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Comparative study of hemicelluloses from wheat straw by alkali and hydrogen peroxide extractions
Polymer Degradation and Stability 66 (1999) 423±432
Comparative study of hemicelluloses from wheat straw by alkali and hydrogen peroxide extractions J.M. Fang, R.C. Sun*, D. Salisbury, P. Fowler, J. Tomkinson The BioComposites Centre, University of Wales, Bangor, Gwynedd, LL57 2UW, UK Received 9 April 1999; accepted 24 May 1999
Abstract As compared to traditional alkaline extractions, alkaline peroxide was used to isolate hemicelluloses from wheat straw. Yields of the solubilized hemicelluloses ranged from 18.9% (2% H2O2 extraction at 90 C for 2 h at pH 11.5) to 26.6% (2% H2O2 extraction at 50 C for 16 h at pH 12.5). The optimum hemicellulose yield (92% of the original hemicelluloses in water treated wheat straw) was obtained when the treatment was performed at 50 C for 16 h at pH 12.5 by use of 2% H2O2. All the hemicellulosic preparations were much lighter in color than those obtained using traditional alkaline extractions in the absence of bleaching. The results, obtained by the destructive method such as acid hydrolysis, showed that extraction of wheat straw with aqueous 10% KOH following alkaline bleaching released the hemicellulosic fractions, which were enriched in xylose, whereas extraction of the straw with aqueous 2% H2O2 under alkaline conditions (pH 11.5±12.5) solubilized the hemicellulosic fractions, which were relatively higher in arabinose and glucose. The nine isolated hemicellulosic samples were further characterized by non-destructive methods such as Fourier transform infrared (FT±IR), and carbon-13 magnetic resonance spectroscopy (13C-NMR) as well as gel permeation chromatography (GPC). It has been demonstrated that the alkaline peroxide treatments under the conditions used do not aect the overall structure of hemicelluloses. The only one major change of the hemicelluloses was found to be degradation during the beaching of alkali-soluble hemicelluloses with 2% H2O2 at 60 C for a period of 16 h at pH 11.5 or bleaching using a relatively higher concentration (5%) of hydrogen peroxide at 60 C for 8 h at pH 11.5. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Wheat straw is an agricultural by-product that is not used as industrial raw material at a signi®cant scale in developed regions like Europe and North America. As a rough estimation, about 170 million tons of wheat straw are produced yearly in Europe. These amounts are signi®cant enough to consider wheat straw as a generic source of renewable materials, particularly for the production of chemical derivatives from cellulose, hemicelluloses, and lignin [1]. Hemicelluloses are one of the most abundant natural polysaccharides and comprise over 30% of the dry matter of wheat straw [2]. The actual yield of hemicellulose arabinoxylan from wheat straw, even on a dry and wax-free basis, is quite variable and largely a function of the conditions used for its isolation. When
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extracted using relatively mild conditions, hemicellulose yields of 26±28% were obtained. Under more exhaustive conditions of extraction, yields of 31±33% were obtained [3]. The de®nition of hemicelluloses is very generical, but is accepted at present. Under this heading are included those polysaccharides closely linked to cellulose that are easily soluble in dilute alkali after elimination of the lignin [4]. Most of the hemicellulosic preparations are soluble in water after alkaline extraction. Their isolation actually involves alkaline hydrolysis of ester linkages to liberate them from the lignocellulosic matrix followed by extraction into aqueous media. Hemicelluloses are branched polymers of low molecular weight of degree of polymerization of 80±200. Their general formulas are (C5H8O4)n and (C6H10O5)n and they are, respectively, called pentosans and hexosans [5]. The sugar composition of wheat straw hemicelluloses showed that xylose was the predominant sugar component, and glucose, galactose, mannose, and rhamnose were the minor sugar constituents. Arabinose appeared in a noticeable amount. The b-(1!4)-d-xylopyranose backbone is
0141-3910/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0141-3910(99)00095-6
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substituted by a-l-arabinofuranose and a-d-glucuronic acid, mainly 4-O-methyl-d-glucuronic acid (MeGlcA) at C-3 and C-2 of xylose residues in the main chain, respectively. Galactose and xylose residues are potentially linked to arabinofuranosyl branches [6]. The hemicelluloses are potentially very useful. Properties of wheat straw hemicelluloses worth exploiting are their ability to serve as adhesives, thickeners, and stabilisers, and as ®lm formers and emulsi®ers [7]. Recently, we found that native hemicelluloses latexes showed good properties for making decorative paints, which indicated a possibility of using hemicelluloses for real commercial decorative paint systems [8]. However, the hemicelluloses prepared by aqueous alkali extraction from ligni®ed wheat straw, were, in general, brown, which impedes their industrial use. The aim in our laboratory is to develop a commercial process for the extraction of hemicelluloses from wheat straw and other agricultural residues. A traditional hypochlorite bleaching process as used in chemical pulp bleaching, causes a serious environmental problem. It is, therefore, necessary to develop an environmentally friendly procedure for isolation of hemicelluloses on a large scale for industries. In this study, several procedures for preparing wheat straw hemicelluloses using hydrogen peroxide were compared in eorts to lighten the color of their solutions because the usefulness of the straw hemicelluloses to the paint industry would be greatest for products with the least color. The hydrogen peroxide deligni®cation of agricultural residues is strongly pH-dependent, with an optimum pH of 11.5±11.6, pKa for the dissociation reaction of H2O2: H2 O2 HOÿ $ H2 O HOOÿ During the treatment, alkaline peroxide reacts rapidly with lignin to form low molecular weight, water-soluble oxidation products. The lignin oxidizing species in these reactions is apparently the highly reactive hydroxyl radical (HO.) that is formed during the degradation of H2O2 in a reaction with the hydroperoxy anion (HOOÿ): H2 O2 HOOÿ ! HOOÿ 2 H2 O Hydroperoxide anion (HOOÿ) is the active species and is responsible for the bleaching action of hydrogen peroxide under alkaline conditions. On the other hand, hydroperoxyl and hydroxyl radicals generated by the decomposition of hydrogen peroxide are responsible for deligni®cation and solubilization of hemicelluloses [9]. In this study, several procedures for alkaline peroxide treatments of wheat straw were examined. The eects of pretreatment using metal chelating agents, addition of stabilizer during the treatments, and treating temperature and pH on the yield, color, and physico-
chemical properties of the solubilized hemicelluloses are comparatively studied and the results are reported. 2. Experimental 2.1. Material Wheat straw was obtained from Compak Co. (Gainsborough, England). The straw was ®rst cut to 1±2 cm lengths by hand and then ground to pass a 0.7 mm size screen. The ground straw was dried in an oven for 16 h at 50 C before treatment. All chemicals used were of analytical or reagent grade. 2.2. Extraction of hemicelluloses To prepare hemicellulosic fraction 1, a sample of water-treated (50 C, 1 h) wheat straw was treated with alkaline peroxide by placing 150 g of the substrate to be treated in 3000 ml of distilled water containing 2% H2O2 (w/v) in a jacketed reaction vessel heated with water from a thermostatically-controlled circulating bath. The suspension was adjusted to pH 11.5 with 4 M NaOH and allowed to stir gently at 60 C for 16 h. For preparing fraction 2, the straw was ®rst treated with deionised water at 90 C for 1 h, and then treated with alkaline peroxide as the method described for fraction 1 above. During initial stages of stirring, oxygen evolution was active, and substantial frothing occurred, requiring that extractions were conducted in vessels with volumes two to three times those of extraction mixtures. No further adjustments in pH were made during the course of the treatment. Under these conditions, the reaction pH remained nearly constant for 2 h before slowly rising to a ®nal value of ca 12.8. The insoluble residue was collected by ®ltration, washed with distilled water until the pH of the ®ltrate was neutral, and then dried at 60 C. The supernatant ¯uid was adjusted to pH 5.5 with 10% HCl and then concentrated. The solubilized hemicelluloses were precipitated by pouring the concentrated supernatant ¯uid into 3 volumes of ethanol, from which they were settled out as a white ¯occulent precipitate. The hemicelluloses were collected after carefully decanting o the supernatant ¯uid and washing with 70% ethanol. Next the hemicelluloses were airdried in a fume hood overnight, ®nely fragmented with a conventional chopper-grinder, and dried to constant weight in an oven at 50 C. Prior to extraction with potassium hydroxide solution, the straw was washed with 0.2% (w/v) ethylenediamine tetraacetic acid (EDTA) solution in deionised water at pH 6 for 1 h at 90 C. The solid material was isolated and then washed thoroughly with deionised water. After extraction with 10% KOH (w/v) at room temperature for 16 h, The solubilized hemicelluloses was bleached by 2% H2O2 at
J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
60 C and pH 11.5, adjusted with acetic acid, for 16 h. Neutralization and precipitation followed according to the procedure described above, and the isolated hemicellulosic preparation was labeled as fraction 3. The hemicellulosic fraction 4 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (90 C, pH 6. 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2 at 90 C and pH 11.5, adjusted by acetic acid, for 2 h. In fraction 5, the straw was ®rst washed with 0.2% EDTA solution in deionised water at pH 6 for 1 h at 90 C. The hemicelluloses were then extracted from the residues with 2% H2O2 at 90 C for 2 h at pH 11.5 adjusted by 10% KOH. Fraction 6 was extracted with 10% KOH at room temperature for 16 h from watertreated (90 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 5% H2O2 at 60 C and pH 11.0, adjusted by acetic acid, for 8 h. Fraction 7 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2±0.2% MgSO4 at 85 C and pH 11.5, adjusted by acetic acid, for 1 h. Fraction 8 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M NaOH, for 16 h from 0.2% EDTA aqueous solution treated (85 C, pH 6 1 h) wheat straw, and the scheme for isolation of the solubilized hemicelluloses was illustrated in Fig. 1. Fraction 9 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M KOH, for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6, 1 h) wheat straw. All the procedures for isolation of the solubilized hemicelluloses were carried out as described for the fractions 1 and 2 above. 2.3. Characterization of the solubilized hemicelluloses The neutral sugar composition of the isolated hemicelluloses was determined by gas chromatography (GC)
Fig. 1. Scheme for extraction of hemicellulosic fraction 8 from wheat straw.
