Structural Variation of Lignin and LigninâCarbohydrate Complex in

Research Article pubs.acs.org/journal/ascecg

Structural Variation of Lignin and Lignin−Carbohydrate Complex in Eucalyptus grandis × E. urophylla during Its Growth Process Bao-Cheng Zhao,† Bo-Yang Chen,† Sheng Yang,† Tong-Qi Yuan,*,† Adam Charlton,‡ and Run-Cang Sun*,† †

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Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, No. 35 Tsinghua East Road Haidian District, 100083 Beijing, China ‡ The BioComposites Centre, Bangor University, Deiniol Road, Bangor, Gwynedd LL57 2UW, U.K. S Supporting Information *

ABSTRACT: Milled wood lignin (MWL), lignin−carbohydrate complex-rich fraction (LCC-AcOH), celluloytic enzyme lignin (CEL), and enzymatic hydrolysis residue (EHR) were sequentially isolated from Eucalyptus grandis × E. urophylla under mild conditions, and the variations of lignin− carbohydrate complex (LCC) linkages and lignin structures during the eucalyptus growth were investigated. The 2D HSQC NMR analysis showed that β-O-4′ and β−β′ were the main linkages in lignin, while other substructures were present in much lower amounts. The amounts of β-O-4′ in the MWL and LCC-AcOH fractions showed an increased tendency and those in the CEL and EHR fractions had no obvious variation with the eucalyptus growth. The S/G ratios of the MWL, LCC-AcOH, and CEL fractions increased first and then decreased, whereas those of the EHR fractions decreased with the tree age. The amount of phenyl glycoside (PhGlc) in the LCC-AcOH fractions varied consistent with the S/G ratio. The variation of the amount of benzyl ether (BE) in the MWL fractions was parallel to the S/G ratio, while that in the CEL fractions was contrary to it. These findings will provide some evidence for the structural variation of lignin and LCC in Eucalypt during its growth process. KEYWORDS: Eucalyptus grandis × E. urophylla, Lignin−carbohydrate complex, Lignin structure, LCC linkages, 2D HSQC NMR

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INTRODUCTION Cellulose, hemicelluloses, and lignin are the major components in lignocellulosic biomass. Lignin, the third most abundant biopolymeron earth after cellulose and hemicelluloses, is composed of three units: guaiacyl (G), sinapyl (S), and phydroxyphenyl (H), which are linked by aryl ether and carbon− carbon bonds.1,2 There are numerous pieces of evidence that lignin and carbohydrates (mainly hemicelluloses) are linked by chemical bonds, forming a special compoundlignin carbohydrate complex (LCC).3,4 Although less LCC exists in the plant, it plays a very important role and almost all wood lignin is associated with polysaccharides.5,6 The linkage types and numbers of LCC are still not well understood, although they can cause technical difficulties during the processing of biomass, limiting the separation of lignin and carbohydrates in chemical pulping and biorefining.7,8 Therefore, in view of theory and practice, it is vitally important to understand the structure of native LCC in the lignocellulosic biomass. It is generally believed that there are three types of LCC linkages in the lignocellulosic biomass, which are phenyl glycoside (PhGlc), benzyl ether (BE), and ester.3,9 In hardwood and softwood, benzyl ether and phenyl glycoside linkages are the main bonds in LCC. On the other hand, in herbaceous, © 2016 American Chemical Society

ferulate and p-coumarate link hemicelluloses (mainly arabinoxylan) and lignin components together forming LCC.10,11 To investigate the linkages, LCC preparations are usually isolated from lignocellulosic materials. LCC preparations can be classified into carbohydrate-rich LCC (Björkman LCC and similar ones, enzymatic LCC fractions), and lignin-rich LCC (cellulolytic enzyme lignin and crude milled wood lignin). Milled wood lignin (MWL), which was extracted from ball milled wood with 96% aqueous dioxane, was first proposed by Björkman,12 whereas the yield of MWL (based on Klason lignin) is relatively low. To improve the yield, celluloytic enzyme lignin (CEL), which was extracted from the enzymatically hydrolyzed ball milled wood residue, was developed by Pew.13 The structure of CEL is similar to MWL, and it is more representative of total lignin in wood than MWL.13,14 Recently, the features of wet chemistry and NMR analysis methods for LCC structure have been summarized.15 Wet chemistry analysis techniques include cleavage of lignin− carbohydrate linkages and detection of the resulting products Received: October 5, 2016 Revised: November 14, 2016 Published: November 18, 2016 1113

