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Fractionation and characterization of alkali-extracted hemicelluloses from peashrub
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
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Fractionation and characterization of alkali-extracted hemicelluloses from peashrub Feng Peng a, Jing Bian b, Jun-Li Ren a, Pai Peng a, Feng Xu b,*, Run-Cang Sun a,b,* a b
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China
article info
abstract
Article history:
Alkali-soluble hemicelluloses were extracted from delignified peashrub (Caragana kor-
Received 1 February 2010
shinskii) by sequential treatments with 0.5, 1.0 and 2.0 mol L1 KOH under a solid to liquid
Received in revised form
ratio of 1:25 (g mL1) at 25 C for 10 h. The successive treatments resulted in a total
2 May 2010
dissolution of 64.1, 15.6, and 12.5% of the original hemicelluloses, respectively. The
Accepted 4 August 2010
released hemicelluloses were separated into linear and branched hemicellulosic sub-
Available online 30 August 2010
fractions by the iodine-complex precipitation technique. Their chemical and physical
Keywords:
was found that the hemicelluloses precipitated by iodine-potassium iodide solution were
C. korshinskyii
more linear as shown they contained more xylose and less uronic acid than the hemi-
Hemicelluloses
celluloses remaining in the solution, which were more branched and contained higher
Alkali
amounts of arabinose, galactose and glucose. In addition, the linear hemicellulosic sub-
Fractionation
fractions with a low arabinose/xylose ratio had higher molecular weights than those of the
Iodine-complex precipitation
branched hemicellulosic subfractions in solution.
characteristics were determined by HPAEC, GPC, FT-IR and 1H and 13C NMR spectroscopy. It
ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
In recent years, biomass has been pointed out as the most important source for a broad variety of advanced polymeric products such as fuels, paints, detergents, biodegradable polymers, textile fibers, fiber composites, and various other commodity and special chemicals. The biomass, such as shrubs, grass, essentially consisted of three different polymer entities (cellulose, hemicelluloses and lignin). The use of all the three polymeric components has been discussed since several decades. The discussion had been intensified in connection with emerging biorefinery strategies [1,2]. Recently, much attention has been paid to utilizing forest and agricultural residues as raw material for industries. Caragana korshinskii is a widely distributed and drought-resistant
leguminous shrub, which is widely used for vegetation rehabilitation in arid and semiarid regions of China, for its high ecological and economic value [3]. This shrub generates a large amount of residues, because its stems are cut once every 3 years to make it flourish. At present, only a small amount of the cut peashrub materials is used for the production of fiberboard, but a huge amount is not used and constitutes an environmental problem. Peashrub as an agroindustrial residue, therefore, can be valuable by developing chemical methods in providing transport fuels or chemical feedstock. In the current trend for a complex and more effective utilization of biomass, increasing attention has been paid during the last few years to exploitation of hemicelluloses as biopolymer resources. Hemicelluloses are the second most
* Corresponding author at: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China. Tel./fax: þ86 10 62336972. E-mail addresses: [email protected], [email protected] (F. Xu). 0961-9534/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2010.08.034
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
abundant biopolymer in the plant kingdom. They are lowmolecular-weight polysaccharides, associated in plant cell walls with celluloses and lignin. In plant cell walls large amounts of hemicelluloses occur with a wide variation in content and chemical structure. In hardwoods, the principal hemicelluloses are O-acetyl-4-O-methylglucuronoxylan with amounts between 10% and 35% depending on the species. This polysaccharide consists of about 200-b-xylopyranose residues, linked together by 1, 4-glycosidic bonds. Approximately every tenth xylose unit carries a single, terminal side chain, and consists of 4-O-methylglucuronic acid attached directly to the 2- position of xylose. Seven out of 10 xylose residues contain an O-acetyl group at C-2, at C-3, or at both positions [4]. Further on small portions of glucomannan (3e5%) are found in hardwoods [5,6]. Nowadays, some important applications for hemicelluloses have been discovered. In monomeric form, they can be fermented to ethanol, acetone, butanol [7], or xylitol [8], in oligomeric form, which can be used as functional-food additive [9e11], and in polymeric form, they are can be used as hydrogels [12], biodegradable barrier films for food packing [13,14], and wet strength additives [15]. The components in lignocellulosic materials are tightly associated and in several processes it has been proved to difficult to remove the lignin from the hemicelluloses and cellulose without modifying the hemicelluloses [16]. Therefore, in order to obtain the hemicellulosic polymers having a high yield and purity, the material must be delignified and/or pre-treated in some way prior to extraction. In particular, a delignification step using chlorite prior to extraction of hemicelluloses can significantly facilitate the extraction. In this case, most of the lignin can be removed without any noticeable degradation of hemicellulosic polymers. In addition, different fractionation techniques, such as graded ethanol precipitation [17], ammonium sulphate precipitation and anion-exchange chromatography [18,19], have been used to obtain deeper insight into the diversity of hemicelluloses. Another fractionation technique was that linear polysaccharides can be separated from branched polymers by precipitation as iodine complexes in the presence of CaCl2, which was discovered by Gaillard [20]. The aim of the fractionation was to obtain more homogeneous fractions and thus explore structureeproperty relationships of the polymers, which might obtain the low-branched xylans for further producing the xylose, an intermediate for the production of xylitol, and a variety of xylo-oligosaccharides. The present work was primarily carried out to gain a new knowledge on the structure features of hemicelluloses from the delignified C. korshinskii by applying the iodine-complex precipitation technique in the presence of KCl, and investigate the structural variation and physicochemical properties of hemicellulosic subfractions.
2.
Materials and methods
2.1.
Materials
Peashrub (C. Korshinskii), 5 years old, with an average stem height of 2.8 m, was harvested in October 2004 in Yanchi
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County, Ningxia province, China. The place is located in the west of the loess plateau and lies in the transitional zone between arid and semiarid regions (107 240 E, 37 470 N) at an altitude of 1349 m. The soil is silt loam of loess origin, which can be classified as Calciorthid. Generally, peashrub turns green in April, blooms in May, and sets seed and senesces in July. The leaves and the bark of peashrub were removed at the harvest time. The trunks were dried in sunlight, and then chipped into small pieces about 1e2 cm with axe. The chips (200 g) were ground to pass a 0.8-mm-size screen. Ground samples (150 g) were obtained and stored in plastic containers at room temperature. Fats, waxes and oils were removed from the ground materials in a Soxhlet apparatus refluxing for 6 h with 2:1 (v/v) tolueneeethanol. The dewaxed sample was further dried in a cabinet oven with air circulation at 60 C for 16 h and stored. The components (%, w/w) of the C. Korshinskii were cellulose 41.6%, hemicelluloses 31.5%, lignin 21.7%, ash 0.7%, and wax 4.5% on a dry weight basis, which was determined by the method for measuring the chemical composition of wheat straw described previously [21]. All standard chemicals, such as sugars and phenolics, were analytical grade, purchased from Sigma Chemical Company (Beijing).
2.2.
Isolation and fractionation of hemicelluloses
In order to study structural differences of the hemicelluloses present in Caragana Korshinski, hemicellulosic fractions were obtained by sequential extraction and fractionation according to the scheme in Fig. 1. The dewaxed powder (15 g) was delignified with 6% sodium chlorite at pH 3.6e3.8, adjusting with 10% acetic acid, at 75 C for 2 h [22]. The residue, holocellulose, was subsequently washed with distilled water and ethanol, and dried at 60 C for 16 h. Then the holocellulose was successively extracted by 0.5, 1.0 and 2.0 mol L1 KOH solution with a solid to liquid ratio of 1: 25 (g mL1) at 25 C for 10 h under stirring. After the indicated period treatment, the insoluble residues were filtered through a nylon cloth on a Bu¨chner funnel, washed with distilled water until the pH of filtrates was neutral, and dried at 60 C. Each of filtrates was adjusted to pH 5.5 with 6 mol L1 HCl. Then the filtrate was concentrated to about 100 mL. After standing for 12 h, the solution was centrifuged (2594 g, 15 min). The precipitate obtained was washed with 70% ethanol, and freeze-dried, and named as hemicelluloses A (HA). Then 10 mL of iodine solution (3 g iodine and 4 g KI in 100 mL water) was added in the supernatant. In this case, the solution of hemicelluloses was aqueous potassium chloride, instead of aqueous calcium chloride [20]. After stirring and standing for 12 h, the solution was centrifuged (2594 g, 15 min). The precipitate was washed with 70% ethanol, freeze-dried, and referred to as the hemicelluloses B (HB). The supernatant were then titrated with Na2S2O3 solution, dialyzed and precipitated with three volumes of ethanol. The precipitated hemicelluloses were named as hemicelluloses C (HC), washed with 70% ethanol and freeze-dried. Note that hemicelluloses A is precipitated by neutralization of the 0.5, 1.0 and 2.0 mol L1 KOH extracts to pH 5.5 with hydrochloric acid (named as HA1, HA2 and HA3), hemicelluloses B is isolated by precipitation with iodinepotassium iodide solution (labeled as HB1, HB2 and HB2), and
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Fig. 1 e Scheme for fractionation of alkali-soluble hemicelluloses from C. korshinskii.
the remaining portion in solution is obtained by precipitation with 3 volumes of ethanol (named as HC1, HC2 and HC3).