425
analysis of their alditol acetates [10]. Alkaline nitrobenzene oxidation of associated lignin from solubilized hemicelluloses was performed at 170 C for 3 h. The lignin content in hemicellulosic fractions was calculated by multiplying the yield of phenolics obtained by nitrobenzene oxidation by 2.41 [8]. Methods of uronic acid analysis, determination of phenolic acids and aldehydes in nitrobenzene oxidation mixtures with high performance liquid chromatography (HPLC), and measurement of the molecular weights have been described in previous papers [3,6]. The FT±IR spectra were obtained on an FT±IR spectrophotometer (Nicolet, 750) using KBr discs containing 1% ®nely ground samples. The solution-state 13C-NMR spectrum was obtained on a Bruker 250 AC spectrometer operating in the FT mode at 62.4 MHz under total proton decoupled conditions. They are recorded at 25 C from 140 mg of sample dissolved in 1 ml D2O after 10,000 scans. A 60 pulse ¯ipping angle, a 3.9 ms pulse width and 0.85 s acquisition time were used. 3. Results and discussion 3.1. Yield of solubilized hemicelluloses The yields of solubilized hemicelluloses (on a dry, water-soluble free basis of the straw) present in Table 1. The content of original hemicelluloses in water treated (50 C, 1 h) residue was measured to be 28.8%. As can be seen from Table 1, treatments from nine fractionation procedures released 88, 83, 91, 93, 66, 85, 86, 92, and 92% of the original hemicelluloses, respectively, which contained 2.8±5.1% of the associated lignin. These end products of the solubilized hemicelluloses were white even though the extracts by alkali prior to alkaline peroxide bleaching were brownish in color. The pigmentation associated with the hemicellulosic fractions is likely to be due to the presence of bound lignin, which linked to hemicelluloses through ether bonds. When wheat straw was treated with 2% H2O2 at pH 11.5 for 16 h at 50 C, slightly more than 80% of the lignin and most of the hemicelluloses were solubilized, leaving a cellulose-enriched residue for paper making [11]. The process incorporating alkaline peroxide in the treatment medium to convert or degrade the lignin portion into water-soluble fractions showed that the deligni®cation was most eective at pH 11.5, the pKa for the dissociation of H2O2, and that the concentration of . the species active in deligni®cation, HO. and Oÿ 2 were optimal at pH 11.6 [12]. Similar results were observed in our studies. As shown in Table 1, treatments with 2% H2O2 at pH 11.5 both in extraction and in bleaching led to not only substantial lignin degradation but also signi®cant hemicellulose solubilization. The released hemicelluloses contained minimal associated lignin (2.8±5.1%).
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Table 1 The yield of hemicelloluses (% water-soluble free basis of the dry straw) and content of associated lignin in solubilized hemicelluloses (% hemicelluloses, w/w) obtained from the various extraction procedures of wheat straw Hemicellulose fractionsa Yield/content
1
2
3
4
5
6
7
8
9
Yield of hemicelluloses Content of associated lignin
25.2 2.80
23.8 3.06
26.1 3.86
26.7 3.88
18.9 5.08
24.4 4.05
24.8 3.64
26.4 3.49
26.6 4.75
The hemicellulosic fraction 1 was extracted with 2% H2O2±0.2% EDTA at 60 C and pH 11.5, adjusted by 4 M NaOH, for 16 h from water treated (50 c, 1 h) wheat straw, fraction 2 was extracted as described in the above fraction 1 from water treated (90 C, 1 h) wheat straw, fraction 3 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (90 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2 at 60 C and pH 11.5, adjusted by acetic acid, for 16 h, fraction 4 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (90 C, pH 6 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2 at 90 C and pH 11.5, adjusted by acetic acid, for 2 h, fraction 5 was extracted with 2% H2O2 at 90 C and pH 11.5, adjusted by 10% KOH, for 2 h from 0.2% EDTA aqueous solution treated (90 C, pH 6 1 h) wheat straw, fraction 6 was extracted with 10% KOH at room temperature for 16 h from water treated (90 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 5% H2O2 at 60 C and pH 11.5, adjusted by acetic acid, for 8 h, fraction 7 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2±0.2% MgSO4 at 85 C and pH 11.5, adjusted by acetic acid, for 1 h, fraction 8 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M NaOH, for 16 h from 0.2% EDTA aqueous solution treated (85 C, pH 6, 1 h) wheat straw ( Fig . 1), fraction 9 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M KOH, for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6.0, 1 h) wheat straw. a
The lowest yield of hemicellulosic fraction 5 (18.9%), which was extracted with 2% H2O2 at 90 C and pH 11.5 for a short period of treatment (2 h), however, had relatively the highest amount of associated lignin (5.1%). This indicated that lignin removal and hemicellulose solubilization were not maximized in 2 h at 90 C and pH 11.5 with 2% H2O2 treatment, and a prolonged treatment duration is needed. Interestingly, as the pH rose above 11.5, increased solubilization of hemicelluloses was noted, supporting the results in Table 1 for fractions 8 and 9. An increase of treatment pH to 12.5 resulted in over 90% of the original hemicellulose release during the 2% H2O2 treatments at 50 C for 16 h. This phenomenon indicated that the amounts of solubilized hemicelluloses increased as the reaction pH become more alkaline. Additionally, partial solubilization of lignin and hemicelluloses was also associated with a severe disruption of the physical integrity of the straw during the alkaline peroxide treatment [13]. Previous studies on the reaction pH indicated that the amount of hemicelluloses solubilized was greatly reduced as compared to the treatments performed without continuous pH control. Nearly 90% of the original hemicelluloses in water-treated wheat straw was solubilized after 16 h of 2% H2O2 treatment at 60 C and pH 11.5 in the absence of continuous pH control, while only about 60% of the original hemicelluloses were solubilized in 16 h when the reaction pH was maintained at pH 11.5 0.1 (data not shown). The reason for this high solubilization of hemicelluloses at high alkalinity was presumed due to the functions of hydroxyl radicals formed in the alkaline solution at high pH. As mentioned earlier, hydroperoxyl and hydroxyl radicals generated by the decomposition of hydrogen peroxide are responsible for the deligni®cation together
with solubilization of hemicelluloses. Hydroxyl radicals are capable of attacking practically all the ether linkages between lignin and hemicelluloses [14], which subsequently results in a signi®cant increase of the release of hemicelluloses. When the color and yield is considered, the alkaline peroxide treatment is most eective at pH 11.5±12.5, since in a relatively high alkalinity (i.e. for a pH >12.5), especially at high temperature, chromophores are possibly generated by alkali-catalyzed modi®cation of reducing end groups on the hemicelluloses. The hemicelluloses produced at pH 12.5 were owhite, while those exposed to pH value of 12.8 were tannish. During the alkaline peroxide treatment, the rate of O2 evolution is strongly dependent upon the amount of straw present in the reaction mixture. Removal of heavy metal contaminants from wheat straw (for example, by acid pre-wash, by addition of chelators such as EDTA, or alkaline precipitation) essentially eliminated O2 evolution from H2O2 reaction mixture [13]. Nearly an equal amount of hemicelluloses obtained by alkaline peroxide treatment or 10% KOH extraction (Table 1) showed that alkaline peroxide was capable of solubilizing the hemicelluloses from wheat straw signi®cantly under the conditions applied. It was noteworthy that the pre-wash of the straw at high temperature or pre-treatment of the straw with the chelating agent such as EDTA had no signi®cant eect on the release of hemicelluloses. This observation was consistent with the ®nding that improvement in H2O2 stabilization was not a prerequisite for good deligni®cation [15]. Similar results were observed in our experiments on the solubilization of hemicelluloses. The data in Table 1 for preparing hemicellulosic fraction 7 also showed that MgSO4 added in the bleaching stage had little eect on the yield
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and color of the hemicelluloses released, but a slightly higher associated lignin (3.6%) than that of hemicellulosic fraction 1 (2.8%) isolated with 2% H2O2± 0.2% EDTA at 60 C for 16 h at pH 11.5. These ®ndings implied that in bleaching of the solubilized hemicellulose±lignin complex, Epsom salt (MgSO4 (7H2O) added under appropriate pH could deactivate transition metals and stabilize hydrogen peroxide to such an extent that reasonable rates of deligni®cation from the complex can be achieved without excessively degrading and depolymerzing hemicelluloses by hydroxyl radicals. During the bleaching process, hydroperoxide anions (HOOÿ), formed in alkaline media, are the principal active species. These anions are the strongly nucleophiles that preferentially attack ethylenic and carbonyl groups present in hemicellulose-lignin complex, and subsequently convert chromophores such as quinones, cinnamaldehyde, and ring-conjugated ketones into non-chromophoric species. However, hydrogen peroxide is unstable in alkaline conditions and readily decomposes, particularly in the presence of certain transition metals such as manganese, iron, and copper. This metal-catalyzed decomposition of H2O2 results in a loss of bleaching capacity and generates more active radicals such as hydroxyl and hydroperoxyl radicals . (HO., Oÿ 2 ) [16]. In this study, removal of these metals was performed by addition of the chelators or pre-wash of the straw prior to alkaline peroxide treatments. No signi®cant eect on the yield of solubilized hemicelluloses was observed after these pre-treatments, while the pre-treatments in these fractions resulted in more light color in hemicelluloses as compared to the hemicelluloses obtained without pre-treatment (data not shown).
