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Structural and thermal characterization of hemicelluloses isolated by organic solvents and alkaline solutions from Tamarix austromongolica
Bioresource Technology 102 (2011) 5947–5951
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Structural and thermal characterization of hemicelluloses isolated by organic solvents and alkaline solutions from Tamarix austromongolica Yong-Chang Sun a, Jia-Long Wen a, Feng Xu a,⇑, Run-Cang Sun a,b,⇑ a b
Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
a r t i c l e
i n f o
Article history: Received 5 January 2011 Received in revised form 4 March 2011 Accepted 4 March 2011 Available online 10 March 2011 Keywords: Lignocellulosic biomass Tamarix austromongolica Hemicelluloses Structure feature Thermal stability
a b s t r a c t Three organosolv and three alkaline hemicellulosic fractions were prepared from lignocellulosic biomass of the fast-growing shrub Tamarix austromongolica (Tamarix Linn.). Sugar analysis revealed that the organosolv-soluble fractions contained a higher content of glucose (33.7–6.5%) and arabinose (14.8–5.6%), and a lower content of xylose (62.2–54.8%) than the hemicellulosic fractions isolated with aqueous alkali solutions. A relatively high concentration of alkali resulted in a decreasing trend of the xylose/4-O-methyl-D-glucuronic acid ratio in the alkali-soluble fractions. The results of NMR analysis supported a major substituted structure based on a linear polymer of b-(1 ? 4)-linked D-xylopyranosyl residues, having ramifications of a-L-arabinofuranose and 4-O-methyl-D-glucuronic acid residues monosubstituted at O-3 and O-2, respectively. Thermogravimetric analysis revealed that one step of major mass loss occurred between 200–400 °C, as hemicelluloses devolatilized with total volatile yield of about 55%. It was found that organosolv-soluble fractions are more highly ramified, and showed a higher thermal stability than the alkali-soluble fractions. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The perennial shrub, Tamarix austromongolica (TA), has found uses in prevention of wind erosion and control of desertification (Zhang and Zhao, 1989), reforestation of dry steppes, and also as a source of medicine, wood, fuel, and animal feed (Orabi et al., 2010). It has a higher content of hemicelluloses (about 31%) on a dry weight basis compared to sugarcane bagasse (25–34%), sunflower husk (23–26%), and cereal straws (20–35%) (Xu et al., 2008; Ren et al., 2008). Hemicelluloses are the least stable polymers within lignocellulosic biomass and, unlike cellulose, hemicelluloses are not chemically homogeneous. Xylan, the main hemicellulose component, is composed of 1,4-linked b-D-xylopyranose (b-D-Xylp) units which can be substituted at C-2 and/or C-3 by short and flexible side chains. In addition, acetyl groups located at O-2 and/or O-3 are often found on the backbone xylopyranosyl residues (Barbat et al., 2010). Neutral homoxylans contain xylose residues only, and can be either linear, such as the (1 ? 4)-b-D-xylan of guar seed husk, tobacco stalk, and esparto grass, or branched such as the xylans of groundnut seed endosperm and those of the angiosperm (Habibi et al., 2002). Neutral heteroxylans or arabinoxylans contained either single a-L-arabinofuranose ⇑ Corresponding authors. Address: Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China. Tel./fax: +86 10 62336972. E-mail addresses: [email protected] (F. Xu), [email protected] (R.-C. Sun). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.012
(a-L-Araf) residues which are usually attached by (1 ? 3) linkages (Bendahou et al., 2007). The frequency and composition of these pendant substituents varies with the source of xylan, and this variation creates linear homoxylan, homoxylan, arabinoxylan, glucuronoxylan, and glucuronoarabinoxylan. Hemicelluloses are complex components in the cell wall of woods, interconnected together with cellulose by physical intermixing, and linked to lignin by covalent bonds (mainly a-benzyl ether linkage) (Freudenberg, 1965). These bonds restrict the liberation of hemicelluloses from the cell wall matrix, and the hydrogen bond between the cell wall components may impede isolation of the hemicellulosic component (Ebringerová and Heinze, 2000). Before extraction of hemicelluloses, extensive hydrogen bonding between the individual polysaccharide cell wall components must be broken, and the wood flour must be pretreated to remove all lipophilic and hydrophilic non-structural components, and delignified with chlorine (Stephen, 1983), sodium chlorite, chlorine dioxide (Wong et al., 1988) or peroxyacetic acid (Bioly, 1985). Acidcatalyzed steam explosion and sulfite pretreatment to overcome recalcitrance of lignocellulose has been applied in research (Zhu et al., 2009; Yang and Wyman, 2008; Zhu and Pan, 2010), and organosolv and alkaline fractionation methods have attracted growing interest because they can release hemicelluloses from different fiber sources in an environmentally friendly manner. Organosolv extraction opens the possibility of exploiting hemicelluloses and lignin from biomass in addition to cellulose (Villaverde