2.3.
Chemical characterization
The constituent neutral sugars in the isolated hemicellulosic fractions were determined by high performance anionexchange chromatography (HPAEC). The hemicelluloses (4e6 mg) were hydrolyzed by 10% H2SO4 at 105 C in a sealed tube for 2.5 h. After hydrolysis, the samples were diluted 30-fold, filtered and injected into the HPAEC system (Dionex ISC 3000,U.S) with amperometric detector, AS50 autosampler and a CarbopacTM PA1 column (4 250 mm, Dionex) [17]. The
molecular weights of the hemicellulosic fractions were determined by gel-permeation chromatography (GPC) on a PL aquagel-OH 50 column (300 7.7 mm, polymer laboratories Ltd), calibrated with PL pullulan polysaccharide standards (peak average molecular weights 783, 12 200, 100 000, 1600 000) [17]. The measurements were conducted with two parallels and the reproducibility of the values was found within the range of 6%.
2.4.
Spectroscopic characterization
FT-IR spectra of hemicellulosic samples were obtained on an FT-IR spectrophotometer (Nicolet 510) using a KBr disc
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Table 1 e Yields of hemicelluloses solubilized during the successive treatments of delignified C. korshinskyii with 0.5, 1.0 and 2.0 mol LL1 KOH at 25 C for 10 h. Fraction
Yield (% dry matter)
Total solubilized hemicelluloses during the successive treatments with 0.5, 1.0 and 2.0 mol L1 KOH Solubilized hemicelluloses in 0.5 mol L1 KOH treatment Solubilized hemicelluloses in 1.0 mol L1 KOH treatment Solubilized hemicelluloses in 2.0 mol L1 KOH treatment Residue (crude cellulose)
29.1
20.2 4.9 4.0 45.7
containing 1% finely ground samples. Thirty-two scans were taken of each sample recorded from 4000 to 400 cm1 at a resolution of 2 cm1 in the transmission mode. The solutionstate 1H NMR spectrum was recorded on a Bruker MSL300 spectrometer at 300 MHz using 15 mg of hemicelluloses in 1.0 mL of D2O. A 13C NMR spectrum was obtained on a Bruker MSL300 spectrometer at 74.5 MHz. The sample (80 mg) was dissolved in 1 mL D2O (99.8% D) with overnight stirring at room temperature. The spectrum was recorded at 25 C after 30 000 scans. Chemical shifts (d in ppm) are expressed relative to the resonance of Me4Si (d ¼ 0). A 60 pulse flipping angle, a 3.9 ms pulse width and a 0.85 s delay time between scans were used.
3.
Results and discussion
3.1.
Yield of hemicelluloses
Many attempts have been made to isolate hemicelluloses from various biomass resources, in which alkali extraction is the most efficient method for isolating large amounts of hemicellulosic polysaccharides. It is known that hydroxyl ions causes swelling of cellulose, hydrolysis of ester linkage, and disruption of intermolecular hydrogen bonds between celluloses and hemicelluloses, bring a portion of the hemicellulosic material into solution. The isolation and fractionation of hemicelluloses from C. korshinskii is presented schematically in Fig. 1, and the yields of the soluble hemicelluloses are given
in Table 1. Sequential extractions of the delignified C. korshinskii with 0.5, 1.0, 2.0 mol L1 KOH aqueous solution at 25 C for 10 h resulted in dissolution of 20.2, 4.9, and 4.0% hemicelluloses, corresponding to release of 64.1, 15.6, and 12.5% of original hemicelluloses, respectively. Meanwhile, the three treatments together yielded 29.1% hemicelluloses and account for 92.4% of the original hemicelluloses. This indicated that alkali extraction is the efficient method for isolating large amounts of hemicellulosic polysaccharides. Obviously, the majority of the hemicelluloses were obtained by the treatment with 0.5 mol L1 KOH under the condition used. In this study, the three alkali-soluble hemicelluloses were further fractioned into nine subfractions (Fig. 1). The yields of nine hemicellulosic subfractions are shown in Table 2. As can be seen, the yield of the subfractions from the 0.5 mol L1 KOH-soluble hemicelluloses was 6.7, 1.8, 7.0% of the dry materials, corresponding to precipitation of 33.2, 8.9, 34.7% of the total 0.5 mol L1 KOH-soluble hemicelluloses. However, it should be noted that the yields of the hemicellulosic subfractions (HA2 and HA3) obtained by precipitation at pH 5.5 were only a trace amount, thus HA2 and HA3 were not studied in the subsequent analysis. This indicated that the large amount of hemicelluloses A was solubilized only in 0.5 mol L1 KOH aqueous solution. In addition, the dark blue complex of hemicellulosic subfractions HB1, HB2 and HB3 precipitated by iodine solution was separated by high-speed centrifugation. Under this condition, 1.8, 1.6, and 1.2% hemicelluloses (percent dry staring material) were precipitated, corresponding to precipitation of 8.9, 32.6, and 30.0% of the 0.5, 1.0, 2.0 mol L1 KOH-soluble hemicelluloses, respectively. This result revealed the hemicelluloses can form a blue complex by precipitation with iodine in the KCl solution, which did not agree with that alkali metal chloride solution can not replace the CaCl2 solution in this reaction [23,24]. Clearly, the total yield of nine hemicellulosic subfractions accounted for 79.0% of total dissolved hemicelluloses during the sequential fractionations, indicating that 21.0% hemicelluloses, mainly degraded substance such as oligosaccharides, were not recovered.
3.2.
Sugar composition
The neutral sugar composition and the content of uronic acid of seven hemicellulosic subfractions are listed in Table 3.
Table 2 e The yield of hemicellulosic subfractions (% dry starting matter) obtained from 0.5, 1.0 and 2.0 mol LL1 KOHsoluble hemicelluloses. Yield/content
1.0 mol L1 KOH-soluble hemicelluloses
0.5 mol L1 KOH-soluble hemicelluloses
2.0 mol L1 KOH-soluble hemicelluloses
Subfraction
HA1a
HB1b
HC1c
HA2a
HB2b
HC2c
HA3a
HB3b
HC3c
Yield of hemicelluloseic subfraction
6.7
1.8
7.0
Trd
1.6
2.8
Trd
1.2
2.0
a b c d
Represents the hemicelluloses obtained by precipitation at pH 5.5 in aqueous solution. Represents the hemicellulosic subfractions obtained by precipitation with iodine-potassium iodide solution. Represents the hemicellulosic subfractions obtained by precipitation with 3 volumes of ethanol. Tr ¼ trace.
Total
23.0
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Table 3 e Contents of neutral sugars and uronic acids (relative percent dry hemicelluloses, w/w) in hemicellulosic subfractions. Hemicellulosic subfractionsa
Sugars (%) HA1 Rhamnose Arabinose Galactose Glucose Xylose Uronic acid Ara/Xyl
HB1
HC1
HB2
HC2
HB3
HC3
0.10 1.09 1.04 0.75 0.62 0.60 6.02 6.91 11.43 1.73 6.10 2.17 1.33 1.91 4.14 0.37 3.43 0.22 0.56 1.31 8.97 1.26 4.87 1.59 91.09 88.78 74.42 94.92 84.97 95.42 2.75 1.35 2.52 1.15 2.58 0.97 0.067 0.078 0.15 0.018 0.072 0.023
Trb 5.66 5.05 9.00 80.30 1.94 0.070
a Corresponding to the hemicellulosic subfractions in Table 2. b Tr ¼ trace.
