of SSB pretreated with Ca(OH)2 increased, the S unit of SSB pretreated with NaOH increased, while G unit of both decreased. Consequently, the S/G ratio increased from 0.63 for both to 0.72 and 0.70 for SSB pretreated with NaOH and Ca(OH)2 respectively. Meanwhile, the H/G ratio increased from 0.67 for both to 0.71 and 0.81 for SSB pretreated with NaOH and Ca(OH)2, respectively. Other researchers reported
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For grass, cell wall digestibility was strongly correlated to the content of H unit as well [31–33]. However, the effect of H unit on the cell wall digestibility under the same lignin content was not reported. Therefore, the higher proportion of H unit in lignin of SSB pretreated with Ca(OH)2 (Table 2) may be the reason for the higher effectiveness of cellulose digestibility of SSB pretreated with Ca(OH)2. The inhibition effect of lignin on the enzymatic hydrolysis of cellulose mainly are: (1) non-productive adsorption of the enzyme by lignin through hydrophobic interactions, electrostatic interactions, and/or hydrogen-bonding interactions; (2) lignin in lignocellulosic materials acting as a surface barrier to block the accessible surface of carbohydrates through physical blockage on the surface and chemical blockage through lignin carbohydrate complex; (3) soluble lignin derivatives can deactivate enzymes in liquid phase [12]. In order to investigate the possible adsorption and deactivate enzymes of cellulases by lignin, the glucose conversion rate of Avicel in the presence of lignin prepared from SSB treated with NaOH or Ca(OH)2 was investigated. The results showed that the enzymatic hydrolysis of Avicel did not decrease with the addition of isolated lignin (Fig. 3). This suggested that the non-productive adsorption and deactivate enzymes of cellulases by lignin was not a factor in the cases of SSB pretreated with NaOH or Ca(OH)2. Whether pretreatment of lignin caused condensation onto the substrate and prevented the physical accessible of enzyme still needs to be investigated. 3.3. Porosity of SSB pretreated with NaOH or Ca(OH)2 Since the non-productive adsorption and deactivate enzymes of cellulase on the lignin could be excluded, cellulose accessibility was studied. The prerequisite of cellulose hydrolysis by cellulases is the adsorption of enzymes onto the substrate. Therefore, the size and volume of pores in the biomass is one of the critical factors limiting the enzymatic hydrolysis of the polysaccharides [19]. Thus, the difference in porosity of SSB pretreated with NaOH and Ca(OH)2 was studied by the Brunauer–Emmett–Teller (BET) method. Table 3 listed the effect of NaOH and Ca(OH)2 pretreatments on the surface area, pore volume and pore size of pretreated SSB. Table 3 showed that both surface area and pore volume of SSB were increased after alkaline pretreatment, and the values of SSB Fig. 2. ATR–FT/IR spectra of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2 (a) was the ATR–FT/IR spectra of the untreated SSB; (b) was the ATR-FT/IR spectra of the SSB pretreated with NaOH; (c) was the ATR–FT/IR spectra of the SSB pretreated with Ca(OH)2.ATR–FT/IR spectra of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2. The black line was the ATR–FT/IR spectra of the untreated SSB; the red line was the ATR–FT/IR spectra of the SSB pretreated with NaOH; the blue line was the ATR–FT/IR spectra of the SSB pretreated with Ca(OH)2.
that G unit was more easily degraded than S unit during pretreatment [27] and S unit [28] or S/G [29] increased after pretreatment. Li et al. [30] revealed that high content of S unit had a positive effect on the effectiveness of enzymatic hydrolysis. But the S/G ratio of samples pretreated with NaOH and Ca(OH)2 was similar, therefore the increase of the S/G ratio did not contribute to the different enzymatic efficiency of the alkaline treated SSB.
Table 2 The abundance and proportion of lignin monomer for untreated SSB and SSB pretreated with NaOH or Ca(OH)2.
Untreated NaOH Ca(OH)2
H (lmol/g)
G (lmol/g)
S (lmol/g)
S/G
H/G
S/H
29.18 ± 0.31 29.29 ± 0.22 32.23 ± 0.43
43.32 ± 0.41 41.16 ± 0.35 39.98 ± 0.28
27.50 ± 0.11 29.55 ± 0.21 27.79 ± 0.18
0.63 0.72 0.70
0.67 0.71 0.81
0.94 1.01 0.86
Fig. 3. Effect of isolated lignin on the enzymatic hydrolysis of Avicel. The triangle symbol was cellulose conversion of control; the circular symbol was effect of lignin from SSB pretreated with Ca(OH)2 on the cellulose conversion of Avicel; the square symbol was effect of lignin from SSB pretreated with NaOH on the cellulose conversion of Avicel.Effect of isolated lignin on the enzymatic hydrolysis of Avicel. The black line was cellulose conversion of control; the red line was effect of lignin from SSB pretreated with Ca(OH)2 on the cellulose conversion of Avicel; the blue line was effect of lignin from SSB pretreated with NaOH on the cellulose conversion of Avicel.
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Table 3 The effect of NaOH and Ca(OH)2 pretreatment on the surface area, pore volume and pore size.
