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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 ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02396 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 21, 2016

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ACS Sustainable Chemistry & Engineering

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Structural variation of lignin and lignin-carbohydrate complex in Eucalyptus

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grandis×E.urophylla during its growth process

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Bao-Cheng Zhao†, Bo-Yang Chen†, Sheng Yang†, Tong-Qi Yuan†,*, Adam Charlton‡, and

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Run-Cang Sun†,*

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†

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Tsinghua East Road Haidian District, 100083, Beijing, China.

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‡

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, No.35

The BioComposites Centre, Bangor University, Deiniol Road, Bangor, Gwynedd, LL57 2UW,

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UK

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*Corresponding Author: Tel.: +86-10-62336903; Fax: +86-10-62336903.

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Email addresses: [email protected] (R.-C. Sun), [email protected] (T.-Q. Yuan).

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ACS Paragon Plus Environment


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ABSTRACT:

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Milled wood lignin (MWL), lignin-carbohydrate complex-rich fraction (LCC-AcOH),

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celluloytic enzyme lignin (CEL) and enzymatic hydrolysis residue (EHR) were sequentially

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isolated from Eucalyptus grandis×E.urophylla under mild conditions, and the variations of

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lignin-carbohydrate complex (LCC) linkages and lignin structures during the eucalyptus

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growth were investigated. The 2D HSQC NMR analysis showed that β-O-4′ and β-β′ were the

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main linkages in lignin, while other substructures were present in much lower amounts. The

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amounts of β-O-4′ in the MWL and LCC-AcOH fractions showed an increased tendency and

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those in the CEL and EHR fractions had no obvious variation with the eucalyptus growth.

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The S/G ratios of the MWL, LCC-AcOH and CEL fractions increased firstly and then

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decreased, whereas those of the EHR fractions decreased with the tree age. The amount of

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phenyl glycoside (PhGlc) in the LCC-AcOH fractions varied in consistent with the S/G ratio.

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The variation of the amount of benzyl ether (BE) in the MWL fractions was paralleled to the

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S/G ratio, while that in the CEL fractions was contrary to it. These findings will provide some

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

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KEYWORDS: Eucalyptus grandis×E.urophylla, lignin–carbohydrate complex, lignin

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structure, LCC linkages, 2D HSQC NMR

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2


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INTRODUCTION Cellulose, hemicelluloses and lignin are the major components in lignocellulosic biomass.

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Lignin, the third most abundant biopolymeron earth after cellulose and hemicelluloses, is

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composed of three units: guaiacyl (G), sinapyl (S), and p-hydroxyphenyl (H),which linked by

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aryl ether and carbon-carbon bonds.1,2 There are numerous evidences that lignin and

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carbohydrates (mainly hemicelluloses) are linked by chemical bonds, forming a special

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compound—lignin carbohydrate complex (LCC).3,4 Although less LCC exists in the plant, it

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plays a very important role and almost all wood lignin is associated with polysaccharides.5,6

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The linkage types and numbers of LCC are still not well understood, although they can cause

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technical difficulties during the processing of biomass, limiting the separation of lignin and

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carbohydrates in chemical pulping and biorefining.7,8 Therefore, in view of theory and

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practice, it is vitally important to understand the structure of native LCC in the lignocellulosic

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biomass.

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It is generally believed that there are three types of LCC linkages in the lignocellulosic

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biomass, which are phenyl glycoside (PhGlc), benzyl ether (BE) and ester.3,9 In hardwood

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and softwood, benzyl ether and phenyl glycoside linkages are the main bonds in LCC. On the

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other hand, in herbaceous, ferulate and p-coumarate link hemicelluloses (mainly arabinoxylan)

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and lignin components together forming LCC.10,11 To investigate the linkages, LCC

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preparations are usually isolated from lignocellulosic materials. LCC preparations can be

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classified into carbohydrate-rich LCC (Björkman LCC and similar ones, enzymatic LCC

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fractions), and lignin-rich LCC (cellulolytic enzyme lignin and crude milled wood lignin).