Obviously, xylose was the dominant sugar component (74.4e95.4%). Arabinose and glucose appeared as the second and the third major sugars, comprising 1.7e11.4% and 0.6e9.0% of total sugars, respectively. Galactose (0.2e5.1%), rhamnose (0.1e1.1%) and uronic acid (mainly glucuronic acid or its 4-O-methyl derivative, 0.9e2.8%) were observed as minor constituents. The dominance of xylose residues probably were originated from the backbone of xylan. The presence of a noticeable amount of arabinose and minor quantity of uronic acids implied that the alkali-soluble hemicelluloses consisted of side chains of arabinose and glucuronic acid, which need to be further investigated, since based on the sugar composition alone it is difficult to draw conclusions on the structural patterns of the hemicelluloses. Moreover, a noticeable amount of glucose from the hemicellulosic subfractions (HC1, HC2, HC3) may be a part of the xylan or present as a glucan, since they are precipitated only on addition of alcohol after iodine-precipitation. Additionally, compared with HB1 and HC1, hemicellulosic subfraction HA1 obtained by precipitation at pH 5.5 from the 0.5 mol L1 NaOH-soluble hemicelluloses, have a higher contents of xylose (91.1%) and uronic acid (2.75%), but the ratio of arabinose to xylose (Ara/Xyl, 0.07) of HA1 is lower than HB1 (0.08) and HC1 (0.15). This indicated that hemicelluloses B and C were more highly branched than hemicelluloses A, and the hemicelluloses A consisted of a very lowly-substituted population, since Ara/Xyl ratios are indicative of the degree of linearity or branching of hemicelluloses [25]. Moreover, from the composition data of HB1 and HC1, the xylose decreased from 88.8% (HB1) to 74.4% (HC1), whereas the arabinose, galactose, glucose, uronic acid and Ara/Xyl increased. Simliar
results were found in the four hemicellulosic subfractions (HB2, HC2, and HB3, HC3) obtained from the 1.0 and 2.0 mol L1 KOH-soluble hemicelluloses. Thus, it can be concluded that the linear hemicelluloses are precipitated by iodine-potassium iodide solution, and the branched ones remain in the solution, which would be isolated by precipitation with 3 volumes of ethanol.
3.3.
Molar mass
The molecular weights of seven hemicellulosic subfractions were investigated by gel-permeation chromatography (GPC), and their weight-average (Mw) and number-average (Mn) molecular weights, the polydispersity (Mw/Mn) and the molecular weight distributions are given in Table 4 and Fig. 2. The molecular weight data were based on the amount of soluble material passing the column, which was denoted as GPC-recovery. Obviously, the first three hemicellulosic subfractions (HA1, HB1 and HC1), obtained from 0.5 mol L1 KOHsoluble hemicelluloses, showed a relative lower degree of polymerization with Mw values between 38 810 and 50 170 g mol1 than the other four hemicellulosic subfractions (HB2, HC2, HB3 and HC3) obtained from 1.0 to 2.0 mol L1 KOHsoluble hemicelluloses with Mw from 56 720 to 83 460 g mol1. This observation suggested that extraction with relative higher concentration of alkaline solution can result in dissolution of larger hemicellulosic polymers from the delignified materials. However, in this case, the degradation of polymers by relatively strong alkali can not be neglected. As can be seen from the Table 4, compared to the Mw values of the two hemicellulosic subfractions (HB2 and HC2) obtained from 1.0 mol L1 KOH-soluble hemicelluloses, the hemicellulosic subfractions (HB3 and HC3) obtained from 2.0 mol L1 KOHsoluble hemicelluloses showed lower molecular weights, which may be ascribe to the slight degradation of macromolecular structure of hemicelluloses under the treatment with a relative higher concentration of alkali used. In addition, the analysis of the first three hemicellulosic subfractions (HA1, HB1 and HC1), showed that the first hemicellulosic subfraction HA1 obtained by precipitation at pH 5.5 had higher molecular weights (50 170 g mol1) than the hemicellulosic subfractions HB1 (43 920 g mol1) and HC1 (38 810 g mol1), indicating that the hemicellulosic A subfraction had a higher molecular weight. Interestingly, compared with the hemicellulosic subfractions (HC1 HC2 and HC3) precipitated with 3 volumes of ethanol, the linear hemicellulosic subfractions (HB1, HB2 and HB3) obtained by
Table 4 e Weight-average (Mw) and number-average (Mn) molecular weights and polydispersity (Mw/Mn) of the hemicellulosic subfractions. Hemicellulosic subfractionsa
Mw Mn Mw/Mn
HA1
HB1
HC1
HB2
HC2
HB3
HC3
50170 29820 1.68
43920 28000 1.57
38810 21640 1.79
83460 68610 1.22
58970 37960 1.55
69150 60369 1.15
56720 38050 1.49
a Corresponding to the hemicellulosic subfractions in Table 2.
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Fig. 2 e Molecular weight distributions of the seven hemicellulosic subfractions.
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Fig. 3 e FT-IR spectra of the hemicellulosic subfractions HA1 (spectrum a), HB1 (spectrum b), and HC1 (spectrum c) obtained from the 0.5 mol LL1 KOH-soluble hemicelluloses.
precipitation with iodine-potassium iodide solution had a somewhat higher proportion of high molecular weight components. Based on this observation and Ara/Xyl ratio in the sugar composition (Table 3), it may be conclude that the hemicellulosic subfractions with a higher Ara/Xyl ratio had lower molecular weights. This result was in agreement with the previous study on water-extractable arabinoxylans by graded ethanol precipitation [26], and in contrast with that found by Gruppen and Dervilly [27,28], who used stepwise ethanol precipitation and chromatography to fractionate water- and alkali-soluble arabinoxylans. The relationship between Ara/Xyl ratio and molecular weight might be attributed to different methods of fractionation and different polymers. It should be noted that molecular weights of polymers varied depending on the method, solvent quality, and chain aggregation for their estimation [29]. Furthermore, all the subfractions showed a relatively low index of polydispersity (1.2e1.8), indicating a chemical and structural homogeneity within each subfraction.
3.4.
FT-IR spectra
FT-IR is one of the most widely used methods to identify chemical constituents and to elucidate their structures. Fig. 3 shows the FT-IR spectra of the hemicellulosic subfractions HA1 (spectrum a) obtained by precipitation at pH 5.5, HB1 (spectrum b) obtained by precipitation with iodine solution, and HC1 (spectrum c) obtained by precipitation with 3 volumes of
ethanol from the 0.5 mol L1 KOH-soluble hemicelluloses. The absorption at 1648 cm1 is principally associated with absorbed water, since the hemicelluloses usually give strong affinity for water [30]. The spectra have bands at 3405 cm1 of OeH stretching and at 2916 and 2846 cm1 of the CeH stretching [31]. The prominent band at 1043 cm1 is attributed to the CeO, CeC stretching or CeOH bending typical of xylans [30], and the absorption intensities decrease in the following order: HA1, HB1, and HC1, which correspond to the result obtained by sugar analysis. In the anomeric region (950e700 cm1), a small sharp band at 895 cm1 is clearly visible in spectrum of HA1, indicating that the presence of dominant b-glycosidic linkages between the sugar units in all the hemicellulosic subfractions, whereas the intensity of such signal becomes weak in the HB1 and HC1 spectra. The presence of the arabinosyl side chains is supported by the two bands at 1150 or 1140 and 986 cm1 [32]. However, the intensity of these two signals becomes strong in the spectrum of HB1 and HC1, which is probably due to the more arabinosyl unit and uronic acid attached xylopyranosyl constituents. The weak absorbance at 1509 cm1 in HB1 is originated from aromatic skeletal vibrations in associated lignin, indicating that HB1 is slightly contaminated with minimal amounts of bound lignin [33]. As expected, the absence of a signal at about 1730 cm1 in all the three spectra revealed that the alkaline extraction completely saponified the ester bonds, such as acetyl and uronic ester groups from the hemicelluloses. The small band at 1413 cm1 relates to the eCOO symmetric stretching of uronic acid
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Fig. 4 e FT-IR spectra of the hemicellulosic subfractions HB2 (spectrum a), HC2 (spectrum b), HB3 (spectrum c), and HC3 (spectrum d) obtained from the 1.0 and 2.0 mol LL1 KOH-soluble hemicelluloses.