Untreated NaOH Ca(OH)2
Surface area (m2/g)
Pore volume (cc/g)
Pore diameter Dv(d) (nm)
2.988 3.737 4.972
0.003 0.004 0.005
2.462 2.448 1.675
pretreated with Ca(OH)2 were significantly higher than NaOH. That suggested that the structure of the SSB became loosen after the alkaline pretreatment. The SSB pretreated with Ca(OH)2 was more porous than that treated by NaOH although they had similar lignin content. Yu et al. reported that the accessible pore volume of pulps delignified by sodium chlorite was higher than that delignified by
ozone when the lignin content was similar, which was consistent with the results of enzymatic hydrolysis [20]. Xu et al. [34] also reported that the digestibility of biomass was proportional to its porosity of biomass pretreated with Ca(OH)2. Thus, the difference in porosity should be the direct reason for the different enzymatic hydrolysis of SSB pretreated with NaOH and Ca(OH)2. However, the pore of SSB pretreated with Ca(OH)2 had higher surface area and volume while the diameter was smaller. We hypothesize that more lignin was removed from its original position. 3.4. Surface topography of SSB pretreated with NaOH or Ca(OH)2 In order to verify our hypothesis, the surface topography of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were compared utilizing the scanning electron microscope (SEM)
Fig. 4. SEM micrographs of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2. (a1) SEM micrographs of the untreated SSB magnified 250 times; (a2) SEM micrographs of the untreated SSB magnified 10,000 times; (b1) SEM micrographs of the SSB pretreated with NaOH magnified 250 times; (b2) SEM micrographs of the SSB pretreated with NaOH magnified 10,000 times; (c1) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 250 times; (c2) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 10,000 times.SEM micrographs of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2. (a1) SEM micrographs of the untreated SSB magnified 250 times; (a2) SEM micrographs of the untreated SSB magnified 10,000 times; (b1) SEM micrographs of the SSB pretreated with NaOH magnified 250 times; (b2) SEM micrographs of the SSB pretreated with NaOH magnified 10,000 times; (c1) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 250 times; (c2) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 10,000 times.
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micrographs (Fig. 4). The vascular bundles observed were formed by vessels surrounded by fibers and parenchyma cells [35]. After NaOH pretreatment, the vascular bundles lost their highly compact structure and had a greater surface area through the removal of lignin and/or hemicellulose (Fig. 4b2). After Ca(OH)2 pretreatment, the vascular bundles also lost their highly compact structure. It was interesting that some droplets were observed on the surface of the pretreated SSB (Fig. 4c2). Though lignin coalescence and migration through maize cell walls following thermochemical pretreatment were reported [36], this phenomenon at room temperature had not been previously reported in literatures. In order to analyze the droplets, energy dispersive spectral analysis (EDS) was performed on the droplets and smooth surface of untreated SSB and SSB pretreated with NaOH or Ca(OH)2. The data obtained by EDS consisted of the O/C ratio and elemental surface composition. The elemental composition detected by EDS was O, C and a trance of Ca. The O/C ratio was used for the estimation of droplets composition. The O/C ratio of these droplet was 0.58, while the O/C ratio of SSB pretreated with NaOH and the smooth surface of SSB pretreated with Ca(OH)2 were both 0.45. The theoretical values of O/C for carbohydrates is 0.83, and the value for lignin is 0.33 [37]. The result of EDS revealed that these droplets were richer in carbohydrate as compared to the original biomass. All the samples were washed completely, and no calcium was observed on the smooth surface of the substrates. Therefore, the small amount of Ca in the droplet was unlikely to have originated from any residual Ca(OH)2. The elemental analysis results showed the formation of a lignin–carbohydrate complex (LCC) with calcium ions and the relocation of hemicellulose and lignin on the surface of the substrates. The formation of the droplets not only lessened the recalcitrant of the plant cell, but also provided an explanation as to why less carbohydrate was loss from Ca(OH)2 pretreatment than NaOH pretreatment. Xu et al. [34] previously predicted that more carbohydrates was retained in the Ca(OH)2 pretreatment was possibly due to the calcium ions crosslinked with lignin and/or carbohydrates. The tiny droplets with rich carbohydrates and calcium ions supported the finding. It also provided an explanation that the porosity of SSB treated by Ca(OH)2 increased while the pore diameter decreased. For all the result listed in Table 3, the decrease of pore diameter of SSB pretreated with Ca(OH)2 was likely due to a carbohydrate rich substance that was dissolved and redeposited on the surface of the pore. This led to more exposed carbohydrates and higher pore volume, thereby improved the efficiency of enzymatic hydrolysis. This observation also explained why the SSB pretreated with Ca(OH)2 had higher enzymatic hydrolysis efficiency than NaOH even if the removal of lignin was similar. Some literatures also reported that the dominant factor to increase the enzymatic hydrolysis of lignocellulose pretreated by Ca(OH)2 was pretreatment time [11, 38]. They hypothesized the formation of the complex between lignin and Ca(OH)2 via the calcium ion adsorption experiment [34,39,40]. In this paper, the formation of the complex was confirmed by the SEM and EDS analysis of lignocellulosic substrates. 4. Conclusions The relocation of LCC in Ca(OH)2 pretreatment was visualized and shown to contribute to the improvement of lignocellulose enzymatic hydrolysis according to the SEM and EDS results. Compared to Ca(OH)2, these was no deposit of LCC in NaOH pretreatment. The results reasonably explained the smaller carbohydrate loss in Ca(OH)2 compared with NaOH pretreatment. In addition, the effect of lignin on non-productive adsorption of cellulase was confirmed to be negligible. According to the findings in this paper, addition of calcium ion could be considered to reduce the carbohydrates loss in pretreatments.
