Isolation and characterization of hemicelluloses and cellulose from rye straw by alkaline peroxide extraction

Transcription

1 Cellulose 7: , Kluwer Academic Publishers. Printed in the Netherlands. Isolation and characterization of hemicelluloses and cellulose from rye straw by alkaline peroxide extraction J. M. FANG, R. C. SUN and J. TOMKINSON The BioComposites Centre, University of Wales, Bangor, Gwynedd, LL57 2UW, UK Received 8 June 1999; accepted 29 February 2000 Abstract. Extraction of water-treated rye straw with 2% H 2 O 2 at ph 11.5 for 12 h at 20 C, 30 C, 40 C, 50 C, 60 C, and 70 C released % of the original hemicelluloses and % of the original lignin. As a comparison, treatment of the straw with a dilute alkaline solution at ph 11.5 for 12 h at 50 C in the absence of H 2 O 2 yielded only 7.3% of the original hemicelluloses and 7.4% of the original lignin. Xylose was the predominant sugar constituent in the seven solubilized hemicellulosic preparations, and arabinose, glucose, and galactose were present in small amounts. The hemicellulosic samples were further characterized by Fourier transform infrared (FT-IR), and carbon-13 magnetic resonance spectroscopy ( 13 C-NMR), gel permeation chromatography (GPC), and nitrobenzene oxidation of the associated lignin, and the results are reported. The most obvious feature was found that the alkaline peroxide treatment of the straw under the conditions used did not affect the overall structure of the hemicelluloses as compared to the hemicelluloses isolated with alkali from delignified rye straw. Key words: hemicelluloses, hydrogen peroxide, lignin, rye straw Introduction Rye is one of the major cereal crops with a world-wide annual production of over 30 million tonnes which yields about million tonnes of straw (Ghaly and Ergudenler, 1994). This straw and other agricultural residues are abundant renewable resources for animal feed, bioconversion to sugars, and paper making, especially in the developing countries such as China and India (Patel and Bhatt, 1992). However, the effluents from both pulping and bleaching processes present serious environmental problems due to the utilization of chlorine and chlorine derivatives which can generate large amounts of chlorinated organic compounds. Due to the increasingly stricter environmental laws and a growing demand for chlorine free bleached pulps, many alternative bleaching processes are being investigated, and great attention has been Author for correspondence.

2 88 devoted to the use of oxygen, ozone, and hydrogen peroxide (Nascimento et al., 1995). Hydrogen peroxide is a mild oxidant which is largely used in the bleaching of high yield pulps, and also in chemical bleaching sequence. Its highest efficiency in bleaching and delignification is observed when the reaction is conducted in alkaline medium. Under these conditions, the active species responsible for the elimination of chromophoric groups from lignin are hydroperoxide anion (HOO ). This anion was found to be a strong nucleophile that, during bleaching, preferentially attacks ethylenic and carbonyl groups present in lignin. As a consequence, such chromophores as quinones, cinnamaldehyde, and ring-conjugated ketoses are converted to nonchromophoric species. On the other hand, radical species such as hydroxyl radicals (HO ) generated from the hydrogen peroxide alkaline decomposition, are responsible for delignification and solubilization of hemicelluloses (Pan et al., 1998). Gould (1984) reported that relatively dilute alkaline solutions of hydrogen peroxide removed one-half of the lignin present in materials such as wheat straw, yielding a cellulose-rich insoluble residue that can be enzymatically converted to glucose. Kerley et al. (1987) indicated that treatment of lignocellulosic materials such as wheat straw with alkaline solutions of hydrogen peroxide significantly increased susceptibility of plant structural carbohydrates to fibre-digesting microorganisms in the ruminant digestive tract. Apparently alkaline peroxide treatment resulted in both partial delignification of the cell wall and at least partial decrystalization of cellulose microfibrils. Hydrogen peroxide has been used to bleach sulfite and kraft pulps for many years, but only a few studies have been performed on delignification and solubilization of hemicelluloses from lignocellulosic materials with alkaline peroxide (Lachenal et al., 1992). Gould (1985) mentioned that approximately half the lignin present in wheat straw could be solubilized when the residue was treated at 25 C with an alkaline solution of hydrogen peroxide. The delignification was most effective at ph With the studies of delignification of southern pine kraft pulp with alkaline hydrogen peroxide, McDonough et al. (1989) found that approximately half the lignin present in the pulp could be removed. Treatment of the wood with hydrogen peroxide at optimum ph (ph 11) followed by alkaline extraction, removed at most 36% of the original lignin (Springer, 1990). For the first time, we studied the extraction of hemicelluloses from rye straw with alkaline peroxide. Hemicelluloses are a large group of well-characterized polysaccharides found in the primary and secondary cell walls in all land and fresh water plants, and in some seaweeds. They are classically defined as the alkali soluble material after removal of the pectic substances, and have a much lower degree of polymerisation ( U) as compared to that of cellulose