sugars. Arabinose appeared as another major sugar constituent. Glucose, galactose, rhamnose, and mannose were observed as minor constituents. The content of xylose was higher in the four hemicellulosic fractions (78.6±79.6%), extracted with 10% KOH, than in the other ®ve hemicellulosic preparations (64.1±76.9%), isolated with 2% H2O2, while the relative amounts of arabinose and glucose in the alkaline peroxide-soluble hemicelluloses were higher than in the alkali-soluble hemicelluloses. This phenomenon provides evidence that in wheat straw cell walls arabinose and glucose, probably as a side chain in hemicelluloses, are easily solubilized during the mild extraction process such as alkaline peroxide treatment, whereas these side chains are partially cleaved or degraded in the strong alkaline solution such as in 10% KOH extraction process for 16 h even though it was performed at room temperature. This was particularly true in the hemicellulosic fraction 5, extracted with 2% H2O2 at 90 C for 2 h at pH 11.5, which had the lowest xylose content (64.1%) and the highest in arabinose (20.2%), suggesting that treatment of wheat straw with 2% H2O2 in a relatively short period such as 2 h was favor extraction of hemicelluloses having more branched structures. The data in Table 2 also showed that no signi®cant dierence in the content of uronic acid among the nine hemicellulosic fractions was observed, as shown by its values which ranged between 4.4 and 5.5%. These ®ndings are consistent with our early study reporting that d-glucopyranosyluronic acid or MeGlcA group attached at position 2 in the main chain of the hemicelluloses obtained from wheat straw [6].
3.2. Content of neutral sugars and uronic acids
3.3. Composition of phenolic acids and aldehydes
To characterize the solubilized hemicelluloses, the nine fractions were prepared for determination of their substituent sugars, and the results are given in Table 2. Xylose was the predominant sugar composition of the hemicelluloses, comprising 64.1±79.6% of the total
The phenolic acids and aldehydes, released from the nine hemicellulosic fractions by alkaline nitrobenzene oxidation of the associated lignin, were analyzed by HPLC, and the results are summarized in Table 3. As expected, compared to the lignin content (8.9%) in the
Table 2 The content of neutral sugars (relative % hemicellulosic sample, w/w) and uronic acids (% hemicellulosic sample, w/w) in isolated hemicellulosic fractions Hemicellulosic fractionsa Neutral sugars/uronic acids
1
2
3
4
5
6
7
8
9
Rha Ara Xyl Man Glc Gal Uronic acids
0.66 13.68 76.61 0.67 4.45 3.93 4.75
0.51 14.00 76.45 1.02 6.71 1.31 4.75
1.43 14.16 78.56 0.46 3.10 2.30 4.75
0.80 13.75 79.08 0.33 2.69 3.34 4.50
1.50 20.15 64.08 0.86 7.78 5.63 5.50
0.62 14.10 79.56 0.27 3.52 1.95 4.50
0.87 14.09 76.92 0.73 4.58 2.81 4.63
0.65 15.46 72.11 0.21 8.94 2.62 4.38
0.92 18.07 68.90 0.15 6.25 5.71 4.78
a
Corresponding to the fractions in Table 1.
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Table 3 The content (% hemicellulosic sample, w/w) of phenolic acids and aldehydes from nitrobenzene oxidation of the associated lignin in various isolated hemicellulosic fractions Hemicellulosic fractionsa Phenolic acids and aldehydes
1
2
3
4
5
6
7
8
9
Gallic acid Protocatechuic acid p-Hydroxybenzoic acid p-Hydroxybenzaldehyde Vanillic acid Syringic acid Vanillin Syringaldehyde Acetovanillone p-Coumaric acid Acetosyringone Ferulic acid Total
0.012 0.009 0.038 0.049 0.030 0.15 0.38 0.35 0.026 0.016 0.056 0.044 1.16
0.023 0.021 0.027 0.060 0.038 0.016 0.40 0.38 0.022 0.013 0.089 0.037 1.27
0.011 0.012 0.040 0.069 0.041 0.18 0.56 0.51 0.024 0.016 0.097 0.041 1.60
0.029 0.019 0.049 0.070 0.038 0.18 0.56 0.50 0.022 0.016 0.088 0.034 1.61
0.022 0.020 0.048 0.078 0.047 0.22 0.76 0.70 0.028 0.018 0.14 0.031 2.11
0.019 0.010 0.042 0.078 0.035 0.10 0.59 0.62 0.017 0.014 0.13 0.029 1.68
0.018 0.018 0.045 0.058 0.037 0.14 0.49 0.51 0.019 0.010 0.14 0.029 1.51
0.019 0.018 0.037 0.072 0.040 0.11 0.49 0.48 0.016 0.010 0.14 0.021 1.45
0.028 0.030 0.049 0.10 0.060 0.24 0.62 0.60 0.033 0.019 0.15 0.038 1.97
a
Corresponding to the fractions in Table 1.
hemicelluloses isolated with 10% KOH in the absence of alkaline peroxide bleaching from ligni®ed wheat straw, extraction with 10% KOH and then bleaching with alkaline peroxide, or direct treatment with 2% H2O2 at pH 11.5±12.5 solubilized the hemicelluloses having a much lower content of associated lignin as shown by the lower content of phenolics (1.3±2%), indicating that alkaline peroxide treatment or bleaching can signi®cantly break the ether bonds between lignin and polysaccharides from wheat straw. This once again suggested that a mild alkaline peroxide was an ecient agent for both deligni®cation and solubilizing hemicelluloses from wheat straw. As can be seen in Table 3, the major products, obtained from alkaline nitrobenzene oxidation, were identi®ed to be vanillin and syringaldehyde, which ranged between 31.5±36.1% and 30.2±36.9% of the total phenolic monomers, respectively. This suggested that the associated lignin in the hemicelluloses contained roughly equal amounts of non-condensed guaiacyl and syringyl units, which was in good agreement with the results obtained from the pure solubilized lignin preparations obtained by alkaline peroxide treatment of wheat straw [11]. A noticeable amount of syringic acid and traces of p-hydroxybenzaldehyde, vanillic acid, phydroxybenzoic acid, acetosyringone, acetovanillone, ferulic acid, p-coumaric acid, gallic acid, and protocatechuic acid were also found to be present in the nitrobenzene oxidation mixtures. No signi®cant dierence in the phenolic molar ratios was found among the nine solubilized hemicellulosic fractions. Occurrence of ferulic and p-coumaric acids in cell walls of wheat straw has been reported to have a profound in¯uence on the growth of the plant cell wall and its mechanical properties and biodegradability [16±19]. Our earlier studies showed that p-coumaric acid was mostly esteri®ed to
lignin or polysaccharides, while ferulic acid appeared almost equally in esteri®ed bonds to arabinose in hemicelluloses and in etheri®ed linkages with lignin [20]. This occurrence of traces of esteri®ed or etheri®ed ferulic and p-coumaric acids in the hemicellulosic fractions, obtained by alkaline peroxide treatment, indicated that these two phenolic acids are strongly associated with polysaccharides or lignin in the cell walls of wheat straw. Similar results have been reported by Billa et al. [21] in the hydrogen peroxide bleaching of wheat straw mechanical pulps. The authors demonstrated that the bleaching resulted in a moderate reduction in esteri®ed ferulic and p-coumaric acids but had little eect on etheri®ed ones. Obviously, treatment of wheat straw with alkaline peroxide can only result in a partial cleavage of the esteri®ed linkages such as those between ferulic acid and hemicelluloses or between p-coumaric acid and lignin/or hemicelluloses, and the solubilization of hemicellulose and lignin fragments bearing ferulic and p-coumaric acids. 3.4. Molecular weight distribution The molecular weight distribution of the hemicellulosic fraction 8 extracted with 2% H2O2 at 50 C for 12 h and pH 12.5, is shown in Fig. 2. As can be seen from the diagram, the molecular weight distribution ranged between 1174890 and 3020 g molÿ1 with two main peaks having the molecular weights of 27670 and 10840 g molÿ1, which showed a wide polymolecularity. The weight- (Mw) and number- (Mn) molar mass averages of the solubilized hemicellulosic fractions, calculated from the GPC chromatograms, are listed in Table 4. As can be seen from the table, an increase in the pre-washing temperature from 50 to 90 C for 1 h prior to 2% H2O2 treatment at pH 11.5 for 16 h at 60 C
J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
resulted in growth of Mw from 20870 to 32830 g molÿ1 between fractions 1 and 2, indicating that pre-washing at a relatively high temperature at least in part, enhanced dissolution of large molecular size hemicelluloses in the next step of alkaline peroxide treatment of the straw. This release of large molecular size of hemicelluloses was also partially due to a severe disruption of the physical integrity of the straw during the prewashing at high temperature. Treatment with 2% H2O2 at pH 11.5 at a high temperature of 90 C for a short period of 2 h from 0.2% EDTA aqueous solution treated straw led to a release of the hemicellulosic fraction 5 having a relatively higher Mw over 40,000 g molÿ1. Additionally, treatment with 2% H2O2 at 50 C for 16 h at a higher pH 12.5 solubilized the hemicellulosic fractions 8 and 9 having not only high yields (26.4 and 26.6%) but also high Mw (55,000 and 63,700 g molÿ1). These observations implied that alkaline peroxide treatment did not signi®cantly degrade the macromolecule of hemicelluloses, and the treatment favored release of large molecular size of hemicelluloses at a relatively higher pH such as at pH 12.5 or at pH 11.5 and 90 C for a short period (2 h) from wheat straw. On the other hand, as compared to the Mw in fractions 4 and 7, the bleaching of the hemicellulosic fractions 3 and 6, isolated with 10% KOH at room temperature for 16 h, with 2% H2O2 at pH 11.5 and 60 C for a long period of 16 h or at 60 C for 8 h at pH 11.5 with a relatively
Fig. 2. GPC molecular weight distribution of hemicellulosic fraction 8.