DOI: 10.1021/acssuschemeng.6b02396 ACS Sustainable Chem. Eng. 2017, 5, 1113−1122

Research Article

ACS Sustainable Chemistry & Engineering

Figure 1. Scheme for isolation of milled wood lignin (MWL), lignin-carbohydrate complex-rich fraction (LCC-AcOH), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residue (EHR) from Eucalyptus grandis × E. urophylla. Isolation of lignin and LCC preparations. The lignin and LCC preparations were isolated from ball-milled wood according to the method proposed by Balakshin al.4 with some modifications. The scheme is described in Figure 1, and the detailed procedures are as follows. The ball-milled wood sample was extracted by 96% aqueous dioxane (v/v) with a solid to liquid ratio of 1:10 (g/mL) at room temperature in the dark for 24 h under stirring. The extraction procedure was repeated twice, and all supernatants were concentrated under reduced pressure to obtain crude MWL. The crude MWL was dissolved in 90% acetic acid (v/v, 20 mL/g crude MWL) and precipitated drop by drop into water. The precipitate was MWL (marked as MWL-2, MWL-3, and MWL-4, based on the age of the tree, respectively). Subsequently, the supernatant was collected, concentrated under reduced pressure, and freeze-dried to obtain LCC-AcOH. The LCC-AcOH fractions isolated from 2, 3, and 4 years old wood were labeled as LCC-AcOH-2, LCC-AcOH-3, and LCCAcOH-4, respectively. After the extraction of MWL and LCC-AcOH, the residue was suspended in an acetate buffer solution (pH 4.8). Cellulase was added to the suspension, which was then incubated at 50 °C for 48 h. Cellulase was added at 30 FPU/g substrate with 5% solid loading. After enzymatic hydrolysis, the solid was separated by centrifugation and washed with buffer solution and deionized water. Then the insoluble residue was extracted twice (24 h each time) with 80% aqueous dioxane (v/v) with a solid to liquid ratio of 1:20 (g/mL). All supernatants were collected and concentrated by the same procedure as before, regenerated in acidic water (pH = 2), and freeze-dried to obtain CEL fractions (labeled as CEL-2, CEL-3, and CEL-4, respectively). The residue after CEL isolation (residue-4) was further treated with a celluloytic enzyme in an acetate buffer solution (pH 4.8) at 50 °C for 48 h. Cellulase was added at 50 FPU/g substrate with 2% solid loading. After enzymatic hydrolysis, the solution was centrifuged and the hydrolyzed solid was washed with buffer solution and deionized water, and then freeze-dried to obtain EHR fractions (marked as EHR-2, EHR-3, and EHR-4, respectively). Analytical methods. The main chemical components (cellulose, hemicelluloses, and lignin) of 2, 3, and 4 years old Eucalypt were measured according to the NREL method.19 The analysis of the carbohydrate moieties associated with the MWL, LCC-AcOH, CEL, and EHR fractions was conducted by hydrolysis with dilute sulfuric acid according to previous literature.20 The weight-average (Mw) and number-average (Mn) molecular weights of all the acetylated lignin and LCC preparations were detected by gel permeation chromatography (GPC) with a UV detector on a PL-gel 10 μm mixed-B 7.5 mm i.d. column.20,21 A 4 mg sample was dissolved in 2 mL of tetrahydrofuran (THF), and then a 10 μL solution was injected. The column was operated at ambient temperature and eluted with THF at a flow rate of

by Smith degradation or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation, etc.15,16 However, these methods cannot directly detect LCC structure. Interestingly, the 2D NMR method performs direct observation of LCC structure, which can elucidate the mechanism of plant cell growth. Lignin structure is also very important to understand LCC preparations. It has been reported that LCC linkages were related to the content and structure of lignin, especially the S to G ratio in the LCC preparations.17 Generally, the major linkages within lignin are β-O-4′, β−β′, β-5′, and β-1′.1 Earlier lignin structural characterization of Eucalyptus globulus indicated that the content and structure of lignin were varied during the plant growth.18 Therefore, the structural characterization of LCC and lignin are important to elucidate the mechanism of plant cell growth. In the present study, MWL, the lignin-carbohydrate complexrich fraction (LCC-AcOH), CEL, and the enzymatic hydrolysis residue (EHR) were isolated from 2, 3, and 4 years old Eucalyptus grandis × E. urophylla for speculating the variation of chemical linkages during its growth. This work will provide some evidence for the structural variation of lignin and LCC in Eucalypt during its growth process. The chemical linkages and structural changes of lignin and LCC preparations were characterized by high performance anion exchange chromatography (HPAEC), Fourier transform infrared (FT-IR), twodimensional heteronuclear single-quantum coherence (2DHSQC), and 31P NMR spectroscopies, as well as gel permeation chromatography (GPC).

