Ind. Eng. Chem. Res. 2010, 49, 8797–8804
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Influence of Incubation Time on the Physicochemical Properties of the Isolated Hemicelluloses from Steam-Exploded Lespedeza Stalks Kun Wang,† Feng Xu,† Run-Cang Sun,*,†,‡ and Gwynn L. Jones§ Institute of Biomass Chemistry and Technology, Beijing Forestry UniVersity, Beijing 100083, China, State Key Laboratory of Pulp and Paper Engineering, South China UniVersity of Technology, Guangzhou 510640, China, and The BioComposites Centre, UniVersity of Wales, Bangor, Gwynedd LL57 2UW, U.K.
Hemicellulosic polysaccharides were isolated from the raw and steam-exploded Lespedeza crytobytrya stalks by sequential 1 M NaOH extraction and fractionated into Hemi-a and Hemi-b fractions by acidification and ethanol precipitation, respectively. The Hemi-a fractions had relatively lower molecular weights and took up 84.6-95.5% of the total released hemicelluloses. After steam explosion, both hemicellulosic fractions were obviously degraded, and a certain amount of glucose or oligosaccharides derived from the amorphous cellulose was coisolated. The influence of increasing incubation time from 2 to 10 min is further discussed, based on the physicochemical properties of the obtained hemicelluloses characterized in terms of sugar component, gel permeation chromatography (GPC), thermal stability, and 13C NMR spectroscopy analysis. Introduction The widespread availability of biomass, which is also renewable and potentially neutral in relation to global warming, motivated the extensive research undertaken in the past decade for industrial development. The natural polymers, due to their abundance, renewability, and biodegradation, have received considerable interest as a source of chemicals in recent years. Hemicelluloses, also termed heteropolysaccharides or polyoses, are noncellulosic polysaccharides consisting of D-xylose, Dmannose, L-arabinose, D-glucose, D-galactose, and 4-O-methylD-glucuronic acid residues, arranged in different fractions and with different substituents. The major structure of hemicelluloses in the agricultural crop residues such as wheat straw is arabino4-O-methylglucuronoxylan, which has a backbone of 1,4-linked β-D-xylopyranosyl units and a lower degree of polymerization (100-200) than cellulose (∼1500).1 The usefulness of the hemicelluloses-based or -derived products in an industrial and biomedical context is beyond dispute and will stimulate further activities in basic and applied research.2 Before the lignocellulosic materials can be utilized, a pretreatment process is necessary to break down the lignocellulose into the three major polymeric constituents, cellulose, hemicelluloses, and lignin, where the lignin binds the cellulose and hemicelluloses together in the vascular tissue of the plant.3 Biorefinery, as a new concept, can sustainably produce chemicals from biomass while being more cost-effective. Ideally all of the fractions of crops, like grass, clover, and alfalfa, are converted into valuable products without leaving any waste materials. In several processes, it has been proved that it is impossible to keep the original structure of hemicelluloses since the components in the lignocellulosic materials are tightly associated. Historically, steam pretreatment regimes have been optimized for enhancing the digestibility of the cellulose component but not for making efficient use of the hemicelluloses component. More recently, with the recognition that the complete utilization of the entire substrate will be required for * To whom correspondence should be addressed. E-mail: rcsun3@ bjfu.edu.cn. Tel: +86-10-62336972. Fax: +86-10-62336972. † Beijing Forestry University. ‡ South China University of Technology. § University of Wales.
improving the process economics, pretreatment strategies have been focused on both the cellulose hydrolysability and the recovery of hemicelluloses and lignin. This has been accomplished through the use of less severe, single-stage pretreatment and/or two-stage pretreatment processes, which facilities efforts to optimize both the hemicelluloses and lignin recovery and the cellulose treatment.4 In this study, according to the concept of biorefinery, we proposed a two-stage approach using steam explosion pretreatment and following alkaline solution extraction to isolate hemicellulosic fractions from Lespedeza stalks (Lespedeza crytobotrya). Depending on the sequence of precipitation in weak acidic aqueous solution and ethanol, the hemicellulosic polymers are categorized as hemicelluloses A and B (designated Hemi-a and Hemi-b, respectively). Their physicochemical properties such as monosaccharide components, molecular weight, content of the associated lignin, and thermal stability were comparatively studied, aiming to investigate the influence of steam explosion pretreatment with increasing incubation time. Materials and Methods Materials. Lespedeza stalks used in this experiment were obtained from the experimental farm of Beijing Forestry University. The chemical compositions of the stalks were cellulose (44.6%), hemicelluloses (29.3%), lignin (17.0%), wax (4.8%), and ash (4.3%). The deviations of these contents from their respective means were all less than 5%. All chemicals used were of analytical grade. Steam Explosion Pretreatment and Isolation Process. The steam explosion experiments were carried out in a flash hydrolysis laboratory pilot unit (7.5 L reactor) designed especially for the process of lignocellulosic materials and can be used to study the effects of the parameters, that is, time and pressure. High-temperature steam was applied to reach the predetermined pressure (22.5 kg/m2) and held for the desired time (2, 3, 4, 5, 6, and 10 min), and then, a ball valve was suddenly opened to release the pressure. The steam-exploded material was collected, oven-dried, and ground to 40 mesh before further separation. The whole procedure for isolating and fractionating hemicelluloses was shown in Figure 1. Briefly, the exploded samples were post-treated with 1 M NaOH at 50
10.1021/ie101180p 2010 American Chemical Society Published on Web 08/05/2010
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Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010 j w), Number Average Table 1. Yield (weight %), Weight Average (M j n), and Molecular Weight and Polydispersity (M j w/M j n) of the (M Obtained Hemicellusic Fractions Hemicellulosic Fractionsa
yield (%) jw M jn M j w/M jn M
yield (%) jw M jn M j w/M jn M Figure 1. Scheme for isolation of the degraded hemicellulosic fractions from the steam-exploded Lespedeza stalks.