Fig. 5 e 1H NMR spectrum of hemicellulosic subfraction HB1.
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Fig. 6 e 13C NMR spectrum of hemicellulosic subfraction HB1.
carboxylate [34], which is in accordance with the data obtained by sugar analysis. The remaining bands at 1461, 1321, 1248 and 1207 cm1 represent eCH2 symmetric bending, OH or CH2 bending vibrations. Fig. 4 illustrates the infrared absorption spectra of HB2 (spectrum a) and HB3 (spectrum c) obtained by precipitation with iodine-potassium iodide solution, HC2 (spectrum b) and HC3 (spectrum d) obtained by precipitation with 3 volumes of ethanol from the 1.0 and 2.0 mol L1 KOH-soluble hemicelluloses. Obviously, not much difference in the four spectra could be observed, indicating similar structures between the hemicellulosic subfractions, corresponding to the result of the sugar composition. The absorbances at 1623, 1463, 1317, 1244, 1161, 1039 and 900 cm1 are associated with the hemicelluloses, in which the band at 1039 cm1 is typical of xylans. Evidently, the presence of the arabinosyl side chains is documented by the low-intensity shoulder at 1161 cm1 [33]. In addition, the low intensity of the band at 984 cm1 also indicates the presences of arabinose, which have been reported to be attached the position 3 of the xylopyranosyl constituents [35]. The sharp absorption occurring at 900 cm1 shows the b configuration of the xylan linkages.
3.5.
1
H and
13
C NMR spectra
Nuclear magnetic resonance (NMR) spectroscopy was used to obtain more structural information on high molecular weight polysaccharides. 1H NMR spectrum of the hemicellulosic subfraction HB1, precipitated with iodine-potassium iodide solution from the 0.5 mol L1 alkali-soluble hemicelluloses is given in Fig. 5. The signals for 1H NMR were assigned based on the previously reported literature data [25, 36e43]. Obviously, the two regions of anomeric protons of arabinofuranosyl and glucuronic acid residues (5.0e5.4 ppm) and xylopyranosyl residues (4.4e4.6 ppm) are clearly discernible [36,37]. The signal at 5.33 ppm is assigned to terminal arabinose residues linked to
O-3 (mono-substituted) of the branched xylose units. Whereas, the signals at 5.29 and 5.24 ppm are due to arabinose linked to O-2 and O-3 of the double-branched of xylose residues. The signal at 5.03 ppm is attributed to glucuronic acid residues linked to the branched xylose units. The signals at 4.59, 4.57, and 4.42 ppm are indicative of the anomeric protons of b-Dxyloses substituted at C-2 and C-3 (di-substituted), C-3 (monosubstituted) and unsubstituted residues, respectively [38,39]. The other main chemical shifts at 4.42, 4.08, 3.71, 3.50, 3.30 and 3.24 ppm are assigned to the H-1, H-5 equatorial, H-4, H-3, H-5 axial and H-2 protons of the b-D-xylp units of hemicelluloses, respectively [40]. The methyl protons of 4-O-methyl-D-glucuronic acid produce peaks at 3.41 ppm [40]. The signals at 2.67, 1.21, and 1.13 ppm correspond to methylene and methyl groups in the residual ethanol. The peak at 1.82 ppm represents protons in CH3 from acetyl groups. A strong signal at 4.70 ppm arises from the residual solvent (HDO). To obtain further information about the anomeric linkage configuration of hemicelluloses, the 13C NMR spectroscopic analysis of the hemicellulosic subfraction HB1 was performed and the spectrum is shown in Fig. 6. The main 1, 4-linked xylopyranosyl units are obviously characterized by five strong signals at 101.5, 76.2, 73.5, 72.5 and 62.8 ppm, which respectively are assigned to C-1, C-4, C-3, C-2, and C-5 of the b-D-Xylp units. The signals at 107.8 and 60.9 ppm correspond to C-1 and C-5 of a-L-arabinofuranosyl residues, respectively. Other signals at 96.9, 82.2, 72.0, 71.1 and 59.6 ppm are characteristic of C-1, C-4, C-3, C-2, and 4-O-methoxyl group of glucuronic acid residue, respectively [40,41]. The signal at 22.8 ppm is most likely due to CH3 in acetyl in hemicelluloses [25]. Therefore, the hemicellulosic subfraction HB1, precipitated with iodine-potassium iodide solution from the 0.5 mol L1 alkali-soluble hemicelluloses, had a structure composed of a main of (1/4)-linked b-D-xylopyranosyl residues, with L-arabinofuranosyl and 4-O-methyl-a-D-glucuronic acid linked as side chains.
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
4.
Conclusions
On the basis of above results, it can be concluded that the alkali-soluble hemicelluloses would be successfully fractionated into linear and branched hemicellulosic subfractions by applying the iodine-complex precipitation technique, in which the alkali-soluble hemicelluloses of C. korshinskii can form a blue complex precipitation with iodine-potassium iodide in the KCl solution. It was found that the linear hemicellulosic subfractions were precipitated by iodine-potassium iodide solution and the branched ones remained in the solution. In addition, the linear hemicellulosic subfractions with a low Ara/Xyl ratio had higher molecular weights than those of the branched hemicellulosic subfractions. Furthermore, these alkali-soluble hemicellulosic polymers from delignified C. korshinskii comprised a backbone of b-(1/4)-linked xylosyl residues substituted with arabinose and 4-O-methyl-D-glucuronic acid at C-2 and/or C-3 of the main chain.
Acknowledgements The authors wish to express their gratitude for the financial support from the Major State Basic Research Projects of China (973-2010CB732204), the National Natural Science Foundation of China (30930073, 30710103906), Ministries of Education (111, NCET-08-0728), State Forestry Administration (200804015), and Hei Long Jiang Province for Distinguished Young Scholars (JC200907).
references
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[10] Parajo JC, Garrote G, Cruz JM, Dominguez H. Production of xylooligosaccharides by autohydrolysis of lignocellulosic materials. Trends Food Sci Technol 2004;15:115e20. [11] Garrote G, Dominguez H, Parajo JC. Production of substituted oligosaccharides by hydrolytic processing of barley husks. Ind Eng Chem Res 2004;43:1608e14. [12] Lindblad MS, Ranucci E, Albertsson AC. Biodegradable polymers from renewable sources. New hemicellulose-based hydrogels. Macromol Rapid Comm 2001;22:962e7. [13] Grondahl M, Eriksson L, Gatenholm P. Material properties of plasticized hardwood xylans for potential application as oxygen barrier films. Biomacromolecules 2004;5: 1528e35. [14] Hartman J, Albertsson AC, Lindblad MS, Sjoberg J. Oxygen barrier materials from renewable sources: material properties of softwood hemicellulose-based films. J Appl Polym Sci 2006;100:2985e91. [15] Watson SA. Corn hull gum, in industrial gums. In: Whistler RL, BeMiller JN, editors. Polysaccharides and their derivatives. New York: Academic Press; 1959. p. 299e306. [16] Hoije A, Grondahl M, Tommeraas K, Gatenholm P. Isolation and characterization of physicochemical and material properties of arabinoxylans from barley husks. Carbohydr Polym 2005;61:266e75. [17] Peng F, Ren JL, Xu F, Bian J, Peng P, Sun RC. Comparative study of hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. J Agric Food Chem 2009;57:6305e17. [18] Cyran MR, Saulnier L. Association and structural diversity of hemicelluloses in the cell walls of rye outer layers: comparison between two ryes with opposite breaking quality. J Agric Food Chem 2007;55:2329e41. [19] Subba Rao MVSST, Muralikrishna G. Hemicelluloses of ragi (finger millet, eleusine coracana, indaf-15): isolation and purification of an alkali-extractable arabinoxylan from native and malted hemicellulose B. J Agric Food Chem 2006; 54:2342e9. [20] Gaillard BGE. Separation of linear from branched polysaccharides by precipitation as iodine complexs. Nature 1961;191:1295e6. [21] Lawther JM, Sun RC, Banks WB. Extraction, fractionation, and characterization of structural polysaccharides from wheat-straw. J Agric Food Chem 1995;43:667e75. [22] Lunsqvist J, Teleman A, Junel L, Zacchi G, Dahlman O, Tjerneld F, et al. Isolation and characterization of acetylated galactoglucomannan. Carbohydr Polym 2002;48:29e39. [23] Gaillard BDE, Thompson NS. Interaction of polysaccharides with iodine. 2. Behavior of xylans in different salt solutions. Carbohydr Res 1971;18:137e46. [24] Gaillard BDE, Thompson NS, Morak AJ. Interaction of polysaccharides with iodine. 1. Investigation of general nature of reaction. Carbohydr Res 1959;11:509e15. [25] Chaikumpollert O, Methacanon P, Suchiva K. Structural elucidation of hemicelluloses from Vetiver grass. Carbohydr Polym 2004;57:191e6. [26] Roels SP, Collado M, Loosveld AM, Grobet PJ, Delcour JA. Variation in the degree of D-xylose substitution in waterextractable European Durum Wheat (Triticum durum Desf.) Semolina arabinoxylans. J Agric Food Chem 1999;47:1813e6. [27] Dervilly G, Saulnier L, Roger P, Thibault JF. Isolation of homogeneous fractions from wheat water-soluble arabinoxylans. Influence of the structure on their macromolecular characteristics. J Agric Food Chem 2000;48: 270e8. [28] Gruppen H, Hamer RJ, Voragen AGJ. Water-unextractable cell wall material from wheat. 2. Fraction of alkali-extracted polymers and comparison with water-exactable arabinoxylans. J Cereal Sci 1992;16:53e67.