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MATERIALS AND METHODS

Materials. Eucalyptus grandis × E. urophylla wood preparations of 2, 3, and 4 years old were collected from Guangxi Province, China. The samples were dried in an oven at 60 °C and ground into small pieces. The 40−60 mesh wood powders were extracted with toluene/ ethanol (2:1, v/v) in a Soxhlet instrument for 12 h to remove wax residues. Subsequently, the dewaxed samples were dried at 60 °C for 16 h and then milled with a planetary ball mill for 5 h (a 10 min lull after every 10 min of milling) under N2. Cellulase (Cellic@ CTec2, 100 FPU/mL) was kindly provided by Novozymes, Beijing, China. All other chemicals used were purchased from Beijing Chemical Works without further purification. 1114


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ACS Sustainable Chemistry & Engineering Table 1. Chemical Composition (%) of the Extractive-free Eucalypt Sample

Cellulose

Hemicelluloses

Klason lignin

Acid-soluble lignin

Ash

2-year-old 3-year-old 4-year-old

38.4 ± 1.2 41.4 ± 1.4 44.2 ± 1.7

15.4 ± 0.7 17.0 ± 0.8 18.3 ± 0.8

27.6 ± 0.3 27.9 ± 0.7 29.2 ± 0.9

4.4 ± 0.1 4.9 ± 0.2 4.5 ± 0.2

0.54 ± 0.02 0.44 ± 0.02 0.43 ± 0.02

Table 2. Yield and Carbohydrate Content of Lignin and LCC Fractions Relative carbohydrate content (%) a

Sample

Yield (%)

MWL-2 MWL-3 MWL-4 LCC-AcOH-2 LCC-AcOH-3 LCC-AcOH-4 CEL-2 CEL-3 CEL-4 EHR-2 EHR-3 EHR-4

8.0 9.2 8.1 15.8 16.9 15.2 18.3 23.2 22.1 35.2 35.5 36.4

Total sugar content (%)

Ara

Gal

Glc

Xyl

Man

GlcA

± ± ± ± ± ± ± ± ± ± ± ±

3.2 3.0 2.1 4.7 4.9 4.5 12.0 12.5 11.3 12.1 14.5 12.0

5.3 4.9 4.1 5.1 4.1 3.0 19.0 23.7 19.0 21.2 28.2 20.1

8.6 5.2 8.1 22.3 14.0 16.2 29.5 26.5 29.2 14.1 8.0 9.6

68.7 70.8 72.7 57.7 63.8 64.8 28.2 27.2 29.2 40.4 36.5 42.1

N.D. N.D. N.D. N.D. N.D. N.D. 4.3 3.3 3.8 4.3 2.5 5.4

14.2 16.1 13.0 10.2 13.1 11.5 7.0 6.8 7.6 7.8 10.3 10.9

5.7 5.5 3.9 8.9 11.6 10.5 4.4 6.2 5.4 4.0 4.4 4.7

0.28 0.26 0.19 0.44 0.57 0.52 0.22 0.31 0.26 0.20 0.22 0.23

a

Based on Klason lignin of dewaxed wood. Ara = arabinose, Gal = galacose, Glc = glucose, Xyl = xylose, Man = mannose, GlcA = glucuronic acid, N.D. = not detected.

1 mL/min. The polystyrene was used as the standard for the molecular weight of lignin. The 2D HSQC NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer at 25 °C. About 40 mg of sample was dissolved in 0.5 mL of DMSO-d6 (99.8% D). The spectral widths for HSQC were 5000 and 20000 Hz for the 1H- and 13C-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 always recorded in the 13Cdimension. The 1JC−H used was 146 Hz. Prior to Fourier transformation, the data matrixes were zero filled up to 1024 points in the 13 C-dimension. Data processing was performed using standard Bruker Topspin-NMR software.22 The standard parameters of 31P NMR experiment were listed as follows: pulse angle 30°, relaxation delay (d1) 2 s, data points 64 K, and scan 1024. Lignin (20 mg) was dissolved in 500 μL of anhydrous CDCl3/pyridine (1:1.6, v/v, liquid A). Cyclohexanol solution (100 μL, 10.85 mg/mL, in liquid A) and 100 μL of chromium(III) acetylacetonate solution (5 mg/mL, in liquid A) was added. Phosphorylating agents (100 μL, 2-chloro-4,4,5,5tetramethyl-1,3,2-dioxaphospholane, TMDP) was added into the above solution, and the mixture was kept for 10 min. The final phosphatized sample was transferred into a 5 mm NMR tube for subsequent determination. FT-IR spectra of the LCC preparations were collected on a Thermo Scientific Nicolet iN10 FT-IR microscope (Thermo Nicolet Corporation, Madison, WI) equipped with a liquid nitrogen-cooled MCT detector. Dried samples were ground and spread on a plant, and the spectra were recorded in the range from 4000 to 700 cm−1 at 4 cm−1 resolution and 128 scans. Before data collection, background scanning was performed for correction.