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Milled wood lignin (MWL),which was extracted from ball milled wood with 96% aqueous 3



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dioxane, was firstly proposed by Björkman,12 whereas the yield of MWL (based on Klason

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lignin) is relatively low. To improve the yield, celluloytic enzyme lignin (CEL), which was

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extracted from the enzymatically hydrolyzed ball milled wood residue, was developed by

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Pew.13 The structure of CEL is similar to MWL, and it is more representative of total lignin in

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wood than MWL.13,14

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Recently, the features of wet chemistry and NMR analysis methods for LCC structure have

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been summarized.15 Wet chemistry analysis techniques include cleavage of

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lignin-carbohydrate linkages and detection of the resulting products by Smith degradation or

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2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation, etc.15,16 However, these

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methods can not directly detect LCC structure. Interestingly, 2D NMR method performs

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direct observation of LCC structure, which can elucidate the mechanism of plant cell growth.

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Lignin structure is also very important to understand LCC preparations. It has been

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reported that LCC linkages were related to the content and structure of lignin, especially the S

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to G ratio in the LCC preparations.17 Generally, the major linkages within lignin are β-O-4′,

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β-β′, β-5′ and β-1′.1 Earlier lignin structural characterization of Eucalyptus globulus indicated

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that the content and structure of lignin were varied during the plant growth.18 Therefore, the

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structural characterization of LCC and lignin is important to elucidate the mechanism of plant

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cell growth.

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In the present study, MWL, lignin-carbohydrate complex-rich fraction (LCC-AcOH), CEL

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and enzymatic hydrolysis residue (EHR) were isolated from 2, 3 and 4 years old Eucalyptus

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grandis×E.urophylla for speculating the variation of chemical linkages during its growth.

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This work will provide some evidence for the structural variation of lignin and LCC in 4


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Eucalypt during its growth process. The chemical linkages and structural changes of lignin

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and LCC preparations were characterized by high performance anion exchange

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chromatography (HPAEC), Fourier transform infrared (FT-IR), two-dimensional

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heteronuclear single-quantum coherence (2D-HSQC) and 31P-NMR spectroscopies, as well as

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gel permeation chromatography (GPC).

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MATERIALS AND METHODS

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Materials. Eucalyptus grandis×E.urophylla wood preparations of 2, 3 and 4 years old

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were collected from Guangxi Province, China. The samples were dried in an oven at 60 °C

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and ground into small pieces. The 40-60 mesh wood powders were extracted with

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toluene/ethanol (2:1, v/v) in a Soxhlet instrument for 12 h to remove wax residues.

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Subsequently, the dewaxed samples were dried at 60 °C for 16 h and then milled by a

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planetary ball mill for 5 h (a 10 min lull after every 10 min milling) under N2.Cellulase

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(Cellic@ CTec2, 100 FPU/mL) was kindly provided by Novozymes, Beijing, China. All other

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chemicals used were purchased from Beijing Chemical Works without further purification.

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Isolation of lignin and LCC prepatations. The lignin and LCC preparations were isolated

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from ball-milled wood according to the method proposed by Balakshinet al.4 with some

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modifications. The scheme is described in Figure 1, and the detailed procedures are as

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follows. The ball-milled wood sample was extracted by 96% aqueous dioxane (v/v) with a

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solid to liquid ratio of 1:10 (g/mL) at room temperature in the dark for 24 h under stirring.

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The extraction procedure was repeated twice and all supernatants were concentrated under

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reduced pressure to obtain crude MWL. The crude MWL was dissolved in 90% acetic acid 5



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(v/v, 20mL/g crude MWL) and precipitated drop by drop into water. The precipitate was

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MWL (marked as MWL-2, MWL-3 and MWL-4, based on the age of the tree, respectively).

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Subsequently, the supernatant was collected, concentrated under reduced pressure, and freeze

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dried to obtain LCC-AcOH. The LCC-AcOH fractions isolated from 2, 3 and 4 years old

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wood were labeled as LCC-AcOH-2, LCC-AcOH-3 and LCC-AcOH-4, respectively. After

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the extraction of MWL and LCC-AcOH, the residue was suspended in an acetate buffer

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solution (pH 4.8). Cellulase was added to the suspension, which was then incubated at 50 °C

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for 48 h. Cellulase was added at 30 FPU/g substrate with 5% solid loading. After enzymatic

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hydrolysis, the solid was separated by centrifugation and washed with buffer solution and