3 (10,000 14,000). The principle sugar components are D-xylose, D-mannose, D-glucose, D-galactose, L-arabinose, D-glucuronic acid, 4-O-methyl-D-glucuronic acid (MeGlcA), D-galacturonic acid, and to a lesser extent, L-rhamnose, L-fucose, and various O-methylated sugars. Annual plants such as straw and grass contain 20 35% hemicelluloses. The hemicelluloses of straw from the Gramineae family have a backbone of β-1,4-linked xylopyranosyl units, and have mostly single arabinofuranosyl units attached to some C-3 position of the main xylan chain and glucuronic acid and/or its MeGlcA linked to some xylose units. The latter are probably mainly linked to the C-2 position (Sun et al., 1996). These large amounts of hemicelluloses are worth exploiting to serve as adhesives, thickeners, and stabilizers, and as film formers and emulsifiers (Doner and Hicks, 1997). This report is the first paper concerning hemicellulose details in rye straw and it also provides evidence of hemicellulose solubilization in an alkaline solution of hydrogen peroxide. Effect of treating temperature on the solubilization of the hemicelluloses and chemical composition of the residues, mainly cellulose, was examined. Their chemical composition, content of associated lignin, molecular weights, and structural features, studied by 13 C-NMR, are reported. 89 Materials and methods Materials Rye straw was obtained from Compak Co. (Gainsborough, England), and was ground to pass a 0.7 mm size screen. All weights and calculations were made on an oven-dried (50, 16 h) weight basis. The composition (w/w) of the rye straw used was: 37.9% cellulose, 36.9% hemicelluloses, 17.6% lignin, 3.3% protein, 3.0% ash, and 2.0% wax. All chemicals used were of analytical or reagent grade. Alkaline peroxide treatment The dried powder was first extracted with toluene ethanol (2:1,v/v) in a Soxhlet apparatus for 6 h. The dewaxed straw was then treated with water at 50 C for 2 h. The water-soluble hemicelluloses were isolated by precipitation in ethanol. Samples free of wax and water solubles (10.0 g) were added to 250 ml of distilled water containing 2% H 2 O 2 (w/v) in a jacketed reaction vessel heated with water from a thermostat-controlled circulating bath. The suspension was adjusted to ph 11.5 with 4 M NaOH and allowed to stir gently for 12 h at 20 C, 30 C, 40 C, 50 C, 60 C, and 70 C, respectively (Figure 1).

4 90 Figure 1. Scheme for isolation of hemicelluloses from the hydrolysates of 2% H 2 O 2 treatment of dewaxed and water-treated rye straw. For comparison of the effect of H 2 O 2 on the yield of hemicelluloses solubilized, one sample was treated with dilute alkaline solution at ph 11.5 for 12 h at 50 C in the absence of H 2 O 2 from water-soluble free and dewaxed rye straw. 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 final value of ca The insoluble residue was collected by filtration, washed with distilled water until the ph of the filtrate was neutral, and then dried at 60 C. The supernatant fluid was adjusted to ph 5.5 with 10% HCl and then concentrated. The solubilized hemicelluloses were precipitated by pouring the concentrated supernatant fluid into three volumes of ethanol, from which they were settled out as a white flocculent precipitate and air-dried. For comparison of the hemicelluloses isolated from

5 91 Figure 2. Scheme for isolation of hemicelluloses and cellulose from delignified rye straw the delignified straw, the lignins in water-treated sample were removed with 1.3% NaClO 2 in acidic solution (ph 4.0, adjusted by 10% acetic acid) at 75 C for 2 h. The hemicelluloses were extracted from the holocellulose sample with 24% KOH 2% H 3 BO 3 at 20 C for 2 h in the absence of H 2 O 2, and isolated as the method above (Figure 2). Characterization of the solubilized hemicelluloses The neutral sugar composition of the isolated hemicelluloses and residues, was determined by gas chromatography (GC) analysis of their alditol acetates (Blakeney et al., 1983). Alkaline nitrobenzene oxidation of associated lignin from solubilized hemicelluloses and residues was performed at 170 C for 3 h. The lignin content in hemicellulosic and residual fractions was calculated multiplying by 2.5 the yield of phenolics, obtained by nitrobenzene oxidation (Sun et al., 1998). The method of determination of phenolic acids and aldehydes in nitrobenzene oxidation mixtures with high performance

6 92 liquid chromatography (HPLC) has been described in previous papers (Sun et al., 1995; Lawther et al., 1995). The content of uronic acids in solubilized hemicelluloses was assayed colorimetrically as glucuronic acid using 3-phenylphenol color reagent according to the procedure outlined by Blumenkrantz and Asboe-Hanson (1973). The molecular-average weights of the hemicelluloses were determined by gel permeation chromatography on a PL aquagel-oh 50 column. The samples were dissolved with 0.02 M NaCl in M sodium phosphate butter, ph 7.5, at a sample concentration of 0.1%, and 200 µl of this solution was injected. The columns was operated at 40 C, and eluted with 0.02 M NaCl in M sodium phosphate buffer, ph 7.5, at a flow rate of 0.3 ml min 1.The column was calibrated using PL pullulan polysaccharide. FT-IR spectra were obtained on an FT-IR spectrophotometer (Nicolet, 750) using a KBr disc containing 1% finely ground samples. The solutionstate 13 C-NMR spectrum was obtained on a Bruker 250 AC spectrometer operating in the FT mode at 62.4 MHz under total proton decoupled conditions. It is recorded at 25 C from 150 mg of sample dissolved in 1.0 ml D 2 O after 10,000 scans. A 60E pulse flipping angle, a 3.9 µs pulse width and 0.85 s acquisition time were used. Results and discussion Yield of solubilized hemicelluloses The yields of hemicelluloses and lignin, solubilized during the alkaline peroxide treatment processes, are given in Table 1. The values of hemicelluloses and lignin are calculated on the basis of water-treated rye straw, while the yield of residues was expressed as the untreated starting material. The rye straw mainly contained 36.9% hemicelluloses, 37.9% cellulose, and 17.6% lignin. Treatment of the dewaxed straw with distilled water at 50 Cfor2h solubilized 6.6% hemicelluloses and 2.8% lignin (% dry matter). The effect of alkaline peroxide treatment temperature on the release of hemicelluloses and lignin is shown in Table 1 for treatment of the water-extracted straw with 2% H 2 O 2 at ph 11.5 for 12 h at various temperatures. As can be seen in Table 1, increasing temperature from 20 Cto40 C, the yield of solubilized hemicelluloses increased significantly from 13.4% to 21.2%, and the yield of residue subsequently decreased from 66.4% to 53.1%. This was largely due to the solubilization of hemicelluloses and lignin during the alkaline peroxide treatment processes. Further increasing temperature from 40 Cto70 Cled to slight increasing yields of hemicelluloses from 21.2% to 21.8% and lignin from 12.1% to 13.0%. This indicated that the reaction of alkaline peroxide

