Sequential extractions and structural characterization of lignin with ethanol and alkali from bamboo (Neosinocalamus affinis)

Industrial Crops and Products 37 (2012) 51–60

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Sequential extractions and structural characterization of lignin with ethanol and alkali from bamboo (Neosinocalamus affinis) Shao-Ni Sun a , Ming-Fei Li a , Tong-Qi Yuan a , Feng Xu a,∗ , Run-Cang Sun a,b a b

Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China

a r t i c l e

i n f o

Article history: Received 24 September 2011 Received in revised form 26 November 2011 Accepted 29 November 2011 Available online 11 January 2012 Keywords: Bamboo culms Lignin GPC FT-IR 13 C NMR HSQC

a b s t r a c t A sequential process with the combination of ethanol and alkali aqueous solutions was utilized to extract lignin from bamboo (Neosinocalamus affinis), a potential lignocellulosic material. In this case, the successive treatments of dewaxed bamboo with 70% ethanol at 80 ◦ C, 0.2 and 0.5 M NaOH, 70% ethanol containing 0.6 M NaOH, and 1.0, 2.0, and 3.0 M NaOH at 50 ◦ C, resulted in a total yield of acid-insoluble lignin fractions of 10.06%, corresponding to release of 62.25% original lignin from the cell walls. The lignin fractions obtained were then characterized by GPC, FT-IR, NMR spectroscopy, and sugar analysis. As compared to the alkali lignin fractions, the ethanol-soluble lignin fraction had a relatively higher molecular weight (2670 g/mol) and the content of carbohydrates primarily consisted of glucose 2.01% and xylose 1.90%. This suggested that the carbohydrate chains linked to lignin may increase the hydrodynamic volume of lignin and therefore increase the apparent molecular weight of the ethanol-soluble lignin. HSQC spectra analysis revealed that the alkali lignin fractions consisted mainly of ˇ-O-4 linkages combined with small amounts of ˇ-ˇ , ˇ-5 , ˇ-1 linkages, and p-hydroxycinnamyl alcohol end groups. Furthermore, minor amounts of esterified p-coumaric and ferulic acids were also detected in the lignins isolated. © 2011 Elsevier B.V. All rights reserved.

1. Introduction In recent decades, the world has been confronted with an energy crisis, associated with irreversible depletion of fossil fuels and the quest for energy security (Goh et al., 2010). Fossil fuel combustion is singled as the most important driver of anthropogenic climate change with their scarcity and uneven geographical distribution coupled with geopolitical factors, which severely affects the national economies and international markets (Gasparatos et al., 2011). Furthermore, the demand for petroleum-derived fuels is not slowing down but instead increases substantially over the past few decades. China’s energy consumption amounted to 1678 million tonnes coal equivalent in 2003 and maintains an annual growth rate of 3.8% (Crompton and Wu, 2005). Therefore, a significant attention to utilization of lignocellulosic materials (LCMs) as a feedstock for biofuels has become a world priority for producing environmentally friendly renewable energy and provides opportunities for economic development. LCMs are composed of carbohydrate polymers (cellulose and hemicelluloses), lignin and small amounts of extractives and

∗ Corresponding author. Tel.: +86 10 62336903; fax: +86 10 62336903. E-mail address: [email protected] (F. Xu). 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.11.033

minerals in an intrinsic three-dimensional cell-wall structure (Gírio et al., 2010), which are difficult to be separated into readily utilizable components due to their recalcitrant nature. After cellulose and hemicelluloses, lignin is the third most abundant constituent of LCMs. It is well known that lignin is a polyphenolic amorphous material derived primarily from the dehydrogenative radical polymerization of monolignols (p-coumaryl-, coniferyl-, and sinapyl-alcohols) and each of these monolignols results in a different type of lignin units called p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively (Boerjan et al., 2003; Davin and Lewis, 2005). Lignin composition varies in different groups of vascular plants being G, GS, and HGS lignin characteristic for softwoods (woody gymnosperms), hardwoods (woody angiosperms), and graminaceous plants (non-woody angiosperms), respectively. These lignins include both ether (ˇ-O-4 , ˛-O-4 , and 4-O-5 ) and C–C (ˇ-5 , ˇ-1 , ˇ-ˇ , and 5-5 ) interunit linkages mediated by laccases and/or peroxidases (del Río et al., 2008; Martínez et al., 2008). Moreover, hydroxycinnamic acids, particularly ferulic acid (FA) and p-coumaric acid (PCA), are found in the cell walls of graminaceous plants and are linked to carbohydrates by ester bonds and to lignin by ester and/or ether bonds (Sun et al., 2002). Currently, lignin can be utilized in polymeric materials (e.g., manufacturing adhesives), biochemicals and biofuels, epoxiand p-henolic-resins, and a variety of nonspecific and novel