429
higher concentration (5%) of hydrogen peroxide resulted in a noticeable degradation of the solubilized hemicelluloses as shown by the relatively lower Mw at 21,790 ÿ1 and 20,370 g mol . 3.5. FT±IR spectra Fig. 3 shows the FT±IR spectra of hemicellulosic fractions 1 (spectrum F1), 2 (spectrum F2), 4 (spectrum F4), and 5 (spectrum F5). As can be seen, the four spectral pro®les and relative intensities of the bands were rather similar, indicating similar structures of the hemicelluloses isolated in various conditions used. This observation once again demonstrated that alkaline peroxide treatment did not result in any signi®cant change in the macromolecular structure of hemicelluloses. The absorption at 1629 cmÿ1 is principally associated with absorbed water, since the hemicelluloses usually have a strong anity for water, and in the solid state these macromolecules may have disordered structures which can easily be hydrated [22]. Bands between 1125 and 1000 cmÿ1 are typical of xylans. The prominent band at 1050 cmÿ1 is attributed to the C±O, C±C stretching or C±OH bending in hemicelluloses [23]. The band at 1096 cmÿ1 corresponds to the C±OH bending, which is
Fig. 3. FT±IR spectra of hemicellulosic fractions 1 (F1), 2(F2), 4(F4), and 5(F5).
Table 4 Weight-average (Mw) and number-average (Mn) molecular weights and polydispersity (Mw/Mn) of the hemicellulosic fractions extracted from wheat straw Hemicellulosic fractionsa Mw Mn Mw/Mn a
1
2
3
4
5
6
7
8
9
20870 10770 1.94
32830 14020 2.34
21790 11190 1.95
36040 15550 2.32
43420 18010 2.41
20370 10660 1.91
31730 12930 2.45
54980 17150 3.21
63730 19950 3.14
Corresponding to the fractions in Table 1.
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strongly in¯uenced by degree of branching. The sharp band at 894 cmÿ1, which corresponds to the C1 group frequency or ring frequency, is characteristic of b-glycosidic linkages between the sugar units [24]. The bands at 1467, 1330, 1260, and 1213 cmÿ1 represent C±H, OH or CH2 bendings. An intensive band at 1421 cmÿ1 corresponds to the C±O stretch and CH or OH bending in
hemicelluloses [22]. The occurrence of a small band at 1510 cmÿ1 in spectra F4 and F5 is undoubtedly due to the presence of small amounts of associated lignin in the hemicelluloses, which corresponded to the results obtained by alkaline nitrobenzene oxidation. The FT±IR spectra of the hemicellulosic fractions 6 (spectrum F6), 8 (spectrum F8), and 9 (spectrum F9) are shown in Fig. 4. The most obvious feature is the similarity of these spectra. The absorbances at 1470, 1412, 1319, 1266, 1219, 1095, 1049, and 892 cmÿ1 in the spectra are associated with hemicelluloses. All the spectra have an intense absorbed water-related absorbance at 1624 cmÿ1. The lignin-related absorbance at 1515 cmÿ1 is rather weak in the spectrum 9 and poorly resolved in the other two spectra. This is in accordance with the content of associated lignin in the isolated hemicelluloses. 3.6.
Fig. 4. FT±IR spectra of hemicellulosic fractions 6 (F6), 8(F8), and 9 (F9).
Fig. 5.
13
13
C-NMR spectra
In order to characterize the structural features of the isolated hemicelluloses, the hemicellulosic fractions 7 and 8 were analyzed by 13C-NMR spectroscopy (Fig. 5). The spectra were interpreted on the basis of reported data for structurally-de®ned arabinoxylan-type, glucoronoxylantype and l-arabino-(4-O-methyl-d-glucurono)-d-xylan, as well as those of wheat straw hemicelluloses extracted
C-NMR spectra of hemicellulosic fractions 7(a) and 8(b).
J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
before deligni®cation [6, 25±27]. The main 1,4-linked bd-Xylp units are obviously characterized by the signals at 104.9, 78.4, 77.5-77.6, 75.8±75.9, and 65.8 ppm, which attributes to C-1, C-4, C-3, C-2, and C-5 of the bd-Xylp units, respectively. The signals at 111.2±111.8, 89.0±89.1, 82.8±83.0, 81.0±81.2, and 64.1±64.3 ppm correspond to C-1, C-4, C-2, C-3, and C-5 of a-l-Araf residues, respectively. Three signals at 176.1, 85.0 (data not shown in the spectra), and 58.8 ppm originate from C-6, C-4 and 4-O-methoxyl group of glucuronic acid residue in the xylan which are very weak and in accord with the low uronic acid content. The signal at 26.8 ppm related to -CH3 in Ar±COCH3, indicating the associated lignins. The presence of esteri®ed acetyl group was identi®ed by a noticeable signal for CH3 in CH3COgroup at 19.6 ppm in the hemicellulosic fraction 8, while it was completely disappeared in the fraction 7. This suggested that treatment with alkaline peroxide under the conditions given only partially broke the esteri®ed linkages between acetyl group and hemicelluloses, while alkaline extraction with 10% KOH under the conditions used completely removed this ester bond. The signal at 179.2 ppm originates the carbonyl group in associated lignin. The near disappearance of the signals at 179.2 and 26.8 ppm for associated lignins in the fraction 8 indicated that alkaline peroxide had more eect on the deligni®cation than the potassium hydroxide under the conditions used in this study. Clearly, the above similar spectra between the alkali-soluble hemicelluloses and the alkaline peroxide-soluble ones demonstrated that treatment of wheat straw with alkaline peroxide did not signi®cantly change the macromolecular structure of hemicelluloses from wheat straw. Schonebaum [28] reached similar conclusions as early as 1922 in the studies of mono- and di-saccharide behaviours in hydrogen peroxide solution. He demonstrated that d-glucose, d-fructose, d-xylose, d-arabinose, lactose, and maltose, at low levels of hydrogen peroxide, even at 70 C, are not signi®cantly changed. In summary, treatment of wheat straw with alkaline peroxide under the conditions used resulted in a partial reduction of esteri®ed acetic acid, ferulic and p-coumaric acids but had a signi®cant eect on the etheri®ed linkages between lignin and hemicelluloses as shown by a release of 83±93% of the originally present hemicelluloses. The treating process did not aect the macromolecular structure of hemicelluloses to any noticeable extent, as supported by 13C-NMR analyses which indicated that no substantial changes occurred during the treatment. Under an optimum condition (2% H2O2, 50 C, pH 12.5, 16 h), the treatment solubilized 92% of the original hemicelluloses, which had a large molecular size (Mw=54,980±63,730 g molÿ1). Furthermore, in comparison, we also tested the possibility of extracting with alkali such as 10% KOH, and then separately bleaching the solubilized hemicelluloses with
431
alkaline peroxide, This two-step treating process minimised the use of hydrogen peroxide and allowed the use of more extreme extraction conditions to increase the hemicellulose yield. However, a prolonging bleaching period up to 16 h with 2% H2O2 at pH 11.5 and 60 C or bleaching using a relatively higher concentration (5%) of hydrogen peroxide at 60 C for 8 h at pH 11.5 resulted in a noticeable degradation of the solubilized hemicelluloses as shown by the relatively lower Mw of 21,790 and 20,370 g ÿ1 mol in fractions 3 and 6, respectively. Colours of the hemicelluloses and its solution will be measured, as well as viscosity-concentration relationships at various temperatures. The chemical modi®cation of the isolated hemicelluloses as a novel material for industries is currently under investigation in our laboratory. Acknowledgements The authors are grateful for the ®nancial support of this research from the European Community under the Industrial and Materials Technologies Programme (Brite-EuRam III)-Depolymerisation, Polymerisation and Applications of Biosustainable Raw Materials for Industrial End Uses.
References [1] Montane D, Farriol X, Salvado J, Jollez P, Chornet E. J Wood Chem Technol 1998;18:171. [2] Sun RC, Lawther JM, Banks WB. Industrial Crops and Products 1995;4:127. [3] Heredia A, Jimenez A, Guillen RZ. Lebensm Unters Forsch 1995;200:24. [4] Lawther JM, Sun RC, Banks WB. J Agric Food Chem 1995;43:667. [5] Cai ZS, Paszner L. Holzforschung 1988;42:11. [6] Sun RC, Lawther JM, Banks WB. Carbohydr Polym 1996;29:325. [7] Doner LW, Hicks K. Cereal Chem 1997;74:176. [8] Sun RC, Fang JM, Goodwin A, Lawther JM, Bolton J. J Agric Food Chem 1998;46:2817. [9] Lewis SM, Holzgraefe DP, Berger LL, Fahey GC, Gould JM, Fanta GF. Animal Feed Sci Technol 1987;17:179. [10] Blakeney AB, Harris PJ, Henry RJ, Stone BA. Carbohydr Res 1983;113:291. [11] Sun RC, Tomkinson J. Characterization of lignins from wheat straw by alkaline peroxide treatment. Submitted for publication. [12] Gould JM. Biotechnology and Bioengineering 1984;26:46. [13] Gould JM. Biotechnology and Bioengineering 1985;27:225. [14] Parthasarathy VR, Klein R, Sundaram VSM, Jameel H, Gratzl JS. Tappi J 1990;7:177. [15] Lachenal D, Choudens CD, Monzie P. Tappi 1980;63:119. [16] Pan GX, Bolton JL, Leary GJ. J Agric Food Chem 1998;46:5283. [17] Iiyama K, Lam TBT, Stone BA. Plant Physiol 1994;104:315. [18] Jung HG, Deetz DA. Forage cell wall structure and digestibility. In: Jung HG, Buxton DR, Hat®eld RD, Ralph J, editors. ASACSSA-SSA. WI: Madison, 1993. p. 315. [19] Fry SC. Annu Rev Plant Physiol 1986;37:165.