°C for 3 h with a shrub-to-water ratio of 1:20 (g/mL) and then filtered in a 100% polyester cloth to separate the insoluble richin-cellulose fraction. Afterward, the filter liquor was neutralized to pH 5-6 with 6 M HCl. The precipitated polysaccharides (Hemi-a) were recovered by filtering with filter paper, freezedried, and labeled Ha2, Ha3, Ha4, Ha5, Ha6, and Ha10, corresponding to the steaming times of 2, 3, 4, 5, 6, and 10 min, respectively. The supernatant was concentrated under vacuum and mixed with three volumes of 95% ethanol. The precipitated polysaccharides (Hemi-b) were filtrated and freeze-dried. Similarly, the recovered hemicellulosic fractions were tagged as Hb2, Hb3, Hb4, Hb5, Hb6, and Hb10, respectively. Ha0 and Hb0, which represent the hemicellulosic fractions released by the same post-treatment process from the raw material without steam pretreatment, were taken as comparisons. All of the samples were kept in a desiccator at room temperature before further analysis. All experiments were performed at least in duplicate, and analyses were carried out at least three times for each of the sample. Chemical Characterization. The analysis of neutral sugars and the associated lignin was described in a previous paper.5 The molecular weights of the hemicellulosic fractions were determined by gel permeation chromatography (GPC) on a PL Aquagel-OH mixed column (300 × 7.5 mm, Agilent). The eluent was 0.02 M NaCl in 5 mM sodium phosphate buffer, pH 7.5, with a flow rate of 0.1 mL/min. Detection was achieved using a Knauer differential refractometer. The column oven was maintained at 30 °C. The samples were dissolved in the buffer at a concentration of 0.1%. To calibrate the column, monodisperse polysaccharide of known molecular weight was used as the standard for calculating the molecular weight of hemicelluloses. The FT-IR spectra of the hemicellulosic fractions were recorded in the transmission mode on a KBr disk containing 1% finely ground samples. The infrared spectra were collected on a Tensor 27 spectrophotometer (Bruker, Germany) equipped with a DTGS detector.6 The solution-state 13C NMR spectrum was obtained on a Bruker DRX-400 spectrometer (Germany) at 75.5 MHz. A sample concentration of approximately 80 mg in 1.0 mL of D2O was placed in 5 mm i.d. glass tube and run at a temperature of 25 °C for 18 h. A 60° pulse flipping angle, a 3.9 µs pulse width, and a 0.85 s delay time between scans were used. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of the hemicellulosic fractions were performed with a simultaneous thermal analyzer (DTG-60, Shimadzu, Japan). This apparatus provides for a continuous measurement of sample weight at a range of temperatures
Ha0
Ha2
Ha3
Ha4
Ha5
Ha6
Ha10
19.1 63300 20400 3.1
11.8 20300 8200 2.5
20.5 19300 8300 2.3
21.1 18400 8000 2.3
22.5 16500 7400 2.2
22.1 8300 4400 1.9
22.5 8100 4300 1.9
Hb0
Hb2
Hb3
Hb4
Hb5
Hb6
Hb10
0.9 102400 29000 3.5
1.6 32700 9300 3.5
2.4 29200 16200 1.8
2.6 22400 12200 1.8
2.4 22100 12600 1.7
2.7 18400 10900 1.6
4.1 16200 10300 1.6
a
Ha2, Ha3, Ha4, Ha5, Ha6, and Ha10 represent the hemicellulosic fractions obtained during the post-treatment with 1 M NaOH at 50 °C for 3 h with a solid-to-water ratio of 1:20 (g/mL) and precipitated in the weak acid aqueous solution from the corresponding steam explosion conditions at 22.5 kg/m2 for 2, 3, 4, 5, 6, and 10 min, respectively, while Ha0 represents the hemicellulosic fraction isolated from the raw Lespedeza crytobotrya stalks. Hb2, Hb3, Hb4, Hb5, Hb6, and Hb10 represent the hemicellulosic fractions obtained during the same post-treatment process but precipitated in the ethanol solution from the corresponding steam explosion conditions at 22.5 kg/m2 for 2, 3, 4, 5, 6, and 10 min, respectively, while Hb0 represents the hemicellulosic fraction precipitated by ethanol from the raw Lespedeza crytobotrya stalks.