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deionized water. Then the insoluble residue was extracted twice (24 h each time) with 80%

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aqueous dioxane (v/v) with a solid to liquid ratio of 1:20 (g/mL). All supernatants were

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collected and concentrated by the same procedure as before, regenerated in acidic water

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(pH=2), and freeze dried to obtain CEL fractions (labeled as CEL-2, CEL-3 and CEL-4,

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respectively). The residue after CEL isolation (residue-4) was further treated with a

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celluloytic enzyme in an acetate buffer solution (pH 4.8) at 50 °C for 48 h. Cellulase was

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added at 50 FPU/g substrate with 2% solid loading. After enzymatic hydrolysis, the solution

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was centrifuged and the hydrolyzed solid was washed with buffer solution and deionized

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water, then freeze dried to obtain EHR fractions (marked as EHR-2, EHR-3 and EHR-4,

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respectively).

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Analytical methods. The main chemical components (cellulose, hemicelluloses and lignin)

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of 2, 3 and 4 years old Eucalypt were measured according to the NREL method.19 The

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analysis of the carbohydrate moieties associated with the MWL, LCC-AcOH, CEL and EHR 6


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fractions were conducted by hydrolysis with dilute sulfuric acid according to a previous

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literature.20 The weight-average (Mw) and number-average (Mn) molecular weights of all the

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acetylated lignin and LCC preparations were detected by gel permeation chromatography

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(GPC) with a UV detector on a PL-gel 10 µm Mixed-B 7.5 mm i.d. column.20,21 A 4 mg

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sample was dissolved in 2 mL tetrahydrofuran (THF), and then a 10 µL solution was injected.

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The column was operated at ambient temperature and eluted with THF at a flow rate of 1

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mL/min. The polystyrene was used as the standard for the molecular weight of lignin. The 2D

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HSQC NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer at 25°C. About

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40 mg of sample was dissolved in 0.5 mL of DMSO-d6 (99.8% D). The spectral widths for

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HSQC were 5000 Hz and 20000 Hz for 1H- and 13C-dimension, respectively. The number of

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collected complex points was 1024 for 1H-dimension with a recycle delay of 5s. The number

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of transients was 128, and 256 time increments were always recorded in the 13C-dimension.

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The 1JC-H used was 146 Hz. Prior to Fourier transformation, the data matrixes were zero filled

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up to 1024 points in the 13C-dimension. Data processing was performed using standard

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Bruker Topspin-NMR software.22 The standard parameters of 31P-NMR experiment was

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listed as follows: pulse angle 30°, relaxation delay (d1) 2 s, data points 64 K, and scan 1024.

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Lignin (20 mg) was dissolved in 500 µL anhydrous CDCl3/pyridine (1:1.6, v/v, liquid A). 100

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µL cyclohexanol solution (10.85 mg/mL, in liquid A) and 100 µl chromium (III)

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acetylacetonate solution (5 mg/mL, in liquid A) were added. 100 µL phosphorylating agents

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(2-chloro-4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaphospholane, TMDP) was added into the above

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solution and the mixture was kept for 10 min. The final phosphatized sample was transferred

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into a 5 mm NMR tube for subsequent determination. 7



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FT-IR spectra of the LCC preparations were collected on a Thermo Scientific Nicolet iN10

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FT-IR Microscope (Thermo Nicolet Corporation, Madison, WI) equipped with a liquid

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nitrogen-cooled MCT detector. Dried samples were ground and spread on a plant and the

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spectra were recorded in the range from 4000 to 700 cm-1 at 4 cm-1 resolution and 128 scans.

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Before data collection, background scanning was performed for correction.

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RESULTS AND DISCUSSION Chemical composition. Table 1 displays the chemical composition of the dewaxed

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Eucalypt with different growth years. As shown, the Klason lignin content increased from

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27.6% in 2 years old wood to 29.2% in 4 years old sample, while acid-soluble lignin content

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exhibited no obvious variation. The contents of cellulose and hemicelluloses presented an

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increased tendency with the growth of Eucalypt. Interestingly, the contents of Klason lignin,

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cellulose, and hemicelluloses increased with maturity. This result indicated that lignification

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degree increased with maturity. The ash content in 2, 3 and 4 years old wood was 0.54, 0.44

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and 0.43%, respectively, which was low and slightly decreased with growth. The potential

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reason for this decrease is that the deposition of inorganic mineral decreased with the increase

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of age.