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http://www.elsevier.com/locate/biombioe
Fractionation and characterization of alkali-extracted hemicelluloses from peashrub Feng Peng a, Jing Bian b, Jun-Li Ren a, Pai Peng a, Feng Xu b,*, Run-Cang Sun a,b,* a b
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China
article info
abstract
Article history:
Alkali-soluble hemicelluloses were extracted from delignified peashrub (Caragana kor-
Received 1 February 2010
shinskii) by sequential treatments with 0.5, 1.0 and 2.0 mol L1 KOH under a solid to liquid
Received in revised form
ratio of 1:25 (g mL1) at 25 C for 10 h. The successive treatments resulted in a total
2 May 2010
dissolution of 64.1, 15.6, and 12.5% of the original hemicelluloses, respectively. The
Accepted 4 August 2010
released hemicelluloses were separated into linear and branched hemicellulosic sub-
Available online 30 August 2010
fractions by the iodine-complex precipitation technique. Their chemical and physical
Keywords:
was found that the hemicelluloses precipitated by iodine-potassium iodide solution were
C. korshinskyii
more linear as shown they contained more xylose and less uronic acid than the hemi-
Hemicelluloses
celluloses remaining in the solution, which were more branched and contained higher
Alkali
amounts of arabinose, galactose and glucose. In addition, the linear hemicellulosic sub-
Fractionation
fractions with a low arabinose/xylose ratio had higher molecular weights than those of the
Iodine-complex precipitation
branched hemicellulosic subfractions in solution.
characteristics were determined by HPAEC, GPC, FT-IR and 1H and 13C NMR spectroscopy. It
ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
In recent years, biomass has been pointed out as the most important source for a broad variety of advanced polymeric products such as fuels, paints, detergents, biodegradable polymers, textile fibers, fiber composites, and various other commodity and special chemicals. The biomass, such as shrubs, grass, essentially consisted of three different polymer entities (cellulose, hemicelluloses and lignin). The use of all the three polymeric components has been discussed since several decades. The discussion had been intensified in connection with emerging biorefinery strategies [1,2]. Recently, much attention has been paid to utilizing forest and agricultural residues as raw material for industries. Caragana korshinskii is a widely distributed and drought-resistant
leguminous shrub, which is widely used for vegetation rehabilitation in arid and semiarid regions of China, for its high ecological and economic value [3]. This shrub generates a large amount of residues, because its stems are cut once every 3 years to make it flourish. At present, only a small amount of the cut peashrub materials is used for the production of fiberboard, but a huge amount is not used and constitutes an environmental problem. Peashrub as an agroindustrial residue, therefore, can be valuable by developing chemical methods in providing transport fuels or chemical feedstock. In the current trend for a complex and more effective utilization of biomass, increasing attention has been paid during the last few years to exploitation of hemicelluloses as biopolymer resources. Hemicelluloses are the second most
* Corresponding author at: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China. Tel./fax: þ86 10 62336972. E-mail addresses: [email protected], [email protected] (F. Xu). 0961-9534/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2010.08.034
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
abundant biopolymer in the plant kingdom. They are lowmolecular-weight polysaccharides, associated in plant cell walls with celluloses and lignin. In plant cell walls large amounts of hemicelluloses occur with a wide variation in content and chemical structure. In hardwoods, the principal hemicelluloses are O-acetyl-4-O-methylglucuronoxylan with amounts between 10% and 35% depending on the species. This polysaccharide consists of about 200-b-xylopyranose residues, linked together by 1, 4-glycosidic bonds. Approximately every tenth xylose unit carries a single, terminal side chain, and consists of 4-O-methylglucuronic acid attached directly to the 2- position of xylose. Seven out of 10 xylose residues contain an O-acetyl group at C-2, at C-3, or at both positions [4]. Further on small portions of glucomannan (3e5%) are found in hardwoods [5,6]. Nowadays, some important applications for hemicelluloses have been discovered. In monomeric form, they can be fermented to ethanol, acetone, butanol [7], or xylitol [8], in oligomeric form, which can be used as functional-food additive [9e11], and in polymeric form, they are can be used as hydrogels [12], biodegradable barrier films for food packing [13,14], and wet strength additives [15]. The components in lignocellulosic materials are tightly associated and in several processes it has been proved to difficult to remove the lignin from the hemicelluloses and cellulose without modifying the hemicelluloses [16]. Therefore, in order to obtain the hemicellulosic polymers having a high yield and purity, the material must be delignified and/or pre-treated in some way prior to extraction. In particular, a delignification step using chlorite prior to extraction of hemicelluloses can significantly facilitate the extraction. In this case, most of the lignin can be removed without any noticeable degradation of hemicellulosic polymers. In addition, different fractionation techniques, such as graded ethanol precipitation [17], ammonium sulphate precipitation and anion-exchange chromatography [18,19], have been used to obtain deeper insight into the diversity of hemicelluloses. Another fractionation technique was that linear polysaccharides can be separated from branched polymers by precipitation as iodine complexes in the presence of CaCl2, which was discovered by Gaillard [20]. The aim of the fractionation was to obtain more homogeneous fractions and thus explore structureeproperty relationships of the polymers, which might obtain the low-branched xylans for further producing the xylose, an intermediate for the production of xylitol, and a variety of xylo-oligosaccharides. The present work was primarily carried out to gain a new knowledge on the structure features of hemicelluloses from the delignified C. korshinskii by applying the iodine-complex precipitation technique in the presence of KCl, and investigate the structural variation and physicochemical properties of hemicellulosic subfractions.
2.
Materials and methods
2.1.
Materials
Peashrub (C. Korshinskii), 5 years old, with an average stem height of 2.8 m, was harvested in October 2004 in Yanchi
21
County, Ningxia province, China. The place is located in the west of the loess plateau and lies in the transitional zone between arid and semiarid regions (107 240 E, 37 470 N) at an altitude of 1349 m. The soil is silt loam of loess origin, which can be classified as Calciorthid. Generally, peashrub turns green in April, blooms in May, and sets seed and senesces in July. The leaves and the bark of peashrub were removed at the harvest time. The trunks were dried in sunlight, and then chipped into small pieces about 1e2 cm with axe. The chips (200 g) were ground to pass a 0.8-mm-size screen. Ground samples (150 g) were obtained and stored in plastic containers at room temperature. Fats, waxes and oils were removed from the ground materials in a Soxhlet apparatus refluxing for 6 h with 2:1 (v/v) tolueneeethanol. The dewaxed sample was further dried in a cabinet oven with air circulation at 60 C for 16 h and stored. The components (%, w/w) of the C. Korshinskii were cellulose 41.6%, hemicelluloses 31.5%, lignin 21.7%, ash 0.7%, and wax 4.5% on a dry weight basis, which was determined by the method for measuring the chemical composition of wheat straw described previously [21]. All standard chemicals, such as sugars and phenolics, were analytical grade, purchased from Sigma Chemical Company (Beijing).