that the lignification degree increased with maturity. The ash contents in 2, 3, and 4 years old wood were 0.54, 0.44 and 0.43%, respectively, which were low and slightly decreased with growth. The potential reason for this decrease is that the deposition of inorganic mineral decreased with the increase of age. Yield and carbohydrate composition in lignin and LCC preparations. The yield and composition of the lignin and LCC preparations are shown in Table 2. As can be seen, the yields of MWL-2, MWL-3, and MWL-4 were 8.0, 9.2, and 8.1%, respectively, based on the weights of Klason lignin, which were lower than those of the LCC-AcOH fractions (15.2− 16.9%). In addition, the contents of the total sugar in the MWL fractions were also lower than those in the LCC-AcOH fractions, which were consistent with that from the 2D NMR spectra. The MWL and LCC-AcOH fractions contained a large percentage of xylose among the total sugars and uronic acids. In other words, xylose was the predominant sugar composition among the five kinds of sugars and uronic acids. These results suggested that xylans in the plant wall were the predominant hemicelluloses which crossly linked with lignin, according to the previous study.23 Other sugars, such as arabinose, galactose, and glucose, were also observed in noticeable amounts. It should be noted that both the MWL and LCC-AcOH fractions had a low glucose content, while the glucose contents in the LCC-AcOH fractions were higher than those in the MWL fractions. The CEL fractions were isolated from the residual ball-milled wood (residue-1) and were extracted with 80% aqueous dioxane. The yields of CEL-2, CEL-3, and CEL-4 were 18.0, 23.2, and 22.1%, respectively, obviously higher than those of the MWL and LCC-AcOH fractions in the present study. The yield of the CEL fractions increased first and then decreased with the growth of Eucalypt. Some carbohydrates remained in these fractions, such as arabinose, galactose, glucose, xylose, mannose, and glucuronic acid. Xylose and glucose were the major sugars in the CEL fractions. The high glucose content in

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RESULTS AND DISCUSSION Chemical composition. Table 1 displays the chemical composition of the dewaxed Eucalypt with different growth years. As shown, the Klason lignin content increased from 27.6% in 2 years old wood to 29.2% in a 4 years old sample, while the acid-soluble lignin content exhibited no obvious variation. The contents of cellulose and hemicelluloses presented an increased tendency with the growth of Eucalypt. Interestingly, the contents of Klason lignin, cellulose, and hemicelluloses increased with maturity. This result indicated 1115