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applications (Villaverde et al., 2009). Despite extensive investigations of lignin have been reported, the complex and irregular structure of lignin has not been completely understood up to now. Lignin generally shows an irregular structure with a highly condensed cross-linked polymer network. On the other hand, lignin is bound physically/chemically to cellulose/hemicelluloses by phenyl glycoside bonds, esters, and benzyl ethers, forming lignin-carbohydrate complexes (LCC) in plant cell walls (Baucher et al., 1998; Fengel and Wegener, 1989; Koshijima and Watanabe, 2003). Recently, it has been reported that about 50% of the original lignin in wheat straw was extracted with alkali at temperatures lower than 100 ◦ C (Lu and Ralph, 2010). Such alkaline treatments did not cause much chemical modification beyond the saponification of ester and ether bonds between lignin and hemicelluloses. More effective extraction of lignin from sweet sorghum stem has been accomplished by dilute alkali due to the enhancement of cleavage of ester linkages (She et al., 2010). More recently, a chemically essentially unchanged lignin was extracted with aqueous ethanol at low temperature (below boiling point), but the yield was very low (5.1% of the original lignin) (Xiao et al., 2011). It was hoped that a combination of alkali with ethanol treatments would liberate the recalcitrant structure of lignocelluloses, achieving a relatively high yield of lignin. Bamboo is a perennial woody grass belonging to the graminaceous plants and distributed widely in Asia (Kobayashi et al., 2004; Scurlock et al., 2000). It has been widely used as feedstocks for paper, textile, food, and reinforcing fibers. Recently, some studies implied that bamboo culms could be exploited to produce ethanol and methanol (Kobayashi et al., 2004; Tsuda et al., 1998). The lignin in graminaceous plants is generally 11–27% on a dried basis (Bagby et al., 1971). Despite lignin presents in bamboo acting as a perfect renewable natural resource, the information about its composition and structure is poor owing to its inherent complex nature. Therefore, the aim of the present study was to examine a sequential process to extract lignin from bamboo with ethanol, aqueous alkaline ethanol, and aqueous alkaline solution at different concentrations. In addition, the lignin fractions obtained were characterized by gel permeation chromatography (GPC), carbohydrate analysis, and advanced spectroscopic techniques, including Fourier transform infrared (FT-IR), 1 H, 13 C NMR, and heteronuclear single quantum coherence (HSQC) spectroscopies.

Dried sample Extracted with toluene-ethanol (2:1, v/v) for 6 h . Dewaxed bamboo sample Successively extracted with 70% ethanol at 80 oC, 0.2 and 0.5 M NaOH, 70% ethanol containning 0.6 M NaOH, and 1.0, 2.0, and 3.0 M NaOH at 50 oC f or 3 h with a solid to li quor ratio of 1: 25 (g/mL).

Residue

Filtrate Neutralized with 6 M HCl solution to pH 5.5, concentrated to about 50 mL, and then precipitated in 2 volumes of 95% ethanol.

Filtrate

Pallet

Concentrated to 20-30 mL, acidized with 6 M HCl to pH 1.5-2.0, and then centrifuged and freeze-dried. Lignin fractions (L1, L2, L3, L4, L5, L6, and L7 ) Fig. 1. Scheme for successive extractions of acid-insoluble lignin from Neosinocalamus affinis.

combined, neutralized to pH 5.5 with 6 M HCl, and concentrated with a rotary evaporator under reduced pressure to about 50 mL. Then the solubilized hemicelluloses were isolated by precipitation of the concentrated liquor with 2 volumes of 95% ethanol (25 ◦ C, 0.5 h) and then hemicellulosic pellet were recovered by filtering, washing with 70% ethanol, and freeze-dried. The filtrate was concentrated to 20–30 mL, and then the pH was adjusted to 1.5–2.0 with 6 M HCl. The acid-insoluble lignin fractions were obtained by centrifugation and freeze-drying. All samples were stored in a desiccator for further characterization. All experiments were performed in duplicate, and the deviation was less than 4.8%. The acid-insoluble lignin fractions extracted with ethanol at 80 ◦ C, 0.2 and 0.5 M NaOH aqueous solution, 70% ethanol containing 0.6 M NaOH, and 1.0, 2.0 and 3.0 M NaOH aqueous solutions at 50 ◦ C for 3 h were labeled as L1 , L2 , L3 , L4 , L5 , L6 , and L7 , respectively. 2.3. Sugar analysis

Bamboo culms (Neosinocalamus affinis) were obtained from Sichuan province, China. The dried raw material was cut into small pieces (1–3 cm) and then ground to pass a 0.8 mm screen. The dried powder was first extracted with toluene/ethanol (2:1, v/v) in a Soxhlet extractor for 6 h, and then dried in an oven at 60 ◦ C for 16 h before use. The lignin content of the dried bamboo was 18.53% (acid-insoluble lignin 16.16% and acid-soluble lignin 2.37%). All chemicals used were of analytical grade.

The acid-insoluble lignin fractions (5 mg) were hydrolyzed with 1.475 mL of 6% sulphuric acid at 105 ◦ C for 2.5 h. After hydrolysis, the samples were diluted 50-fold, and analyzed by high performance anion exchange chromatography (HPAEC) system (Dionex ISC 3000) with an amperometric detector, an AS50 autosample, a CarbopacTM PA-20 column (4 × 250 mm, Dionex), and a guard PA-20 column (3 × 30 mm, Dionex). The sugars were separated in 18 mM NaOH (carbonate free and purged with nitrogen) with post-column addition of 0.3 M NaOH at a rate of 0.5 mL/min. Run time was 45 min, followed by 10 min elution with 0.2 M NaOH to wash the column and then a 15 min elution with 18 mM NaOH to re-equilibrate the column. Calibration was performed with standard solution of L-rhamnose, L-arabinose, D-glucose, D-xylose, D-mannose, D-galactose, glucuronic acid and galacturonic acid.