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Authors’ contributions ZY designed, carried out the experiments, interpreted data and drafted the manuscript. JL contributed to the original conception of the study, advised on the design and progress of the experimentation, performed the SEM lyses, and helped draft and revises the manuscript. SL conceived idea, supervised the work and edited the manuscript. SC revised the manuscript. YJ helped the HPLC analyses. TC, MY, LZ, GZ, PQ helped the analysis of results. All authors critically revised the draft and approved the final manuscript. Acknowledgements This work was supported by National High Technology Research and Development Program of China (Grant No. 2012AA101805), International Scientific and Technological Cooperation Program (Grant Nos. 2013DFA60470, 2012DFG61700), and Independent Research and Development Program (Grant No. 2012Z08127). We thank Novozymes China Research Center for generously providing the cellulase. We also thank the Professor Peng of Hua Zhong Agricultural University for helped the analysis of lignin monomer. References [1] Hu F, Ragauskas A. Pretreatment and lignocellulosic chemistry. Bioenergy Res 2012;5:1043–66. [2] Ding SY, Liu YS, Zeng YN, Himmel ME, Baker JO, Bayer EA. How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 2012;338:1055–60. [3] Kumar R, Mago G, Balan V, Wyman CE. Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies. Bioresource Technol 2009;100:3948–62. [4] Mood SH, Golfeshan AH, Tabatabaei M, Jouzani GS, Najafi GH, Gholami M, et al. Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment. Renew Sustain Energy Rev 2013;27:77–93. [5] Hsu TA. Handbook on bioethanol, production and utilization: pretreatment of biomass. Washington DC: Taylor and Francis; 1996. p. 179–212. [6] Zhang YHP, Lynd LR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng 2004;88:797–824. [7] Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Biotechnol Prog 2009;100:10–8. [8] Buranov AU, Mazza G. Lignin in straw of herbaceous crops. Ind Crops Prod 2008;28:237–59. [9] Wu L, Arakane M, Ike M, Wada M, Takai T, Gau M, et al. Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production. Bioresource Technol 2011;102(7):4793–9. [10] Cao WX, Sun C, Liu RH, Yin RZ, Wu XW. Comparison of the effects of five pretreatment methods on enhancing the enzymatic digestibility and ethanol production from sweet sorghum bagasse. Bioresource Technol 2012;111:215–21. [11] Kim S, Holtzapple MT. Lime pretreatment and enzymatic hydrolysis of corn stover. Bioresource Technol 2005;96:1994–2006. [12] Zhao XB, Zhang LH, Liu DH. Biomass recalcitrance Part I: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose. Biofuels Bioprod Bioref 2012;6:465–82. [13] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Determination of Structural Carbohydrates and Lignin in Biomass. Laboratory Analytical Procedure (LAP). Technical Report NREL/TP-510-42618 Revised April 2008. [14] Li JH, Li SZ, Han B, Yu MH, Li GM, Jiang Y. A novel cost-effective technology to convert sucrose and homocelluloses in sweet sorghum stalks into ethanol. Biotechnol Biofuels 2013;6:174. [15] Foster CE, Martin TM, Pauly M. Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) Part I lignin. J Vis Exp 2010;37:5–8. [16] Hu ZJ, Yeh TF, Chang HM, Matsumoto Y, Kadla JF. Elucidation of the structure of cellulolytic enzyme lignin. Holzforschung 2006;60(4):389–97. [17] Yan Z, Li J, Li S, Chang S, Cui T, Jiang Y, et al. Impact of lignin removal on the enzymatic hydrolysis of fermented sweet sorghum bagasse. Appl Energy 2015. http://dx.doi.org/10.1016/j.apenergy.2015.02.070. [18] Studer MH, DeMartini JD, Davis MF, Sykes RW, Davison B, Keller M, et al. Lignin content in natural Populus variants affects sugar release. Proc Natl Acad Sci USA 2011;108:6300–5. [19] Santi C, Milagres A, Ferraz A, Carvalho W. The effects of lignin removal and drying on the porosity and enzymatic hydrolysis of sugarcane bagasse. Cellulose 2013;20:3165–77. [20] Yu ZY, Jameel H, Chang HM, Park S. The effect of delignification of forest biomass on enzymatic hydrolysis. Bioresour Technol 2011;102:9083–9.
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Lignin relocation contributed to the alkaline pretreatment efficiency of sweet sorghum bagasse Zhipei Yan a,b, Jihong Li a,b,⇑, Sandra Chang a,b, Ting Cui a,b, Yan Jiang a,b, Menghui Yu a,b, Lei Zhang a,b, Gang Zhao b, Panlun Qi c, Shizhong Li a,b,⇑ a b c
Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, China Beijing Engineering Research Center of Biofuels, Tsinghua University, Beijing 100084, China Sinopec Research Institute of Petroleum Processing, Petro China Company Limited, Beijing 100083, China
h i g h l i g h t s The relocation of LCC occurred in Ca(OH)2 pretreatment. Complex formation of LCC and Ca
2+
resulted in the relocation.
Lignin did not adsorb cellulase during enzymatic hydrolysis.
a r t i c l e
i n f o
Article history: Received 15 January 2015 Received in revised form 23 April 2015 Accepted 15 May 2015 Available online 23 May 2015 Keywords: Sodium hydroxide pretreatment Calcium hydroxide pretreatment Lignin Enzymatic hydrolysis Relocation
a b s t r a c t Sodium hydroxide and calcium hydroxide are used to improve enzymatic hydrolysis of lignocellulose. Compared to NaOH, less carbohydrates was lost in Ca(OH)2 pretreatment. It was found that the substrates with similar lignin content prepared from NaOH and Ca(OH)2 pretreatments had distinct cellulose conversion rate. To understand the mechanism of action, two sweet sorghum bagasse (SSB) samples with similar lignin content were prepared. The enzymatic hydrolysis result showed that the cellulose conversion rate of SSB treated by Ca(OH)2 was about 1.62 times that of SSB treated by NaOH. SEM results showed tiny droplets on the SSB surface pretreated with Ca(OH)2. EDS analysis revealed that the droplets were a complex of lignin–carbohydrate and calcium ions. The results suggested that Ca(OH)2 not only increased the porosity of substrates, but also reduced carbohydrates loss by forming a complex with lignin and calcium ions. This demonstrated that calcium ions could be used to reduce the carbohydrates loss from the pretreatment procedure. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose is the most abundant renewable resources on the earth and a unique renewable feedstock to produce liquid fuels. Biofuel production from lignocellulose using bioconversion technology is low-energy and environmentally friendly [1]. However, the release of fermentable sugars from lignocellulose has become Abbreviations: ATR–FT/IR, attenuated total reflectance–fourier transform infrared spectroscopy; BET, Brunauer–Emmett–Teller; EDS, energy dispersive spectral analysis; G, guaiacyl propane; H, p-hydroxyl phenyl propane; LCC, lignin–carbohydrate complexes; S, syringyl propane; SEM, scanning electron microscope; SSB, sweet sorghum bagasse. ⇑ Corresponding authors at: Institute of New Energy Technology, Tsinghua University, Beijing 100084, China. Tel./fax: +86 10 80194050. E-mail addresses: [email protected] (J. Li), [email protected] (S. Li). http://dx.doi.org/10.1016/j.fuel.2015.05.029 0016-2361/Ó 2015 Elsevier Ltd. All rights reserved.