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ACS Sustainable Chemistry & Engineering the CEL fractions was likely originated from the remaining glucan after enzymatic hydrolysis, although a small part of the residual lignin was linked to cellulose via a covalent bond in a previous study.24 It has been reported that xylans are the predominant hemicellulosic components in the cell walls of hardwood, and most hemicelluloses are located in the secondary wall.25 Meanwhile, CEL was isolated from the secondary wall of the plant cell walls.26 This was the reason for the high contents of xylose and galactose in the CEL fractions. Because the elimination of lignin in the plant cell walls could improve the accessibility of cellulase to cellulose,27,28 the residue after extraction of the CEL was treated with cellulose again to isolate EHR. The yields of EHR-2, EHR-3, and EHR-4 were 35.2, 35.5, and 36.4%, respectively. The sugar content in the EHR fractions was lower than that in the CEL fractions. This was because further enzymatic treatment removed a part of the remaining carbohydrates in the residue. As shown in Table 2, the yields of the MWL, LCC-AcOH, and CEL fractions were all increased first and then decreased with the eucalyptus growth, while the yield of the EHR fractions increased with the maturation. The carbohydrate contents in the EHR fractions were lower than those of other LCC fractions, since further enzymatic hydrolysis removed the majority of carbohydrates in the residue after CEL extraction. FT-IR analysis. The FT-IR spectra of the MWL, LCCAcOH, CEL, and EHR fractions from Eucalypt are shown in Figure S1, and the bands were assigned according to the previous literature.29−31 Apparently, all the lignin and LCC preparations showed a similar band at 1726 cm−1, which is assigned to the ester bonds in hemicelluloses (mainly acetylated xylans). In addition, the band at 1665 cm−1 is probably due to the conjugated carbonyl groups, and the content is higher in MWL, CEL, and EHR samples than that in LCC-AcOH, suggesting that the ball-milling process (MWL) and enzymatic hydrolysis (CEL and EHR) induce the formation of conjugated carbonyl groups in lignin samples rather than LCC samples. Moreover, the MWL, LCC-AcOH, CEL, and EHR fractions showed stronger absorbances at 1595, 1505, and 1420 cm−1 (aromatic ring), indicating that these samples are lignin-rich fractions. Furthermore, an observable band at 1036 cm−1 (typical signal from hemicelluloses) appeared for all the samples, indicating that the samples contained some carbohydrates, as also revealed by the aforementioned carbohydrate composition in lignin and LCC preparations. Molecular weight distributions. The results of the weight-average (Mw) and number-average (Mn) molecular weights and the polydispersity (Mw/ Mn) of all the lignin and LCC preparations are shown in Table 3. It can be seen that the molecular weights of the LCC preparations ranged from 2580 to 8500 g/mol. Because the molecular weights of lignin and the LCC preparation were related to the isolation methods and the raw material, thus the molecular weights of the lignin and LCC preparations in this study were different from those from the previous literature.32 The high molecular weight was found in the CEL and EHR-2 fractions. For the sample of the same year, the molecular weight of the MWL fraction was lower than that of the CEL fraction. This may be because MWL was isolated from the middle lamella, while CEL originated from the secondary wall. Besides, the MWL, LCC-AcOH, and CEL fractions exhibited relatively narrow molecular weight distributions with Mw/Mn< 2.0. The molecular weight of the MWL fractions increased with the growth, while the molecular weight

Table 3. Weight-Average (Mw) and Number Average (Mn) and Mw/Mn of Lignin and LCC Fractions Sample MWL-2 MWL-3 MWL-4 LCC-AcOH-2 LCC-AcOH-3 LCC-AcOH-4 CEL-2 CEL-3 CEL-4 EHR-2 EHR-3 EHR-4

Mw 5510 5630 5890 2790 2660 2580 8500 8070 7920 8440 6680 6830

± ± ± ± ± ± ± ± ± ± ± ±

Mn 30 50 40 20 25 30 60 70 40 55 40 50

3480 3540 3690 2490 2370 2320 4810 4420 4360 3540 2540 2480

± ± ± ± ± ± ± ± ± ± ± ±

Mw/Mn 20 25 20 25 30 30 40 50 25 35 20 30

1.58 1.59 1.60 1.12 1.12 1.11 1.77 1.83 1.82 2.38 2.63 2.75

of the LCC-AcOH and CEL fractions, in general, decreased with the growth of Eucalypt. The different tendencies of the molecular weight are probably related to the isolation method applied in the present study. 2D HSQC NMR analysis. Two-dimensional 1H−13C NMR (2D NMR) spectroscopy provides important information about the lignin carbohydrate complex and lignin.4,8 The application of 2D NMR can provide a direct proof about the linkages of LCC. In the present study, the main substructures are shown in Figure 2 and the side-chain and aromatic regions of the 2D HSQC NMR spectra of the MWL, LCC-AcOH, CEL, and EHR fractions are shown in Figures 3 and 4, respectively. Table S1 lists the main lignin and associated carbohydrate crosssignals assigned in the HSQC spectra. In the present study, the semiquantification method was adopted according to the literature.22 The amounts of the main LCC and lignin linkages were calculated by the mean of parallel samples, and the results were expressed as how many linkages per 100 aromatic rings. The formula was listed as follows: IC9 units = 0.5IS2,6 + IG2 (hardwood lignin)