2.2. Extraction and purification of lignin fractions

2.4. Determination of molecular weights

Fig. 1 illustrates the extraction sequence of the lignin fractions. The power (10 g) was successively treated with 70% ethanol at 80 ◦ C, 0.2 and 0.5 M NaOH, 70% ethanol containing 0.6 M NaOH, and 1.0, 2.0, and 3.0 M NaOH at 50 ◦ C for 3 h with a solid to liquor ratio of 1:25 (g/mL). After the indicated period of treatment, the insoluble residue was collected by filtration a Buchner funnel, washed with distilled water until the pH of filtrates was neutral, and then ovendried at 60 ◦ C for 16 h. The supernatant and washing fluid were

The molecular-average weights and molecular weight distribution of the acid-insoluble lignin fractions were examined by gel permeation chromatograph (GPC, Agilent 1200, USA) using a PL-gel 10 ␮m Mixed-B 7.5 mm ID column. The detector was a differential refractive index detector (RID). A 4 mg sample was dissolved in 2 mL tetrahydrofuran (THF), and 20 ␮L lignin solutions were injected. The column was operated at ambient temperature and eluted with THF at a flow rate of 1.0 mL/min.

2. Materials and methods 2.1. Materials

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2.5. Fourier transformation infrared spectroscopy (FT-IR) The FT-IR spectra of the acid-insoluble lignin fractions were performed on a FT-IR spectrophotometer (Bruker Tensor 27) using KBr pellet containing 1% finely ground samples. Each spectrum was recorded 32 scans in the range from 4000 cm−1 to 400 cm−1 with a resolution 2 cm−1 in the transmission mode. 2.6. NMR spectroscopy The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV III 400 MHz spectrometer. The 1 H NMR spectrum was obtained at 400 MHz using 10 mg acid-insoluble lignin in 0.5 mL of dimethylsulfoxide-d6 (DMSO-d6 ). The chemical shifts of 1 H NMR spectrum were calibrated with reference to DMSO, used as an internal standard, at 2.49 ppm. The acquisition time was 3.9 s, and relaxation time was 1.0 s. The 13 C NMR spectrum was obtained on a Bruker spectrometer at 100 MHz. The acid-insoluble lignin sample (80 mg) was dissolved in 0.5 mL of DMSO-d6 , and the spectrum was recorded at 25 ◦ C after 30,000 scans. A 30◦ pulse flipping angle, a 9.2 ␮s pulse width, 1.36 s acquisition time, and 1.89 s relaxation delay time were used. The heteronuclear single quantum coherence (HSQC) spectra were recorded at 25 ◦ C with the same lignin concentration for 13 C NMR. The spectral widths for the HSQC were 5000 and 20,000 Hz for the 1 H and 13 C dimensions, respectively. The number of collected complex points was 1024 for the 1 H dimension with a recycle delay of 5 s. The number of transients was 128, and 256 time increments were recorded in 13 C-dimension. The 1 JC–H used was 146 Hz. Prior to Fourier transformation, the data matrixes were zero filled up to 1024 points in the 13 C-dimension. Date processing was performed using standard Bruker Topspin-NMR software. A semi-quantitative analysis of the intensities of the HSQC cross-signal was performed in this study. A direct analysis of the cross-signal intensities was elusive, which was due to the intensities depending on the particular 1 JC–H value and the T2 relaxation time. Thus, the integration of the cross-signals was performed separately for the different regions of the HSQC spectra, which contain signals that correspond to chemically analogous carbon–proton pairs. For these signals, the 1 JC–H coupling value was relatively similar and therefore was used semiquantitatively to estimate the different C–H correlations. In the aliphatic oxygenated region, interunit linkages were estimated from C˛ –H˛ correlations to avoid possible interference from homonuclear 1 H–1 H couplings. In the aromatic region, C2,6 –H2,6 correlations from S units and C2 –H2 plus C6 –H6 correlations from G units were used to calculate the S/G ratio of lignin. 3. Results and discussion