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et al., 2009), and alkaline extraction is used for lower temperature and pressure extractions, and is a promising process to achieve complete utilization of lignocelluloses (Chaa et al., 2008). Hemicelluloses have been shown to have biological activities including decreasing blood cholesterol and preventing colon cancer (Hu et al., 2007). They can also be used for the production of high value-added chemicals such as xylitol (Rao et al., 2006) and furfural (Karimi et al., 2006), and for food, and non-food applications, and for the synthesis of thermoplastic xylan derivatives (Jain et al., 2000). The aim of the present study was to develop an efficient strategy to disrupt the cross-linked matrix of lignin and hemicelluloses. The hemicelluloses released from the lignocellulosic biomass was characterized with high-performance anionexchange chromatography (HPAEC), and Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopies. In addition, the pyrolysis mechanism of the fractions was investigated by thermogravimetric analysis (TGA) and differential thermal analysis (DTA). 2. Experimental
2.2. Sugar composition and content of uronic acids The chemical compositions of the six hemicellulosic fractions were determined by hydrolysis with dilute sulfuric acid. A 4–6 mg sample was hydrolyzed with 1.475 mL of 10% H2SO4 for 2.5 h at 105 °C. After hydrolysis, the mixture was filtered and diluted 60-fold, and analyzed by high-performance anion-exchange chromatography (HPAEC) seytem (Dionex ICS3000, US) with an amperometric detector, AS50 autosamper, a Carbopac™ PA-20 column (4 250 mm, Dionex) and a guard PA-20 column (3 30 mm, Dionex). Neutral sugars and uronic acids were separated in an isocratic 5 mM NaOH solution (carbonate free and purged with nitrogen) for 20 min, followed by a 0–75 mM NaAc gradient in 5 mM NaOH solution for 15 min. Calibration was performed with standard solutions of L-arabinose, D-glucose, D-xylose, D-rhamnose, D-mannose, D-galactose, glucuronic acid, and galacturonic acid. All experiments were performed at least in duplicate, and the average values were calculated for all of the hemicellulosic fractions. 2.3. FT-IR analysis
2.1. Materials and preparation TA was harvested in October 2009 in Inner Mongolia, China. Leaves and bark were removed, and the stems were chipped into small pieces (1–3 cm). After drying at 60 °C for 16 h in an oven, the chips were sieved through a 40–60 mm mesh. The scheme for successive extractions with DMSO, alkaline ethanol, and aqueous alkaline solutions is shown in Fig. 1. The pH value was adjusted with 10% acetic acid in the delignification process, and a material to liquor ratio of 1:25 (g/mL) was used in the experiment. Note that H1, H2, and H3 represent the hemicellulosic fractions isolated with DMSO, 70% ethanol containing 2% triethylamine (TEA), and 70% ethanol containing 0.5% KOH, respectively. H4, H5, and H6 represent the hemicellulosic fractions isolated with 1%, 3%, and 5% KOH aqueous solution, respectively.
The FT-IR spectra were obtained from a KBr disc using Tensor 27 FT-IR spectrophotometer in the range of 400–4000 cm1. The finely grounded sample was mixed with high purity KBr (1:100, w/w). The spectrum of pure KBr was used as background. Each spectrum was recorded with a total of 32 scans. 2.4. Molecular weight determination W Þ and number-average ðM n Þ molecular The weight-average ðM weights of the hemicellulosic fractions were determined by gel permeation chromatography (GPC) with a refraction index detector (RID) on a PL aquagel-OH 50 column (300 7.7 mm, Polymer Laboratories Ltd.), calibrated with PL pullulan polysaccharide stan W = 738, 12,200, 100,000, 1600,000, Polymer Laboratories dards ðM
Fig. 1. Scheme for isolation of the hemicellulosic fractions from Tamarix austromongolica.
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Ltd.). A 2 mg sample was dissolved with 0.02 N NaCl in 0.005 M sodium phosphate buffer, pH 7.5 at a concentration of 0.1% before measurement.
Table 2 Neutral sugar and uronic acid content (relative mmol% ± standard deviation 0.1%) in the isolated hemicellulosic fractions and the residue. Sugars
2.5. 1D and 2D NMR analysis The solution-state 1H and 13C NMR spectra were obtained on a Bruker AVIII 400 MHz spectrometer operating in the FT mode at 100.6 MHz. The hemicellulosic fractions (20 mg for 1H, 80 mg for 13 C) were dissolved in 1 mL D2O. The 1H NMR spectrum was recorded at 25 °C after 128 scans. The 13C NMR spectrum was recorded at 25 °C after 30,000 scans. A 30° pulse flipping angle, a 9.2 ls pulse width, a 1.37 s acquisition time and 2 s relaxation delay time were used. 1H–13C correlation 2D (HSQC) NMR spectrum was also recorded at 25 °C on the same apparatus equipped with a z-gradient double resonance probe. The spectral widths were 3268 and 18,116 Hz for the 1H- and 13C- dimensions, respectively. The number of collected complex points was 1024 for 1H-dimension with a relaxation delay of 1.5 s. The number of scans was 128 and 256 time increments were always recorded in 13C-dimension. The 1JCH used was 145 Hz. Prior to Fourier transformation, the data matrixes were zero filled up to 1024 points in the 13C-dimension. 2.6. Thermogravimetric analysis The thermal degradation of the hemicellulosic fractions was investigated using both thermogravimetric (TGA) and differential thermal analyses (DTA) on a simultaneous thermal analyzer (DTG-60 Shimadzu, Japan) from 40 to 600 °C. Samples of approximately 10 mg weight were heated in an aluminum crucible with the heating rate of 10 °C/min while the apparatus was continually flushed with a nitrogen flow of 30 mL/min. Calcined alumina (Al2O3) was used as a reference material in all experiments. 3. Results and discussion 3.1. Yield and sugar composition The hemicellulose yield and content of neutral sugars of the six hemicellulosic fractions are shown in Table 1 and Table 2, respectively. DMSO extraction removed 5.6% of the original hemicelluloses from the crude holocellulose, but preserved the acetyl groups. The DMSO-soluble hemicellulosic fraction contained much structural information as it had a diverse composition with different types of sugars substituted at different positions of the xylan backbone, which was revealed by the structural analysis. The low yield might mainly have been due to the extraction ability DMSO, a neutral and mild organic solvent. Evidently, glucose is the major sugar component of the DMSO-soluble fraction compared to the other five hemicellulosic fractions. This suggested that a relatively higher amount of b-glucan or starch was removed by DMSO than Table 1 Yield of hemicelluloses (% original components) extracted with organic solvents and alkaline solutions. Hemicellulosic fractions