a bottleneck for bioconversion produced bioethanol due to its recalcitrance [1]. The natural recalcitrance of lignocellulosic materials to enzymatic hydrolysis is due to the structure of the plant cell wall, which is composed of cellulose, hemicellulose and lignin [2,3]. Lignin is relatively hydrophobic and covalently linked to hemicelluloses and fills the spaces in the cell wall between cellulose and hemicellulose. This structural arrangement protects the carbohydrates from degradation by micro-organisms or enzymes [1]. Thus, a pretreatment process is required to remove the recalcitrant physical and chemical barriers and increase the enzymatic digestibility of cellulose [4]. Among various pretreatments, numerous studies have focused on alkaline pretreatments because they have some practical operational advantages including lower reaction temperature and pressure, no need for complicated reactor, and allowing the reuse of residual alkali [1]. Alkali pretreatments weaken the recalcitrance of cell wall to cellulases by cleaving the
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hydrolyzable linkages between lignin and glycosidic bonds of polysaccharides, as well as disruption of the lignin structure [5]. In addition, alkaline saponification of acetyl and uronic ester bonds also improved the enzymatic accessibility of the polysaccharides [6,7]. Sodium hydroxide (NaOH) and calcium hydroxide (Ca(OH)2) are two common chemicals studied extensively in alkaline pretreatments. Mild NaOH pretreatment could effectively improve the enzymatic hydrolysis of herbaceous crops due to their lower lignin content, special lignin and lignin-carbohydrate complexes (LCC) structures which are more susceptible to alkaline pretreatment [8]. The glucan saccharification yield of sweet sorghum bagasse (SSB) pretreated with 2.5 M or 20% NaOH at room temperature for 120 min increased significantly compared to the control [9,10]. Ca(OH)2 could also successfully improve the enzymatic hydrolysis of lignocellulose. Corn stover pretreated with 0.5 g Ca(OH)2/g raw biomass at 55 °C for 4 weeks with aeration converted 91.3% of glucan and 51.8% of xylan to glucose and xylose, respectively [11]. The time of Ca(OH)2 pretreatment needed was longer than NaOH pretreatment due to its poor solubility in water (1.73 g/L at 20 °C). However, it is widely used because it is inexpensive (about 6% cost of NaOH), easier to handle and recover [1]. Most of the previous literatures were focused on process optimization of pretreatment. Although some papers discussed the changes to the substrates by NaOH or Ca(OH)2 pretreatment, the sensitivity of the substrates to these different alkalis was not studied. Moreover, due to most of the attention were focused on the enzymatic hydrolysis, comparison of the substrates with similar key features after pretreatment has not been investigated up to now. Lignin content is an important component influenced by alkaline pretreatment because lignin not only is the barrier to cellulose, but also can absorb cellulases [12]. However, other factors may play an important role in the bioconversion of cellulose. In previous studies the differences in lignin content of the substrate can obscure and make comparison of other factors difficult. In order to exclude the influence of the lignin content, two alkaline samples with similar lignin content were prepared by NaOH and Ca(OH)2 pretreatment. The changes to SSB after pretreatment, including chemical structure, porosity, surface topography, and surface composition of pretreated SSB were characterized in detail. The changes to the properties were correlated to the enzymatic digestibility of the samples. The differences between NaOH and Ca(OH)2 pretreatment on enzymatic hydrolysis and effect on SSB were discussed.
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2.4. Characterization of chemical structure The chemical structure of untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were characterized by attenuated total reflectance–fourier transform infrared spectroscopy (ATR–FT/IR). ATR–FT/IR was conducted using a Spectrum One FTIR system (Perkin Elmer) with a universal ATR (Attenuated Total reflection) accessory. Samples were washed completely with DI water and dried to constant mass at 45 °C in vacuum oven. Sample spectra were obtained using an average of 32 scans over the range between 600 cm 1 and 4500 cm 1 with a spectral resolution of 2 cm 1. Baseline and ATR corrections for penetration depth and frequency variations were applied using Spectrum software supplied with the equipment. 2.5. Thioacidolysis Thioacidolysis was used to estimate the relative abundance and proportion of S, G, and H monolignols according to Foster et al. [15]. 2.6. Preparation of lignins The lignin was isolated from SSB pretreated with NaOH or Ca(OH)2 according to the method reported by Hu et al. [16]. 2.7. Enzymatic hydrolysis of Avicel The hydrolysis of Avicel was carried out in 50 mM sodium acetate buffer (pH 4.8, 50 °C) at 2% consistency in the presence of the isolated lignin. The lignin loading was 1/4 of cellulose. The cellulase (CTec 3) loading for the Avicel was 10 FPU/g cellulose. The hydrolysis of Avicel, without the addition of isolated lignin, was performed as control. Aliquots were taken at the indicated time from the reaction mixture and centrifuged to remove the insoluble materials. The sugar content was measured according to the method reported by Li et al. [14]. All samples were measured in triplicates. 2.8. Porosity
2. Methods
Specific surface area determinations were performed using the Brunauer–Emmett–Teller (BET) method with an adsorptionmeter (Quadrasorb SI-MP, Quantachrome). Porosity was determined by BJH/DH method. Outgas Temp: 120.0 °C, Analysis gas: nitrogen.