AX = IX/IC9 × 100%

where IS2,6 and IG2 are the integrations of S2,6 (including S and S′) and G2, respectively. IC9 and IX represent the integration of the aromatic ring and the objective linkages, respectively. AX represents the amount of the main LCC and lignin linkages. All the integrations were performed in the same contour level. Characterization of LCC preparations. It has been reported that PhGlc linkages can be detected in the area of δC/δH 104−99/4.8−5.2 according to model compound data.4,33 In the present study, PhGlc linkages were observed in the LCCAcOH fractions (Table 4). It is well-known that the PhGlc linkages are the important bonds between lignin and carbohydrates (xylan and glucan). According to semiquantitative results, the amounts of PhGlc moieties in LCC-AcOH-2, LCC-AcOH-3, and LCC-AcOH-4 were 3.4, 7.9, and 5.0 per 100Ar (monomeric lignin unit), respectively. Obviously, the LCC-AcOH-3 contained the highest relative amount of PhGlc linkages among the LCC-AcOH fractions with the highest yield of 16.9% (Table 2). It was about 2.3 and 1.6 times higher than that in LCC-AcOH-2 and LCC-AcOH-4, respectively. The amount of PhGlc moieties was increased first and then decreased with the maturation of Eucalypt. Meanwhile, the absence of PhGlc structures in CEL samples suggested that the subsequently extracted CEL is not cross-linked to the 1116


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Figure 2. Main lignin−carbohydrate complex (LCC) linkages and substructures of lignin: (PhGlc) phenyl glycoside; (Est) γ-ester; (BE) benzyl ether; (A) β-O-4′ linkages; (B) resionl substructures formed by β−β′,α-O-γ′, and γ-O-α′ linkages; (C) phenylcoumarance structures formed by β-5′ and α-O-4′ linkages; (D) spirodienone structures formed by β-1′ and α-O-α′ linkages; (I) p-hydroxycinnamyl alcohol end groups; (S) syringyl units; (S′) oxidized syringyl units; and (G) guaiacyl units.

in the area of δC/δH 81−80/4.5−4.7 (at C-6 of Glc, Gal, and Man, and C-5 of Ara); and (b) BE2 linkages between the αposition of lignin and secondary OH groups of carbohydrates, mainly of lignin-xylan type (at C-2 or C-3 of Xyl), giving a cross-peak at δC/δH 81−80/4.9−5.1.4 However, the signal of BE2 was markedly lower than that of BE1 in the plant cell walls,34,35 which is overlapped with the correlations of Cα-Hα in the spirodienone structure (D) at δC/δH 81.3/5.07. For the LCC fractions, the amount of BE moieties was decreased in the sequence of LCC-AcOH-2 > LCC-AcOH-3 > LCC-AcOH-4, and it was lower than the amount of PhGlc moieties. However,

carbohydrates with phenyl glycoside linkages, but covalently linked with carbohydrates via benzyl ether (BE1) linkages (see below), as revealed by the high content of glucose, galactose, and arabinose, which generally act as the glycoside for BE1. Besides, other lignin-rich LCC fractions were free of PhGlc linkages. It can be inferred that the LCC-AcOH fraction was preferable for the analysis of phenyl glycoside. According to lignin−carbohydrate model compounds, benzyl ether LCC structures can be subdivided into two types: (a) BE1 linkages between the α-position of lignin and the primary OH groups of carbohydrates, which can be observed in the signals 1117


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Figure 3. Side-chain region in 2D HSQC NMR of milled wood lignin (MWL), lignin−carbohydrate complex-rich fraction (LCC-AcOH), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residue (EHR) fractions isolated from Eucalyptus grandis × E. urophylla at different growth stages.

the amount of BE linkages in the MWL fractions decreased in the row MWL-3 > MWL-4 > MWL-2. The contents of BE linkages in CEL-2, CEL-3, and CEL-4 were 0.8, 0.4, and 1.0/ 100Ar, respectively, which decreased first and then increased with the tree age. The signal of the LCC γ-ester should be observed in the area of δC/δH 65−62/4.0−4.5.4,36 However, these signals were not easily distinguished from some overlapped signal at these regions, especially in LCC-AcOH samples. Consequently, the exact amount of γ-ester linkages was difficult to determine in

the present study. In addition, various signals from the associated carbohydrates could be found in the 2D HSQC NMR spectra of the LCC-AcOH fractions, including β-Dxylopyranoside units (X), β-D-glucopyranoside units (Glc), and 4-O-methyl-α-D-glucuronic acid units (U).20 The signals for the C5−H5 (X5), C4−H4 (X4), C3−H3 (X3), and C2−H2 (X2) from X units were found at δC/δH 62.6/3.40 and 3.72, 75.4/3.60, 73.7/3.22, and 72.5/3.02, respectively. The C2−H2 correlations from 2-O-acetyl-β-D-xylopyranoside units (X′2) and C3−H3 correlations from 3-O-acetyl-β-D-xylopyranoside units (X′3) 1118