Extractant
H1 H2
DMSO 70% ethanol containing 2% triethylamine 70% ethanol containing 0.5% KOH 1% KOH 3% KOH 5% KOH
H3 H4 H5 H6 Total
Reaction conditions
Yield (%)
Temperature (°C)
Time (h)
75 75
3 3
5.6 7.3
75
3
9.8
30 30 30
3 3 3
6.2 4.4 11.1 44.4
Rhamnose Arabinose Galactose Glucose Xylose Uronic acids
Hemicellulosic fractions H1
H2
H3
H4
H5
H6
Residue
0.8 5.6 2.4 33.7 54.8 3.6
1.9 14.8 2.7 17.5 55.6 7.4
2.1 7.3 4.2 6.5 62.2 17.7
0.9 1.9 0.5 4.4 86.7 5.6
0.7 1.1 0.5 1.8 88.1 7.8
0.7 0.2 0.2 0.7 89.6 8.5
0.0 0.0 0.0 74.7 24.1 1.2
with the other solvents, since the glucose liberated was partially attributed to the hydrolysis of the b-glucan or starch. This observation was in agreement with the trend reported from wheat straw DMSO-soluble hemicelluloses (Xu et al., 2007). DMSO might also swell up the cell walls and promote solubilization of the hemicelluloses (Hägglund et al., 1956). Therefore, after the DMSO extraction process, the residue was successively extracted with 70% ethanol containing 2% triethylamine (TEA), 70% ethanol containing 0.5% KOH, and 1%, 3% and 5% KOH aqueous solution. Organic solvent extraction released higher content of glucose and arabinose than alkaline extraction, suggesting that organosolv-soluble hemicelluloses are much more ramified than the alkali-soluble hemicelluloses. These two different types of xylans might occur in structural variations differing in side-chain types, distribution, localization and/or types and distribution of glycoside linkages in the macromolecular backbone. The alkaline extraction yielded a high content of xylose from 86.7 to 88.1, and to 89.6% (Table 2). In contrast to 1% and 3% KOH extractions, 5% KOH extraction led to a high amount of hemicelluloses released from the cell wall (Table 1). The alkali-soluble fractions contained lower amounts of arabinose, rhamnose, and glucose than the organosolv-soluble fractions, indicating that the alkaline extraction under the given conditions promoted the release of xylose (main-chain), but not the ramified monosaccharide (side-chain). Furthermore, the about 28% (dry matter) residue left after the sequential extractions contained mainly glucose (74.7%) and xylose (24.1%) as revealed by sugar analysis in Table 2. The small amount of hemicelluloses remained in the residue as compared with the organosolv- and alkali-soluble hemicellulosic fractions is indicative of the hemicellulosic fractions being be tightly bound to the cell wall component. In addition, mannose was not detected in all of the fractions and residues. Most of the hemicelluloses have 4-O-methyl-a-D-glucuronic acid residue (MeGlcA) attached always at position 2 of the main chain Xylp units (Ebringerová et al., 2005). 4-O-methyl-a-glucurono-D-xylan (MGX) represents the main hemicellulose component of hardwood, showing Xyl/4-O-MeGlcA ratios from 4:1 to 16:1 depending on the extraction conditions used (Ebringerová et al., 2005); on average, the ratio is about 10:1. The Xyl/4-O-MeGlcA ratios are indicative of the degree of linearity or substitution of hemicellulosic fractions. A high Xyl/4-O-MeGlcA ratio would indicate a high degree of polymerization with little bonding to other monosaccharide constituents. In this study, the molar ratios of Xyl/4O-MeGlcA were 15:1, 8:1, and 4:1 for fractions of H1, H2, and H3, respectively, and 15:1, 11:1, and 11:1 for fractions of H4, H5, and H6. Therefore, the organosolv-soluble hemicellulosic fractions have a lower molar ratio than the alkali-soluble fractions except for the DMSO-soluble fraction. Moreover, a decreasing trend of the Xyl/4O-MeGlcA ratio can be found from the alkali-soluble fractions. 3.2. Molecular mass Gel-permeation charomatography demonstrated that the six hemicellulosic fractions had different molecular weights
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(Table 3), and it can therefore be expected that these fractions would have different solubility and rheological and thermal prop W, erties. The average molar mass of alkali-soluble fraction H6 (M 1065,000 g/mol) was nearly two hundred times as high as that of W ; 46,780 g/mol). This phenomenon DMSO-soluble fraction (M can be explained by the fact that DMSO is a neutral solvent, and released mainly low molecular weight polysaccharides, as well as a large amount of b-glucan or starch, as revealed by sugar analysis (Table 2). The alkali could dissolve the hemicelluloses with high molecular weights. As extractions were performed sequentially, the accessibility to higher linear molecular weight hemicellulosic fragments, e.g. xylan, was increased. The hemicellulosic fractions were not substantially degraded under the alkaline extraction conditions. Interestingly, as shown in Table 3, an increase in KOH con W value (H4, centration (from 1% to 5%) led to an increase in M W = 1065,000 g/mol), indicating that W = 419,900 g/mol; H6, M M less content of xylan was solubilized with increasing alkali concentrations, and aggregation of the xylan fractions occured (Ebringerova et al., 1994). The 70% ethanol-containing 2% TEA extraction W = 89,430 g/mol) of the released a higher molecular weight (M hemicelluloses than that of the other organosolv-soluble fractions.