7 Table 1. The yields of hemicelluloses and lignin (% dry matter) solubilized in water, dilute alkaline solution, and 2% H 2 O 2 treatment of water-soluble-free and dewaxed rye straw at ph 11.5 for 12 h at different temperatures Yield 2% H 2 O 2 (ph 11.5, 12 h) treatment WS b DAS c PH d (% dry matter) temperature ( C) 20 a 30 a 40 a 50 a 60 a 70 a Hemicelluloses e Lignin Residue a The hemicellulosic and lignin fractions obtained by treatment of water-soluble-free and dewaxed rye straw with 2% H 2 O 2 at ph 11.5 for 12 h at different temperatures. b Represent the water-soluble hemicellulosic and lignin fractions obtained by treatment of the dewaxed rye straw with distilled water at 50 Cfor2h. c Represent the hemicellulosic and lignin fractions extracted with dilute alkaline solution (ph 11.5) at 50 C for 12 h in the absence of H 2 O 2 from water-soluble-free and dewaxed rye straw. d Represent the aqueous potassium hydroxide solubilized hemicellulosic fraction extracted with 24% KOH 2% H 3 BO 3 at 20 C for 2 h from the dewaxed and delignified rye straw. e Obtained by precipitation of the neutralized extracts with 3 volumes of ethanol. 93 with rye straw under the conditions used proceeded at a relatively rapid rate at 40 C if the requirement for energy consumption needs to be minimized. The isolated hemicelluloses were white except for those released at the high temperature of 70 C, which were slightly brownish. All the recovered residues were off-white. The degree of solubilization of various components from rye straw in alkaline peroxide treatments depended upon the reaction ph, duration, H 2 O 2 concentration, and temperature being the four main parameters. Our earlier studies (Fang et al., 1999) on wheat straw found that over 90% of the original hemicelluloses were solubilized during the treatment with 2% H 2 O 2 at ph 11.5 for 12 h at 50 C, and hence a period of 12 h was used for studying the effect of alkaline peroxide treatment on the solubilization of hemicelluloses at six different temperatures from rye straw (Table 1). As can be seen in Table 1, more rapid increasing rate of solubilizing hemicelluloses appeared as the temperature rose from 20 Cto40 C, while a slight increase in the solubilization of hemicelluloses was found during the further increment of treating temperature between 40 C and 70 C. The cellulose originally present in the straw was recovered in the insoluble residue after the treatment. As a result of the treatment, the straw residue was substantially enriched in cellulose over the starting material (53.1% residue versus 37.9% cellulose originally, obtained at 40 C).

8 94 The extent of the delignification and solubilization of hemicelluloses by alkaline peroxide treatment was found to be strongly dependent on the reaction ph. Below ph 11.0, the efficiency of delignification and solubilization of hemicelluloses declined significantly. Above ph 11.5, the rate of delignification was improved greatly, and the solubilization of hemicelluloses increased substantially. When properly adjusted, the total alkalinity of the solution should be high enough to ensure an adequate concentration of hydroperoxide anion (HOO ), the active bleaching species, throughout the course of reaction as dictated by the equilibrium: H 2 O 2 + HO HOO + H 2 O. As mentioned above, this reaction is strongly ph-dependent, with an optimum at ph , the pka for the dissociation reaction of H 2 O 2 (Gould, 1984). At the same time, the alkalinity must be low enough to minimize peroxide decomposition and chromophore-forming reactions by the highly reactive hydroxyl radical (HO ) that are prone to occur at high ph during the degradation of H 2 O 2 in a reaction with the hydroperoxide anion (Doner and Hicks, 1997). H 2 O 2 + HOO HO +O 2 +H 2O. To obtain maximum rate of delignification and yield of solubilized hemicelluloses, it is, therefore, not necessary to continuously regulate the reaction ph, even though over the course of the treatment the reaction ph increased from initial ph 11.5 to final ca As the reaction ph became more alkaline, more hemicelluloses were solubilized. About 70% of the hemicelluloses originally present in rye straw were solubilized within 12 h of 2% H 2 O 2 treatment in the absence of continuous ph control, while only 48% of the original hemicelluloses were solubilized in 12 h when the reaction ph was maintained at ph 11.5 (data not shown). Further studies showed that the introduction of hydrogen peroxide stabilizer such as sodium silicate had no effect (or an adverse effect, data not shown) on the straw delignification and hemicelluloses solubilization. This observation is consistent with our earlier findings from wheat straw by alkaline treatment (Fang et al., 1999). We indicated that improvement in H 2 O 2 stabilization was not a prerequired condition for good delignification and solubilization of hemicelluloses. Alkaline peroxide is an effective treating agent for both delignification and solubilization of hemicelluloses from agricultural residues such as rye straw. More than 70% of the original hemicelluloses and 80% of the original lignin in water-treated rye straw were solubilized during the treatment with 2% H 2 O 2 at 50 C for 12 h at ph 11.5, while only about 7.3% hemicelluloses and 7.4% lignin solubilized during the treatment of water-extracted straw at