2.1. Alkali treatment
2.9. Surface topography
NaOH pretreatment of SSB was carried out by the following conditions other than stated specially: 10 g NaOH/100 g SSB, 95 °C, 0.5 h, 1 g SSB/20 mL water; The condition for Ca(OH)2 pretreatment of SSB was: 10 g Ca(OH)2/100 g SSB, 25 °C, 24 h, 1 g SSB/10 mL water. The pretreated solids were washing to pH 7.0 by deionized water.
SEM images were taken at 250 and 10,000 times using JSM-6460/LV SEM (Hitachi, Tokyo, Japan) at 10 kV; the samples were sputter-coated with a thin layer of gold. 3. Results and discussion 3.1. Effect of lignin on the enzymatic hydrolysis
2.2. Composition analysis Compositions of samples were determined according to the NREL method [13]. All samples were measured in triplicates. 2.3. Enzymatic hydrolysis of the pretreated samples Enzymatic hydrolysis of the SSB pretreated with NaOH or Ca(OH)2 were carried out according to the method reported by Li et al. [14]. All samples were measured in triplicates.
NaOH and Ca(OH)2 are two important alkalis in alkaline pretreatment protocols. Although their basicity is different, each reacts with the biomass to open the natural recalcitrance of lignocellulose by solvation and saponification of lignin structures [1]. After alkaline pretreatment, the structure of lignocelluloses changes in many aspects. Among them, the lignin content changed significantly. Since the effect of lignin content is well established and studied [17–20], other changes influencing enzymatic hydrolysis caused by alkaline pretreatment were investigated. In order to
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exclude the effect of lignin content, SSB samples with about 17.3–17.4% lignin content resulting from NaOH and Ca(OH)2 pretreatment were prepared. The pretreatment conditions for NaOH was 10% NaOH at 95 °C for 30 min, and for Ca(OH)2 was 10% Ca(OH)2 at 25 °C for 24 h. The composition of these two samples was listed in Table 1. The enzymatic hydrolysis result showed that the cellulose conversion rate of SSB treated with Ca(OH)2 (61.11%) was 1.62 times of that for SSB treated with NaOH (37.73%) (Fig. 1). The result revealed that the enzymatic digestibility of substrates pretreated with the two different chemicals had no correlation with the lignin content. A similar result was reported by Yu et al. [20], that the lignin content of loblolly pine delignified by ozone and sodium chlorite was 12.8% and 18.0%, respectively. However, the carbohydrate conversion was 58% and 80%, respectively. It was believed that the different delignification mechanism between sodium chlorite and ozone caused the difference in enzymatic hydrolysis efficiency. Therefore, the pretreatment mechanism of NaOH and Ca(OH)2 was likely different as well. The properties of lignocellulosic substrates including chemical structure, porosity, surface topography, and surface composition of SSB pretreated with NaOH or Ca(OH)2 were systematically studied. 3.2. Comparison of chemical structure of SSB pretreated with NaOH or Ca(OH)2 In addition to lignin content, the structure of the substrate contributed to the efficiency of enzymatic hydrolysis of the lignocellulose. The acetyl groups of hemicellulose can interfere with enzyme recognition [1], inhibit hydrogen bonds between cellulose and the catalytic domain of cellulases, and increase the diameter of the cellulose chain [12]. Lignin is a phenolic polymer composed of three major types of building blocks: p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units. In general, S/G ratios and/or S content are indicative of efficiency of pretreatment [18]. The chemical structure of SSB pretreated with NaOH or Ca(OH)2 was characterized by attenuated total reflectance–fourier transform infrared spectroscopy (ATR–FT/IR). Normalized ATR–FT/IR spectra at a band position of 1510 cm 1 [21] of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were presented in Fig. 2. NaOH and Ca(OH)2 pretreatments did not change the cellulose structure significantly, but altered certain functional groups and linkages. The peak near 3348 cm 1 represented –OH stretching. The intensity of the peak of SSB pretreated with NaOH increased, suggested the increase of hydrogen bonds in the cellulose. The peak for SSB pretreated with Ca(OH)2 reduced in the intensity of this peak, indicated that hydrogen bonds of cellulose were disrupted [2,22,23]. The band position at 2900 cm 1 is attributed to C–H stretching, and both NaOH and Ca(OH)2 pretreatments reduced the intensity, indicated that the methyl and methylene portions of cellulose were reduced. Both pretreatments caused significant changes in the 1735 cm 1 band which is attributed to ester carbonyl vibration in acetyl, feruloyl, and p-coumaroyl groups in lignin and hemicellulose, indicated that the ester bonds between hydroxycinnamic acids and lignin were ruptured [24]. The intensity of SSB pretreated with NaOH was reduced significantly while the peak for the SSB pretreated with Ca(OH)2 disappeared. The disappearance of the Table 1 The composition for SSB pretreated with NaOH and Ca(OH)2.