2.2.
Isolation and fractionation of hemicelluloses
In order to study structural differences of the hemicelluloses present in Caragana Korshinski, hemicellulosic fractions were obtained by sequential extraction and fractionation according to the scheme in Fig. 1. The dewaxed powder (15 g) was delignified with 6% sodium chlorite at pH 3.6e3.8, adjusting with 10% acetic acid, at 75 C for 2 h [22]. The residue, holocellulose, was subsequently washed with distilled water and ethanol, and dried at 60 C for 16 h. Then the holocellulose was successively extracted by 0.5, 1.0 and 2.0 mol L1 KOH solution with a solid to liquid ratio of 1: 25 (g mL1) at 25 C for 10 h under stirring. After the indicated period treatment, the insoluble residues were filtered through a nylon cloth on a Bu¨chner funnel, washed with distilled water until the pH of filtrates was neutral, and dried at 60 C. Each of filtrates was adjusted to pH 5.5 with 6 mol L1 HCl. Then the filtrate was concentrated to about 100 mL. After standing for 12 h, the solution was centrifuged (2594 g, 15 min). The precipitate obtained was washed with 70% ethanol, and freeze-dried, and named as hemicelluloses A (HA). Then 10 mL of iodine solution (3 g iodine and 4 g KI in 100 mL water) was added in the supernatant. In this case, the solution of hemicelluloses was aqueous potassium chloride, instead of aqueous calcium chloride [20]. After stirring and standing for 12 h, the solution was centrifuged (2594 g, 15 min). The precipitate was washed with 70% ethanol, freeze-dried, and referred to as the hemicelluloses B (HB). The supernatant were then titrated with Na2S2O3 solution, dialyzed and precipitated with three volumes of ethanol. The precipitated hemicelluloses were named as hemicelluloses C (HC), washed with 70% ethanol and freeze-dried. Note that hemicelluloses A is precipitated by neutralization of the 0.5, 1.0 and 2.0 mol L1 KOH extracts to pH 5.5 with hydrochloric acid (named as HA1, HA2 and HA3), hemicelluloses B is isolated by precipitation with iodinepotassium iodide solution (labeled as HB1, HB2 and HB2), and
22
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Fig. 1 e Scheme for fractionation of alkali-soluble hemicelluloses from C. korshinskii.
the remaining portion in solution is obtained by precipitation with 3 volumes of ethanol (named as HC1, HC2 and HC3).
2.3.
Chemical characterization
The constituent neutral sugars in the isolated hemicellulosic fractions were determined by high performance anionexchange chromatography (HPAEC). The hemicelluloses (4e6 mg) were hydrolyzed by 10% H2SO4 at 105 C in a sealed tube for 2.5 h. After hydrolysis, the samples were diluted 30-fold, filtered and injected into the HPAEC system (Dionex ISC 3000,U.S) with amperometric detector, AS50 autosampler and a CarbopacTM PA1 column (4 250 mm, Dionex) [17]. The
molecular weights of the hemicellulosic fractions were determined by gel-permeation chromatography (GPC) on a PL aquagel-OH 50 column (300 7.7 mm, polymer laboratories Ltd), calibrated with PL pullulan polysaccharide standards (peak average molecular weights 783, 12 200, 100 000, 1600 000) [17]. The measurements were conducted with two parallels and the reproducibility of the values was found within the range of 6%.
2.4.
Spectroscopic characterization
FT-IR spectra of hemicellulosic samples were obtained on an FT-IR spectrophotometer (Nicolet 510) using a KBr disc
23
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Table 1 e Yields of hemicelluloses solubilized during the successive treatments of delignified C. korshinskyii with 0.5, 1.0 and 2.0 mol LL1 KOH at 25 C for 10 h. Fraction
Yield (% dry matter)
Total solubilized hemicelluloses during the successive treatments with 0.5, 1.0 and 2.0 mol L1 KOH Solubilized hemicelluloses in 0.5 mol L1 KOH treatment Solubilized hemicelluloses in 1.0 mol L1 KOH treatment Solubilized hemicelluloses in 2.0 mol L1 KOH treatment Residue (crude cellulose)
29.1
20.2 4.9 4.0 45.7
containing 1% finely ground samples. Thirty-two scans were taken of each sample recorded from 4000 to 400 cm1 at a resolution of 2 cm1 in the transmission mode. The solutionstate 1H NMR spectrum was recorded on a Bruker MSL300 spectrometer at 300 MHz using 15 mg of hemicelluloses in 1.0 mL of D2O. A 13C NMR spectrum was obtained on a Bruker MSL300 spectrometer at 74.5 MHz. The sample (80 mg) was dissolved in 1 mL D2O (99.8% D) with overnight stirring at room temperature. The spectrum was recorded at 25 C after 30 000 scans. Chemical shifts (d in ppm) are expressed relative to the resonance of Me4Si (d ¼ 0). A 60 pulse flipping angle, a 3.9 ms pulse width and a 0.85 s delay time between scans were used.
3.
Results and discussion
3.1.
Yield of hemicelluloses
Many attempts have been made to isolate hemicelluloses from various biomass resources, in which alkali extraction is the most efficient method for isolating large amounts of hemicellulosic polysaccharides. It is known that hydroxyl ions causes swelling of cellulose, hydrolysis of ester linkage, and disruption of intermolecular hydrogen bonds between celluloses and hemicelluloses, bring a portion of the hemicellulosic material into solution. The isolation and fractionation of hemicelluloses from C. korshinskii is presented schematically in Fig. 1, and the yields of the soluble hemicelluloses are given
in Table 1. Sequential extractions of the delignified C. korshinskii with 0.5, 1.0, 2.0 mol L1 KOH aqueous solution at 25 C for 10 h resulted in dissolution of 20.2, 4.9, and 4.0% hemicelluloses, corresponding to release of 64.1, 15.6, and 12.5% of original hemicelluloses, respectively. Meanwhile, the three treatments together yielded 29.1% hemicelluloses and account for 92.4% of the original hemicelluloses. This indicated that alkali extraction is the efficient method for isolating large amounts of hemicellulosic polysaccharides. Obviously, the majority of the hemicelluloses were obtained by the treatment with 0.5 mol L1 KOH under the condition used. In this study, the three alkali-soluble hemicelluloses were further fractioned into nine subfractions (Fig. 1). The yields of nine hemicellulosic subfractions are shown in Table 2. As can be seen, the yield of the subfractions from the 0.5 mol L1 KOH-soluble hemicelluloses was 6.7, 1.8, 7.0% of the dry materials, corresponding to precipitation of 33.2, 8.9, 34.7% of the total 0.5 mol L1 KOH-soluble hemicelluloses. However, it should be noted that the yields of the hemicellulosic subfractions (HA2 and HA3) obtained by precipitation at pH 5.5 were only a trace amount, thus HA2 and HA3 were not studied in the subsequent analysis. This indicated that the large amount of hemicelluloses A was solubilized only in 0.5 mol L1 KOH aqueous solution. In addition, the dark blue complex of hemicellulosic subfractions HB1, HB2 and HB3 precipitated by iodine solution was separated by high-speed centrifugation. Under this condition, 1.8, 1.6, and 1.2% hemicelluloses (percent dry staring material) were precipitated, corresponding to precipitation of 8.9, 32.6, and 30.0% of the 0.5, 1.0, 2.0 mol L1 KOH-soluble hemicelluloses, respectively. This result revealed the hemicelluloses can form a blue complex by precipitation with iodine in the KCl solution, which did not agree with that alkali metal chloride solution can not replace the CaCl2 solution in this reaction [23,24]. Clearly, the total yield of nine hemicellulosic subfractions accounted for 79.0% of total dissolved hemicelluloses during the sequential fractionations, indicating that 21.0% hemicelluloses, mainly degraded substance such as oligosaccharides, were not recovered.