has been developed. It has been found that the treatment with 70% ethanol at boiling point for 3 h is a good solution to release lignin with a slight change of its structure (Xiao et al., 2011). In addition, the extractions with solutions of NaOH in water and/or ethanol at mild temperatures for 3 h are effective for disrupting the recalcitrant nature of the plant cell wall (Sun et al., 2010; Xiao et al., 2011). For instance, the treatment of sweet sorghum stem with dilute alkali solution can release lignin only resulting in a slight saponification of the ester groups (She et al., 2010). Owning to the good solubility of lignin in ethanol solution, an ethanol solution with a relatively higher concentration of alkali librated a higher amount of lignin (Xiao et al., 2011; Yuan et al., 2011). Under the relatively concentrated alkali solutions (1–3 M NaOH), more lignin in the cell wall of Caragana sinica has been extracted due to the more severe cleavage of the linkages between lignin and hemicelluloses (Xiao et al., 2011). Therefore, the optimum extraction condition aforementioned for extraction of lignin was adopted in the present study. In this case, Neosinocalamus affinis was subjected to the sequential extractions with increased alkaline concentrations, i.e., 70% ethanol at 80 ◦ C, 0.2 and 0.5 M NaOH, 70% ethanol containing 0.6 M NaOH, and 1.0, 2.0 and 3.0 M NaOH at 50 ◦ C for 3 h. The yields of the acidinsoluble lignin fractions (%, dried material) from Neosinocalamus affinis are given in Table 1. As can be seen, the sequential treatments of the dewaxed bamboo yielded 0.38, 1.96, 2.20, 1.42, 1.29, 1.81, and 1.00% of the acid-insoluble lignin fractions labeled as L1 , L2 , L3 , L4 , L5 , L6 , and L7 , respectively. The total yield of the seven lignin fractions accounted for 10.06%, corresponding to release of 62.25% of the original lignin from the cell walls. It has been reported that alkali treatment of lignocelluloses disrupted the plant cell walls and resulted in the dissolution of lignin and polysaccharides (Jackson, 1977). In addition, alkali treatment cleaved the linkages between lignin and hemicelluloses, such as ester bonds between ferulic acid and hemicelluloses or between p-coumaric acid and lignin, and ␣aryl ether linkages between lignin and hemicelluloses (Sun et al., 2010). To verify the purity of the acid-insoluble lignin fractions, the composition of the carbohydrates in the seven lignin fractions was determined. As can be seen from Table 1, all of the lignin fractions except L1 contained small amounts of bound carbohydrates (0.22–0.94%). This low content of sugar residues in alkaline lignin fractions might be explained by the cleavage of alkali-labile linkages, especially the ester linkages between lignin and hemicelluloses (Spencer and Akin, 1980). Among all lignin fractions extracted with alkali, xylose was identified to be the major sugar component. However, it is worthy to note that glucose (2.01%) occupied a dominant proportion of the sugar in lignin fraction L1 , which Table 1 Yield of acid-insoluble lignin and content of neutral sugars and uronic acids in the lignin fractions obtained from Neosinocalamus affinis. Lignin fractiona

3.1. Fractional yield and content of carbohydrates Alcohols (mainly methanol and ethanol) are good neutral solvents for lignin and have been extensively used to extract lignin from lignocelluloses. In addition, alkali solutions, such as aqueous NaOH, KOH, and Ca(OH)2 , are widely used to disrupt the rigid structure of the cell wall for the separation of the main components of lignocellulosic materials. An increase in the strength of alkali can result in a more release of lignin embedded compactly in the lignocellulosic matrix. This is mainly because the concentration of alkali can effectively reduces the gigantic molecular size of lignin and disrupts the bonds between lignin and carbohydrates (Wen et al., 2010; Xiao et al., 2011). In order to achieve an effective isolation of lignin from lignocellulosic materials, a serious of primary studies has been conducted in our laboratory and the optimized condition

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Arabinose Galactose Glucose Xylose Uronic acid Total sugarsb Yieldc

L1

L2

L3

L4

L5

L6

L7

0.27 0.22 2.01 1.90 0.10 4.50 0.38

0.04 NDd 0.19 0.63 0.08 0.94 1.96

0.02 ND 0.02 0.20 0.06 0.30 2.20

0.02 ND 0.02 0.34 0.05 0.43 1.42

0.01 ND 0.03 0.46 0.05 0.55 1.29

ND ND 0.01 0.18 0.03 0.22 1.81

ND ND ND 0.35 0.04 0.39 1.00

a L1 , L2 , L3 , L4 , L5 , L6 , and L7 represent the lignin fractions isolated by successive treatments with 70% ethanol at 80 ◦ C, 0.2 and 0.5 M NaOH, 70% ethanol containing 0.6 M NaOH, and 1.0, 2.0 and 3.0 M NaOH at 50 ◦ C for 3 h, respectively. b Represent the total associated carbohydrates in the lignin fractions (% dry lignin sample, w/w). c Represent the yield of the acid-insoluble lignin fractions (% dry dewaxed sample, w/w). d ND, not detectable.

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Fig. 2. Molecular weight distributions of the acid-insoluble lignin fractions extracted from Neosinocalamus affinis.

arose probably from the degradation of associated hemicelluloses or ␤-glucan under the condition given (Sun et al., 2010).

Table 2 Weight-average (Mw ) and number-average (Mn ) molecular weights and polydispersity (Mw /Mn ) of the acid-insoluble lignin fractions. Lignin fractiona

3.2. Molecular weight distribution Weight-average (Mw ) and number-average (Mn ) molecular weight, and polydispersity (Mw /Mn ) of all the acid-insoluble lignin fractions were determined to estimate the effects of aqueous ethanol and/or alkali solutions on the polymer structures (Table 2). It should be noted that the data shown in this table can only be used for comparison since the calibration was carried out using polystyrene standards. As can be seen, for the fraction extracted with 70% ethanol, the Mw and Mn were 2670 and 1430 g/mol, respectively. However, the alkali lignin fractions exhibited low Mw (ranging from 1400 to 1680 g/mol) and Mn (ranging from 740 to 840 g/mol). Clearly, the ethanol-soluble lignin had a higher molecular weight as compared to those extracted with alkaline solutions

Mw Mn Mw /Mn a

L1

L2

L3

L4

L5

L6

L7

2670 1430 1.86

1580 790 1.99

1670 770 2.22

1620 740 2.19

1400 720 1.95

1570 790 2.00

1680 840 2.01

Corresponding to the lignin fractions in Table 1.