between ambient and 600 °C. Samples were heated in a platinum crucible to 600 °C at a heating rate of 10 °C/min under nitrogen. Results and Discussion Yield and Neutral Sugar Composition. As mentioned previously, it is important that the optimal pretreatment condition is defined to ensure the efficient hydrolysis of the water-insoluble cellulose stream with the acceptable recovery of the total carbohydrates. The yields of the isolated hemicellulosic fractions are listed in Table 1. Clearly, the yields (11.8-22%) of the weak-acid-insoluble hemicellulosic fractions (Ha) were much higher than those (0.9-4.1%) precipitated in the ethanol solution (Hb), covering about 84.6-95.5% of the total released hemicelluloses. When a range of incubation times for pretreating Lespedeza stalks was assessed, it was apparent that the severity had a substantial effect on the total recovery of the hemicellulosic fractions. Overall, by comparing the data from the raw materials, the extraction efficiency of the alkali solution was improved by the steam explosion pretreatment process. However, we can also observe an obvious decrease (11.8%) of the recovered Hemi-a fraction at the lowest severity (Ha2). As wellknown, autohydrolysis was generated by the organic acids, mainly acetic acid derived from the acetylated hemicelluloses in the lignocellulosic materials during the steam explosion. Hence, the unavoidable losses were generated by solubilizing the degraded hemicelluloses in the present water. In comparison, the yield of Hemi-b fraction (Hb2) was increased by 1.6% at the same condition, probably explained by the deeper distribution of this fraction. Under such a mild condition, the saturated steam could not reach and hydrolyze the “core” of hemicelluloses but could expose the intermacromolecule and, consequently, improve the extraction efficiency. As the steaming time further increased from 3 to 10 min, the yields of the Hemi-a fractions increased from 20.5 to 22.5%, and the yields of the Hemi-b fractions increased from 2.4 to 4.1%, correspondingly. This trend was probably related to the comprehensive hydrolysis of the high molecular weights of hemicelluloses and the cleavage of the bonds between hemicelluloses and lignin.
Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010 Table 2. Content of Neutral Sugars (relative %, w/w) and Uronic Acid (weight %, w/w) in the Degraded Hemicellulosic Fractions Hemicellulosic Fractionsa
rhamnose arabinose galactose glucose mannose xylose ara/xylc galacturonic acid
rhamnose arabinose galactose glucose mannose xylose ara/xylc galacturonic acid b
Ha0
Ha2
Ha3
Ha4
Ha5
Ha6
Ha10
2.0 5.5 4.9 17.9 0.8 66.4 0.08 1.9
NDb 3.3 2.5 19.6 1.2 74.3 0.04 ND
ND 1.1 2.2 57.7 2.3 30.0 0.04 ND
ND 0.8 2.0 57.0 2.6 35.3 0.02 ND
ND 0.1 3.1 57.6 1.8 33.5 Td ND
ND ND 2.9 58.2 1.9 32.6 0 ND
ND ND 1.1 76.7 1.5 16.1 0 ND
Hb0
Hb2
Hb3
Hb4
Hb5
Hb6
Hb10
2.4 5.5 4.7 17.0 1.4 67.1 0.08 1.9
0.6 0.9 3.0 11.4 0.7 84.5 0.01 0.2
0.5 0.6 3.0 9.3 1.5 83.8 0.01 ND
0.4 0.3 2.7 6.1 0.9 90.5 T ND
0.1 0.5 4.0 32.0 0.7 61.5 0.01 ND
ND 0.3 3.1 33.4 0.5 59.5 0.01 ND
ND 0.1 1.9 36.6 1.8 55.8 T ND
a Corresponding to the degraded hemicellulosic fractions in Table 1. Not detectable. c Present the ratio of arabinose to xylose. d Trace.
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Once the influence of incubation time on the recovery of hemicellulosic polymers is tested, the nature and concentration of sugars in the hemicellulosic fractions should be investigated subsequently. In Table 2, the sugar (relative weight %) and uronic acids (weight %) composition of the isolated hemicellulosic fractions are presented. The standard deviation was less than 5%. It was surprising to find that the components in the Hemi-a and Hemi-b fractions were almost the same, demonstrating that the different physicochemical characteristics between these two fractions mainly depended on the structural variance. Xylose (∼67%), followed by glucose (∼17%) and arabinose (∼5.5%) were the three most abundant monosaccharides in the original material. A certain amount of galactose and small quantities of rhamnose and mannose were also detected. This indicated that the main hemicellulosic component in Lespedeza crytobotrya stalks was arabinoxylans, assumably together with a small amount of β-glucans or xyloglucans, as previously reported in the cereal straws.7-9 Although arabinoxylans from various materials share the same basic chemical structure, they differ in the manner of substitution of the xylan backbone. The main differences were found in the ratio of arabinose to xylose, the sequence of the various linkages between these two sugars, and the presence of other substituents.