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Yield and carbohydrate composition in lignin and LCC preparations. The yield and

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composition of the lignin and LCC preparations are shown in Table 2. As can be seen, the

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yields of MWL-2, MWL-3 and MWL-4 were 8.0, 9.2 and 8.1%, respectively, based on the

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weight of Klason lignin, which were lower than those of the LCC-AcOH fractions

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(15.2-16.9%). In addition, the contents of the total sugar in the MWL fractions were also 8


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lower than those in the LCC-AcOH fractions. which were consistent with that from the 2D

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NMR spectra. The MWL and LCC-AcOH fractions contained a large percentage of xylose

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among the total sugars and uronic acids. In other words, xylose was the predominant sugar

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composition among the five kinds of sugars and uronic acids. These results suggested that

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xylans in the plant wall were the predominant hemicelluloses which crossly linked with

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lignin, according to the previous study.23 Other sugars, such as arabinose, galactose and

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glucose, were also observed in noticeable amounts. It should be noted that both the MWL and

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LCC-AcOH fractions had a low glucose content, while the glucose contents in the

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LCC-AcOH fractions were higher than those in the MWL fractions.

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The CEL fractions were isolated from the residual ball-milled wood (residue-1), which

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were extracted with 80% aqueous dioxane. The yields of CEL-2, CEL-3 and CEL-4 were

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18.0, 23.2 and 22.1%, respectively, obviously higher than those of the MWL and LCC-AcOH

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fractions in the present study. The yield of the CEL fractions increased firstly and then

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decreased with the growth of Eucalypt. Some carbohydrates remained in these fractions, such

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as arabinose, galactose, glucose, xylose, mannose and glucuronic acid. Xylose and glucose

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were the major sugars in the CEL fractions. The high glucose content in the CEL fractions

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was likely originated from the remained glucan after enzymatic hydrolysis, although a small

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part of residual lignin was linked to cellulose via covalent bond in a previous study.24

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It has been reported that xylans are the predominant hemicellulosic components in the cell

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walls of hardwood, and most of hemicelluloses are located in secondary wall.25 Meanwhile,

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CEL was isolated from secondary wall of the plant cell walls.26 This was the reason why the

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high contents of xylose and galactose in the CEL fractions. Because the elimination of lignin 9



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in the plant cell walls could improve the accessibility of cellulase to cellulose,27,28 the residue

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after extraction of the CEL was treated with cellulose again to isolate EHR. The yields of

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EHR-2, EHR-3 and EHR-4 were 35.2, 35.5 and 36.4%, respectively. The sugar content in the

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EHR fractions was lower than that in the CEL fractions. This was because further enzymatic

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treatment removed a part of remained carbohydrates in the residue.

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As shown in Table 2, the yields of the MWL, LCC-AcOH and CEL fractions were all

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increased firstly and then decreased with the eucalyptus growth. While the yield of the EHR

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fractions increased with the maturation. The carbohydrate contents in the EHR fractions were

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lower than those of other LCC fractions since further enzymatic hydrolysis removed the

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majority of carbohydrates in the residue after CEL extraction.

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FT-IR analysis. The FT-IR spectra of the MWL, LCC-AcOH, CEL and EHR fractions

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from Eucalypt are shown in Figure S1 and the bands were assigned according to the previous

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literatures.29-31 Apparently, all the lignin and LCC preparations showed a similar band at

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1726 cm-1, which is assigned to the ester bonds in hemicelluloses (mainly acetylated xylans).

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In addition, the band at 1665 cm-1 is probably due to the conjugated carbonyl groups, and the

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content is higher in MWL, CEL, and EHR samples than that in LCC-AcOH, suggesting that

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ball-milling process (MWL) and enzymatic hydrolysis (CEL and EHR) induces the formation

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of conjugated carbonyl groups in lignin samples rather than LCC samples. Moreover, the

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MWL, LCC-AcOH, CEL and EHR fractions showed stronger absorbances at 1595, 1505 and

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1420 cm-1 (aromatic ring), indicating that these samples are lignin-rich fractions. Furthermore,

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an observable band at 1036 cm-1 (typical signal from hemicelluloses) appeared at all the 10


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samples, indicating that the samples contained some carbohydrates, as also revealed by afore

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mentioned carbohydrate composition in lignin and LCC preparations.