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Figure 4. Aromatic region in 2D HSQC NMR of milled wood lignin (MWL), lignin−carbohydrate complex-rich fraction (LCC-AcOH), and cellulolytic enzyme lignin (CEL) and enzymatic hydrolysis residue (EHR) fractions isolated from Eucalyptus grandis × E. urophylla at different growth stages.

were found at δC/δH 73.2/4.49 and 74.7/4.80, respectively. These results confirmed that xylan was the major polysaccharide linked with lignin.37 Major lignin structural characterization. Except for the main LCC linkages, the structure of lignin was characterized. All the spectra showed prominent signals corresponding to β-

O-4′ substructures (A). The Cα-Hα correlations in the β-O-4′ substructures were observed at δC/δH 71.8/4.86, while the CβHβ correlations of S and G-type β-O-4′ were observed at δC/δH 86/4.1 and 84/4.3, respectively. The Cγ-Hγ correlations in the β-O-4′ substructures were observed at δC/δH 59.5−59.7/3.40− 3.63. In addition, strong signals for β−β′ substructures (B) 1119


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Table 4. Quantification of LCC and Lignin Substructures from Lignin and LCC Fractions by 2D-HSQC (%, based on 100Ar)a

a

Sample

β-O-4′

β-β′

β-1′

β-5′

Phenyl glycoside

Benzyl ether (BE1)

S/G

MWL-2 MWL-3 MWL-4 LCC-AcOH-2 LCC-AcOH-3 LCC-AcOH-4 CEL-2 CEL-3 CEL-4 EHR-2 EHR-3 EHR-4

49.5 53.0 55.2 53.5 62.3 70.1 60.0 60.1 58.3 65.9 63.3 62.1

14.5 14.1 14.7 10.0 3.8 4.4 14.9 15.9 12.7 8.9 12.2 11.5

1.2 1.9 0.8 N.D. N.D. N.D. 2.0 2.3 1.7 0.9 1.2 1.3

2.6 2.8 2.3 0.9 0.7 0.9 1.4 1.3 1.9 0.6 0.8 1.0

N.D. N.D. N.D. 3.4 7.9 5.0 N.D. N.D. N.D. N.D. N.D. N.D.

0.3 1.0 0.5 0.9 0.6 0.3 0.8 0.4 1.0 N.D. N.D. N.D.

1.99 2.55 2.43 3.01 4.47 3.86 3.38 3.55 3.36 3.82 3.71 3.67

N.D. = not detected.

were observed in all spectra, with their Cα-Hα, Cβ-Hβ, and the double Cγ-Hγ correlations at δC/δH 84.8/4.65, 53.5/3.06, and 71.0/4.18 and 3.82, respectively. The Cα-Hα and Cβ-Hβ correlations in phenylcoumaran (β-5′) substructures (C) were also found at δC/δH 86.8/5.46 and 53.3/3.46, respectively, and the Cγ-Hγ correlations overlap with other signals around δC/δH 62.5/3.73. Moreover, small signals corresponding to spirodienone (β-1′) substructures (D) were also observed in the spectra, with their Cα-Hα, Cβ-Hβ, and Cβ′-Hβ′ correlations being at δC/δH 81.2/5.07, 59.7/2.77, and 79.5/4.12, respectively. Other signals were observed in the side chain region of the 2D HSQC NMR spectra corresponding to Cγ-Hγ (δC/δH 61.4/4.0) in p-hydroxycinnamyl alcohol end groups (I). The main cross-signals in the aromatic region of the 2D HSQC NMR spectra correspond to the aromatic rings of the different lignin units. S and G lignin units could be found in the spectra of all the LCC preparations, while H lignin units could not be observed in this study. The relative abundances of the S and G lignin units and the main interunit linkages (per 100 aromatic units) are shown in Table 4. The S/G ratios of the MWL, LCC-AcOH, CEL, and EHR fractions were 1.99−2.55, 2.43−3.01, 3.38−3.55, and 3.67−3.82, respectively. The S/G ratios of the MWL, LCC-AcOH, and CEL fractions were increased first and then decreased, while those of the EHR fractions were decreased with the wood maturation. For the same year samples, the S/G ratio in the MWL fraction was lower than that in the CEL fraction. This indicated that the MWL fraction had lower β-O-4′ and higher β-5′ linkage content. It has been reported that β-O-4′ linkages in hard eucalyptus lignin are mainly formed by S units and the G lignin unit is the premise for obtaining the β-5′ linkage.22,38 In addition, the MWL fraction was mainly from the middle lamella of the plant cell walls and more G lignin units were located in this region.26 31 P NMR analysis. To investigate the changes of functional groups in the MWL and CEL fractions, the quantitative 31P NMR technique was also applied (Table 5, Figure S2). For MWL fractions, the contents of S−OH groups were lower than those of G−OH groups. This suggested that most S-type lignin units are involved in the formation of β-O-4′ linkages in these lignins and that only a small amount of S−OH could be reacted with TMDP and detected by the 31P NMR technique. For CEL samples, the contents of S-type lignin units were higher than those of G-type lignin units, except for CEL-2. The higher Stype phenolic OH in CEL-3 is related to its higher S/G ratio and β-O-4′ linkages. In addition, higher S-type phenolic OH is