Figure 2. Molecular weight distribution curves of the Hemi-a fractions (Ha0, Ha2, Ha4, and Ha10) and Hemi-b fractions (Hb0, Hb2, Hb4, and Hb10).
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The arabinose to xylose (ara/xyl) ratio is indicative of the degree of branching of hemicelluloses.10 As the data shows in Table 2, a rather low value (0.08) of the ara/xyl ratio in both hemicellulosic fractions manifested a relatively linear structure of the polymer. As a result of the steam explosion on the Hemi-a fractions, the content of xylose was first increased to 74.3% (Ha2) and then dropped to 30.0 (Ha3) and 16.1% (Ha10). According to the data, it is not hard to find that the comprehensive hydrolysis of hemicelluloses led to the partial losses but improved the “purity” of the isolated hemicellulosic polymers to a certain extent in the first 2 min. When the steaming time reached 3 min, almost half of the hemicelluloses were extensively autohydrolyzed into monosaccharide or low molecular oligosaccharides and even further degraded into furfural.11 Meanwhile, a certain amount of the degraded cellulosic components were mixed with the isolated polymers, which actually cannot be called hemicelluloses. Therefore, for the better recovery of hemicellulosic polymer under the present steam explosion pressure, the cooking time was not prolonged to as much as 3 min. At the longest cooking time (10 min), most of the hemicelluloses were lost. In terms of the content of glucose, there are two obvious booms during the range of 2-10 min. One is at 3 min, probably due to the hydrolysis of the amorphous cellulose, and the other is at 10 min, presumably related to the partial hydrolysis of the crystalline cellulose. In comparison, the main neutral sugars in the Hemi-b fractions, xylose and glucose, appeared to have similar trends. As the data shows in Table 2, the content of glucose in the Hemi-b fraction was suddenly rocketed to an extremely high level at 22.5 kg/m2 for 5 min. Due to the deeper distribution and a relatively higher molecular weight (discussed in the next section), the critical point of the comprehensive degradation for the Hemi-b fraction was 2 min later than that of the Hemi-a fraction. Additionally, the linearly decrease of the ara/xyl ratio from 0.08 to 0 both in the Hemi-a and Hemi-b fractions suggested that the noticeable debranching reactions occurred during the steam explosion pretreatment. The galacturonic acid (1.9%) was presented in both hemicellulosic fractions of the original hemicelluloses and almost completely disappeared in the steam-exploded samples. Distribution of Molecular Weight. The average molecular weight values of all of the hemicellulosic fractions were estimated by GPC in aqueous medium, and the values of the j n), molecular weight j w), number average (M weight average (M j n) of the hemicelluloses are listed in j w/M and polydispersity (M Table 1. In order to intuitively illustrate the extent of degradation that occurred during the steam explosion and the following alkaline extraction, the molecular weight distribution curves of the four respective Hemi-a and Hemi-b fractions under the same conditions are also shown in Figure 2. From the data of the raw material, the molecular weight of the Hemi-a fraction was obviously lower than that of the Hemi-b fraction. Although the value of Ha0 is 63 300 g/mol, it was probably overestimated because of the minimum presence of the very high molecular weight material as the “tail” extended to higher than 1 × 106 g/mol shown in Figure 2. Differing from the relatively concentrating distribution pattern of Ha0, Hb0 shows a boarder distribution scale and correspondingly has a higher polydispersity value (3.5) than that of Ha0 (3.1). As the function of steam explosion, the comprehensive hydrolysis of hemicelluloses significantly reduced the molecular weight, even at the lowest severity. For both hemicellulosic fractions, increasing the incubation time from 3 to 10 min further degraded the macromolecule into smaller ones by hydrolysis or debranching reactions. This result
Table 3. Yield of Phenolic Acids and Aldehydes (weight %, w/w) Obtained by Alkaline Nitrobenzene Oxidation of the Isolated Hemicellulosic Fractions Hemicellulosic Fractionsa phenolic acids and aldehydes
Ha0
Ha2
Ha3
Ha4
Ha5
Ha6
Ha10
p-hydroxybezonic acid p-hydroxybenzaldehyde vanillic acid vanillin acetovaillone syringic acid syringaldehyde acetosyringone p-coumaric acid ferulic acid total
0.31 0.03 0.08 1.82 0.03 0.16 2.08 0.07 0.05 0.04 4.67
0.16 0.03 0.09 1.20 0.03 0.10 1.73 0.11 0.05 0.03 3.53
0.09 0.04 0.05 1.21 0.03 0.04 1.64 0.16 0.01 NDb 3.27
0.02 0.03 0.11 1.05 0.02 0.06 2.32 0.05 0.03 0.03 3.72
0.05 0.02 0.01 1.04 0.02 0.01 2.16 0.08 0.01 ND 3.40
0.02 0.04 0.01 0.83 0.05 0.05 2.52 0.05 0.05 ND 3.62
0.05 0.01 0.01 0.75 0.01 0.02 1.32 0.04 0.02 ND 2.23
Hb0
Hb2
Hb3
Hb4
Hb5
Hb6
Hb10
0.26 0.19 0.03 0.30 ND 0.05 0.36 ND 0.19 0.07 1.45
0.14 0.16 0.06 0.30 0.01 0.21 0.35 0.01 0.23 0.04 1.51
0.05 0.13 0.05 0.32 ND 0.11 0.37 ND 0.12 0.02 1.17
0.08 0.18 0.29 0.28 ND 0.16 0.32 ND 0.19 ND 1.50
0.11 0.24 0.34 0.60 0.01 0.37 0.41 0.03 0.25 0.01 2.37
0.08 0.19 0.34 0.39 ND 0.39 0.39 0.05 0.20 0.03 2.06
0.06 0.17 0.28 0.43 0.01 0.37 0.33 0.03 0.22 0.01 1.91
p-hydroxybezonic acid p-hydroxybenzaldehyde vanillic acid vanillin acetovaillone syringic acid syringaldehyde acetosyringone p-coumaric acid ferulic acid total b
a Corresponding to the degraded hemicellulosic fractions in Table 1. Not detectable.