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[20] Sun RC, Xiao B, Lawther JM. J Appl Polym Sci 1998;68:1633. [21] Billa E, Monties B, de Choudens C. Proceedings of PIRA International Conference on `Straw: a valuable material', Paper Industry research Association (PIRA): Surrey, UK, 1993. p. 4. [22] Kacurakova M, Belton PS, Wilson RH, Hirsch J, Ebringerova A. J Sci Food Agric 1998;77:38. [23] Marchessault RH, Liang CY. J Polym Sci 1962;59:367.
[24] Gupta S, Madan RN, Bansal MC. Tappi J 1987;70:113. [25] Kato A, Azuma J, Koshijima T. Agric Bio Chem 1987;51:1691. [26] Ebringerova A, Hromadkova Z, Alfoldi J, Berth G. Carbohydr Polym 1992;19:99. [27] Imamura T, Watanabe T, Kuwahara M, Koshijima T. Phytochemistry 1994;37:1165. [28] Schonebaum CW. Rec Trav Chim 1922;41:503.
Comparative study of hemicelluloses from wheat straw by alkali and hydrogen peroxide extractions J.M. Fang, R.C. Sun*, D. Salisbury, P. Fowler, J. Tomkinson The BioComposites Centre, University of Wales, Bangor, Gwynedd, LL57 2UW, UK Received 9 April 1999; accepted 24 May 1999
Abstract As compared to traditional alkaline extractions, alkaline peroxide was used to isolate hemicelluloses from wheat straw. Yields of the solubilized hemicelluloses ranged from 18.9% (2% H2O2 extraction at 90 C for 2 h at pH 11.5) to 26.6% (2% H2O2 extraction at 50 C for 16 h at pH 12.5). The optimum hemicellulose yield (92% of the original hemicelluloses in water treated wheat straw) was obtained when the treatment was performed at 50 C for 16 h at pH 12.5 by use of 2% H2O2. All the hemicellulosic preparations were much lighter in color than those obtained using traditional alkaline extractions in the absence of bleaching. The results, obtained by the destructive method such as acid hydrolysis, showed that extraction of wheat straw with aqueous 10% KOH following alkaline bleaching released the hemicellulosic fractions, which were enriched in xylose, whereas extraction of the straw with aqueous 2% H2O2 under alkaline conditions (pH 11.5±12.5) solubilized the hemicellulosic fractions, which were relatively higher in arabinose and glucose. The nine isolated hemicellulosic samples were further characterized by non-destructive methods such as Fourier transform infrared (FT±IR), and carbon-13 magnetic resonance spectroscopy (13C-NMR) as well as gel permeation chromatography (GPC). It has been demonstrated that the alkaline peroxide treatments under the conditions used do not aect the overall structure of hemicelluloses. The only one major change of the hemicelluloses was found to be degradation during the beaching of alkali-soluble hemicelluloses with 2% H2O2 at 60 C for a period of 16 h at pH 11.5 or bleaching using a relatively higher concentration (5%) of hydrogen peroxide at 60 C for 8 h at pH 11.5. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Wheat straw is an agricultural by-product that is not used as industrial raw material at a signi®cant scale in developed regions like Europe and North America. As a rough estimation, about 170 million tons of wheat straw are produced yearly in Europe. These amounts are signi®cant enough to consider wheat straw as a generic source of renewable materials, particularly for the production of chemical derivatives from cellulose, hemicelluloses, and lignin [1]. Hemicelluloses are one of the most abundant natural polysaccharides and comprise over 30% of the dry matter of wheat straw [2]. The actual yield of hemicellulose arabinoxylan from wheat straw, even on a dry and wax-free basis, is quite variable and largely a function of the conditions used for its isolation. When
* Corresponding author. Tel.:+44-(0)1248-370588; Fax:+44(0)1248370594.
extracted using relatively mild conditions, hemicellulose yields of 26±28% were obtained. Under more exhaustive conditions of extraction, yields of 31±33% were obtained [3]. The de®nition of hemicelluloses is very generical, but is accepted at present. Under this heading are included those polysaccharides closely linked to cellulose that are easily soluble in dilute alkali after elimination of the lignin [4]. Most of the hemicellulosic preparations are soluble in water after alkaline extraction. Their isolation actually involves alkaline hydrolysis of ester linkages to liberate them from the lignocellulosic matrix followed by extraction into aqueous media. Hemicelluloses are branched polymers of low molecular weight of degree of polymerization of 80±200. Their general formulas are (C5H8O4)n and (C6H10O5)n and they are, respectively, called pentosans and hexosans [5]. The sugar composition of wheat straw hemicelluloses showed that xylose was the predominant sugar component, and glucose, galactose, mannose, and rhamnose were the minor sugar constituents. Arabinose appeared in a noticeable amount. The b-(1!4)-d-xylopyranose backbone is
0141-3910/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0141-3910(99)00095-6
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substituted by a-l-arabinofuranose and a-d-glucuronic acid, mainly 4-O-methyl-d-glucuronic acid (MeGlcA) at C-3 and C-2 of xylose residues in the main chain, respectively. Galactose and xylose residues are potentially linked to arabinofuranosyl branches [6]. The hemicelluloses are potentially very useful. Properties of wheat straw hemicelluloses worth exploiting are their ability to serve as adhesives, thickeners, and stabilisers, and as ®lm formers and emulsi®ers [7]. Recently, we found that native hemicelluloses latexes showed good properties for making decorative paints, which indicated a possibility of using hemicelluloses for real commercial decorative paint systems [8]. However, the hemicelluloses prepared by aqueous alkali extraction from ligni®ed wheat straw, were, in general, brown, which impedes their industrial use. The aim in our laboratory is to develop a commercial process for the extraction of hemicelluloses from wheat straw and other agricultural residues. A traditional hypochlorite bleaching process as used in chemical pulp bleaching, causes a serious environmental problem. It is, therefore, necessary to develop an environmentally friendly procedure for isolation of hemicelluloses on a large scale for industries. In this study, several procedures for preparing wheat straw hemicelluloses using hydrogen peroxide were compared in eorts to lighten the color of their solutions because the usefulness of the straw hemicelluloses to the paint industry would be greatest for products with the least color. The hydrogen peroxide deligni®cation of agricultural residues is strongly pH-dependent, with an optimum pH of 11.5±11.6, pKa for the dissociation reaction of H2O2: H2 O2 HOÿ $ H2 O HOOÿ During the treatment, alkaline peroxide reacts rapidly with lignin to form low molecular weight, water-soluble oxidation products. The lignin oxidizing species in these reactions is apparently the highly reactive hydroxyl radical (HO.) that is formed during the degradation of H2O2 in a reaction with the hydroperoxy anion (HOOÿ): H2 O2 HOOÿ ! HOOÿ 2 H2 O Hydroperoxide anion (HOOÿ) is the active species and is responsible for the bleaching action of hydrogen peroxide under alkaline conditions. On the other hand, hydroperoxyl and hydroxyl radicals generated by the decomposition of hydrogen peroxide are responsible for deligni®cation and solubilization of hemicelluloses [9]. In this study, several procedures for alkaline peroxide treatments of wheat straw were examined. The eects of pretreatment using metal chelating agents, addition of stabilizer during the treatments, and treating temperature and pH on the yield, color, and physico-
chemical properties of the solubilized hemicelluloses are comparatively studied and the results are reported. 2. Experimental 2.1. Material Wheat straw was obtained from Compak Co. (Gainsborough, England). The straw was ®rst cut to 1±2 cm lengths by hand and then ground to pass a 0.7 mm size screen. The ground straw was dried in an oven for 16 h at 50 C before treatment. All chemicals used were of analytical or reagent grade. 2.2. Extraction of hemicelluloses To prepare hemicellulosic fraction 1, a sample of water-treated (50 C, 1 h) wheat straw was treated with alkaline peroxide by placing 150 g of the substrate to be treated in 3000 ml of distilled water containing 2% H2O2 (w/v) in a jacketed reaction vessel heated with water from a thermostatically-controlled circulating bath. The suspension was adjusted to pH 11.5 with 4 M NaOH and allowed to stir gently at 60 C for 16 h. For preparing fraction 2, the straw was ®rst treated with deionised water at 90 C for 1 h, and then treated with alkaline peroxide as the method described for fraction 1 above. During initial stages of stirring, oxygen evolution was active, and substantial frothing occurred, requiring that extractions were conducted in vessels with volumes two to three times those of extraction mixtures. No further adjustments in pH were made during the course of the treatment. Under these conditions, the reaction pH remained nearly constant for 2 h before slowly rising to a ®nal value of ca 12.8. The insoluble residue was collected by ®ltration, washed with distilled water until the pH of the ®ltrate was neutral, and then dried at 60 C. The supernatant ¯uid was adjusted to pH 5.5 with 10% HCl and then concentrated. The solubilized hemicelluloses were precipitated by pouring the concentrated supernatant ¯uid into 3 volumes of ethanol, from which they were settled out as a white ¯occulent precipitate. The hemicelluloses were collected after carefully decanting o the supernatant ¯uid and washing with 70% ethanol. Next the hemicelluloses were airdried in a fume hood overnight, ®nely fragmented with a conventional chopper-grinder, and dried to constant weight in an oven at 50 C. Prior to extraction with potassium hydroxide solution, the straw was washed with 0.2% (w/v) ethylenediamine tetraacetic acid (EDTA) solution in deionised water at pH 6 for 1 h at 90 C. The solid material was isolated and then washed thoroughly with deionised water. After extraction with 10% KOH (w/v) at room temperature for 16 h, The solubilized hemicelluloses was bleached by 2% H2O2 at
J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
60 C and pH 11.5, adjusted with acetic acid, for 16 h. Neutralization and precipitation followed according to the procedure described above, and the isolated hemicellulosic preparation was labeled as fraction 3. The hemicellulosic fraction 4 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (90 C, pH 6. 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2 at 90 C and pH 11.5, adjusted by acetic acid, for 2 h. In fraction 5, the straw was ®rst washed with 0.2% EDTA solution in deionised water at pH 6 for 1 h at 90 C. The hemicelluloses were then extracted from the residues with 2% H2O2 at 90 C for 2 h at pH 11.5 adjusted by 10% KOH. Fraction 6 was extracted with 10% KOH at room temperature for 16 h from watertreated (90 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 5% H2O2 at 60 C and pH 11.0, adjusted by acetic acid, for 8 h. Fraction 7 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2±0.2% MgSO4 at 85 C and pH 11.5, adjusted by acetic acid, for 1 h. Fraction 8 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M NaOH, for 16 h from 0.2% EDTA aqueous solution treated (85 C, pH 6 1 h) wheat straw, and the scheme for isolation of the solubilized hemicelluloses was illustrated in Fig. 1. Fraction 9 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M KOH, for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6, 1 h) wheat straw. All the procedures for isolation of the solubilized hemicelluloses were carried out as described for the fractions 1 and 2 above. 2.3. Characterization of the solubilized hemicelluloses The neutral sugar composition of the isolated hemicelluloses was determined by gas chromatography (GC)
Fig. 1. Scheme for extraction of hemicellulosic fraction 8 from wheat straw.