Cellulose (%) Hemicellulose (%) Lignin (%)
Untreated
NaOH
Ca(OH)2
39.42 ± 0.73 25.25 ± 0.26 18.93 ± 0.31
44.66 ± 0.70 28.23 ± 0.54 17.32 ± 0.19
48.17 ± 0.88 25.72 ± 0.00 17.45 ± 0.40
Fig. 1. Comparison of enzymatic hydrolysis of SSB pretreated with NaOH and Ca(OH)2. The white column was the cellulose conversion; the black column was the hemicellulose conversion; the gray column was the utilization of substrate.Comparison of enzymatic hydrolysis of SSB pretreated with NaOH and Ca(OH)2. The black column was the cellulose conversion; the red column was the hemicellulose conversion; the blue column was the utilization of substrate.
peak indicated that the ester bonds between hydroxycinnamic acids and lignin were cleaved during the pretreatment or the content of these groups were so low that they were below the detection limit for the spectroscopical methods employed. Both pretreatments reduced the intensity of the 1245 cm 1 band, which is attributed to the cleavage and/or alterations of acetyl groups [24]. The acetyl group was likely removed from the hemicellulose in both NaOH and Ca(OH)2 pretreatments. These changes likely contributed to the enzymatic hydrolysis but did not explain the difference in enzymatic hydrolysis efficiency. In both pretreatments the peak at the 1600 cm 1 band which is associated with aromatic skeletal vibration [24] did not change much. It suggested that skeleton of the benzene ring of lignin did not change during NaOH and Ca(OH)2 pretreatments. The band at 1462 cm 1 is associated with C–H deformation by methyl and methylene. The band at 1425 cm 1 is associated with C–H in-plane deformation by aromatic ring stretching [25]. With both peaks the SSB pretreated with NaOH did not change as compared to the untreated SSB, while the sample pretreated with Ca(OH)2 were significantly higher. This suggested that the proportion of G or H unit increased in lignin of the sample pretreated with Ca(OH)2 [25]. Furthermore, the C–H asymmetric deformation occurs at 1370 cm 1 is associated with the G unit. The band at 1323 cm 1 is attributed to the C–C and C–O skeletal vibrations, which is associated with S unit and G unit condensation. The peak at 1164 cm 1 is related to HGS lignin [26]. The relative reductions of all the above peaks by the Ca(OH)2 pretreatment pointed to the removal of the aromatic ring on lignin and solubilization of lignin polymers, especially of the G unit. Through ATR–FT/IR analysis, it was suggested that Ca(OH)2 pretreatment removed the G unit and increased the H unit. In order to quantify the changes, the lignin monomer of untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were analyzed (Table 2). Table 2 shows that the H unit of SSB pretreated with Ca(OH)2 increased, the S unit of SSB pretreated with NaOH increased, while G unit of both decreased. Consequently, the S/G ratio increased from 0.63 for both to 0.72 and 0.70 for SSB pretreated with NaOH and Ca(OH)2 respectively. Meanwhile, the H/G ratio increased from 0.67 for both to 0.71 and 0.81 for SSB pretreated with NaOH and Ca(OH)2, respectively. Other researchers reported
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For grass, cell wall digestibility was strongly correlated to the content of H unit as well [31–33]. However, the effect of H unit on the cell wall digestibility under the same lignin content was not reported. Therefore, the higher proportion of H unit in lignin of SSB pretreated with Ca(OH)2 (Table 2) may be the reason for the higher effectiveness of cellulose digestibility of SSB pretreated with Ca(OH)2. The inhibition effect of lignin on the enzymatic hydrolysis of cellulose mainly are: (1) non-productive adsorption of the enzyme by lignin through hydrophobic interactions, electrostatic interactions, and/or hydrogen-bonding interactions; (2) lignin in lignocellulosic materials acting as a surface barrier to block the accessible surface of carbohydrates through physical blockage on the surface and chemical blockage through lignin carbohydrate complex; (3) soluble lignin derivatives can deactivate enzymes in liquid phase [12]. In order to investigate the possible adsorption and deactivate enzymes of cellulases by lignin, the glucose conversion rate of Avicel in the presence of lignin prepared from SSB treated with NaOH or Ca(OH)2 was investigated. The results showed that the enzymatic hydrolysis of Avicel did not decrease with the addition of isolated lignin (Fig. 3). This suggested that the non-productive adsorption and deactivate enzymes of cellulases by lignin was not a factor in the cases of SSB pretreated with NaOH or Ca(OH)2. Whether pretreatment of lignin caused condensation onto the substrate and prevented the physical accessible of enzyme still needs to be investigated. 3.3. Porosity of SSB pretreated with NaOH or Ca(OH)2 Since the non-productive adsorption and deactivate enzymes of cellulase on the lignin could be excluded, cellulose accessibility was studied. The prerequisite of cellulose hydrolysis by cellulases is the adsorption of enzymes onto the substrate. Therefore, the size and volume of pores in the biomass is one of the critical factors limiting the enzymatic hydrolysis of the polysaccharides [19]. Thus, the difference in porosity of SSB pretreated with NaOH and Ca(OH)2 was studied by the Brunauer–Emmett–Teller (BET) method. Table 3 listed the effect of NaOH and Ca(OH)2 pretreatments on the surface area, pore volume and pore size of pretreated SSB. Table 3 showed that both surface area and pore volume of SSB were increased after alkaline pretreatment, and the values of SSB Fig. 2. ATR–FT/IR spectra of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2 (a) was the ATR–FT/IR spectra of the untreated SSB; (b) was the ATR-FT/IR spectra of the SSB pretreated with NaOH; (c) was the ATR–FT/IR spectra of the SSB pretreated with Ca(OH)2.ATR–FT/IR spectra of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2. The black line was the ATR–FT/IR spectra of the untreated SSB; the red line was the ATR–FT/IR spectra of the SSB pretreated with NaOH; the blue line was the ATR–FT/IR spectra of the SSB pretreated with Ca(OH)2.
that G unit was more easily degraded than S unit during pretreatment [27] and S unit [28] or S/G [29] increased after pretreatment. Li et al. [30] revealed that high content of S unit had a positive effect on the effectiveness of enzymatic hydrolysis. But the S/G ratio of samples pretreated with NaOH and Ca(OH)2 was similar, therefore the increase of the S/G ratio did not contribute to the different enzymatic efficiency of the alkaline treated SSB.