could also be confirmed by the distribution curves in Figure 2, in which a noticeable shift of the main distribution region to the relative lower molecular weight region was observed (pay attention to the X-axis, which is not the same and is automatically generated by software). Meanwhile, the molecular weight distributions were more concentrated with the disappearance of the small amount of the large molecules in Ha0 and Hb0, especially for the Hemi-b fractions, which showed a much simpler double-apex curve under the conditions at 22.5 kg/m2 for 4 min. Correspondingly, the value of polydispersity of the Hemi-b fractions decreased to a greater extent than that of the Hemi-a fractions. Content of the Bound Lignin and Its Phenolic Composition. The pigmentation associated with the hemicellulosic fractions is likely due to the presence of the strongly associated lignin, which was linked to arabinoxylans by ether bonds.12 The results concerning the characterization of the associated lignin are listed in Table 3. The method of alkaline nitrobenzene oxidation provides an estimate of the total amount of lignin and an indication of the composition of phenolic units. As the data show, the major products were identified to be syringaldehyde and vanillin, which together represented 91.6-98.2% of the total phenolic monomers in the Hemi-a fractions and 55.9-77.2% of the total phenolic monomers in the Hemi-b fractions. This suggested that the associated lignin in the hemicellulosic fractions contained roughly equal amounts of noncondensed syringyl and guaiacyl units. Small amounts of p-hydroxybezonic acid, p-hydroxybenzaldehyde, vanillic acid, syringic acid, and p-coumaric acid and traces of acetovaillone, acetosyringone, and ferulic acid were also found to be present in the nitrobenzene oxidation mixtures. In comparison, the Hemi-a fractions had an obviously higher content of phenolic compounds than the Hemi-b fractions, which was further confirmed by the FT-IR spectra analysis below. Considering the structural difference between these two fractions, this result was probably ascribable
Ind. Eng. Chem. Res., Vol. 49, No. 18, 2010
Figure 3. The FT-IR spectra of the alkaline-soluble hemicellulosic fractions precipitated under the weak acidic condition from the raw material (Ha0) and steam-exploded samples at 22.5 kg/m2 for 2 (Ha2), 3 (Ha3), and 4 (Ha4) min.
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Figure 5. The FT-IR spectra of the alkaline-soluble hemicellulosic fractions precipitated in ethanol from the samples steam-exploded at 22.5 kg/m2 for 5 (Hb5), 6 (Hb6), and 10 (Hb10) min.
Figure 6. TGA/DTA curves of the Hemi-a fractions Ha0, Ha3, Ha6, and Ha10. Figure 4. The FT-IR spectra of the alkaline-soluble hemicellulosic fractions precipitated in ethanol from the raw material (Hb0) and steam-exploded samples at 22.5 kg/m2 for 2 (Hb2), 3 (Hb3), and 4 (Hb4) min.