3.3. FT-IR spectra The FT-IR spectra of H1, H3, and H4 are illustrated (see Fig. S1 in Supplementary data). Each isolated hemicellulosic fraction spectra clearly showed the typical signal of polysaccharides. A small band at 896 or 897 cm1 is characteristic of b-glycosidic linkages
Table 3 W Þ, number-average ðM n Þ molecular weights and polydispersity Weight-average ðM W =M n Þ of the hemicellulosic fractions. ðM Hemicellulosic fractions
W M n M n W =M M
H1
H2
H3
H4
H5
H6
46,780 9000 5.19
89,430 11,450 7.81
68,650 11,880 5.78
419,900 61,880 6.79
1197,100 334,490 3.58
1065,000 180,220 5.91
between the sugar units, and a specific maximum band at 1047 cm1 is dominated by the glycosidic bond stretching (C–O– C) (Sun et al., 1996). An intensive band at 1741 cm1 in the spectrum of DMSO-soluble fraction is originated from the acetyl and uronic ester groups of the hemicelluloses. However, the absence of this signal in spectra H3 and H4 suggested that the ethanol containing KOH and KOH aqueous solution could completely cleave this ester bond from the hemicelluloses. The occurrence of two noticeable bands at 1047 and 897 cm1 can be explained by the xylose content in the fractions of H1, H3, and H4. This result was correlated with the data obtained by sugar analysis of the hemicellulosic fractions, in which the xylose content increased from 54.8% (H1) to 62.2% (H3), and to 86.7% (H4) (Table 2). 3.4. 1D and 2D NMR spectra The 13C NMR spectra (see Fig. S2 in Supplementary data) showed that signal at 168.5 ppm in H1 are characteristic for the carbonyl signal of esterified ferulic acid in DMSO-soluble hemicelluloses (Sun et al., 2005), however, the absence of this signal suggested that 70% ethanol containing 0.5% KOH and 1% KOH extractions led to the cleavage of the esterified groups. The cross-signals observed in the HSQC spectra (see Fig. S3 in Supplementary data) showed prominent signals (see Table S1 in Supplementary data) corresponding to (1 ? 4)-b-D-Xylp linkages. The absence signals of a-L-Araf residues of the alkali-soluble fraction H4 suggested that the Ara-substituted xylans have already been extracted, and the alkali-extracted xylan was found to be less ramified. These observations indicated that the linear xylan monomer units were partially substituted by a-L-Araf residues either on O3 or O-2 (monosubstitution) or on both O-2 and O-3 (disubstitution) of xylose (Ebringerová et al., 2005). The molar ratios of Xyl/Ara and Xyl/4-O-MeGlcA of fraction H1 were approximately 14:1 and 15:1, respectively. However, the values obtained by sugar analysis were 10:1 and 15:1, respectively. This discrepancy is presumably due to the hemicellulosic fractions completely hydrolyzed in the sulfuric acid medium, and released high amounts of arabinose and 4-O-MeGlcA. The molar ratios of Xyl/Ara and Xyl/4-O-MeGlcA of fraction H3 were 7:1 and 5:1,
Fig. 2. Proposed structure of the hemicellulosic fractions H1, H3, and H4.
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respectively, which is similar with the results obtained by sugar analysis (Table 2). In contrast, fraction H4 has no arabinose signal appearance in the HSQC spectra, and giving the ratio of Xyl/4-OMeGlcA of 18:1, implying that the trace amount of arabinose in H4 can be neglected. In short, the potential ratios of Xyl/4-O-MeGlcA/Ara were estimated to be 14:1:1 and 7:2:1 of fractions H1 and H3, respectively. Based on monosaccharide composition, FT-IR and NMR analyses, a theoretical structural model can be reasonably proposed for the 4-O-MGX repeating unit of TA as in Fig. 2. These results indicated that the organosolv-soluble fractions are more highly ramified than the alkali-soluble hemicellulosic fractions. We can conclude that the higher arabinose and uronic acid content is, the higher the degree of substitution of xylan chains is. 3.5. Thermal stability The degradation mechanism of the hemicellulosic fractions H1, H3, and H4 were comparatively studied (see Fig. S4 in Supplementary data). In the first step (40–200 °C), approximately 10% of the initial sample weight was lost, which was mainly due to the moisture evaporation (Yang et al., 2006). The weight loss mainly happened at 200–400 °C. At 50% weight loss, the decomposing temperature of the hemicellulosic fractions H1, H3, and H4 occurred at 310, 307, and 298 °C, respectively. This minor difference was owing to the structural inhomogeneity of the three fractions, suggesting that more ramified polysaccharide degraded as the decomposition temperature increased. The weight loss ranging from 400 to 560 °C was suggested to proceed through the degradation of the volatile components (Yang et al., 2007). In the last step (>560 °C), the three fractions could be explained by the decomposition of the ‘‘char residue’’ which formed during the former step (Yang et al., 2007). In the DTA curves, fractions H1 and H3 produced more exothermic peaks than the fraction H4, suggesting a complex structure of the fractions H1 and H3. The first exothermic step displayed the degradation of the volatile products, while the late step implied the degradation of the char residue. It could be concluded that a small weight-loss changed and more heat produced in the late step than the first step. The DMSO-soluble fraction H1 and ethanol containing KOH-soluble fraction H3 were found to have a higher thermal stability than the alkali-soluble fraction H4. 4. Conclusions The present study demonstrated that DMSO, 70% ethanol containing 2% TEA, 70% ethanol containing 0.5% KOH, and 1%, 3% and 5% KOH solutions are effective in isolating hemicelluloses from lignocellulosic biomass. The results indicated that the organosolv-soluble fractions are more highly ramified than the alkali-soluble hemicellulosic fractions. In particular, the alkali treatment led to dissolution of the hemicelluloses having a higher content of xylose than that of the polymers released in the organic solvents treatment. It was also found that the inherent variations in the chemical structure of different hemicellulosic fractions played an important role in their thermal stability. Acknowledgements The authors gratefully acknowledge the financial support from the Major State Basic Research Projects of China (9732010CB732204), the National Natural Science Foundation of China (30930073 and 31070526), the State Forestry Administration (200804015, 948-2010-4-16), and China Ministry of Education (111).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.03.012.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Structural and thermal characterization of hemicelluloses isolated by organic solvents and alkaline solutions from Tamarix austromongolica Yong-Chang Sun a, Jia-Long Wen a, Feng Xu a,⇑, Run-Cang Sun a,b,⇑ a b
Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
a r t i c l e
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Article history: Received 5 January 2011 Received in revised form 4 March 2011 Accepted 4 March 2011 Available online 10 March 2011 Keywords: Lignocellulosic biomass Tamarix austromongolica Hemicelluloses Structure feature Thermal stability
a b s t r a c t Three organosolv and three alkaline hemicellulosic fractions were prepared from lignocellulosic biomass of the fast-growing shrub Tamarix austromongolica (Tamarix Linn.). Sugar analysis revealed that the organosolv-soluble fractions contained a higher content of glucose (33.7–6.5%) and arabinose (14.8–5.6%), and a lower content of xylose (62.2–54.8%) than the hemicellulosic fractions isolated with aqueous alkali solutions. A relatively high concentration of alkali resulted in a decreasing trend of the xylose/4-O-methyl-D-glucuronic acid ratio in the alkali-soluble fractions. The results of NMR analysis supported a major substituted structure based on a linear polymer of b-(1 ? 4)-linked D-xylopyranosyl residues, having ramifications of a-L-arabinofuranose and 4-O-methyl-D-glucuronic acid residues monosubstituted at O-3 and O-2, respectively. Thermogravimetric analysis revealed that one step of major mass loss occurred between 200–400 °C, as hemicelluloses devolatilized with total volatile yield of about 55%. It was found that organosolv-soluble fractions are more highly ramified, and showed a higher thermal stability than the alkali-soluble fractions. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The perennial shrub, Tamarix austromongolica (TA), has found uses in prevention of wind erosion and control of desertification (Zhang and Zhao, 1989), reforestation of dry steppes, and also as a source of medicine, wood, fuel, and animal feed (Orabi et al., 2010). It has a higher content of hemicelluloses (about 31%) on a dry weight basis compared to sugarcane bagasse (25–34%), sunflower husk (23–26%), and cereal straws (20–35%) (Xu et al., 2008; Ren et al., 2008). Hemicelluloses are the least stable polymers within lignocellulosic biomass and, unlike cellulose, hemicelluloses are not chemically homogeneous. Xylan, the main hemicellulose component, is composed of 1,4-linked b-D-xylopyranose (b-D-Xylp) units which can be substituted at C-2 and/or C-3 by short and flexible side chains. In addition, acetyl groups located at O-2 and/or O-3 are often found on the backbone xylopyranosyl residues (Barbat et al., 2010). Neutral homoxylans contain xylose residues only, and can be either linear, such as the (1 ? 4)-b-D-xylan of guar seed husk, tobacco stalk, and esparto grass, or branched such as the xylans of groundnut seed endosperm and those of the angiosperm (Habibi et al., 2002). Neutral heteroxylans or arabinoxylans contained either single a-L-arabinofuranose ⇑ Corresponding authors. Address: Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China. Tel./fax: +86 10 62336972. E-mail addresses: [email protected] (F. Xu), [email protected] (R.-C. Sun). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.012
(a-L-Araf) residues which are usually attached by (1 ? 3) linkages (Bendahou et al., 2007). The frequency and composition of these pendant substituents varies with the source of xylan, and this variation creates linear homoxylan, homoxylan, arabinoxylan, glucuronoxylan, and glucuronoarabinoxylan. Hemicelluloses are complex components in the cell wall of woods, interconnected together with cellulose by physical intermixing, and linked to lignin by covalent bonds (mainly a-benzyl ether linkage) (Freudenberg, 1965). These bonds restrict the liberation of hemicelluloses from the cell wall matrix, and the hydrogen bond between the cell wall components may impede isolation of the hemicellulosic component (Ebringerová and Heinze, 2000). Before extraction of hemicelluloses, extensive hydrogen bonding between the individual polysaccharide cell wall components must be broken, and the wood flour must be pretreated to remove all lipophilic and hydrophilic non-structural components, and delignified with chlorine (Stephen, 1983), sodium chlorite, chlorine dioxide (Wong et al., 1988) or peroxyacetic acid (Bioly, 1985). Acidcatalyzed steam explosion and sulfite pretreatment to overcome recalcitrance of lignocellulose has been applied in research (Zhu et al., 2009; Yang and Wyman, 2008; Zhu and Pan, 2010), and organosolv and alkaline fractionation methods have attracted growing interest because they can release hemicelluloses from different fiber sources in an environmentally friendly manner. Organosolv extraction opens the possibility of exploiting hemicelluloses and lignin from biomass in addition to cellulose (Villaverde