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Molecular weight distributions. The results of the weight-average (Mw) and

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number-average (Mn) molecular weights and the polydispersity (Mw/ Mn) of all the lignin and

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LCC preparations are shown in Table 3. It can be seen that the molecular weights of the LCC

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preparations ranged from 2580 to 8500 g/mol. Because the molecular weight of lignin and

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LCC preparation was related to the isolation methods and the raw material, thus the

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molecular weights of the lignin and LCC preparations in this study were different from those

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from a previous literature.32 The high molecular weight was found in CEL and EHR-2

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fractions. For the sample of the same year, the molecular weight of the MWL fraction was

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lower than the CEL fraction. This may be because MWL was isolated from middle lamella,

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while CEL originated from the secondary wall. Besides, the MWL, LCC-AcOH and CEL

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fractions exhibited relatively narrow molecular weight distributions with Mw/Mn< 2.0. The

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molecular weight of the MWL fractions increased with the growth, while the molecular

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weight of the LCC-AcOH and CEL fractions in general decreased with the growth of

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Eucalypt. The different tendency of molecular weight is probably related to the isolation

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method applied in the present study.

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2D HSQC NMR analysis. Two-dimensional 1H-13C NMR (2D NMR) spectroscopy

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provides important information about lignin carbohydrate complex and lignin.4,8 The

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application of 2D NMR can provide a direct proof about the linkages of LCC. In the present

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study, main substructures are shown in Figure 2 and the side-chain and aromatic regions of

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the 2D HSQC NMR spectra of the MWL, LCC-AcOH, CEL and EHR fractions are shown in 11



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Figures 3 and 4, respectively. Table S1 lists the main lignin and associated carbohydrate

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cross-signals assigned in the HSQC spectra. In the present study, the semi-quantification

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method was adopted according to the literature.22 The amounts of the main LCC and lignin

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linkages were calculated by the mean of parallel samples and the results were expressed as

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how many linkages per 100 aromatic rings. The formula was listed as follows:

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IC9 units = 0.5IS2,6+IG2 (hardwood lignin)

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AX = IX/IC9 *100%

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Where IS2,6 and IG2 are the integration of S2,6 (including S and S′ ) and G2, respectively. IC9

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and IX represent the integration of the aromatic ring and the objective linkages, respectively.

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AX represents the amount of the main LCC and lignin linkages. All the integrations were

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performed in the same contour level.

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Characterization of LCC preparations. It has been reported that PhGlc linkages can be

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detected in the area of δC/δH 104-99/4.8-5.2 according to model compound data.4,33 In the

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present study, PhGlc linkages were observed in the LCC-AcOH fractions (Table 4). It is well

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known that the PhGlc linkages are the important bonds between lignin and carbohydrates

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(xylan and glucan). According to semi-quantitative results, the amounts of PhGlc moieties in

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LCC-AcOH-2, LCC-AcOH-3 and LCC-AcOH-4 were 3.4, 7.9 and 5.0 per 100Ar

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(monomeric lignin unit), respectively. Obviously, the LCC-AcOH-3 contained the highest

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relative amount of PhGlc linkages among the LCC-AcOH fractions with the highest yield of

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16.9% (Table 2). It was about 2.3 and 1.6 times higher than that in LCC-AcOH-2 and

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LCC-AcOH-4, respectively. The amount of PhGlc moieties was increased firstly and then 12


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decreased with the maturation of Eucalypt. Meanwhile, absence of PhGlc structures in CEL

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samples suggested that the subsequently extracted CEL is not cross-linked to the

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carbohydrates with phenyl glycoside linkages, but covalently linked with carbohydrates via

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benzyl ether (BE1) linkages (see below), as revealed by the high content of glucose, galactose,

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and arabinose, which generally act as the glycoside for BE1. Besides, other lignin-rich LCC

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fractions were free of PhGlc linkages. It can be inferred that the LCC-AcOH fraction was

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preferable for the analysis of phenyl glycoside.