425
analysis of their alditol acetates [10]. Alkaline nitrobenzene oxidation of associated lignin from solubilized hemicelluloses was performed at 170 C for 3 h. The lignin content in hemicellulosic fractions was calculated by multiplying the yield of phenolics obtained by nitrobenzene oxidation by 2.41 [8]. Methods of uronic acid analysis, determination of phenolic acids and aldehydes in nitrobenzene oxidation mixtures with high performance liquid chromatography (HPLC), and measurement of the molecular weights have been described in previous papers [3,6]. The FT±IR spectra were obtained on an FT±IR spectrophotometer (Nicolet, 750) using KBr discs containing 1% ®nely ground samples. The solution-state 13C-NMR spectrum was obtained on a Bruker 250 AC spectrometer operating in the FT mode at 62.4 MHz under total proton decoupled conditions. They are recorded at 25 C from 140 mg of sample dissolved in 1 ml D2O after 10,000 scans. A 60 pulse ¯ipping angle, a 3.9 ms pulse width and 0.85 s acquisition time were used. 3. Results and discussion 3.1. Yield of solubilized hemicelluloses The yields of solubilized hemicelluloses (on a dry, water-soluble free basis of the straw) present in Table 1. The content of original hemicelluloses in water treated (50 C, 1 h) residue was measured to be 28.8%. As can be seen from Table 1, treatments from nine fractionation procedures released 88, 83, 91, 93, 66, 85, 86, 92, and 92% of the original hemicelluloses, respectively, which contained 2.8±5.1% of the associated lignin. These end products of the solubilized hemicelluloses were white even though the extracts by alkali prior to alkaline peroxide bleaching were brownish in color. The pigmentation associated with the hemicellulosic fractions is likely to be due to the presence of bound lignin, which linked to hemicelluloses through ether bonds. When wheat straw was treated with 2% H2O2 at pH 11.5 for 16 h at 50 C, slightly more than 80% of the lignin and most of the hemicelluloses were solubilized, leaving a cellulose-enriched residue for paper making [11]. The process incorporating alkaline peroxide in the treatment medium to convert or degrade the lignin portion into water-soluble fractions showed that the deligni®cation was most eective at pH 11.5, the pKa for the dissociation of H2O2, and that the concentration of . the species active in deligni®cation, HO. and Oÿ 2 were optimal at pH 11.6 [12]. Similar results were observed in our studies. As shown in Table 1, treatments with 2% H2O2 at pH 11.5 both in extraction and in bleaching led to not only substantial lignin degradation but also signi®cant hemicellulose solubilization. The released hemicelluloses contained minimal associated lignin (2.8±5.1%).
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Table 1 The yield of hemicelloluses (% water-soluble free basis of the dry straw) and content of associated lignin in solubilized hemicelluloses (% hemicelluloses, w/w) obtained from the various extraction procedures of wheat straw Hemicellulose fractionsa Yield/content
1
2
3
4
5
6
7
8
9
Yield of hemicelluloses Content of associated lignin
25.2 2.80
23.8 3.06
26.1 3.86
26.7 3.88
18.9 5.08
24.4 4.05
24.8 3.64
26.4 3.49
26.6 4.75
The hemicellulosic fraction 1 was extracted with 2% H2O2±0.2% EDTA at 60 C and pH 11.5, adjusted by 4 M NaOH, for 16 h from water treated (50 c, 1 h) wheat straw, fraction 2 was extracted as described in the above fraction 1 from water treated (90 C, 1 h) wheat straw, fraction 3 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (90 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2 at 60 C and pH 11.5, adjusted by acetic acid, for 16 h, fraction 4 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (90 C, pH 6 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2 at 90 C and pH 11.5, adjusted by acetic acid, for 2 h, fraction 5 was extracted with 2% H2O2 at 90 C and pH 11.5, adjusted by 10% KOH, for 2 h from 0.2% EDTA aqueous solution treated (90 C, pH 6 1 h) wheat straw, fraction 6 was extracted with 10% KOH at room temperature for 16 h from water treated (90 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 5% H2O2 at 60 C and pH 11.5, adjusted by acetic acid, for 8 h, fraction 7 was extracted with 10% KOH at room temperature for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6, 1 h) wheat straw and bleached by treatment of the alkaline extracts with 2% H2O2±0.2% MgSO4 at 85 C and pH 11.5, adjusted by acetic acid, for 1 h, fraction 8 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M NaOH, for 16 h from 0.2% EDTA aqueous solution treated (85 C, pH 6, 1 h) wheat straw ( Fig . 1), fraction 9 was extracted with 2% H2O2 at 50 C and pH 12.5, adjusted by 4 M KOH, for 16 h from 0.2% EDTA aqueous solution treated (80 C, pH 6.0, 1 h) wheat straw. a
The lowest yield of hemicellulosic fraction 5 (18.9%), which was extracted with 2% H2O2 at 90 C and pH 11.5 for a short period of treatment (2 h), however, had relatively the highest amount of associated lignin (5.1%). This indicated that lignin removal and hemicellulose solubilization were not maximized in 2 h at 90 C and pH 11.5 with 2% H2O2 treatment, and a prolonged treatment duration is needed. Interestingly, as the pH rose above 11.5, increased solubilization of hemicelluloses was noted, supporting the results in Table 1 for fractions 8 and 9. An increase of treatment pH to 12.5 resulted in over 90% of the original hemicellulose release during the 2% H2O2 treatments at 50 C for 16 h. This phenomenon indicated that the amounts of solubilized hemicelluloses increased as the reaction pH become more alkaline. Additionally, partial solubilization of lignin and hemicelluloses was also associated with a severe disruption of the physical integrity of the straw during the alkaline peroxide treatment [13]. Previous studies on the reaction pH indicated that the amount of hemicelluloses solubilized was greatly reduced as compared to the treatments performed without continuous pH control. Nearly 90% of the original hemicelluloses in water-treated wheat straw was solubilized after 16 h of 2% H2O2 treatment at 60 C and pH 11.5 in the absence of continuous pH control, while only about 60% of the original hemicelluloses were solubilized in 16 h when the reaction pH was maintained at pH 11.5 0.1 (data not shown). The reason for this high solubilization of hemicelluloses at high alkalinity was presumed due to the functions of hydroxyl radicals formed in the alkaline solution at high pH. As mentioned earlier, hydroperoxyl and hydroxyl radicals generated by the decomposition of hydrogen peroxide are responsible for the deligni®cation together
with solubilization of hemicelluloses. Hydroxyl radicals are capable of attacking practically all the ether linkages between lignin and hemicelluloses [14], which subsequently results in a signi®cant increase of the release of hemicelluloses. When the color and yield is considered, the alkaline peroxide treatment is most eective at pH 11.5±12.5, since in a relatively high alkalinity (i.e. for a pH >12.5), especially at high temperature, chromophores are possibly generated by alkali-catalyzed modi®cation of reducing end groups on the hemicelluloses. The hemicelluloses produced at pH 12.5 were owhite, while those exposed to pH value of 12.8 were tannish. During the alkaline peroxide treatment, the rate of O2 evolution is strongly dependent upon the amount of straw present in the reaction mixture. Removal of heavy metal contaminants from wheat straw (for example, by acid pre-wash, by addition of chelators such as EDTA, or alkaline precipitation) essentially eliminated O2 evolution from H2O2 reaction mixture [13]. Nearly an equal amount of hemicelluloses obtained by alkaline peroxide treatment or 10% KOH extraction (Table 1) showed that alkaline peroxide was capable of solubilizing the hemicelluloses from wheat straw signi®cantly under the conditions applied. It was noteworthy that the pre-wash of the straw at high temperature or pre-treatment of the straw with the chelating agent such as EDTA had no signi®cant eect on the release of hemicelluloses. This observation was consistent with the ®nding that improvement in H2O2 stabilization was not a prerequisite for good deligni®cation [15]. Similar results were observed in our experiments on the solubilization of hemicelluloses. The data in Table 1 for preparing hemicellulosic fraction 7 also showed that MgSO4 added in the bleaching stage had little eect on the yield
J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
427
and color of the hemicelluloses released, but a slightly higher associated lignin (3.6%) than that of hemicellulosic fraction 1 (2.8%) isolated with 2% H2O2± 0.2% EDTA at 60 C for 16 h at pH 11.5. These ®ndings implied that in bleaching of the solubilized hemicellulose±lignin complex, Epsom salt (MgSO4 (7H2O) added under appropriate pH could deactivate transition metals and stabilize hydrogen peroxide to such an extent that reasonable rates of deligni®cation from the complex can be achieved without excessively degrading and depolymerzing hemicelluloses by hydroxyl radicals. During the bleaching process, hydroperoxide anions (HOOÿ), formed in alkaline media, are the principal active species. These anions are the strongly nucleophiles that preferentially attack ethylenic and carbonyl groups present in hemicellulose-lignin complex, and subsequently convert chromophores such as quinones, cinnamaldehyde, and ring-conjugated ketones into non-chromophoric species. However, hydrogen peroxide is unstable in alkaline conditions and readily decomposes, particularly in the presence of certain transition metals such as manganese, iron, and copper. This metal-catalyzed decomposition of H2O2 results in a loss of bleaching capacity and generates more active radicals such as hydroxyl and hydroperoxyl radicals . (HO., Oÿ 2 ) [16]. In this study, removal of these metals was performed by addition of the chelators or pre-wash of the straw prior to alkaline peroxide treatments. No signi®cant eect on the yield of solubilized hemicelluloses was observed after these pre-treatments, while the pre-treatments in these fractions resulted in more light color in hemicelluloses as compared to the hemicelluloses obtained without pre-treatment (data not shown).