9 Table 2. The content of neutral sugars (relative% dry weight of hemicelluloses, w/w) and uronic acids (% dry weight of hemicelluloses, w/w) in isolated hemicellulosic fractions solubilized in water, dilute alkaline solution, and 2% H 2 O 2 treatment of water-soluble-free and dewaxed rye straw at ph 11.5 for 12 h at different temperatures Sugar (%) 2% H 2 O 2 (ph 11.5, 12 h) treatment WS b DAS c PH d temperature ( C) 20 a 30 a 40 a 50 a 60 a 70 a Rhamnose Fucose ND d ND ND ND ND ND 0.47 ND ND Arabinose Xylose Mannose Glucose Glactose Uronic acids a,b,c,d Corresponding to hemicellulosic fractions in Table 1. ND = not detected. 95 ph 11.5 for 12 h at 50 C in the absence of H 2 O 2. With the study of alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification, Gould (1984) found that approximately one-half of the lignin and most of the hemicelluloses present in agricultural residues such as wheat straw and corn stover were solubilized when the residue was treated at room temperature with 1% H 2 O 2 at ph 11.5 for 24 h. He also indicated that the alkaline peroxide treatment was most efficient for substrate concentration up to 4 g/100 ml with a minimum H 2 O 2 concentration of at least 1%. To achieve maximum delignification and solubilization of hemicelluloses, a substrate concentration of 10 g/250 ml and H 2 O 2 level of 2% were used in this study. As expected, the increasing peroxide concentration enhanced not only the rate of brightening but also efficiency of delignification and the solubilization of hemicelluloses. Sugar composition and content of uronic acids Table 2 shows the monosaccharide composition and content of uronic acids in nine recovered polysaccharide fractions. The water-soluble polysaccharide fraction (WS) mainly comprised neutral chains, including glucose (33.1%), xylose (27.5%), galactose (19.3%), and arabinose (13.6%). This high percentage of glucose and xylose was taken to indicate correspondingly more glucans and xylans. Similarly, the hemicellulosic fraction, extracted with dilute alkaline solution (ph 11.5) at 50 C for 12 in the absence of H 2 O 2 from water-

10 96 treated and dewaxed rye straw, contained relatively higher xylose. Glucose, arabinose, and galactose appeared as the other major sugar constituents. Mannose and rhamnose were detected in trace amounts. These data showed that the water-soluble and dilute alkali-soluble hemicellulosic fractions were probably similar in the structure. Xylose was the predominant sugar in other seven hemicellulosic fractions, extracted with 2% H 2 O 2 at ph 11.5 for 12 h at C from the water-treated straw and with 24% KOH 2% H 3 BO 3 at 20 C for 2 h from the dewaxed and delignified holocellulose, comprising 71.1% 77.2% of the total sugars. Arabinose and glucose appeared as noticeable amounts. Galactose, mannose, and rhamnose were observed as minor constituents. An increase of treating temperature from 20 Cto40 C resulted in an increment of xylose from 72.2% to 77.2% and a nearly constant value of arabinose, but a decrement of glucose from 10.0% to 6.1% in hemicellulosic fractions, extracted by alkaline peroxide. These data provided evidence that in rye straw cell walls, glucose and arabinose, probably present in side chains of hemicelluloses, are bound to ferulic acid or directly to lignin and easily released at low temperature, while xylose in the main chain of hemicelluloses are favourably extracted at high temperature or at low temperature from delignified straw. It is very likely that the hemicelluloses, extracted by alkaline peroxide, are mainly composed of a xylan structure similar to that found in wheat straw (Sun et al., 1996). As the temperature was further increased to 50 C, 60 C, and 70 C, the content of xylose decreased to 75.0%, 73.5%, and 71.1%, respectively. However, no further decrease in the contents of arabinose and glucose was observed in the isolated hemicellulosic fractions when the temperature rose between 40 C and 70 C. This phenomenon indicated that the treatments with 2% H 2 O 2 in alkaline solution at relatively high temperature such as between 50 C and 70 C could result in some degradation of the macromolecular hemicelluloses by partial hydrolysis of the main chains. This is particularly true during the treatment at 70 C. In addition, as can be seen in Table 2, all the hemicellulosic fractions contained small proportions of uronic acids, mainly glucuronic acid or MeGlcA, ranging between 5.1% and 8.5%, which was slightly higher than observed in the hemicellulosic preparations extracted with alkali from wood or wheat straw samples. These findings were consistent with those of an earlier study reporting that more acidic groups were found in the hemicellulosic fractions, extracted by hydrogen peroxide in alkaline solution, as compared to use of alkali only (Fang et al., 1999). The data on sugar composition in the residues are summarized in Table 3. Glucose was the predominant sugar component and increased in the following order: water-treated residue (WR, 65.6%), dilute alkali-treated residue (DAR, 65.8%), alkaline peroxide-treated residues (69.9% 79.9%), and 24% KOH 2% H 3 BO 3 treated residue (PHR, 95.8%), which corresponded to the