Table 2 The abundance and proportion of lignin monomer for untreated SSB and SSB pretreated with NaOH or Ca(OH)2.
Untreated NaOH Ca(OH)2
H (lmol/g)
G (lmol/g)
S (lmol/g)
S/G
H/G
S/H
29.18 ± 0.31 29.29 ± 0.22 32.23 ± 0.43
43.32 ± 0.41 41.16 ± 0.35 39.98 ± 0.28
27.50 ± 0.11 29.55 ± 0.21 27.79 ± 0.18
0.63 0.72 0.70
0.67 0.71 0.81
0.94 1.01 0.86
Fig. 3. Effect of isolated lignin on the enzymatic hydrolysis of Avicel. The triangle symbol was cellulose conversion of control; the circular symbol was effect of lignin from SSB pretreated with Ca(OH)2 on the cellulose conversion of Avicel; the square symbol was effect of lignin from SSB pretreated with NaOH on the cellulose conversion of Avicel.Effect of isolated lignin on the enzymatic hydrolysis of Avicel. The black line was cellulose conversion of control; the red line was effect of lignin from SSB pretreated with Ca(OH)2 on the cellulose conversion of Avicel; the blue line was effect of lignin from SSB pretreated with NaOH on the cellulose conversion of Avicel.
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Table 3 The effect of NaOH and Ca(OH)2 pretreatment on the surface area, pore volume and pore size.
Untreated NaOH Ca(OH)2
Surface area (m2/g)
Pore volume (cc/g)
Pore diameter Dv(d) (nm)
2.988 3.737 4.972
0.003 0.004 0.005
2.462 2.448 1.675
pretreated with Ca(OH)2 were significantly higher than NaOH. That suggested that the structure of the SSB became loosen after the alkaline pretreatment. The SSB pretreated with Ca(OH)2 was more porous than that treated by NaOH although they had similar lignin content. Yu et al. reported that the accessible pore volume of pulps delignified by sodium chlorite was higher than that delignified by
ozone when the lignin content was similar, which was consistent with the results of enzymatic hydrolysis [20]. Xu et al. [34] also reported that the digestibility of biomass was proportional to its porosity of biomass pretreated with Ca(OH)2. Thus, the difference in porosity should be the direct reason for the different enzymatic hydrolysis of SSB pretreated with NaOH and Ca(OH)2. However, the pore of SSB pretreated with Ca(OH)2 had higher surface area and volume while the diameter was smaller. We hypothesize that more lignin was removed from its original position. 3.4. Surface topography of SSB pretreated with NaOH or Ca(OH)2 In order to verify our hypothesis, the surface topography of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2 were compared utilizing the scanning electron microscope (SEM)
Fig. 4. SEM micrographs of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2. (a1) SEM micrographs of the untreated SSB magnified 250 times; (a2) SEM micrographs of the untreated SSB magnified 10,000 times; (b1) SEM micrographs of the SSB pretreated with NaOH magnified 250 times; (b2) SEM micrographs of the SSB pretreated with NaOH magnified 10,000 times; (c1) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 250 times; (c2) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 10,000 times.SEM micrographs of the untreated SSB and SSB pretreated with NaOH or Ca(OH)2. (a1) SEM micrographs of the untreated SSB magnified 250 times; (a2) SEM micrographs of the untreated SSB magnified 10,000 times; (b1) SEM micrographs of the SSB pretreated with NaOH magnified 250 times; (b2) SEM micrographs of the SSB pretreated with NaOH magnified 10,000 times; (c1) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 250 times; (c2) SEM micrographs of the SSB pretreated with Ca(OH)2 magnified 10,000 times.
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micrographs (Fig. 4). The vascular bundles observed were formed by vessels surrounded by fibers and parenchyma cells [35]. After NaOH pretreatment, the vascular bundles lost their highly compact structure and had a greater surface area through the removal of lignin and/or hemicellulose (Fig. 4b2). After Ca(OH)2 pretreatment, the vascular bundles also lost their highly compact structure. It was interesting that some droplets were observed on the surface of the pretreated SSB (Fig. 4c2). Though lignin coalescence and migration through maize cell walls following thermochemical pretreatment were reported [36], this phenomenon at room temperature had not been previously reported in literatures. In order to analyze the droplets, energy dispersive spectral analysis (EDS) was performed on the droplets and smooth surface of untreated SSB and SSB pretreated with NaOH or Ca(OH)2. The data obtained by EDS consisted of the O/C ratio and elemental surface composition. The elemental composition detected by EDS was O, C and a trance of Ca. The O/C ratio was used for the estimation of droplets composition. The O/C ratio of these droplet was 0.58, while the O/C ratio of SSB pretreated with NaOH and the smooth surface of SSB pretreated with Ca(OH)2 were both 0.45. The theoretical values of O/C for carbohydrates is 0.83, and the value for lignin is 0.33 [37]. The result of EDS revealed that these droplets were richer in carbohydrate as compared to the original biomass. All the samples were washed completely, and no calcium was observed on the smooth surface of the substrates. Therefore, the small amount of Ca in the droplet was unlikely to have originated from any residual Ca(OH)2. The elemental analysis results showed the formation of a lignin–carbohydrate complex (LCC) with calcium ions and the relocation of hemicellulose and lignin on the surface of the substrates. The formation of the droplets not only lessened the recalcitrant of the plant cell, but also provided an explanation as to why less carbohydrate was loss from Ca(OH)2 pretreatment than NaOH pretreatment. Xu et al. [34] previously predicted that more carbohydrates was retained in the Ca(OH)2 pretreatment was possibly due to the calcium ions crosslinked with lignin and/or carbohydrates. The tiny droplets with rich carbohydrates and calcium ions supported the finding. It also provided an explanation that the porosity of SSB treated by Ca(OH)2 increased while the pore diameter decreased. For all the result listed in Table 3, the decrease of pore diameter of SSB pretreated with Ca(OH)2 was likely due to a carbohydrate rich substance that was dissolved and redeposited on the surface of the pore. This led to more exposed carbohydrates and higher pore volume, thereby improved the efficiency of enzymatic hydrolysis. This observation also explained why the SSB pretreated with Ca(OH)2 had higher enzymatic hydrolysis efficiency than NaOH even if the removal of lignin was similar. Some literatures also reported that the dominant factor to increase the enzymatic hydrolysis of lignocellulose pretreated by Ca(OH)2 was pretreatment time [11, 38]. They hypothesized the formation of the complex between lignin and Ca(OH)2 via the calcium ion adsorption experiment [34,39,40]. In this paper, the formation of the complex was confirmed by the SEM and EDS analysis of lignocellulosic substrates. 4. Conclusions The relocation of LCC in Ca(OH)2 pretreatment was visualized and shown to contribute to the improvement of lignocellulose enzymatic hydrolysis according to the SEM and EDS results. Compared to Ca(OH)2, these was no deposit of LCC in NaOH pretreatment. The results reasonably explained the smaller carbohydrate loss in Ca(OH)2 compared with NaOH pretreatment. In addition, the effect of lignin on non-productive adsorption of cellulase was confirmed to be negligible. According to the findings in this paper, addition of calcium ion could be considered to reduce the carbohydrates loss in pretreatments.