to the fact that most of the lignin-carbohydrate complex distributed in the low molecular portion or the edge of the core macromolecule of hemicelluloses. Evidently, the steam explosion pretreatment could partially cleave the linkages between lignin and hemicelluloses. As the data show in Table 3 for the Hemi-a fractions, the total yields of alkaline nitrobenzene oxidation products were significantly decreased to 3.27-3.72% by the steam explosion at 22.5 kg/ m2 for 2-6 min and eventually dropped to 2.23% at the severest condition (10 min). This indicated that the steam explosion broke the ester or ether bonds between lignin and hemicelluloses13 and consequently prevented the lignin from codepositing with hemicelluloses in the weak acid solution. This phenomenon could also be explained by the formation of so-called pseudolignin from the condensation reactions involving lignin and sugar (mainly xylose), as reported by many researchers.14,15 Wayman and Chua16 reported that the decrease in aromatic aldehyde yield was attributed to the condensation reactions involving the formation of new carbon-carbons, which resulted in a much less amenable lignin structure being oxidized to phenolic aldehydes. However, it can be neglected in this study by considering the negligible amount of the associated lignin in all seven Hemi-a fractions. For the content of the associated lignin in the Hemi-b fractions, it was surprising to note that the steam explosion pretreatment conversely increased the amount
of total phenolic acids and aldehydes. The reason for this phenomenon was not clearly understood; it presumably is that the dissociated lignin connected with the lower molecular hemicelluloses was coprecipitated with the higher molecular ones. FT-IR Spectra Analysis. Infrared spectroscopy is quite extensively applied in plant cell wall analysis.17 FT-IR spectroscopy allows solving of the problems of identification of polysaccharides, checking their purity, carrying out semiquantitative functional analyses, determining structure, and investigating complex and intermolecular interactions.18,19 Figure 3 shows the FT-IR spectra of four Hemi-a fractions, labeled as Ha0, Ha2, Ha3, and Ha4. No significant differences in the main absorption intensities could be observed among the four spectra. As can be seen, the signals at 3352, 2924, and 2857 cm-1 are assigned to the stretching -OH and -CH. Disappearance of the ester band at 1740 cm-1 is undoubtedly caused by the full saponification of acetyl groups and methyl esters during the alkali post-treatment process. The occurrence of the obvious bands at 1600, 1510, and 1424 cm-1 in all of the spectra is assigned to the phenolic ring absorbance. The absorption at 1458 cm-1 is indicative of the C-H deformations and aromatic ring vibrations. These phenomena are due to the presence of a considerable amount of the associated lignin in the Hemi-a fractions, which is supported by the oxidative degradation results. The low intensities of the bands at 1372, 1325, 1269, and 1232 cm-1 indicate the methyl C-H wagging and -OH,
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Figure 7. TGA/DTA curves of the Hemi-b fractions Hb3, Hb6, and Hb10.
-CH2, and C-H bending, respectively. Under the detailed examination, the main differences occurred between 1200 and 950 cm-1, which is the characteristic region of hemicelluloses.20,21 As the spectrum of Ha0 shows, the dominant absorption at 1052 cm-1 is the characteristic wavenumber of the typical xylans. However, the steam explosion pretreatment widened this band, and another two remarkable absorptions emerged. One is the signal at 1038 cm-1, which is probably due to a certain amount of glucose that occurred in the steam-exploded samples. The axial and equatorial positions of the -OH groups can affect quite significantly the main band positions.20 The all-equatorial -OH group positions in glucose showed the lowest frequency maximum in the IR spectrum, while amide xylose showed its maximum a little higher. Besides, the signal at 1117 cm-1 derived from the in-plane ring stretching was also observed in the spectra of steam-exploded samples, which commonly occurs in the FT-IR spectra of cellulosic materials.22,23 These results further confirmed the conclusion that a little part of cellulose was degraded into oligosaccharides and monosaccharide during the autohydrolysis process and coextracted during the posttreatment step. The spectra of the four Hemi-b fractions (Hb0, Hb2, Hb3, and Hb4) precipitated in ethanol are present in Figure 4. In comparison to the spectra of the Hemi-a fractions, the major distinction is the absence of some bands in the region of 1600-1200 cm-1. It is probably derived from the disappearance of the associated lignin in this fraction, as analyzed above. The strong absorption at 1650 cm-1 is assigned to the absorbed water since hemicelluloses usually have a strong affinity for water.24 As expected, the absorption at 1045 cm-1 was still taken as the preponderant position due to the much higher content of xylose than other sugars in these four fractions. As the incubation time reached 5 min, the critical time point mentioned above, the fact
Figure 8. 13C NMR spectrum of the Hemi-b fraction Hb2.