11 Table 3. The composition of neutral sugars (relative% dry weight of residues, w/w) in the residues treated by water, dilute alkaline solution, 2% H 2 O 2, and 24% KOH 2% H 3 BO 3 from rye straw Sugar (%) 2% H 2 O 2 (ph 11.5, 12 h) treatment WR b DAR c PHR d temperature ( C) 20 a 30 a 40 a 50 a 60 a 70 a Rhamnose Tr Tr ND ND ND Arabinose Xylose Mannose Glucose Glactose a The residual fractions obtained by treatment of water-soluble-free and dewaxed rye straw with 2% H 2 O 2 at ph 11.5 for 12 h at different temperatures. b The residual fraction obtained by treatment of the dewaxed rye straw with water at 50 C for 2 h. c The residual fraction obtained by extraction of water-soluble-free and dewaxed rye straw with dilute alkaline solution (ph 11.5) at 50 C for 12 h in the absence of H 2 O 2. d Represent the cellulose fraction obtained by extraction with 24% KOH 2% H 3 BO 3 at 20 C for 2 h from the dewaxed and delignified rye straw. Tr = trace, ND = not detected. 97 content of cellulose and the yield of hemicelluloses solubilized during the various treatment processes. The cellulose fraction (PHR),. extracted with 24% KOH-2% H 3 BO 3 at 20 C for 2 h from the dewaxed and delignified straw, still contained a small amount of xylose (3.1%), together with trace amounts of arabinose (0.5%), mannose (0.3%), and galactose (0.3%). These resistances to extraction with high concentration of alkali (24% KOH 2% H 3 BO 3 ) and 2% H 2 O 2 at ph 11.5 for 12 h implied that the hemicelluloses in rye straw cell walls are very strongly associated to the surface of cellulose. In general, alkaline peroxide treatment resulted in a significant delignification together with a substantial solubilization of hemicelluloses, which, however, did not cause detectable changes in the structure of highly polymerized cellulose (Martel and Gould, 1990). Content of associated lignin and composition of phenolic acids and aldehydes The pigmentation associated with the hemicellulosic preparations is likely due to the presence of strongly associated lignin, which was linked to arabinoxylans by ether bonds (Doner and Hicks, 1997). In this study, the hemicelluloses prepared by alkaline peroxide extraction were much lighter in col-

12 98 our than that obtained under similar conditions but in the absence of peroxide. Treatment of the dewaxed and water-treated rye straw with 2% H 2 O 2 at 50 C for 12 h at ph 11.5 released over 83% of the original lignin and 70% of the original hemicelluloses having 5.8% associated lignin, whereas the treatment under similar conditions but in the absence of H 2 O 2 solubilized only 7.4% original lignin and 7.3% original hemicelluloses, which contained over 8% associated lignin. The foregoing results indicated once again that alkaline peroxide appeared as a strong agent both for delignification and for bleaching. To further verify the presence of associated lignin, alkaline nitrobenzene oxidation of bound lignin in the nine isolated hemicellulosic fractions was performed at 170 C for 3 h. This method provides an estimate of the total amount of lignin and an indication of the composition of phenolic units. As can be seen in Table 4, the major products, obtained from alkaline nitrobenzene oxidation, were identified to be syringaldehyde and vanillin, which together represented for 52.3% 77.0% of the total phenolic monomers. This suggested that the associated lignin in the hemicelluloses contained roughly equal amounts of non-condensed syringyl and guaiacyl units. Small amounts of p-hydroxybenzaldehyde, vanillic acid, and syringic acid, and traces of p- hydroxybenzoic acid, p-coumaric acid, and ferulic acid were also found to be present in the nitrobenzene oxidation mixtures. Occurrence of ferulic and p-coumaric acids in cell walls of straw and grass has been reported to have a profound influence on the growth of the plant cell wall and its mechanical properties and biodegradability (Pan et al., 1998). It has found that p-coumaric acid was mostly esterified to lignin or polysaccharides, while ferulic acid appeared almost equally in esterified bonds to arabinose in hemicelluloses and in etherified linkages with lignin. The occurrence of traces of esterified or etherified 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 rye straw. This observation indicated that treatment of rye straw with alkaline peroxide can only result in a partial cleavage of the esterified linkages such as those between ferulic acid and hemicelluloses or between p-coumaric acid and lignin/or hemicelluloses. This treatment had a little effect on the ether bonds between ferulic acid and lignin, but had a significant efficiency on the cleavage of the direct ether bonds between hemicelluloses and lignin, as evidenced by a significant solubilization of hemicellulose and lignin fragments bearing ferulic and p- coumaric acids, and release of 83% of original lignin during the treatment with 2% H 2 O 2 for 12 h at ph 11.5 such as at 50 C. The content of residual lignin and yield of phenolic acids and aldehydes obtained in the products of alkaline nitrobenzene oxidation of residual lignin

13 Table 4. The yield (% hemicellulosic sample, w/w) of phenolic acids and aldehydes from alkaline nitrobenzene oxidation of the associated lignin in isolated hemicellulosic fractions Phenolic acids 2% H 2 O 2 (ph 11.5, 12 h) treatment WS b DAS c PH d and aldehydes temperature ( C) 20 a 30 a 40 a 50 a 60 a 70 a p-hydroxy benzoic acid p-hydroxy benzaldehyde Vanillic acid Syringic acid Vanillin Syring aldehyde p-coumaric acid Ferulic acid Total Lignin content a,b,c,d Corresponding to hemicellulosic fractions in Table 1. from the corresponding residues are given in Table 5. As discussed earlier, due to the significant delignification of H 2 O 2, the content of lignin in the residues, treated by 2% H 2 O 2 at ph 11.5 for 12 h at C, was much lower than that in the residue, treated by dilute alkali at ph 11.5 in the absence of H 2 O 2. Syringaldehyde and vanillin appeared to remain the major components of phenolics in the nitrobenzene oxidation of bound lignin from the residues. It was highly probable that increase of alkaline peroxide treatment temperature favoured the reduction of bound lignin in the residues, indicated by the decrease lignin content from 8.9% to 3.7% with the increase of treatment temperature from 20 Cto70 C. Occurrence of 2% of the bound lignin in the residue, obtained by extraction with 24% KOH 2% H 3 BO 3 at 20 C for 2 h from the almost delignified holocellulose, indicated that the fraction of the cellulose was still contaminated with small amounts of residual lignin. Molecular weight distribution In order to illustrate whether the extent of degradation occurred during the process of alkaline peroxide treatment, all the molecular weights of hemicel-