3.2.
Sugar composition
The neutral sugar composition and the content of uronic acid of seven hemicellulosic subfractions are listed in Table 3.
Table 2 e The yield of hemicellulosic subfractions (% dry starting matter) obtained from 0.5, 1.0 and 2.0 mol LL1 KOHsoluble hemicelluloses. Yield/content
1.0 mol L1 KOH-soluble hemicelluloses
0.5 mol L1 KOH-soluble hemicelluloses
2.0 mol L1 KOH-soluble hemicelluloses
Subfraction
HA1a
HB1b
HC1c
HA2a
HB2b
HC2c
HA3a
HB3b
HC3c
Yield of hemicelluloseic subfraction
6.7
1.8
7.0
Trd
1.6
2.8
Trd
1.2
2.0
a b c d
Represents the hemicelluloses obtained by precipitation at pH 5.5 in aqueous solution. Represents the hemicellulosic subfractions obtained by precipitation with iodine-potassium iodide solution. Represents the hemicellulosic subfractions obtained by precipitation with 3 volumes of ethanol. Tr ¼ trace.
Total
23.0
24
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Table 3 e Contents of neutral sugars and uronic acids (relative percent dry hemicelluloses, w/w) in hemicellulosic subfractions. Hemicellulosic subfractionsa
Sugars (%) HA1 Rhamnose Arabinose Galactose Glucose Xylose Uronic acid Ara/Xyl
HB1
HC1
HB2
HC2
HB3
HC3
0.10 1.09 1.04 0.75 0.62 0.60 6.02 6.91 11.43 1.73 6.10 2.17 1.33 1.91 4.14 0.37 3.43 0.22 0.56 1.31 8.97 1.26 4.87 1.59 91.09 88.78 74.42 94.92 84.97 95.42 2.75 1.35 2.52 1.15 2.58 0.97 0.067 0.078 0.15 0.018 0.072 0.023
Trb 5.66 5.05 9.00 80.30 1.94 0.070
a Corresponding to the hemicellulosic subfractions in Table 2. b Tr ¼ trace.
Obviously, xylose was the dominant sugar component (74.4e95.4%). Arabinose and glucose appeared as the second and the third major sugars, comprising 1.7e11.4% and 0.6e9.0% of total sugars, respectively. Galactose (0.2e5.1%), rhamnose (0.1e1.1%) and uronic acid (mainly glucuronic acid or its 4-O-methyl derivative, 0.9e2.8%) were observed as minor constituents. The dominance of xylose residues probably were originated from the backbone of xylan. The presence of a noticeable amount of arabinose and minor quantity of uronic acids implied that the alkali-soluble hemicelluloses consisted of side chains of arabinose and glucuronic acid, which need to be further investigated, since based on the sugar composition alone it is difficult to draw conclusions on the structural patterns of the hemicelluloses. Moreover, a noticeable amount of glucose from the hemicellulosic subfractions (HC1, HC2, HC3) may be a part of the xylan or present as a glucan, since they are precipitated only on addition of alcohol after iodine-precipitation. Additionally, compared with HB1 and HC1, hemicellulosic subfraction HA1 obtained by precipitation at pH 5.5 from the 0.5 mol L1 NaOH-soluble hemicelluloses, have a higher contents of xylose (91.1%) and uronic acid (2.75%), but the ratio of arabinose to xylose (Ara/Xyl, 0.07) of HA1 is lower than HB1 (0.08) and HC1 (0.15). This indicated that hemicelluloses B and C were more highly branched than hemicelluloses A, and the hemicelluloses A consisted of a very lowly-substituted population, since Ara/Xyl ratios are indicative of the degree of linearity or branching of hemicelluloses [25]. Moreover, from the composition data of HB1 and HC1, the xylose decreased from 88.8% (HB1) to 74.4% (HC1), whereas the arabinose, galactose, glucose, uronic acid and Ara/Xyl increased. Simliar
results were found in the four hemicellulosic subfractions (HB2, HC2, and HB3, HC3) obtained from the 1.0 and 2.0 mol L1 KOH-soluble hemicelluloses. Thus, it can be concluded that the linear hemicelluloses are precipitated by iodine-potassium iodide solution, and the branched ones remain in the solution, which would be isolated by precipitation with 3 volumes of ethanol.
3.3.
Molar mass
The molecular weights of seven hemicellulosic subfractions were investigated by gel-permeation chromatography (GPC), and their weight-average (Mw) and number-average (Mn) molecular weights, the polydispersity (Mw/Mn) and the molecular weight distributions are given in Table 4 and Fig. 2. The molecular weight data were based on the amount of soluble material passing the column, which was denoted as GPC-recovery. Obviously, the first three hemicellulosic subfractions (HA1, HB1 and HC1), obtained from 0.5 mol L1 KOHsoluble hemicelluloses, showed a relative lower degree of polymerization with Mw values between 38 810 and 50 170 g mol1 than the other four hemicellulosic subfractions (HB2, HC2, HB3 and HC3) obtained from 1.0 to 2.0 mol L1 KOHsoluble hemicelluloses with Mw from 56 720 to 83 460 g mol1. This observation suggested that extraction with relative higher concentration of alkaline solution can result in dissolution of larger hemicellulosic polymers from the delignified materials. However, in this case, the degradation of polymers by relatively strong alkali can not be neglected. As can be seen from the Table 4, compared to the Mw values of the two hemicellulosic subfractions (HB2 and HC2) obtained from 1.0 mol L1 KOH-soluble hemicelluloses, the hemicellulosic subfractions (HB3 and HC3) obtained from 2.0 mol L1 KOHsoluble hemicelluloses showed lower molecular weights, which may be ascribe to the slight degradation of macromolecular structure of hemicelluloses under the treatment with a relative higher concentration of alkali used. In addition, the analysis of the first three hemicellulosic subfractions (HA1, HB1 and HC1), showed that the first hemicellulosic subfraction HA1 obtained by precipitation at pH 5.5 had higher molecular weights (50 170 g mol1) than the hemicellulosic subfractions HB1 (43 920 g mol1) and HC1 (38 810 g mol1), indicating that the hemicellulosic A subfraction had a higher molecular weight. Interestingly, compared with the hemicellulosic subfractions (HC1 HC2 and HC3) precipitated with 3 volumes of ethanol, the linear hemicellulosic subfractions (HB1, HB2 and HB3) obtained by
Table 4 e Weight-average (Mw) and number-average (Mn) molecular weights and polydispersity (Mw/Mn) of the hemicellulosic subfractions. Hemicellulosic subfractionsa
Mw Mn Mw/Mn
HA1
HB1
HC1
HB2
HC2
HB3
HC3
50170 29820 1.68
43920 28000 1.57
38810 21640 1.79
83460 68610 1.22
58970 37960 1.55
69150 60369 1.15
56720 38050 1.49
a Corresponding to the hemicellulosic subfractions in Table 2.
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Fig. 2 e Molecular weight distributions of the seven hemicellulosic subfractions.
25
26
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Fig. 3 e FT-IR spectra of the hemicellulosic subfractions HA1 (spectrum a), HB1 (spectrum b), and HC1 (spectrum c) obtained from the 0.5 mol LL1 KOH-soluble hemicelluloses.
precipitation with iodine-potassium iodide solution had a somewhat higher proportion of high molecular weight components. Based on this observation and Ara/Xyl ratio in the sugar composition (Table 3), it may be conclude that the hemicellulosic subfractions with a higher Ara/Xyl ratio had lower molecular weights. This result was in agreement with the previous study on water-extractable arabinoxylans by graded ethanol precipitation [26], and in contrast with that found by Gruppen and Dervilly [27,28], who used stepwise ethanol precipitation and chromatography to fractionate water- and alkali-soluble arabinoxylans. The relationship between Ara/Xyl ratio and molecular weight might be attributed to different methods of fractionation and different polymers. It should be noted that molecular weights of polymers varied depending on the method, solvent quality, and chain aggregation for their estimation [29]. Furthermore, all the subfractions showed a relatively low index of polydispersity (1.2e1.8), indicating a chemical and structural homogeneity within each subfraction.