that the intensity of the band at 1045 cm-1 was significantly weakened and the obvious peak at 1113 cm-1 appeared (Figure 5) indicated that the isolated polymers contained a certain amount of the cellulosic degradation products. Thermal Analysis. The pyrolysis, basically a polymeric structure cracking process, converts the lignocellulosic material into a volatile fraction and char. The knowledge of the thermal degradation of the lignocellulosic material is of relevant important because it is crucial to understand the polymeric thermal stability.25 Thermogravimetric analysis (TGA) is a useful method for the quantitative determination of the degradation behavior and the composition of the materials. The magnitude and location of peaks found in the differential thermal analysis (DTA) curve also provide information on the component and the mutual effect of the material components on the temperature scale. In the pyrolysis reactions of hemicelluloses, acetic acid comes from the elimination of acetyl groups originally linked to the xylose unit, furfural is formed by dehydration of the xylose unit, formic acid proceeds to form carbonxylic groups of uronic acid, and methanol arises from methoxyl groups of uronic acid.26 The data displayed in Figures 6 and 7 correspond to the pyrolysis process of the Hemi-a fractions (Ha0, Ha3, Ha6, and Ha10) and the Hemi-b fractions (Hb3, Hb6, and Hb10) in a nitrogen environment at the heating rate of 10 °C/min, respectively. As can be seen in Figure 6, the TGA curves of the four Hemi-a samples (Ha0, Ha3, Ha6, and Ha10) started to decompose at 180, 182, 130, and 200 °C and yielded 46, 49, 42, and 54% pyrolysis residuals, respectively. Similarly, at 70% weight loss, the degradation temperature occurred at 358 (for Ha0), 366 (Ha3), 342 (Ha6), and 392 °C (Ha10). Accordingly, the thermal stability of the Hemi-a fractions underwent the wavelike process. From another aspect, this trend further confirmed the changes of chemical composition during the steam explosion process. At the lower severity (Ha3), most of the low molecular weight molecules were hydrolyzed, leaving the relative “core” macromolecule, which induced the slight increment of the thermal property. By prolonging the cooking time (Ha6), the comprehensive hydrolysis of hemicelluloses produced a much smaller molecule, which was prone to degradation in the pyrolysis process. At the most severe conditions (Ha10), the much higher thermal stability was probably due to the generation of the condensation substances that required higher temperature ranges for complete decomposition. Additionally, it is worth observing the relatively higher exothermic peak at around 500 °C in the DTA diagram of Ha10. Yang et al.27 studied the pyrolysis characteristics of the three main components in the lignocellulosic material and found that lignin released the highest yield of CH4 at around 500 °C because of its highest -OCH3 content. The exothermic peaks of hemicelluloses appeared at lower
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temperatures than those of lignin, and their thermal decomposition occurred at a lower temperature.28 Thereby, it can be concluded that the condensation products, mainly derived from lignin and hemicelluloses or their own degradation products, were formed at the severest conditions and increased the thermal stability. In comparison, the variety of the thermal stability of the Hemi-b fractions (Hb3, Hb6, and Hb10) appeared simply (Figure 7). The decomposition temperature and pyrolysis residue were steadily increased with the longer incubation time. To be specific, the onset temperature of Hb3 was 214 °C, that of Hb6 was 230 °C, and that of Hb10 was 235 °C, and the final pyrolysis residues for these three fractions were 51 (Hb3), 58 (Hb6), and 61% (Hb10), respectively. Besides, these values were relatively higher than those of the Hemi-a fractions at the same conditions because of the relatively higher molecular weights in the Hemi-b fractions. Furthermore, a slight shift of the exothermic peak toward a high-temperature region was also observed from the DTA curves (Figure 7), indicating a higher thermal stability under the more severe conditions. 13 C NMR Spectra Analysis. The structural features of two Hemi-b fractions (Hb2 and Hb5) were investigated and assigned using 13C NMR spectroscopy. The spectra (Figure 8) were interpreted on the basis of the reported data for structurally defined arabinoxylan-type,1 glucuronoxylan type,29 and L-arabino-(4-O-methyl-D-glucurono)-D-xylan.30 The main 1,4-linked β-D-xylp units in Hb2 were further confirmed by five signals at 102.2, 76.0, 74.6, 73.1, and 64.8 ppm, which correspond to the C-1, C-4, C-3, C-2, and C-5 positions of the β-D-xylp units, respectively. The strong signal at 29.8 ppm is assigned to the γ-methyl and β- and R-methylene groups in lignin associated to hemicelluloses. However, the steam explosion pretreatment at 22.5 kg/m2 for 5 min significantly decreased the intensities of these five signals (Hb5, not shown), demonstrating the overall degradation of the hemicellulosic macromolecule. Conclusions Hemi-a and Hemi-b, two types of the hemicellulosic fractions, were isolated by 1 M NaOH extraction and the following precipitation in weak acidic aqueous solution and ethanol, respectively. By comparing the physicochemical characteristics of each sample from the raw material and steam explosion pretreated materials, the structural differences in these two types the fractions and the influence