14 100 Table 5. The yield (% residual sample, w/w) of phenolic acids and aldehydes from alkaline nitrobenzene oxidation of the associated lignin in the residual fractions Phenolic acids 2% H 2 O 2 (ph 11.5, 12 h) treatment WR b DAR c PHR d and aldehydes temperature ( C) 20 a 30 a 40 a 50 a 60 a 70 a p-hydroxy benzoic acid p-hydroxy benzaldehyde Vanillic acid Syringic acid Vanillin Syringaldehyde p-coumaric acid Ferulic acid Total Lignin content a,b,c,d Corresponding to hemicellulosic fractions in Table 3. Table 6. Weight-average (M w ) and number-average (M n ) molecular weights and polydispersity (M w /M n ) of the hemicellulosic fractions isolated from rye straw 2% H 2 O 2 (ph 11.5, 12 h) treatment WS b DAS c PH d temperature ( C) 20 a 30 a 40 a 50 a 60 a 70 a M w M n M w /M n a,b,c,d Corresponding to hemicellulosic fractions in Table 1. lulosic preparations were calculated from the GPC chromatograms, and the weight-average (M w ) and number-average (M n ) molecular weights and the polydispersity (M w /M n ) of the solubilized hemicellulosic preparations are listed in Table 6. As compared to the molecular weight (M w = 21,150 g mol 1 ) of hemicellulosic fraction, isolated with a traditional method (24% KOH 2% H 3 BO 3 at 20 C for 2 h) from the delignified straw, all the molecular weights of the hemicellulosic fractions, solubilized during the alkaline peroxide treatment except for the fraction, extracted with 2% H 2 O 2 at 20 C for 12 h at ph

15 , showed approximately equal levels, indicating that the alkaline peroxide treatment under the conditions used did not result in any significant degradation of the macromolecular structure of hemicelluloses, and the extraction with 2% H 2 O 2 at 20 C for 12 h at ph 11.5 favoured solubilization of the small molecular size of hemicelluloses. As can be seen from the table, an increase in temperature from 20 C to 50 C during the 2% H 2 O 2 treatment at ph 11.5 for 12 h resulted in the growth of M w from 14,510 to 21,760 g mol 1, indicating that increasing temperature between 20 C and 50 C at least in part, enhanced dissolution of large molecular size hemicelluloses during the alkaline peroxide treatment of the straw. In contrast, as the temperature was further increased from 50 C to 60 C,andto70 C, the M w decreased from 21,760 to 21,210, and to 19,560 g mol 1, suggesting that a minimal degradation occurred during the treatment over 50 C. This degradation was particularly true at high temperature of 70 C, in which the molecular weight obtained was lower than the value measured at 50 C by 10%. As expected, the water-soluble and dilute alkali-soluble polysaccharides had lower M w between 6180 and 6340 g mol 1 as compared to the hemicelluloses, extracted with 2% H 2 O 2 or 24% KOH 2% H 3 BO 3, indicating that the extraction of rye straw with water at 50 Cand dilute alkali in the absence of H 2 O 2 resulted in dissolution of low molecular size polysaccharides. The molecular weight distribution of the hemicellulosic fraction, extracted with 2% H 2 O 2 at 50 C for 12 h and ph 11.5, is shown in Figure 3. As can be seen from the diagram, the molecular weight distribution ranged between over one million and 860 g mol 1 with a main peak having the molecular weight of 10,260 g mol 1. The second small peak had a relatively lower molecular weight value of around 1720 g mol 1, which was presumed to be due to the fragmentation of hemicelluloses during the alkaline peroxide treatment process. The distribution showed a wide polymolecularity. FT-IR spectra FT-IR spectroscopy has been used to identify polysaccharides, to check their purity, to carry out semi-quantitative functional analyses, to determine structure, and to investigate complexing and inter-molecular interactions. Figure 4 shows the FT-IR spectra of hemicellulosic fractions extracted with 2% H 2 O 2 at ph 11.5 for 12 h at 40 C (spectrum a) and at 50 C (spectrum b), with dilute alkaline solution (ph 11.5) at 50 C for 12 h in the absence of H 2 O 2 (spectrum c) from water-treated rye straw, and with 24% KOH 2% H 3 BO 3 at 20 Cfor 2 h from the delignified straw (spectrum d). As expected, the four spectral profiles and relative intensities of the most bands are rather similar, indicating similar structures for the hemicelluloses. This observation once again