and alkaline ethanol solution. It has been reported that the carbohydrates chains linked to lignin can increase the hydrodynamic volume of lignin and therefore increase the apparent molecular weight of the lignin when it was measured using GPC (Jääskeläinen et al., 2003). This was in line with the results of carbohydrate analysis as shown in Table 1. The molecular weight distributions of the seven acid-insoluble lignin fractions are shown in Fig. 2. As can be seen, the lignin

S.-N. Sun et al. / Industrial Crops and Products 37 (2012) 51–60

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Fig. 3. FT-IR spectra of the acid-insoluble lignin fractions extracted with (a) 70% ethanol (L1 ), 0.2 M (L2 ) and 0.5 M NaOH (L3 ), and (b) 70% ethanol aqueous solution containing 0.6 M NaOH (L4 ), 1.0 (L5 ), 2.0 (L6 ), and 3.0 (L7 ) M NaOH.

fraction L1 obtained from aqueous ethanol solution (70% ethanol) had a somewhat higher proportion of high molecular weight component compared with the fractions extracted with aqueous alkaline solutions. Additionally, L1 gave a narrow molecular weight distribution, corresponding to a polydispersity index of 1.86 as compared to the other lignin fractions (1.95–2.22). It was obvious that the alkaline lignin fractions exhibited bimodal distributions, different from that of the ethanol lignin. This was due to the considerable degradation of lignin with high molecular weights in alkaline solution, resulting in heterogeneous distributions of the molecular weights. The first peak (left on the graphs) was probably attributed to dimmers and/or trimers of low molecular components. Similar

result has also been obtained in alkali-soluble lignin isolated from wheat straw (Lawther et al., 1996). 3.3. FT-IR Spectra Fourier transform infrared spectroscopy has been proven to be a useful approach to study physicochemical and conformational properties of lignin in which various functional groups and structural fragments can be characterized. The FT-IR spectra of the acid-insoluble lignin fractions L1 , L2 , and L3 obtained by 70% ethanol at 80 ◦ C, 0.2 and 0.5 M NaOH at 50 ◦ C for 3 h, are shown in Fig. 3(a). As can be seen, a wide absorption band at 3424 cm−1 is assigned to

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Fig. 4.

1

H and 13 C NMR spectra of the acid-insoluble lignin fraction L3 extracted with 0.5 M NaOH at 50 ◦ C.

the O–H stretching vibration in aromatic and aliphatic OH groups, while the bands at 2933 and 2853 cm−1 arise from the C–H symmetrical and asymmetric vibrations in methyl and methylene groups (Labidi et al., 2009). The bands at 1697 and 1659 cm−1 are indicative of carbonyl stretching in non-conjugated ketones and conjugated psubstituted aryl ketones, respectively (Sun and Tomkinson, 2002). The peaks at 1598, 1510, and 1424 cm−1 are assigned to aromatic skeleton vibrations, indicating a primary structure of lignin. The strong band at 1461 cm−1 is attributed to the C–H asymmetric vibrations (asymmetric in methyl, methylene, and methoxyl groups) (Jahan et al., 2007). The band at 1329 cm−1 , corresponding to syringyl and condensed guaiacyl absorptions, and the guaiacyl ring breathing with C O stretching at 1267 cm−1 (Xu et al., 2005), are present. A weak band at 1158 cm−1 in the spectrum of L2 might arise from the absorption of C O in ester groups (conjugated). A remarkable characteristic of lignin is the presence of a strong

band at 1126 cm−1 for syringyl structures, whereas aromatic C–H in-plane deformation in guaiacyl type is observed at 1033 cm−1 (Sun and Tomkinson, 2002). The band at 986 cm−1 belongs to –CH CH– group out of plane deformation (trans), which further indicated that the lignin with conjugated units was isolated by aqueous alkaline solutions. Meanwhile, the signal of aromatic C–H out-of-plane deformation appears at 834 cm−1 (Villaverde et al., 2009). Fig. 3(b) shows the FT-IR spectra of L4 , L5 , L6 , and L7 extracted with 70% ethanol containing 0.6 M NaOH, and 1.0, 2.0, and 3.0 M NaOH, respectively. Some shoulders at 1090 and 1088 cm−1 are assigned to the C–O stretching in the spectra of all alkali-soluble lignin fractions, which are derived from hemicelluloses such as glucans and xylans (She et al., 2010). This was in agreement with the results obtained by sugar analysis. As can be seen, the relative intensities, corresponding to aromatic skeleton vibrations at 1598, 1508,

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Table 3 Assignments of 13 C–1 H correlation signals in the HSQC spectra of the alkaliextractable lignin fraction (L3 ). Label

ıC /ıH

Assignments

Cˇ Bˇ –OCH3 Dˇ A

Cˇ –Hˇ in phenylcoumaran substructures (C) Cˇ –Hˇ in ˇ-ˇ (resinol) substructures (B) C–H in methoxyls Cˇ –Hˇ in spirodienone substructures (D) C –H in ˇ-O-4 substructures (A)

E

52.0/3.50 53.4/3.04 55.9/3.71 59.6/2.75 59.6/3.42 and 3.71 61.2/4.09

A 

65.0/4.21

B

71.3/3.80 and 4.16 70.4/4.76 71.8/4.85 81.0/5.10 81.8/4.91 83.6/4.36 84.3/4.64 86.0/4.09 86.8/5.48 104.3/6.69 106.8/7.22 106.8/7.32 110.9/7.26 111.1/6.90 114.7/6.69 115.8/6.90 115.3/6.27–6.30 116.3/6.77 116.5/6.30 119.8/6.72 122.2/7.10 128.1/7.16 130.5/7.49 144.2/7.49