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et al., 2009), and alkaline extraction is used for lower temperature and pressure extractions, and is a promising process to achieve complete utilization of lignocelluloses (Chaa et al., 2008). Hemicelluloses have been shown to have biological activities including decreasing blood cholesterol and preventing colon cancer (Hu et al., 2007). They can also be used for the production of high value-added chemicals such as xylitol (Rao et al., 2006) and furfural (Karimi et al., 2006), and for food, and non-food applications, and for the synthesis of thermoplastic xylan derivatives (Jain et al., 2000). The aim of the present study was to develop an efficient strategy to disrupt the cross-linked matrix of lignin and hemicelluloses. The hemicelluloses released from the lignocellulosic biomass was characterized with high-performance anionexchange chromatography (HPAEC), and Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopies. In addition, the pyrolysis mechanism of the fractions was investigated by thermogravimetric analysis (TGA) and differential thermal analysis (DTA). 2. Experimental
2.2. Sugar composition and content of uronic acids The chemical compositions of the six hemicellulosic fractions were determined by hydrolysis with dilute sulfuric acid. A 4–6 mg sample was hydrolyzed with 1.475 mL of 10% H2SO4 for 2.5 h at 105 °C. After hydrolysis, the mixture was filtered and diluted 60-fold, and analyzed by high-performance anion-exchange chromatography (HPAEC) seytem (Dionex ICS3000, US) with an amperometric detector, AS50 autosamper, a Carbopac™ PA-20 column (4 250 mm, Dionex) and a guard PA-20 column (3 30 mm, Dionex). Neutral sugars and uronic acids were separated in an isocratic 5 mM NaOH solution (carbonate free and purged with nitrogen) for 20 min, followed by a 0–75 mM NaAc gradient in 5 mM NaOH solution for 15 min. Calibration was performed with standard solutions of L-arabinose, D-glucose, D-xylose, D-rhamnose, D-mannose, D-galactose, glucuronic acid, and galacturonic acid. All experiments were performed at least in duplicate, and the average values were calculated for all of the hemicellulosic fractions. 2.3. FT-IR analysis
2.1. Materials and preparation TA was harvested in October 2009 in Inner Mongolia, China. Leaves and bark were removed, and the stems were chipped into small pieces (1–3 cm). After drying at 60 °C for 16 h in an oven, the chips were sieved through a 40–60 mm mesh. The scheme for successive extractions with DMSO, alkaline ethanol, and aqueous alkaline solutions is shown in Fig. 1. The pH value was adjusted with 10% acetic acid in the delignification process, and a material to liquor ratio of 1:25 (g/mL) was used in the experiment. Note that H1, H2, and H3 represent the hemicellulosic fractions isolated with DMSO, 70% ethanol containing 2% triethylamine (TEA), and 70% ethanol containing 0.5% KOH, respectively. H4, H5, and H6 represent the hemicellulosic fractions isolated with 1%, 3%, and 5% KOH aqueous solution, respectively.
The FT-IR spectra were obtained from a KBr disc using Tensor 27 FT-IR spectrophotometer in the range of 400–4000 cm1. The finely grounded sample was mixed with high purity KBr (1:100, w/w). The spectrum of pure KBr was used as background. Each spectrum was recorded with a total of 32 scans. 2.4. Molecular weight determination W Þ and number-average ðM n Þ molecular The weight-average ðM weights of the hemicellulosic fractions were determined by gel permeation chromatography (GPC) with a refraction index detector (RID) on a PL aquagel-OH 50 column (300 7.7 mm, Polymer Laboratories Ltd.), calibrated with PL pullulan polysaccharide stan W = 738, 12,200, 100,000, 1600,000, Polymer Laboratories dards ðM
Fig. 1. Scheme for isolation of the hemicellulosic fractions from Tamarix austromongolica.
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Ltd.). A 2 mg sample was dissolved with 0.02 N NaCl in 0.005 M sodium phosphate buffer, pH 7.5 at a concentration of 0.1% before measurement.
Table 2 Neutral sugar and uronic acid content (relative mmol% ± standard deviation 0.1%) in the isolated hemicellulosic fractions and the residue. Sugars
2.5. 1D and 2D NMR analysis The solution-state 1H and 13C NMR spectra were obtained on a Bruker AVIII 400 MHz spectrometer operating in the FT mode at 100.6 MHz. The hemicellulosic fractions (20 mg for 1H, 80 mg for 13 C) were dissolved in 1 mL D2O. The 1H NMR spectrum was recorded at 25 °C after 128 scans. The 13C NMR spectrum was recorded at 25 °C after 30,000 scans. A 30° pulse flipping angle, a 9.2 ls pulse width, a 1.37 s acquisition time and 2 s relaxation delay time were used. 1H–13C correlation 2D (HSQC) NMR spectrum was also recorded at 25 °C on the same apparatus equipped with a z-gradient double resonance probe. The spectral widths were 3268 and 18,116 Hz for the 1H- and 13C- dimensions, respectively. The number of collected complex points was 1024 for 1H-dimension with a relaxation delay of 1.5 s. The number of scans was 128 and 256 time increments were always recorded in 13C-dimension. The 1JCH used was 145 Hz. Prior to Fourier transformation, the data matrixes were zero filled up to 1024 points in the 13C-dimension. 2.6. Thermogravimetric analysis The thermal degradation of the hemicellulosic fractions was investigated using both thermogravimetric (TGA) and differential thermal analyses (DTA) on a simultaneous thermal analyzer (DTG-60 Shimadzu, Japan) from 40 to 600 °C. Samples of approximately 10 mg weight were heated in an aluminum crucible with the heating rate of 10 °C/min while the apparatus was continually flushed with a nitrogen flow of 30 mL/min. Calcined alumina (Al2O3) was used as a reference material in all experiments. 3. Results and discussion 3.1. Yield and sugar composition The hemicellulose yield and content of neutral sugars of the six hemicellulosic fractions are shown in Table 1 and Table 2, respectively. DMSO extraction removed 5.6% of the original hemicelluloses from the crude holocellulose, but preserved the acetyl groups. The DMSO-soluble hemicellulosic fraction contained much structural information as it had a diverse composition with different types of sugars substituted at different positions of the xylan backbone, which was revealed by the structural analysis. The low yield might mainly have been due to the extraction ability DMSO, a neutral and mild organic solvent. Evidently, glucose is the major sugar component of the DMSO-soluble fraction compared to the other five hemicellulosic fractions. This suggested that a relatively higher amount of b-glucan or starch was removed by DMSO than Table 1 Yield of hemicelluloses (% original components) extracted with organic solvents and alkaline solutions. Hemicellulosic fractions