16 102 Figure 3. The GPC molecular weight distribution of the hemicellulosic fraction extracted with 2% H 2 O 2 at 50 C and ph 11.5 for 12 h from dewaxed and water-treated rye straw. indicated that alkaline peroxide treatment did not result in any significant change in the macromolecular structure of hemicelluloses. The absorption at 1640 cm 1 is principally associated with absorbed water (Kacurakova et al., 1998). Bands between 1125 and 1000 cm 1 are typical of xylans. The prominent band at 1043 cm 1 is attributed to the C O, C C stretching or C OH bending in hemicelluloses. The sharp band at 894 cm 1, which corresponds to the C 1 group frequency or ring frequency, is characteristic of β-glycosidic linkages between the sugar units (Gupta et al., 1987). The small bands at 1480, 1328, and 1268 cm 1 represent C H, OH or CH 2 bendings. A band at 1428 cm 1 corresponds to the C O stretch and CH or OH bending in hemicelluloses (Sun et al., 1998). The bands at 1388 and 1169 cm 1 attribute to C H deformation and C O C vibration in hemicelluloses, respectively. The acetyl and uronic ester groups of hemicellulose residue absorb at 1745 cm 1 in the spectrum c, and the intensity for this band is quite weak, indicating that the dilute alkali (ph 11.5) treatment of the straw in the absence of H 2 O 2 only partially cleaved the ester linkages, while the treatment with alkaline peroxide under the same alkalinity such as ph 11.5 or extraction with a strong alkaline solution such as 24% KOH completely cleaved these ester bonds. Occurrence of a small band at 1514 cm 1 in the spectra is undoubtedly due to the presence of small amounts of associated lignin in the hemicelluloses, and the band intensity decreased from c to a, to b, and to d, which corresponded to the content of bound lignin, determined by alkaline nitrobenzene oxidation.

17 103 Figure 4. The FT-IR spectra of the hemicellulosic fractions extracted with 2% H 2 O 2 at ph 11.5 for 12 h at 40 C (spectrum a) and 50 C (spectrum b) from the water-treated rye straw, with dilute alkaline solution (ph 11.5) at 50 C for 12 h in the absence of H 2 O 2 (spectrum c) from the water-treated rye straw, and with 24% KOH 2% H 3 BO 3 at 20 C for 2 h from the dewaxed and delignified rye straw (spectrum d) The FT-IR spectra of the residual fractions obtained by treatment with 2% H 2 O 2 at ph 11.5 for 12 h at 20 C (spectrum a) and 60 C (spectrum b), with dilute alkaline solution (ph 11.5) at 50 C for 12 h in the absence of H 2 O 2 (spectrum c), and with 24% KOH 2% H 3 BO 3 at 20 C for 2 h (spectrum d) are shown in Figure 5. The absorbances at 1434, 1381, 1321, 1268, 1170, 1070, 1049, 1023, and 897 cm 1 in the spectra are associated with cellulose. As can be seen from the Figure 5, all the spectra have an intense absorbed water-related absorbance at 1640 cm 1. Analogously, a band at 1740 cm 1 in the residue, obtained by treatment of the water-treated straw with dilute alkaline solution (ph 11.5) in the absence of H 2 O 2, corresponds to the acetyl and uronic ester groups. The lignin-related absorbances at 1514 and 1450 cm 1 is obvious in the spectrum c, suggesting a noticeable amount of bound lignin in the fraction. While in the spectra a and b, this band appears to be rather weak, indicating a small amount of associated lignin in the residues treated by alkaline peroxide. A almost disappearance of these two bands in the spectrum d implied a relatively free of the bound lignin in the residue obtained by 24% KOH 2% H 3 BO 3 extraction at 20 C for 2 h from the holocellulose.

18 104 Figure 5. The FT-IR spectra of the residues obtained by treatment with 2% H 2 O 2 at ph 11.5 for 12 h in 20 C(spectruma)and60 C (spectrum b) from the water-treated rye straw, with dilute alkaline solution (ph 11.5) at 50 C for 12 h in the absence of H 2 O 2 (spectrum c) from the water-treated rye straw, and 24% KOH 2% H 3 BO 3 at 20 C for 2 h from the dewaxed and delignified rye straw (spectrum d). 13 C-NMR spectrum The 13 C-NMR spectroscopy (in D 2 O) of the hemicelluloses, obtained by treatment of the water-extracted rye straw with 2% H 2 O 2 at 40 C for 12 h at ph 11.5, is depicted in Figure 6. It is similar to the spectrum of the hemicellulosic preparation, isolated with 3% NaOH at 40 C for 6 h from lignified rye straw (spectrum not shown), indicating a similar structural feature. The spectrum was interpreted on the basis of reported data for structurally-defined arabinoxylan-type, glucoronoxylan-type, and L-arabino-(4-O-methyl- D-glucurono)-D-xylan (Kate et al., 1987; Ebringerova et al., 1992; Imamura et al., 1994). The main 1,4-linked β-d-xylp units are obviously characterized by the strong signals at 105.1, 78.5, 77.7, 76.1, and 65.9 ppm, which respectively are assigned to C-1, C-4, C-3, C-2, and C-5 of the β-d-xylp units. Signals at 112.3, 89.2, 82.9, 81.0, and 64.4 ppm correspond to C-1, C-4, C-2, C-3, and C-5 of α-l-araf residues, respectively. Signals attributed 4-O-methoxyl group of glucuronic acid residue in the hemicelluloses appear at 85.5 and 58.3 ppm (data not shown). Two weak signals at 73.1 and 68.9 ppm (data not shown) originate C-4 and C-2 of galactopyranosyl residues in hemicelluloses. These signals demonstrated that the alkaline per-

19 105 Figure C-NMR sepctrum of hemicellulosic fraction (in D 2 O extracted with 2% H 2 O 2 at 40 C for 12 h at ph 11.5 from dewaxed and water-treated rye straw. oxide treatment under the conditions used did not affect the overall structure of the macromolecular hemicelluloses. Summary and conclusions From the results discussed above, it is clear that treatments of the rye straw with 2% H 2 O 2 at ph 11.5 for 12 h at C resulted in a solubilization of % of the original hemicelluloses, which had a degree of polymerization giving weight-average molecular weights between 14,510 and 21,760 g mol 1. Xylose was a predominant component sugar in all the hemicellulosic fractions, comprising % of the total sugars. Arabinose, glucose, and galactose were present in small amounts, and rhamnose and mannose were identified as minimal quantities. The data obtained by alkaline nitrobenzene oxidation showed that the six hemicellulosic preparations contained % associated lignin, which mainly consisted of approximately equal amounts of syringyl and guaiacyl units. Under the 2% H 2 O 2 and ph 11.5 used, an optimum treatment temperature was found to be at 40 Cifthe high yield, light colour, and minimal degradation of the hemicelluloses were considered. Further studies showed the hemicellulosic preparations, obtained by alkaline peroxide extraction, had similarly physico-chemical properties as compared to those, isolated by 24% KOH 2% H 3 BO 3 at 20 C for 2 h from the delignified straw in the absence of H 2 O 2. This suggested that the alkaline peroxide treatment of the straw under the conditions used did not result in any significant change in the macromolecular structure of hemicelluloses.