3.4.
FT-IR spectra
FT-IR is one of the most widely used methods to identify chemical constituents and to elucidate their structures. Fig. 3 shows the FT-IR spectra of the hemicellulosic subfractions HA1 (spectrum a) obtained by precipitation at pH 5.5, HB1 (spectrum b) obtained by precipitation with iodine solution, and HC1 (spectrum c) obtained by precipitation with 3 volumes of
ethanol from the 0.5 mol L1 KOH-soluble hemicelluloses. The absorption at 1648 cm1 is principally associated with absorbed water, since the hemicelluloses usually give strong affinity for water [30]. The spectra have bands at 3405 cm1 of OeH stretching and at 2916 and 2846 cm1 of the CeH stretching [31]. The prominent band at 1043 cm1 is attributed to the CeO, CeC stretching or CeOH bending typical of xylans [30], and the absorption intensities decrease in the following order: HA1, HB1, and HC1, which correspond to the result obtained by sugar analysis. In the anomeric region (950e700 cm1), a small sharp band at 895 cm1 is clearly visible in spectrum of HA1, indicating that the presence of dominant b-glycosidic linkages between the sugar units in all the hemicellulosic subfractions, whereas the intensity of such signal becomes weak in the HB1 and HC1 spectra. The presence of the arabinosyl side chains is supported by the two bands at 1150 or 1140 and 986 cm1 [32]. However, the intensity of these two signals becomes strong in the spectrum of HB1 and HC1, which is probably due to the more arabinosyl unit and uronic acid attached xylopyranosyl constituents. The weak absorbance at 1509 cm1 in HB1 is originated from aromatic skeletal vibrations in associated lignin, indicating that HB1 is slightly contaminated with minimal amounts of bound lignin [33]. As expected, the absence of a signal at about 1730 cm1 in all the three spectra revealed that the alkaline extraction completely saponified the ester bonds, such as acetyl and uronic ester groups from the hemicelluloses. The small band at 1413 cm1 relates to the eCOO symmetric stretching of uronic acid
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Fig. 4 e FT-IR spectra of the hemicellulosic subfractions HB2 (spectrum a), HC2 (spectrum b), HB3 (spectrum c), and HC3 (spectrum d) obtained from the 1.0 and 2.0 mol LL1 KOH-soluble hemicelluloses.
Fig. 5 e 1H NMR spectrum of hemicellulosic subfraction HB1.
27
28
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
Fig. 6 e 13C NMR spectrum of hemicellulosic subfraction HB1.
carboxylate [34], which is in accordance with the data obtained by sugar analysis. The remaining bands at 1461, 1321, 1248 and 1207 cm1 represent eCH2 symmetric bending, OH or CH2 bending vibrations. Fig. 4 illustrates the infrared absorption spectra of HB2 (spectrum a) and HB3 (spectrum c) obtained by precipitation with iodine-potassium iodide solution, HC2 (spectrum b) and HC3 (spectrum d) obtained by precipitation with 3 volumes of ethanol from the 1.0 and 2.0 mol L1 KOH-soluble hemicelluloses. Obviously, not much difference in the four spectra could be observed, indicating similar structures between the hemicellulosic subfractions, corresponding to the result of the sugar composition. The absorbances at 1623, 1463, 1317, 1244, 1161, 1039 and 900 cm1 are associated with the hemicelluloses, in which the band at 1039 cm1 is typical of xylans. Evidently, the presence of the arabinosyl side chains is documented by the low-intensity shoulder at 1161 cm1 [33]. In addition, the low intensity of the band at 984 cm1 also indicates the presences of arabinose, which have been reported to be attached the position 3 of the xylopyranosyl constituents [35]. The sharp absorption occurring at 900 cm1 shows the b configuration of the xylan linkages.
3.5.
1
H and
13
C NMR spectra
Nuclear magnetic resonance (NMR) spectroscopy was used to obtain more structural information on high molecular weight polysaccharides. 1H NMR spectrum of the hemicellulosic subfraction HB1, precipitated with iodine-potassium iodide solution from the 0.5 mol L1 alkali-soluble hemicelluloses is given in Fig. 5. The signals for 1H NMR were assigned based on the previously reported literature data [25, 36e43]. Obviously, the two regions of anomeric protons of arabinofuranosyl and glucuronic acid residues (5.0e5.4 ppm) and xylopyranosyl residues (4.4e4.6 ppm) are clearly discernible [36,37]. The signal at 5.33 ppm is assigned to terminal arabinose residues linked to
O-3 (mono-substituted) of the branched xylose units. Whereas, the signals at 5.29 and 5.24 ppm are due to arabinose linked to O-2 and O-3 of the double-branched of xylose residues. The signal at 5.03 ppm is attributed to glucuronic acid residues linked to the branched xylose units. The signals at 4.59, 4.57, and 4.42 ppm are indicative of the anomeric protons of b-Dxyloses substituted at C-2 and C-3 (di-substituted), C-3 (monosubstituted) and unsubstituted residues, respectively [38,39]. The other main chemical shifts at 4.42, 4.08, 3.71, 3.50, 3.30 and 3.24 ppm are assigned to the H-1, H-5 equatorial, H-4, H-3, H-5 axial and H-2 protons of the b-D-xylp units of hemicelluloses, respectively [40]. The methyl protons of 4-O-methyl-D-glucuronic acid produce peaks at 3.41 ppm [40]. The signals at 2.67, 1.21, and 1.13 ppm correspond to methylene and methyl groups in the residual ethanol. The peak at 1.82 ppm represents protons in CH3 from acetyl groups. A strong signal at 4.70 ppm arises from the residual solvent (HDO). To obtain further information about the anomeric linkage configuration of hemicelluloses, the 13C NMR spectroscopic analysis of the hemicellulosic subfraction HB1 was performed and the spectrum is shown in Fig. 6. The main 1, 4-linked xylopyranosyl units are obviously characterized by five strong signals at 101.5, 76.2, 73.5, 72.5 and 62.8 ppm, which respectively are assigned to C-1, C-4, C-3, C-2, and C-5 of the b-D-Xylp units. The signals at 107.8 and 60.9 ppm correspond to C-1 and C-5 of a-L-arabinofuranosyl residues, respectively. Other signals at 96.9, 82.2, 72.0, 71.1 and 59.6 ppm are characteristic of C-1, C-4, C-3, C-2, and 4-O-methoxyl group of glucuronic acid residue, respectively [40,41]. The signal at 22.8 ppm is most likely due to CH3 in acetyl in hemicelluloses [25]. Therefore, the hemicellulosic subfraction HB1, precipitated with iodine-potassium iodide solution from the 0.5 mol L1 alkali-soluble hemicelluloses, had a structure composed of a main of (1/4)-linked b-D-xylopyranosyl residues, with L-arabinofuranosyl and 4-O-methyl-a-D-glucuronic acid linked as side chains.
b i o m a s s a n d b i o e n e r g y 3 9 ( 2 0 1 2 ) 2 0 e3 0
4.
Conclusions
On the basis of above results, it can be concluded that the alkali-soluble hemicelluloses would be successfully fractionated into linear and branched hemicellulosic subfractions by applying the iodine-complex precipitation technique, in which the alkali-soluble hemicelluloses of C. korshinskii can form a blue complex precipitation with iodine-potassium iodide in the KCl solution. It was found that the linear hemicellulosic subfractions were precipitated by iodine-potassium iodide solution and the branched ones remained in the solution. In addition, the linear hemicellulosic subfractions with a low Ara/Xyl ratio had higher molecular weights than those of the branched hemicellulosic subfractions. Furthermore, these alkali-soluble hemicellulosic polymers from delignified C. korshinskii comprised a backbone of b-(1/4)-linked xylosyl residues substituted with arabinose and 4-O-methyl-D-glucuronic acid at C-2 and/or C-3 of the main chain.
Acknowledgements The authors wish to express their gratitude for the financial support from the Major State Basic Research Projects of China (973-2010CB732204), the National Natural Science Foundation of China (30930073, 30710103906), Ministries of Education (111, NCET-08-0728), State Forestry Administration (200804015), and Hei Long Jiang Province for Distinguished Young Scholars (JC200907).
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