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According to lignin-carbohydrate model compounds, benzyl ether LCC structures can be

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subdivided into two types: (a) BE1 linkages between the α-position of lignin and primary OH

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groups of carbohydrates, which can be observed the signals in the area of δC/δH 81-80/4.5-4.7

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(at C-6 of Glc, Gal and Man, and C-5 of Ara); and (b) BE2 linkages between the α-position of

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lignin and secondary OH groups of carbohydrates, mainly of lignin-xylan type (at C-2 or C-3

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of Xyl), giving a cross-peak at δC/δH 81-80/4.9-5.1.4 However, the signal of BE2 was

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markedly lower than that of BE1 in the plant cell walls,34,35 which is overlapped with the

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correlations of Cα-Hα in spirodienone structure (D) at δC/δH 81.3/5.07. For the LCC fractions,

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the amount of BE moieties was decreased in the sequence of LCC-AcOH-2 > LCC-AcOH-3 >

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LCC-AcOH-4, and it was lower than the amount of PhGlc moieties. However, the amount of

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BE linkages in the MWL fractions decreased in the row of MWL-3 > MWL-4 > MWL-2.

270

The contents of BE linkages in CEL-2, CEL-3 and CEL-4 were 0.8, 0.4 and 1.0/100Ar,

271

respectively, which decreased firstly and then increased with the tree age.

272 273

The signal of 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 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

274

regions, especially in LCC-AcOH samples. Consequently, the exact amount of γ-ester

275

linkages was difficult to determine in the present study. In additional, various signals from

276

the associated carbohydrates could be found in the 2D HSQC NMR spectra of the

277

LCC-AcOH fractions, including β-D-xylopyranoside units (X), β-D-glucopyranoside units

278

(Glc), and 4-O-methyl-α-D-glucuronic acid units (U).20 The signals for the C5-H5 (X5), C4-H4

279

(X4), C3-H3 (X3), and C2-H2 (X2) from X units were found at δC/δH 62.6/3.40 and 3.72,

280

75.4/3.60, 73.7/3.22, and 72.5/3.02, respectively. The C2-H2 correlations from

281

2-O-acetyl-β-D-xylopyranoside units (X′2) and C3-H3 correlations from

282

3-O-acetyl-β-D-xylopyranoside units (X′3) were found at δC/δH 73.2/4.49 and 74.7/4.80,

283

respectively. These results confirmed that xylan was the major polysaccharides linked with

284

lignin.37

285

Major lignin structural characterization. Except for main LCC linkages, the structure of

286

lignin was characterized. All the spectra showed prominent signals corresponding to β-O-4′

287

substructures (A). The Cα-Hα correlations in β-O-4′ substructures were observed at δC/δH

288

71.8/4.86, while the Cβ-Hβ correlations of S and G-type β-O-4′ were observed at δC/δH 86/4.1

289

and 84/4.3, respectively. The Cγ-Hγ correlations in β-O-4′ substructures were observed at

290

δC/δH 59.5-59.7/3.40-3.63. In addition, strong signals for β-β′ substructures (B) were observed

291

in all spectra, with their Cα-Hα, Cβ-Hβ, and the double Cγ-Hγ correlations at δC/δH 84.8/4.65,

292

53.5/3.06, and 71.0/4.18 and 3.82, respectively. The Cα-Hα and Cβ-Hβ correlations in

293

phenylcoumaran (β-5´) substructures (C) were also found at δC/δH 86.8/5.46 and 53.3/3.46,

294

respectively, and the Cγ-Hγ correlations overlap with other signals around δC/δH 62.5/3.73.

295

Moreover, small signals corresponding to spirodienone (β-1′) substructures (D) were also 14


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296

observed in the spectra, with their Cα-Hα, Cβ-Hβ, and Cβ´-Hβ´ correlations being at δC/δH

297

81.2/5.07, 59.7/2.77, and 79.5/4.12, respectively. Other signals were observed in the side

298

chain region of the 2D HSQC NMR spectra corresponding to Cγ-Hγ (δC/δH 61.4/4.0) in

299

p-hydroxycinnamyl alcohol end groups (I).