sugars. Arabinose appeared as another major sugar constituent. Glucose, galactose, rhamnose, and mannose were observed as minor constituents. The content of xylose was higher in the four hemicellulosic fractions (78.6±79.6%), extracted with 10% KOH, than in the other ®ve hemicellulosic preparations (64.1±76.9%), isolated with 2% H2O2, while the relative amounts of arabinose and glucose in the alkaline peroxide-soluble hemicelluloses were higher than in the alkali-soluble hemicelluloses. This phenomenon provides evidence that in wheat straw cell walls arabinose and glucose, probably as a side chain in hemicelluloses, are easily solubilized during the mild extraction process such as alkaline peroxide treatment, whereas these side chains are partially cleaved or degraded in the strong alkaline solution such as in 10% KOH extraction process for 16 h even though it was performed at room temperature. This was particularly true in the hemicellulosic fraction 5, extracted with 2% H2O2 at 90 C for 2 h at pH 11.5, which had the lowest xylose content (64.1%) and the highest in arabinose (20.2%), suggesting that treatment of wheat straw with 2% H2O2 in a relatively short period such as 2 h was favor extraction of hemicelluloses having more branched structures. The data in Table 2 also showed that no signi®cant dierence in the content of uronic acid among the nine hemicellulosic fractions was observed, as shown by its values which ranged between 4.4 and 5.5%. These ®ndings are consistent with our early study reporting that d-glucopyranosyluronic acid or MeGlcA group attached at position 2 in the main chain of the hemicelluloses obtained from wheat straw [6].
3.2. Content of neutral sugars and uronic acids
3.3. Composition of phenolic acids and aldehydes
To characterize the solubilized hemicelluloses, the nine fractions were prepared for determination of their substituent sugars, and the results are given in Table 2. Xylose was the predominant sugar composition of the hemicelluloses, comprising 64.1±79.6% of the total
The phenolic acids and aldehydes, released from the nine hemicellulosic fractions by alkaline nitrobenzene oxidation of the associated lignin, were analyzed by HPLC, and the results are summarized in Table 3. As expected, compared to the lignin content (8.9%) in the
Table 2 The content of neutral sugars (relative % hemicellulosic sample, w/w) and uronic acids (% hemicellulosic sample, w/w) in isolated hemicellulosic fractions Hemicellulosic fractionsa Neutral sugars/uronic acids
1
2
3
4
5
6
7
8
9
Rha Ara Xyl Man Glc Gal Uronic acids
0.66 13.68 76.61 0.67 4.45 3.93 4.75
0.51 14.00 76.45 1.02 6.71 1.31 4.75
1.43 14.16 78.56 0.46 3.10 2.30 4.75
0.80 13.75 79.08 0.33 2.69 3.34 4.50
1.50 20.15 64.08 0.86 7.78 5.63 5.50
0.62 14.10 79.56 0.27 3.52 1.95 4.50
0.87 14.09 76.92 0.73 4.58 2.81 4.63
0.65 15.46 72.11 0.21 8.94 2.62 4.38
0.92 18.07 68.90 0.15 6.25 5.71 4.78
a
Corresponding to the fractions in Table 1.
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Table 3 The content (% hemicellulosic sample, w/w) of phenolic acids and aldehydes from nitrobenzene oxidation of the associated lignin in various isolated hemicellulosic fractions Hemicellulosic fractionsa Phenolic acids and aldehydes
1
2
3
4
5
6
7
8
9
Gallic acid Protocatechuic acid p-Hydroxybenzoic acid p-Hydroxybenzaldehyde Vanillic acid Syringic acid Vanillin Syringaldehyde Acetovanillone p-Coumaric acid Acetosyringone Ferulic acid Total
0.012 0.009 0.038 0.049 0.030 0.15 0.38 0.35 0.026 0.016 0.056 0.044 1.16
0.023 0.021 0.027 0.060 0.038 0.016 0.40 0.38 0.022 0.013 0.089 0.037 1.27
0.011 0.012 0.040 0.069 0.041 0.18 0.56 0.51 0.024 0.016 0.097 0.041 1.60
0.029 0.019 0.049 0.070 0.038 0.18 0.56 0.50 0.022 0.016 0.088 0.034 1.61
0.022 0.020 0.048 0.078 0.047 0.22 0.76 0.70 0.028 0.018 0.14 0.031 2.11
0.019 0.010 0.042 0.078 0.035 0.10 0.59 0.62 0.017 0.014 0.13 0.029 1.68
0.018 0.018 0.045 0.058 0.037 0.14 0.49 0.51 0.019 0.010 0.14 0.029 1.51
0.019 0.018 0.037 0.072 0.040 0.11 0.49 0.48 0.016 0.010 0.14 0.021 1.45
0.028 0.030 0.049 0.10 0.060 0.24 0.62 0.60 0.033 0.019 0.15 0.038 1.97
a
Corresponding to the fractions in Table 1.
hemicelluloses isolated with 10% KOH in the absence of alkaline peroxide bleaching from ligni®ed wheat straw, extraction with 10% KOH and then bleaching with alkaline peroxide, or direct treatment with 2% H2O2 at pH 11.5±12.5 solubilized the hemicelluloses having a much lower content of associated lignin as shown by the lower content of phenolics (1.3±2%), indicating that alkaline peroxide treatment or bleaching can signi®cantly break the ether bonds between lignin and polysaccharides from wheat straw. This once again suggested that a mild alkaline peroxide was an ecient agent for both deligni®cation and solubilizing hemicelluloses from wheat straw. As can be seen in Table 3, the major products, obtained from alkaline nitrobenzene oxidation, were identi®ed to be vanillin and syringaldehyde, which ranged between 31.5±36.1% and 30.2±36.9% of the total phenolic monomers, respectively. This suggested that the associated lignin in the hemicelluloses contained roughly equal amounts of non-condensed guaiacyl and syringyl units, which was in good agreement with the results obtained from the pure solubilized lignin preparations obtained by alkaline peroxide treatment of wheat straw [11]. A noticeable amount of syringic acid and traces of p-hydroxybenzaldehyde, vanillic acid, phydroxybenzoic acid, acetosyringone, acetovanillone, ferulic acid, p-coumaric acid, gallic acid, and protocatechuic acid were also found to be present in the nitrobenzene oxidation mixtures. No signi®cant dierence in the phenolic molar ratios was found among the nine solubilized hemicellulosic fractions. Occurrence of ferulic and p-coumaric acids in cell walls of wheat straw has been reported to have a profound in¯uence on the growth of the plant cell wall and its mechanical properties and biodegradability [16±19]. Our earlier studies showed that p-coumaric acid was mostly esteri®ed to
lignin or polysaccharides, while ferulic acid appeared almost equally in esteri®ed bonds to arabinose in hemicelluloses and in etheri®ed linkages with lignin [20]. This occurrence of traces of esteri®ed or etheri®ed ferulic and p-coumaric acids in the hemicellulosic fractions, obtained by alkaline peroxide treatment, indicated that these two phenolic acids are strongly associated with polysaccharides or lignin in the cell walls of wheat straw. Similar results have been reported by Billa et al. [21] in the hydrogen peroxide bleaching of wheat straw mechanical pulps. The authors demonstrated that the bleaching resulted in a moderate reduction in esteri®ed ferulic and p-coumaric acids but had little eect on etheri®ed ones. Obviously, treatment of wheat straw with alkaline peroxide can only result in a partial cleavage of the esteri®ed linkages such as those between ferulic acid and hemicelluloses or between p-coumaric acid and lignin/or hemicelluloses, and the solubilization of hemicellulose and lignin fragments bearing ferulic and p-coumaric acids. 3.4. Molecular weight distribution The molecular weight distribution of the hemicellulosic fraction 8 extracted with 2% H2O2 at 50 C for 12 h and pH 12.5, is shown in Fig. 2. As can be seen from the diagram, the molecular weight distribution ranged between 1174890 and 3020 g molÿ1 with two main peaks having the molecular weights of 27670 and 10840 g molÿ1, which showed a wide polymolecularity. The weight- (Mw) and number- (Mn) molar mass averages of the solubilized hemicellulosic fractions, calculated from the GPC chromatograms, are listed in Table 4. As can be seen from the table, an increase in the pre-washing temperature from 50 to 90 C for 1 h prior to 2% H2O2 treatment at pH 11.5 for 16 h at 60 C
J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
resulted in growth of Mw from 20870 to 32830 g molÿ1 between fractions 1 and 2, indicating that pre-washing at a relatively high temperature at least in part, enhanced dissolution of large molecular size hemicelluloses in the next step of alkaline peroxide treatment of the straw. This release of large molecular size of hemicelluloses was also partially due to a severe disruption of the physical integrity of the straw during the prewashing at high temperature. Treatment with 2% H2O2 at pH 11.5 at a high temperature of 90 C for a short period of 2 h from 0.2% EDTA aqueous solution treated straw led to a release of the hemicellulosic fraction 5 having a relatively higher Mw over 40,000 g molÿ1. Additionally, treatment with 2% H2O2 at 50 C for 16 h at a higher pH 12.5 solubilized the hemicellulosic fractions 8 and 9 having not only high yields (26.4 and 26.6%) but also high Mw (55,000 and 63,700 g molÿ1). These observations implied that alkaline peroxide treatment did not signi®cantly degrade the macromolecule of hemicelluloses, and the treatment favored release of large molecular size of hemicelluloses at a relatively higher pH such as at pH 12.5 or at pH 11.5 and 90 C for a short period (2 h) from wheat straw. On the other hand, as compared to the Mw in fractions 4 and 7, the bleaching of the hemicellulosic fractions 3 and 6, isolated with 10% KOH at room temperature for 16 h, with 2% H2O2 at pH 11.5 and 60 C for a long period of 16 h or at 60 C for 8 h at pH 11.5 with a relatively
Fig. 2. GPC molecular weight distribution of hemicellulosic fraction 8.