20 106 Acknowledgements The authors are grateful for the financial support of this research from the European Community under the Industrial & Materials Technologies Programme (Brite-EuRam III)-Depolymerisation, Polymerisation and Applications of Biosustainable Raw Materials for Industrial End Uses. References Blakeney, A. B., Harris, P. J., Henry, R. J. and Stone, B. A. (1983) A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr. Res. 113, Blumenkrantz, N. and Asboe-Hanson, G. (1973) New method for quantitative determination of uronic acids. Anal. Biochem. 54, Doner, L. M. and Hicks, K. (1997) Isolation of hemicellulose from corn fibre by alkaline hydrogen peroxide extraction. Cereal Chem. 74, Ebringerova, A., Hromadkova, Z., Alfoldi, J. and Berth, G. (1992) Structural and solution properties of corn cob heteroxylans. Carbohydr. Polym. 19, Fang, J. M., Sun, R. C., Salisbury, D., Fowler, P. and Tomkinson, J. (1999) Comparative study of hemicelluloses and lignin from wheat straw by alkali and hydrogen peroxide extractions. Polym. Degrad. Stabil. 66, Ghaly, A. E. and Ergudenler, A. (1994) Reaction kinetics of rye straw for thermochemical conversion. Can. J. Chem. Egnin. 72, Gould, J. M. (1984) Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification. Biotechnol. Bioengin. 26, Gould, J. M. (1985) Studies on the mechanism of alkaline peroxide delignification of agricultural residues. Biotechnol. Bioengin. 27, Gupta, S., Madan, R. N. and Bansal, M. C. (1987) Chemical composition of Pinus caribaea hemicellulose. Tappi J. 70, Imamura, T., Watanabe, T., Kuwahara, M. and Koshijima, T. (1994) Ester linkages between lignin and glucuronic acid in lignin carbohydrate complexes from Fagus crenata. Phytochemistry 37, Kacurakova, M., Belton, P. S., Wilson, R. H., Hirsch, J. and Ebringerova, A. (1998) Hydration properties of xylan-type structures: and FTIR study of xylooligosaccharides. J. Sci. Food Agric. 77, Kato, A., Azuma, J. and Koshijima, T. (1987) Isolation and identification of a new ferulated tetrasaccharide from bagasse lignin carbohydrate complex containing phenolic acid. Agric. Bio. Chem. 51, Kerley, M. S., Fahey, G. C., Berger, J. L. L., Merchem, N. R. and Gould, J. M. (1987) Effect of treating wheat straw with ph regulated solutions of alkaline hydrogen peroxide on nutrient digestion by sheep. J. Dairy Sci. 70, Lachenal, D., Wable, F., Damiens, P. and Ledon, H. (1992) The potential of H 2 O 2 as delignifying and bleaching agent-application to new bleaching sequences. Pan Pacific Pulp and Paper Technology Conference Proceedings, JTAPPI, Tokyo, pp Lawther, J. M., Sun, R. C. and Banks, W. B. (1995) Extraction, fractionation, and characterization of structural polysaccharides from wheat straw. J. Agric. Food Chem. 43,

21 107 Martel, P. and Gould, J. M. (1990) Cellulose stability and delignification after alkaline hydrogen peroxide treatment of straw. J. Appl. Polym. Sci. 39, McDonough, T. J., Kirk, R. C., Backlund, B. and Winter, L. (1989) Oxygen delignification Conference Proceedings, Tappi Press, Atlanta, pp Nascimento, E. A., Machado, A. E. H., Morais, S. A. L., Brasileiro, L. B. and Pilo-Veloso, D. (1995) Photochemical hydrogen peroxide bleaching of Eucalyptus organosolv pulp. J. Braz. Chem. Soc. 6, Pan, G. X., Bolton, J. L. and Leary, G. J. (1998) Determination of ferulic and p-coumaric acids in wheat straw and the amounts released by mild acid and alkaline peroxide treatment. J. Agric. Food Chem. 46, Patel, M. M. and Bhatt, R. M. (1992) Optimisation of the alkaline peroxide pretreatment for the delignification of rice straw and its applications. J. Chem. Tech. Biotechnol. 53, Springer, E. L. (1990) Delignification of aspen wood using hydrogen peroxide and peroxymonosulfate. Tappi J. 73, Sun, R. C., Lawther, J. M. and Banks, W. B. (1995) Influence of alkaline pre-treatment on the cell wall components of wheat straw. Ind. Crops Prod. 4, Sun, R. C., Lawther, J. M. and Banks, W. B. (1996) Fractional and structural characterization of wheat straw hemicelluloses. Carbohydr. Polym. 29, Sun, R. C., Fang, J. M., Goodwin, A., Lawther, J. M. and Bolton, J. (1998) Isolation and characterization of polysaccharides from abaca fibre. J. Agric. Food Chem. 46,