Table 5. Quantification of the MWL and CEL Fractions by Quantitative 31P-NMR Analysis (mmol/g) Guaiacyl OH

a

Samples

Aliphatic OH

Syringyl OH

C

MWL-2 MWL-3 MWL-4 CEL-2 CEL-3 CEL-4

3.95 5.01 4.46 5.24 3.95 5.54

0.23 0.40 0.34 0.27 0.38 0.35

0.09 0.11 0.09 0.06 0.09 0.05

a

NC

b

0.38 0.46 0.47 0.28 0.24 0.30

Carboxylic group

Total phenolic OH

0.19 0.31 0.19 0.28 0.08 0.21

0.69 0.96 0.89 0.61 0.71 0.69

C, condensed, 5-substitued lignin. bNC, noncondensed.

always indicative of the cleavage of β-O-4′ linkages. However, the absence of phenyl glycoside (PhGlc) is the main cause for the higher S-type phenolic OH. With regard to the COOH group, it was found that the COOH group in MWL-3 is higher while that for CEL-3 is lower as compared to those of other lignin samples. The differences in the COOH groups reflected from 31P NMR are in agreement with the content of glucuronic acid (GlcA) in these lignin samples. In summary, lignin and LCC preparations were extracted from E. grandis × E. urophylla to elucidate the variations of chemical linkages (LCC linkages and lignin−lignin crossbonds) during the growth of Eucalypt. The contents of Klason lignin, cellulose, and hemicelluloses increased with the growth, while the ash content was slightly decreased with the maturation of the wood. Except for the EHR fractions, other fractions exhibited relatively narrow molecular weight distributions with Mw/Mn< 2.0. The S/G ratios of the MWL, LCCAcOH, and CEL fractions increased first and then decreased with growth, while those of the EHR fractions decreased with the eucalyptus maturation. The amount of PhGlc in the LCCAcOH fractions varied consistently with the S/G ratio. Meanwhile, the variation of the amount of BE in the MWL fractions was parallel to the S/G ratio, while that in the CEL fractions was contrary to it. The amounts of β-O-4′ in the MWL and LCC-AcOH fractions increased, and those in the CEL and EHR fractions had no obvious variation with the eucalyptus growth. In addition, the LCC-AcOH fraction was a good sample to evaluate PhGlc linkages, while the CEL fraction was a good sample to calculate BE linkages. In short, the fundamental research of LCC and lignin−lignin linkages in different ages of Eucalypt provides some evidence for the 1120


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structural variation of lignin and LCC in Eucalypt during its growth process.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02396. Table S1. Assignment of main lignin and carbohydrate 13 C−1H cross-signal in the 2D HSQC spectra of lignin and LCC fractions; Figure S1. FT-IR spectra of milled wood lignin (MWL), lignin-carbohydrate complex-rich fraction (LCC-AcOH), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residue (EHR) fractions from Eucalyptus grandis × E. urophylla; Figure S2. 31P NMR of milled wood lignin (MWL) and cellulolytic enzyme lignin (CEL) fractions from Eucalyptus grandis × E. urophylla (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-10-62336903; Fax: +86-10-62336903. E-mail address: [email protected] (T.-Q. Yuan). *Tel.: +86-10-62336903; Fax: +86-10-62336903. E-mail address: [email protected] (R.-C. Sun). ORCID

Run-Cang Sun: 0000-0003-2721-6357 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Fundamental Research Funds for the Central Universities 2015ZCQ-CL-02, the National Natural Science Foundation of China (31430092, 31400296), and Program of International S & T Cooperation of China (2015DFG31860).

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REFERENCES

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Structural Variation of Lignin and LigninâCarbohydrate Complex in

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