300

The main cross-signals in the aromatic region of the 2D HSQC NMR spectra correspond to

301

the aromatic rings of the different lignin units. S and G lignin units could be found in the

302

spectra of all the LCC preparations, while H lignin units could not be observed in this study.

303

The relative abundances of the S and G lignin units and the main interunit linkages (per 100

304

aromatic units) are shown in Table 4. The S/G ratios of the MWL, LCC-AcOH, CEL and

305

EHR fractions were 1.99-2.55, 2.43-3.01, 3.38-3.55 and 3.67-3.82, respectively. The S/G

306

ratios of the MWL, LCC-AcOH and CEL fractions were increased firstly and then decreased,

307

while those of the EHR fractions was decreased with the wood maturation. For the same year

308

samples, the S/G ratio in the MWL fraction was lower than that in the CEL fraction. This

309

indicated that the MWL fraction had lower β-O-4′ and higher β-5′ linkage content. It has been

310

reported that β-O-4′ linkages in hard eucalyptus lignin are mainly formed by S units and the

311

G lignin unit is the premise for obtaining the β-5′ linkage.22,38 In addition, the MWL fraction

312

was mainly from the middle lamella of the plant cell walls and more G lignin units were

313

located in this region.26

314

31

P-NMR analysis. To investigate the changes of functional groups in the MWL and CEL

315

fractions, quantitative 31P-NMR technique was also applied (Table 5, Figure S2). For MWL

316

fractions, the contents of S-OH groups were lower than those of G-OH groups. This

317

suggested that most of S-type lignin units involve in the formation of β-O-4′ linkages in these 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

318

lignins and only a small amount of S-OH could be reacted with TMDP and detected by

319

31

320

those of G-type lignin units, except for CEL-2. The higher S-type phenolic OH in CEL-3 is

321

related to its higher S/G ratio and β-O-4′ linkages. In addition, higher S-type phenolic OH is

322

always indicated of the cleavage of β-O-4′ linkages. However, the absence of phenyl

323

glycoside (PhGlc) is the main cause for the higher S-type phenolic OH. With regarding to the

324

COOH group, it was found that the COOH group in MWL-3 is higher while that for CEL-3 is

325

lower as compared to those of other lignin samples. The differences in the COOH groups

326

reflected from 31P-NMR are in agreement with the content of glucuronic acid (GlcA) in these

327

lignin samples.

328

P-NMR technique. For CEL samples, the contents of S-type lignin units were higher than

In summary, lignin and LCC preparations were extracted from E. grandis×E.urophylla to

329

elucidate the variations of chemical linkages (LCC linkages and lignin-lignin cross-bonds)

330

during the growth of Eucalypt. The contents of Klason lignin, cellulose and hemicelluloses

331

increased with the growth, while the ash content was slightly decreased with the maturation

332

of the wood. Except for the EHR fractions, other fractions exhibited relatively narrow

333

molecular weight distributions with Mw/Mn< 2.0. The S/G ratios of the MWL, LCC-AcOH

334

and CEL fractions increased firstly and then decreased with growth, while those of the EHR

335

fractions decreased with the eucalyptus maturation. The amount of PhGlc in the LCC-AcOH

336

fractions varied in consistent with the S/G ratio. Meanwhile, the variation of the amount of

337

BE in the MWL fractions was paralleled to the S/G ratio, while that in the CEL fractions was

338

contrary to it. The amounts of β-O-4′ in the MWL and LCC-AcOH fractions increased, and

339

those in the CEL and EHR fractions had no obvious variation with the eucalyptus growth. In 16


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340

addition, the LCC-AcOH fraction was a good sample to evaluate PhGlc linkages, while the

341

CEL fraction was a well sample to calculate BE linkages. In short, the fundamental research

342

of LCC and lignin-lignin linkages in different ages of Eucalypt provides some evidence for

343

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

344 345

ACKNOWLEDGMENTS This work was financially supported by Fundamental Research Funds for the Central

346 347

Universities 2015ZCQ-CL-02, the National Natural Science Foundation of China (31430092,

348

31400296), and Program of International S & T Cooperation of China (2015DFG31860).