of steam explosion with increasing incubation time were investigated. On the basis of the similar monosaccharide components in the two types of fractions obtained from the raw material, the Hemi-b fractions were considered to be the “core” macromolecule of the hemicelluloses due to its relatively higher molecular weight. Thereby, the Hemi-a fractions were easy to extract and took up 95.5% of the total released hemicelluloses. Although steam explosion pretreatment with increasing incubation time did improve the extraction efficiency of the post-treatment process at a certain degree, it led to the comprehensive degradation of hemicelluloses. Meanwhile, a large amount of glucose derived from cellulose was coisolated at higher severities and made the isolated polymer not be called hemicelluloses. Because of the deeper distribution and higher molecular weight, the critical point of the comprehensive degradation for the Hemi-b fractions was 5 min, 2 min later than that of the Hemi-a fractions. Hence, for the better recovery of hemicelluloses, the incubation time at the pressure of 22.5 kg/m2 was suggested to not exceed 3 min. Additionally, the thermal stabilities of these two hemicel-
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lulosic fractions were finally improved due to the formation of a certain amount of condensation products from hemicelluloses and lignin. Acknowledgment This work was supported by grants from the Natural Science Foundation of China (No. 30930073, 30710103906), China Ministry of Education (No. 111), Ministry of Science and Technology (973-2010CB732204), State Forestry Administration (200804015), and China Scholarship Council (2009651012). Literature Cited (1) Sun, R. C.; Lawther, J. M.; Banks, W. B. Fractional and structural characterization of wheat straw hemicelluloses. Carbohydr. Polym. 1996, 29, 325. (2) Ebringerova´, A. Structural diversity and application potential of hemicelluloses. Macromol. Symp. 2006, 232, 1. (3) Liyama, K.; Lam, T. B.; Stone, B. A. Covalent cross-link in the cell wall. Plant Physiol. 1994, 104, 315. (4) Robinson, J.; Keating, J. D.; Mansfield, S. D.; Saddler, J. N. The fermentability of concentrated softwood-derived hemicellulose fractions with and without supplemental cellulose hydrolysates. Enzyme Microb. Technol. 2003, 33, 757. (5) Wang, K.; Jiang, J. X.; Xu, F.; Sun, R. C. Influence of steaming explosion time on the physic-chemical properties of cellulose from Lespedeza stalks (Lespedeza crytobotrya). Bioresour. Technol. 2009, 100, 5288. (6) Sun, R. C.; Sun, X. F. Fractional and structural characterization of hemicellulosis isolated by alkali and alkaline peroxide from barley straw. Carbohydr. Polym. 2002, 49, 415. (7) Kato, Y.; Iki, K.; Matsuda, K. Cell-wall polysaccharides of immature barley plants. II. Characterization of a xyloglucan. Agric. Biol. Chem. 1981, 45, 2745. (8) Wilkie, K. C. B. The hemicelluloses of grasses and cereals. AdV. Carbohydr. Chem. Biochem. 1979, 36, 215. (9) Woodward, J. R.; Fincher, G. B.; Stone, B. A. Water-soluble (1f3), (1f4)-β-D-glucans from barley (Hordeum Vulgare) endosperm. II. Fine structure. Carbohydr. Polym. 1983, 3, 207. (10) Wedig, C. L.; Jaster, E. H.; Moore, K. J. Hemicellulose monosaccharide composition and in vitro disappearance of orchard grass and alfalfa hay. J. Agric. Food Chem. 1987, 35, 214. (11) Bolan˜os, J. F.; Felizo´n, B.; Heredia, A.; Rodrı´guez, R.; Guille´n, R.; Jime´nez, A. Steam-explosion of olive stones: hemicellulose solubilization and enhancement of enzymatic hydrolysis of cellulose. Bioresour. Technol. 2001, 79, 53. (12) Doner, L. W.; Hicks, K. B. Isolation of hemicellulose from corn fiber by alkaline hydrogen peroxide extraction. Cereal Chem. 1997, 74, 176. (13) Sun, R. C.; Sun, X. F.; Tomkinson, J. Hemicelluloses: Science and Technology; American Chemical Society: Washington DC, 2003. (14) Garrote, G.; Dominguez, H.; Parajo´, J. C. Mild autohydrolysis: an environmentally friendly technology for xylooligosaccharide production from wood. J. Chem. Technol. Biotechnol. 1999, 74, 1101. (15) Hetiz, M.; Capek-Menard, E.; Koeberle, P. G.; Gagne, J.; Chornet, E.; Overend, R. P.; Taylor, J. D.; Yu, E. Fractionation of populus tremuloides at the pilot plant scale: Optimization of steam pretreatment conditions using the STAKE II technology. Bioresour. Technol. 1991, 35, 23. (16) Wayman, M.; Chua, M. G. S. Characterization of autohydrolysis aspen (P. tremuloides) lignins. Part 2. Alkaline-nitrobenzene oxidation of extracted autohydrolysis lignin. Can. J. Chem. 1979, 57, 2599. (17) Kaura´kova´, M.; Wilson, R. H. Developments in mid-infrared FTIR spectroscopy of selected carbohydrates. Carbohydr. Polym. 2001, 44, 291. (18) Chen, L. M.; Wilson, R. H.; McCann, M. C. Investigation of macromolecule orientation in dry and hydrated walls of single onion epidermal cells by FTIR microspectroscopy. J. Mol. Struct. 1996, 408409, 257. (19) Filippov, M. P. Practical infrared spectroscopy of pectic substances. Food Hydrocolloids 1992, 6, 115. (20) Kaura´kova´, M.; Capek, P.; Sasinkova´, V.; Wellner, N.; Ebringerova´, A. FT-IR study of plant cell wall model compounds: Pectic polysaccharides and hemicelluloses. Carbohydr. Polym. 2000, 43, 195. (21) Kaura´kova´, M.; Ebringerova´, A.; Hirsch, J.; Hroma´dkova´, Z. Infrared study of arabinoxylans. J. Sci. Food Agric. 1994, 66, 423.
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ReceiVed for reView June 1, 2010 ReVised manuscript receiVed July 22, 2010 Accepted July 25, 2010 IE101180P