A˛(G) A˛(S) D˛ D˛ Aˇ(G) B˛ Aˇ(S) C˛ S2,6 S 2,6 S 2,6 FE2 G2 G5 PCEˇ PCE3,5 FEˇ G6 FE6 H2,6 PCE2,6 PCE˛

C –H in p-hydroxycinnamyl alcohol end groups (E) C –H in -acylated ˇ-O-4 substructures (A and A ) C –H in ␤-␤ resinol substructures (B) C˛ –H˛ in ˇ-O-4 substructures linked to a G unit (A) C␣ –H␣ in ˇ-O-4 substructures linked to a S unit (A) C˛ –H˛ in spirodienone substructures (D) C˛ –H˛ in spirodienone substructures (D) Cˇ –Hˇ in ˇ-O-4 substructures linked to a G unit (A) C␣ –H␣ in ˇ-ˇ (resinol) substructures (B) Cˇ –Hˇ in ˇ-O-4 substructures linked to a S unit (A) C˛ –H˛ in phenylcoumaran substructures (C) C2,6 –H2,6 in etherified syringyl units (S) C2,6 –H2,6 in C˛ -oxidized syringyl units (S ) C2,6 –H2,6 in C˛ -oxidized phenolic syringyl units (S ) C2 –H2 in FE ester (FE) C2 –H2 in guaiacyl units (G) C5 –H5 in guaiacyl units (G) C␤ –H␤ in p-coumaroylated substructures (A ) C3,5 –H3,5 in p-coumaroylated substructures (A ) C␤ –H␤ in FE ester (FE) C6 –H6 in guaiacyl units (G) C6 –H6 in FE ester (FE) C2,6 –H2,6 in H units (H) C2,6 –H2,6 in p-coumaroylated substructures (A ) C␣ –H␣ in p-coumaroylated substructures (A )

PCE, esterified p-coumaric acid; FE, esterified ferulic acid.

and 1422 cm−1 in Fig. 3(b), were very similar to Fig. 3(a). It indicated that the “core” of the lignin structure did not change and the obtained lignin had same aromaticity during the alkali treatment processes (Sun et al., 2003). 3.4.

1H

and 13 C NMR

The analytical techniques of 1 H and 13 C NMR spectroscopy are powerful methods for determination of lignin structure. To further interpret the structural features of the acid-insoluble lignin fractions isolated by aqueous alkaline solutions, the fraction L3 extracted with 0.5 M NaOH was characterized by NMR spectroscopy. 1 H and 13 C NMR spectra of lignin fraction L3 are shown in Fig. 4, and the resonances were assigned according to literatures (Jahan et al., 2007; Kringstad and Mörck, 1983; Oliveira et al., 2009; Samuel et al., 2010; Sun and Tomkinson, 2002; Xu et al., 2005). The 1 H spectrum showed two weak signals, which corresponded to Hˇ (4.85 ppm) and H (4.09 ppm) of ˇ-O-4 structure, respectively (Xu et al., 2005). Signals between 6.00 and 8.00 ppm are originated from the aromatic protons in syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) units, p-coumarate and ferulate (Jahan et al., 2007). The sharp signals at 6.30 and 6.27, 6.81 and 6.83, and 7.45, 7.47, 7.49 ppm relate to Hˇ , H3/5 , and H2/6 protons of p-coumarate, respectively. The weak signals at 6.34 and 6.37 ppm are attributed to Hˇ of the ferulate. The resonances at 6.69 and 6.81 ppm are originated from aromatic protons in syingylpropane and guaiacylpropane structures, respectively. The Hˇ in ˇ-O-4 structures produces a broad signal at 4.85 ppm, whereas the H˛ in benzyl aryl ethers gives a signal at 5.58 ppm (Xu et al., 2005).

Fig. 5. HSQC spectra of the acid-insoluble lignin fractions L3 (extracted with 0.5 M NaOH) and L5 (extracted with 1.0 M NaOH).