Extractant
H1 H2
DMSO 70% ethanol containing 2% triethylamine 70% ethanol containing 0.5% KOH 1% KOH 3% KOH 5% KOH
H3 H4 H5 H6 Total
Reaction conditions
Yield (%)
Temperature (°C)
Time (h)
75 75
3 3
5.6 7.3
75
3
9.8
30 30 30
3 3 3
6.2 4.4 11.1 44.4
Rhamnose Arabinose Galactose Glucose Xylose Uronic acids
Hemicellulosic fractions H1
H2
H3
H4
H5
H6
Residue
0.8 5.6 2.4 33.7 54.8 3.6
1.9 14.8 2.7 17.5 55.6 7.4
2.1 7.3 4.2 6.5 62.2 17.7
0.9 1.9 0.5 4.4 86.7 5.6
0.7 1.1 0.5 1.8 88.1 7.8
0.7 0.2 0.2 0.7 89.6 8.5
0.0 0.0 0.0 74.7 24.1 1.2
with the other solvents, since the glucose liberated was partially attributed to the hydrolysis of the b-glucan or starch. This observation was in agreement with the trend reported from wheat straw DMSO-soluble hemicelluloses (Xu et al., 2007). DMSO might also swell up the cell walls and promote solubilization of the hemicelluloses (Hägglund et al., 1956). Therefore, after the DMSO extraction process, the residue was successively extracted with 70% ethanol containing 2% triethylamine (TEA), 70% ethanol containing 0.5% KOH, and 1%, 3% and 5% KOH aqueous solution. Organic solvent extraction released higher content of glucose and arabinose than alkaline extraction, suggesting that organosolv-soluble hemicelluloses are much more ramified than the alkali-soluble hemicelluloses. These two different types of xylans might occur in structural variations differing in side-chain types, distribution, localization and/or types and distribution of glycoside linkages in the macromolecular backbone. The alkaline extraction yielded a high content of xylose from 86.7 to 88.1, and to 89.6% (Table 2). In contrast to 1% and 3% KOH extractions, 5% KOH extraction led to a high amount of hemicelluloses released from the cell wall (Table 1). The alkali-soluble fractions contained lower amounts of arabinose, rhamnose, and glucose than the organosolv-soluble fractions, indicating that the alkaline extraction under the given conditions promoted the release of xylose (main-chain), but not the ramified monosaccharide (side-chain). Furthermore, the about 28% (dry matter) residue left after the sequential extractions contained mainly glucose (74.7%) and xylose (24.1%) as revealed by sugar analysis in Table 2. The small amount of hemicelluloses remained in the residue as compared with the organosolv- and alkali-soluble hemicellulosic fractions is indicative of the hemicellulosic fractions being be tightly bound to the cell wall component. In addition, mannose was not detected in all of the fractions and residues. Most of the hemicelluloses have 4-O-methyl-a-D-glucuronic acid residue (MeGlcA) attached always at position 2 of the main chain Xylp units (Ebringerová et al., 2005). 4-O-methyl-a-glucurono-D-xylan (MGX) represents the main hemicellulose component of hardwood, showing Xyl/4-O-MeGlcA ratios from 4:1 to 16:1 depending on the extraction conditions used (Ebringerová et al., 2005); on average, the ratio is about 10:1. The Xyl/4-O-MeGlcA ratios are indicative of the degree of linearity or substitution of hemicellulosic fractions. A high Xyl/4-O-MeGlcA ratio would indicate a high degree of polymerization with little bonding to other monosaccharide constituents. In this study, the molar ratios of Xyl/4O-MeGlcA were 15:1, 8:1, and 4:1 for fractions of H1, H2, and H3, respectively, and 15:1, 11:1, and 11:1 for fractions of H4, H5, and H6. Therefore, the organosolv-soluble hemicellulosic fractions have a lower molar ratio than the alkali-soluble fractions except for the DMSO-soluble fraction. Moreover, a decreasing trend of the Xyl/4O-MeGlcA ratio can be found from the alkali-soluble fractions. 3.2. Molecular mass Gel-permeation charomatography demonstrated that the six hemicellulosic fractions had different molecular weights
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(Table 3), and it can therefore be expected that these fractions would have different solubility and rheological and thermal prop W, erties. The average molar mass of alkali-soluble fraction H6 (M 1065,000 g/mol) was nearly two hundred times as high as that of W ; 46,780 g/mol). This phenomenon DMSO-soluble fraction (M can be explained by the fact that DMSO is a neutral solvent, and released mainly low molecular weight polysaccharides, as well as a large amount of b-glucan or starch, as revealed by sugar analysis (Table 2). The alkali could dissolve the hemicelluloses with high molecular weights. As extractions were performed sequentially, the accessibility to higher linear molecular weight hemicellulosic fragments, e.g. xylan, was increased. The hemicellulosic fractions were not substantially degraded under the alkaline extraction conditions. Interestingly, as shown in Table 3, an increase in KOH con W value (H4, centration (from 1% to 5%) led to an increase in M W = 1065,000 g/mol), indicating that W = 419,900 g/mol; H6, M M less content of xylan was solubilized with increasing alkali concentrations, and aggregation of the xylan fractions occured (Ebringerova et al., 1994). The 70% ethanol-containing 2% TEA extraction W = 89,430 g/mol) of the released a higher molecular weight (M hemicelluloses than that of the other organosolv-soluble fractions.