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Authors’ contributions ZY designed, carried out the experiments, interpreted data and drafted the manuscript. JL contributed to the original conception of the study, advised on the design and progress of the experimentation, performed the SEM lyses, and helped draft and revises the manuscript. SL conceived idea, supervised the work and edited the manuscript. SC revised the manuscript. YJ helped the HPLC analyses. TC, MY, LZ, GZ, PQ helped the analysis of results. All authors critically revised the draft and approved the final manuscript. Acknowledgements This work was supported by National High Technology Research and Development Program of China (Grant No. 2012AA101805), International Scientific and Technological Cooperation Program (Grant Nos. 2013DFA60470, 2012DFG61700), and Independent Research and Development Program (Grant No. 2012Z08127). We thank Novozymes China Research Center for generously providing the cellulase. We also thank the Professor Peng of Hua Zhong Agricultural University for helped the analysis of lignin monomer. References [1] Hu F, Ragauskas A. Pretreatment and lignocellulosic chemistry. Bioenergy Res 2012;5:1043–66. [2] Ding SY, Liu YS, Zeng YN, Himmel ME, Baker JO, Bayer EA. How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 2012;338:1055–60. [3] Kumar R, Mago G, Balan V, Wyman CE. Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies. Bioresource Technol 2009;100:3948–62. [4] Mood SH, Golfeshan AH, Tabatabaei M, Jouzani GS, Najafi GH, Gholami M, et al. Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment. Renew Sustain Energy Rev 2013;27:77–93. [5] Hsu TA. Handbook on bioethanol, production and utilization: pretreatment of biomass. Washington DC: Taylor and Francis; 1996. p. 179–212. [6] Zhang YHP, Lynd LR. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng 2004;88:797–824. [7] Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Biotechnol Prog 2009;100:10–8. [8] Buranov AU, Mazza G. Lignin in straw of herbaceous crops. Ind Crops Prod 2008;28:237–59. [9] Wu L, Arakane M, Ike M, Wada M, Takai T, Gau M, et al. Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production. Bioresource Technol 2011;102(7):4793–9. [10] Cao WX, Sun C, Liu RH, Yin RZ, Wu XW. Comparison of the effects of five pretreatment methods on enhancing the enzymatic digestibility and ethanol production from sweet sorghum bagasse. Bioresource Technol 2012;111:215–21. [11] Kim S, Holtzapple MT. Lime pretreatment and enzymatic hydrolysis of corn stover. Bioresource Technol 2005;96:1994–2006. [12] Zhao XB, Zhang LH, Liu DH. Biomass recalcitrance Part I: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose. Biofuels Bioprod Bioref 2012;6:465–82. [13] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Determination of Structural Carbohydrates and Lignin in Biomass. Laboratory Analytical Procedure (LAP). Technical Report NREL/TP-510-42618 Revised April 2008. [14] Li JH, Li SZ, Han B, Yu MH, Li GM, Jiang Y. A novel cost-effective technology to convert sucrose and homocelluloses in sweet sorghum stalks into ethanol. Biotechnol Biofuels 2013;6:174. [15] Foster CE, Martin TM, Pauly M. Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) Part I lignin. J Vis Exp 2010;37:5–8. [16] Hu ZJ, Yeh TF, Chang HM, Matsumoto Y, Kadla JF. Elucidation of the structure of cellulolytic enzyme lignin. Holzforschung 2006;60(4):389–97. [17] Yan Z, Li J, Li S, Chang S, Cui T, Jiang Y, et al. Impact of lignin removal on the enzymatic hydrolysis of fermented sweet sorghum bagasse. Appl Energy 2015. http://dx.doi.org/10.1016/j.apenergy.2015.02.070. [18] Studer MH, DeMartini JD, Davis MF, Sykes RW, Davison B, Keller M, et al. Lignin content in natural Populus variants affects sugar release. Proc Natl Acad Sci USA 2011;108:6300–5. [19] Santi C, Milagres A, Ferraz A, Carvalho W. The effects of lignin removal and drying on the porosity and enzymatic hydrolysis of sugarcane bagasse. Cellulose 2013;20:3165–77. [20] Yu ZY, Jameel H, Chang HM, Park S. The effect of delignification of forest biomass on enzymatic hydrolysis. Bioresour Technol 2011;102:9083–9.
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