Besides, the signals between 0.8 and 1.6 ppm are attributed to CH3 and CH2 in s aliphatic chains of the lignin (Oliveira et al., 2009). In 13 C NMR spectrum, a small signal at 174.6 ppm confirmed the presence of ester-linked fatty/hydroxyl acids. The S units were identified by signals at 152.1 ppm (C-3/C-5 etherified), 149.7 and 147.1 ppm (C-3/C-5 nonetherified), 138.1 ppm (C-4 etherified), 134.2 ppm (C-1 etherified), and 106.8 and 104.3 ppm (C-2/C-6). The G units give signals at 149.7 ppm (C-3 etherified), 145.3 ppm (C-4 nonetherified), 134.2 ppm (C-1 etherified)), 133.0 ppm (C-1 nonetherified), 119.2 ppm (C-6), 115.8 and 114.7 ppm (C-5), and 111.1 ppm (C-2). The p-hydroxyphenyl (H) unit was found at 128.1 ppm. These signals confirmed that the lignin fraction could be justified as an HGS type. Moreover, the presence of p-coumaric ester is evidenced by six signals at 167.9, 159.8, 145.3, 130.0, 125.1, and 115.8 ppm, assigned to C-, C-4, C-˛, C-2/C-6, C-1, and C-ˇ in the structure, respectively. Meanwhile, esterified ferulic acid presents a signal at 122.2 ppm corresponded to C-6 in the structure. Etherified ferulic acid gives signals at 167.9 and 144.2 ppm originated from C- and C-˛, respectively. The resonance of C- was probably overlapped with the signal of C-4 in ˇ-5 units (Sun et al., 2003). These signals implied that p-coumaric acid was linked to lignin via ester bonds, whereas ferulic acid was linked to lignin by both ether and ester bonds (Sun and Tomkinson, 2002). Resonances between 86 and 50 ppm are assigned to lignin interunit linkages. More importantly, one of the most important reactions for lignin degradation in alkaline media is the cleavage of ˇ-O-4 structures. The ˇ-O-4 linkages were detected by signals at 72.2, 86.0, and 59.6 ppm, attributed to C-˛, C-ˇ, and C-, respectively. These resonances suggested that the extraction with 0.5 M NaOH at 50 ◦ C did not attack the ˇ-aryl ether structure to a significant extent. Besides, certain signals belonging to condensed structures can be

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Fig. 6. Main structures present in the acid-insoluble lignin fraction L3 : (A) ˇ-O-4 linkages; (A ) ␥-acetylated ˇ-O-4 substructures; (A ) -p-coumaroylated ˇ-O-4 linkages; (B) phenylcoumarane structures formed by ˇ-5 /˛-O-4 linkages; (C) resinol structures formed by ˇ-ˇ /˛-O-  /-O-˛ linkages; (D) spirodienone structures formed by ˇ-1 linkages; (E) p-hydroxycinnamyl alcohol end groups; (H) p-hydroxyphenyl unit; (G) guaiacyl unit; (S) syringyl unit; (S ) oxidized syringyl unit linked a carbonyl group at C˛ (phenolic); (S ) oxidized syringyl unit linked a carboxyl group at C˛ .

easily distinguished. The strong signal at 55.9 ppm is attributed to the –OCH3 in the guaiacyl and syringyl units. According to the carbon assignments in the lignin structural elements, the signals for the -methyl, ˛- and ˇ-methylene groups in n-propyl side chains relate to the integration at 33.7–13.9 ppm. 3.5. 2D-NMR The acid-insoluble lignin fractions extracted with aqueous 0.5 and 1.0 M NaOH were subjected to HSQC analysis to acquire detailed information. As shown in Fig. 5, the lignin fractions extracted with 0.5 M NaOH seemingly exhibited more signals than that extracted with 1.0 M NaOH. Therefore, the main crosssignals of L3 assigned in the HSQC spectrum are listed in Table 3 and the main substructures found are depicted in Fig. 6. Generally, the HSQC spectra of lignin could be divided into three regions of 13 C–1 H correlations corresponding to aliphatic region (around ıC /ıH 0–50/0–2.5), oxygenated aliphatic (side chain) region (around ıC /ıH 50–100/2.5–6.5), and aromatic region (around ıC /ıH 100–160/5.5–9.0). Considering that the signals usually originate from certain impurities, the aliphatic (nonoxygenated) region is not shown in this paper. The main lignin cross-signals of the acid-insoluble lignin fraction L3 from the oxygenated aliphatic and aromatic regions were mainly made based on previously reported results (del Río et al., 2009; Martínez et al., 2008; Nakamura and Higuchi, 1976; Rencoret

et al., 2009). Signals from S, G, and H units were observed in the HSQC spectra. The S units showed a prominent resonance for C2,6 –H2,6 correlation at ıC /ıH 104.3/6.69, while the G units exhibited different correlations for C2 –H2 (ıH /ıC 111.1/6.90), C5 –H5 (ıC /ıH 115.8/6.90 and 114.7/6.69), and C6 –H6 (ıC /ıH 119.2/6.81). The double H5 –C5 correlation suggested that some heterogeneity among the G units specially affecting the H5 –C5 correlation, probably due to different substituents at C4 (e.g., phenolic or etherified in different substructures) (del Río et al., 2009). A signal was assigned to C2,6 –H2,6 (ıC /ıH 128.1/7.16) correlation in H units. Signals corresponding to C2,6 –H2,6 correlations of C˛ -oxidized S units (S and S ) were observed at ıC /ıH 106.8/7.32 and 106.8/7.22, respectively (del Río et al., 2008). It has been reported that esterified p-coumaric acid (PCE) existed in the form of p-coumaroylated substructures (A ) (Nakamura and Higuchi, 1976). Aromatic ring cross-signals corresponding to correlations C2,6 –H2,6 and C3,5 –H3,5 in p-coumaric ester were observed at ıC /ıH 130.0/7.49 and 115.8/6.83, respectively. Unsaturated C˛ –H˛ and Cˇ –Hˇ signals of PCE were also observed at ıC /ıH 144.2/7.49 and 115.3/6.27–6.30, respectively (Martínez et al., 2008). In addition, esterified ferulic acid (FE) was also found at C2 –H2 (ıC /ıH 110.9/7.26), C6 –H6 (ıC /ıH 122.2/7.10), and Cˇ –Hˇ (ıC /ıH 116.5/6.30) correlations in the HSQC spectra of lignin fraction extracted with 0.5 M NaOH. Cross-signals of methoxyl substituents (ıC /ıH 55.9/3.71) and ring side-chain in classical lignin substructures were detected at the oxygenated aliphatic region. This region showed useful information