3.3. FT-IR spectra The FT-IR spectra of H1, H3, and H4 are illustrated (see Fig. S1 in Supplementary data). Each isolated hemicellulosic fraction spectra clearly showed the typical signal of polysaccharides. A small band at 896 or 897 cm1 is characteristic of b-glycosidic linkages
Table 3 W Þ, number-average ðM n Þ molecular weights and polydispersity Weight-average ðM W =M n Þ of the hemicellulosic fractions. ðM Hemicellulosic fractions
W M n M n W =M M
H1
H2
H3
H4
H5
H6
46,780 9000 5.19
89,430 11,450 7.81
68,650 11,880 5.78
419,900 61,880 6.79
1197,100 334,490 3.58
1065,000 180,220 5.91
between the sugar units, and a specific maximum band at 1047 cm1 is dominated by the glycosidic bond stretching (C–O– C) (Sun et al., 1996). An intensive band at 1741 cm1 in the spectrum of DMSO-soluble fraction is originated from the acetyl and uronic ester groups of the hemicelluloses. However, the absence of this signal in spectra H3 and H4 suggested that the ethanol containing KOH and KOH aqueous solution could completely cleave this ester bond from the hemicelluloses. The occurrence of two noticeable bands at 1047 and 897 cm1 can be explained by the xylose content in the fractions of H1, H3, and H4. This result was correlated with the data obtained by sugar analysis of the hemicellulosic fractions, in which the xylose content increased from 54.8% (H1) to 62.2% (H3), and to 86.7% (H4) (Table 2). 3.4. 1D and 2D NMR spectra The 13C NMR spectra (see Fig. S2 in Supplementary data) showed that signal at 168.5 ppm in H1 are characteristic for the carbonyl signal of esterified ferulic acid in DMSO-soluble hemicelluloses (Sun et al., 2005), however, the absence of this signal suggested that 70% ethanol containing 0.5% KOH and 1% KOH extractions led to the cleavage of the esterified groups. The cross-signals observed in the HSQC spectra (see Fig. S3 in Supplementary data) showed prominent signals (see Table S1 in Supplementary data) corresponding to (1 ? 4)-b-D-Xylp linkages. The absence signals of a-L-Araf residues of the alkali-soluble fraction H4 suggested that the Ara-substituted xylans have already been extracted, and the alkali-extracted xylan was found to be less ramified. These observations indicated that the linear xylan monomer units were partially substituted by a-L-Araf residues either on O3 or O-2 (monosubstitution) or on both O-2 and O-3 (disubstitution) of xylose (Ebringerová et al., 2005). The molar ratios of Xyl/Ara and Xyl/4-O-MeGlcA of fraction H1 were approximately 14:1 and 15:1, respectively. However, the values obtained by sugar analysis were 10:1 and 15:1, respectively. This discrepancy is presumably due to the hemicellulosic fractions completely hydrolyzed in the sulfuric acid medium, and released high amounts of arabinose and 4-O-MeGlcA. The molar ratios of Xyl/Ara and Xyl/4-O-MeGlcA of fraction H3 were 7:1 and 5:1,
Fig. 2. Proposed structure of the hemicellulosic fractions H1, H3, and H4.
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respectively, which is similar with the results obtained by sugar analysis (Table 2). In contrast, fraction H4 has no arabinose signal appearance in the HSQC spectra, and giving the ratio of Xyl/4-OMeGlcA of 18:1, implying that the trace amount of arabinose in H4 can be neglected. In short, the potential ratios of Xyl/4-O-MeGlcA/Ara were estimated to be 14:1:1 and 7:2:1 of fractions H1 and H3, respectively. Based on monosaccharide composition, FT-IR and NMR analyses, a theoretical structural model can be reasonably proposed for the 4-O-MGX repeating unit of TA as in Fig. 2. These results indicated that the organosolv-soluble fractions are more highly ramified than the alkali-soluble hemicellulosic fractions. We can conclude that the higher arabinose and uronic acid content is, the higher the degree of substitution of xylan chains is. 3.5. Thermal stability The degradation mechanism of the hemicellulosic fractions H1, H3, and H4 were comparatively studied (see Fig. S4 in Supplementary data). In the first step (40–200 °C), approximately 10% of the initial sample weight was lost, which was mainly due to the moisture evaporation (Yang et al., 2006). The weight loss mainly happened at 200–400 °C. At 50% weight loss, the decomposing temperature of the hemicellulosic fractions H1, H3, and H4 occurred at 310, 307, and 298 °C, respectively. This minor difference was owing to the structural inhomogeneity of the three fractions, suggesting that more ramified polysaccharide degraded as the decomposition temperature increased. The weight loss ranging from 400 to 560 °C was suggested to proceed through the degradation of the volatile components (Yang et al., 2007). In the last step (>560 °C), the three fractions could be explained by the decomposition of the ‘‘char residue’’ which formed during the former step (Yang et al., 2007). In the DTA curves, fractions H1 and H3 produced more exothermic peaks than the fraction H4, suggesting a complex structure of the fractions H1 and H3. The first exothermic step displayed the degradation of the volatile products, while the late step implied the degradation of the char residue. It could be concluded that a small weight-loss changed and more heat produced in the late step than the first step. The DMSO-soluble fraction H1 and ethanol containing KOH-soluble fraction H3 were found to have a higher thermal stability than the alkali-soluble fraction H4. 4. Conclusions The present study demonstrated that DMSO, 70% ethanol containing 2% TEA, 70% ethanol containing 0.5% KOH, and 1%, 3% and 5% KOH solutions are effective in isolating hemicelluloses from lignocellulosic biomass. The results indicated that the organosolv-soluble fractions are more highly ramified than the alkali-soluble hemicellulosic fractions. In particular, the alkali treatment led to dissolution of the hemicelluloses having a higher content of xylose than that of the polymers released in the organic solvents treatment. It was also found that the inherent variations in the chemical structure of different hemicellulosic fractions played an important role in their thermal stability. Acknowledgements The authors gratefully acknowledge the financial support from the Major State Basic Research Projects of China (9732010CB732204), the National Natural Science Foundation of China (30930073 and 31070526), the State Forestry Administration (200804015, 948-2010-4-16), and China Ministry of Education (111).
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.03.012.
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