429
higher concentration (5%) of hydrogen peroxide resulted in a noticeable degradation of the solubilized hemicelluloses as shown by the relatively lower Mw at 21,790 ÿ1 and 20,370 g mol . 3.5. FT±IR spectra Fig. 3 shows the FT±IR spectra of hemicellulosic fractions 1 (spectrum F1), 2 (spectrum F2), 4 (spectrum F4), and 5 (spectrum F5). As can be seen, the four spectral pro®les and relative intensities of the bands were rather similar, indicating similar structures of the hemicelluloses isolated in various conditions used. This observation once again demonstrated that alkaline peroxide treatment did not result in any signi®cant change in the macromolecular structure of hemicelluloses. The absorption at 1629 cmÿ1 is principally associated with absorbed water, since the hemicelluloses usually have a strong anity for water, and in the solid state these macromolecules may have disordered structures which can easily be hydrated [22]. Bands between 1125 and 1000 cmÿ1 are typical of xylans. The prominent band at 1050 cmÿ1 is attributed to the C±O, C±C stretching or C±OH bending in hemicelluloses [23]. The band at 1096 cmÿ1 corresponds to the C±OH bending, which is
Fig. 3. FT±IR spectra of hemicellulosic fractions 1 (F1), 2(F2), 4(F4), and 5(F5).
Table 4 Weight-average (Mw) and number-average (Mn) molecular weights and polydispersity (Mw/Mn) of the hemicellulosic fractions extracted from wheat straw Hemicellulosic fractionsa Mw Mn Mw/Mn a
1
2
3
4
5
6
7
8
9
20870 10770 1.94
32830 14020 2.34
21790 11190 1.95
36040 15550 2.32
43420 18010 2.41
20370 10660 1.91
31730 12930 2.45
54980 17150 3.21
63730 19950 3.14
Corresponding to the fractions in Table 1.
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J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
strongly in¯uenced by degree of branching. The sharp band at 894 cmÿ1, which corresponds to the C1 group frequency or ring frequency, is characteristic of b-glycosidic linkages between the sugar units [24]. The bands at 1467, 1330, 1260, and 1213 cmÿ1 represent C±H, OH or CH2 bendings. An intensive band at 1421 cmÿ1 corresponds to the C±O stretch and CH or OH bending in
hemicelluloses [22]. The occurrence of a small band at 1510 cmÿ1 in spectra F4 and F5 is undoubtedly due to the presence of small amounts of associated lignin in the hemicelluloses, which corresponded to the results obtained by alkaline nitrobenzene oxidation. The FT±IR spectra of the hemicellulosic fractions 6 (spectrum F6), 8 (spectrum F8), and 9 (spectrum F9) are shown in Fig. 4. The most obvious feature is the similarity of these spectra. The absorbances at 1470, 1412, 1319, 1266, 1219, 1095, 1049, and 892 cmÿ1 in the spectra are associated with hemicelluloses. All the spectra have an intense absorbed water-related absorbance at 1624 cmÿ1. The lignin-related absorbance at 1515 cmÿ1 is rather weak in the spectrum 9 and poorly resolved in the other two spectra. This is in accordance with the content of associated lignin in the isolated hemicelluloses. 3.6.
Fig. 4. FT±IR spectra of hemicellulosic fractions 6 (F6), 8(F8), and 9 (F9).
Fig. 5.
13
13
C-NMR spectra
In order to characterize the structural features of the isolated hemicelluloses, the hemicellulosic fractions 7 and 8 were analyzed by 13C-NMR spectroscopy (Fig. 5). The spectra were interpreted on the basis of reported data for structurally-de®ned arabinoxylan-type, glucoronoxylantype and l-arabino-(4-O-methyl-d-glucurono)-d-xylan, as well as those of wheat straw hemicelluloses extracted
C-NMR spectra of hemicellulosic fractions 7(a) and 8(b).
J.M. Fang et al. / Polymer Degradation and Stability 66 (1999) 423±432
before deligni®cation [6, 25±27]. The main 1,4-linked bd-Xylp units are obviously characterized by the signals at 104.9, 78.4, 77.5-77.6, 75.8±75.9, and 65.8 ppm, which attributes to C-1, C-4, C-3, C-2, and C-5 of the bd-Xylp units, respectively. The signals at 111.2±111.8, 89.0±89.1, 82.8±83.0, 81.0±81.2, and 64.1±64.3 ppm correspond to C-1, C-4, C-2, C-3, and C-5 of a-l-Araf residues, respectively. Three signals at 176.1, 85.0 (data not shown in the spectra), and 58.8 ppm originate from C-6, C-4 and 4-O-methoxyl group of glucuronic acid residue in the xylan which are very weak and in accord with the low uronic acid content. The signal at 26.8 ppm related to -CH3 in Ar±COCH3, indicating the associated lignins. The presence of esteri®ed acetyl group was identi®ed by a noticeable signal for CH3 in CH3COgroup at 19.6 ppm in the hemicellulosic fraction 8, while it was completely disappeared in the fraction 7. This suggested that treatment with alkaline peroxide under the conditions given only partially broke the esteri®ed linkages between acetyl group and hemicelluloses, while alkaline extraction with 10% KOH under the conditions used completely removed this ester bond. The signal at 179.2 ppm originates the carbonyl group in associated lignin. The near disappearance of the signals at 179.2 and 26.8 ppm for associated lignins in the fraction 8 indicated that alkaline peroxide had more eect on the deligni®cation than the potassium hydroxide under the conditions used in this study. Clearly, the above similar spectra between the alkali-soluble hemicelluloses and the alkaline peroxide-soluble ones demonstrated that treatment of wheat straw with alkaline peroxide did not signi®cantly change the macromolecular structure of hemicelluloses from wheat straw. Schonebaum [28] reached similar conclusions as early as 1922 in the studies of mono- and di-saccharide behaviours in hydrogen peroxide solution. He demonstrated that d-glucose, d-fructose, d-xylose, d-arabinose, lactose, and maltose, at low levels of hydrogen peroxide, even at 70 C, are not signi®cantly changed. In summary, treatment of wheat straw with alkaline peroxide under the conditions used resulted in a partial reduction of esteri®ed acetic acid, ferulic and p-coumaric acids but had a signi®cant eect on the etheri®ed linkages between lignin and hemicelluloses as shown by a release of 83±93% of the originally present hemicelluloses. The treating process did not aect the macromolecular structure of hemicelluloses to any noticeable extent, as supported by 13C-NMR analyses which indicated that no substantial changes occurred during the treatment. Under an optimum condition (2% H2O2, 50 C, pH 12.5, 16 h), the treatment solubilized 92% of the original hemicelluloses, which had a large molecular size (Mw=54,980±63,730 g molÿ1). Furthermore, in comparison, we also tested the possibility of extracting with alkali such as 10% KOH, and then separately bleaching the solubilized hemicelluloses with
431
alkaline peroxide, This two-step treating process minimised the use of hydrogen peroxide and allowed the use of more extreme extraction conditions to increase the hemicellulose yield. However, a prolonging bleaching period up to 16 h with 2% H2O2 at pH 11.5 and 60 C or bleaching using a relatively higher concentration (5%) of hydrogen peroxide at 60 C for 8 h at pH 11.5 resulted in a noticeable degradation of the solubilized hemicelluloses as shown by the relatively lower Mw of 21,790 and 20,370 g ÿ1 mol in fractions 3 and 6, respectively. Colours of the hemicelluloses and its solution will be measured, as well as viscosity-concentration relationships at various temperatures. The chemical modi®cation of the isolated hemicelluloses as a novel material for industries is currently under investigation in our laboratory. Acknowledgements The authors are grateful for the ®nancial support of this research from the European Community under the Industrial and Materials Technologies Programme (Brite-EuRam III)-Depolymerisation, Polymerisation and Applications of Biosustainable Raw Materials for Industrial End Uses.
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