S.-N. Sun et al. / Industrial Crops and Products 37 (2012) 51–60 Table 4 Structural characterisitics (relative abundance of the interunit linkages and S/G weights ranging from 1400 ratio) from integration of 13 C–1 H correlation signals in the HSQC spectra. Linkage relative amount (% of total side chains involved)

L3 a

L5 a

␤-Aryl-ether units (ˇ-O-4 , A/A /A  ) Resinol substructures (ˇ-ˇ , B) Phenylcoumaran substructure (ˇ-5 , C) Spirodienones (ˇ-1 , D) p-Hydroxycinnamyl alcohol end groups (E) S/G ratio

83.1 6.6 5.0 3.0 2.3 2.67

91.3 5.6 NDb 1.1 2.0 2.37

a b

Corresponding to the lignin fractions in Table 1. ND, not detectable.

about the different interunit linkages present in bamboo lignin. The H˛ –C˛ correlations in ˇ-O-4 substructures were observed at ıC /ıH 70.4/4.76 and 71.8/4.85 for structures linked to G or S lignin units, respectively (del Río et al., 2009). Likewise, the Hˇ –Cˇ correlations at ıC /ıH 83.6/4.36 and 86.0/4.09 linked to G units and S units in ˇ-O-4 substructures, respectively. The C –H correlations in ˇ-O-4 substructures were also observed at ıC /ıH 59.6/3.42 and 3.71. Meanwhile, the presence of -acylated ˇ-O-4 , probably acylated with acetate and/or p-coumarate groups (A , A ), is confirmed by a small but clear C –H correlation signal (ıC /ıH 65.0/4.21) (del Río et al., 2008). In addition, other lignin substructures were also observed in the HSQC spectra of L3 . Resinol (ˇ-ˇ /˛-O-  /-O-˛ ) substructures (B) were found with strong signals. These signals were detected with their C˛ –H˛ , Cˇ –Hˇ , and the double C –H correlations at ıC /ıH 84.3/4.64, 53.4/3.04, and 71.3/4.16 and 3.80, respectively. Furthermore, phenylcoumaran (ˇ5 ) units (C) were also detected, and the signals for their C˛ –H˛ and Cˇ –Hˇ correlations were observed at ıC /ıH 86.8/5.48 and 52.2/3.50, respectively. Small signals corresponding to spirodienone (ˇ-1 /˛O-˛ ) substructures (D) could be observed, their C˛ –H˛ , C˛ –H˛ and Cˇ –Hˇ correlations being at ıC /ıH 81.0/5.10, 81.8/4.91 and 59.6/2.75, respectively. Signal of p-hydroxycinnamyl alcohol end groups (E) C –H (ıC /ıH 61.2/4.09) correlation was also detected in the side-chain region. The relative amounts of interunit linkages and molar S/G ratios present in lignin obtained with 0.5 and 1.0 M NaOH are shown in Table 4. As expected, the main lignin substructures were ˇ-O4 aryl ether linkages (over 80% of total side chains), followed by low amounts of ˇ-ˇ resinol-type, ˇ-5 phenylcoumaran-type, ˇ-1 spirodienones-type linkages, and p-hydroxycinnamyl alcohol end groups. Moreover, the ratios of S/G were calculated to be 2.67 and 2.37 in L3 and L5 , respectively. Such S/G ratios implied that S units were more liable to be released than the G units from cell walls under a mild alkaline condition. Based on the analysis above, it could be concluded that the alkaline lignin from Neosinocalamus affinis was an HGS-type. In comparison, the spectra of alkaline lignin fractions exhibited similar structural features. In consideration of aforementioned signal assignments, the acid-insoluble lignin fraction L3 extracted with 0.5 M NaOH had more signals than that extracted with 1.0 M NaOH. This revealed that L3 contained more interunit linkages. The main substructures present in both L3 and L5 were ˇ-O-4 aryl ether structures. In addition, the lignin fraction L3 contained low amount of phenylcoumaran substructure (ˇ-5 ), whereas this one could not be detected in L5 . This difference might be due to the different concentrations of aqueous alkaline solutions used for isolating the lignin fractions. 4. Conclusions The results of the present study showed that the sequential extractions of bamboo yielded 10.06% the acid-insoluble lignin. The lignin fractions extracted with alkali contained small amounts of bound carbohydrates (0.22–0.94%) and exhibited molecular

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to 1680 g/mol and the polydispersities ranging from 1.95 to 2.22. On the other hand, the lignin fraction extracted with aqueous ethanol had a relatively higher molecular weight (2670 g/mol) and content of sugar residues (4.50%), but a low polydispersity (1.86). The spectra analysis indicated that the acid-insoluble lignin fractions extracted with alkali were classified as an HGS-type. The lignin fractions extracted with 0.5 and 1.0 M NaOH had a predominance of ˇ-O-4 (over 80% of all linkages), followed by low amounts of ˇ-ˇ , ˇ-5 , ˇ-1 linkages, and p-hydroxycinnamyl alcohol end groups. Additionally, the lignin side chains were found to be partially acylated at the -carbon with p-coumarate and/or acetate groups.

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