349 350

SUPPORTING INFORMATION Supporting Information Available: Table S1. Assignment of main lignin and carbohydrate

351 352

13

C-1H cross-signal in the 2D HSQC spectra of lignin and LCC fractions; Figure S1. FT-IR

353

spectra of milled wood lignin (MWL), lignin-carbohydrate complex-rich fraction

354

(LCC-AcOH), cellulolytic enzyme lignin (CEL) and enzymatic hydrolysis residue (EHR)

355

fractions from Eucalyptus grandis × E.urophylla; Figure S2. 31P-NMR of milled wood lignin

356

(MWL) and cellulolytic enzyme lignin (CEL) fractions from Eucalyptus grandis ×

357

E.urophylla.

358

17



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Hatfield, R. D.; Ralph, S. A.; Christensen, J. H. Lignins: natural polymers from oxidative

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coupling of 4-hydroxyphenyl-propanoids. Phytochem. Rev. 2004, 3 (1-2), 29-60.

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characterization and valorization. J. Chem. Technol. Biot. 2013, 88 (3), 346-352.

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ultrastructure and reactions,. De Gruyter, W. Berlin, Eds.; Walter de Gruyter: Berlin, New

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lignin-carbohydrate linkages with high-resolution NMR spectroscopy. Planta 2011, 233 (6),

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complexes (LCCs) from lignocellulosic biomass: an example using spruce wood. Plant J.

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lignin-carbohydrate complexes present in wood and in chemical pulps. Biomacromolecules

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lignin-lignin linkages after hydrolase treatment of xylan-lignin, glucomannan-lignin and

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glucan-lignin complexes from spruce wood. Planta 2014, 239 (5), 1079-1090.

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(9) Koshijima, T.; Watanabe, T. E. Association between lignin and carbohydrates in wood and other plant tissues. Springer-verlag: Berlin Heidelberg, New York., 2013. (10) Zeng, J.; Helms, G. L.; Gao, X.; Chen, S. Quantification of wheat straw lignin

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(11) Cornu, A.; Besle, J.; Mosoni, P.; Grenet, E. Lignin-carbohydrate complexes in

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forages: structure and consequences in the ruminal degradation of cell-wall carbohydrates.

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Reprod. Nutr. Dev. 1994, 34 (5), 385-398.

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(12) Björkman, A., Isolation of lignin from finely divided wood with neutral solvents. Nature 1954, 174, 1057-1058. (13) Pew, J. C. Properties of powdered wood and isolation of lignin by cellulytic enzymes. Tappi. 1957, 40 (7), 553-558.

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(14) Chang, H.M.; Cowling, E. B.; Brown, W. Comparative studies on cellulolytic

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enzyme lignin and milled wood lignin of sweetgum and spruce. Holzforschung 1975, 29 (5),

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Lignin–Carbohydrate Complexes Preparations with Traditional and Advanced Methods.

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application to pine wood. J. Wood Sci. 2004, 50 (2), 141-150.

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(17) Min, D.Y.; Yang, C.; Chiang, V.; Jameel, H.; Chang, H.M. The influence of

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lignin–carbohydrate complexes on the cellulase-mediated saccharification II: Transgenic

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hybrid poplars (Populus nigra L. and Populus maximowiczii A.). Fuel 2014, 116, 56-62.

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(18) Rencoret, J.; Gutierrez, A.; Nieto, L.; Jimenez-Barbero, J.; Faulds, C. B.; Kim, H.;

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Ralph, J.; Martinez, A. T.; del Rio, J. C. Lignin Composition and Structure in Young versus

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Adult Eucalyptus globulus Plants. Plant Physiol. 2011, 155 (2), 667-682.

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(19) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D.

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Determination of structural carbohydrates and lignin in biomass. Laboratory analytical

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procedure, 2008, 1617.

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(20) Yuan, T. Q.; Sun, S. N.; Xu, F.; Sun, R. C. Characterization of lignin structures and

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lignin-carbohydrate complex (LCC) linkages by quantitative 13C and 2D HSQC NMR

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spectroscopy. J. Agric. Food Chem. 2011, 59 (19), 10604-10614.

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(21) Wen, J.L.; Sun, S.L.; Xue, B.L.; Sun, R.C. Quantitative structural characterization

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of the lignins from the stem and pith of bamboo (Phyllostachys pubescens). Holzforschung

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2013, 67 (6), 613-627.

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(22) Wen, J.L.; Sun, S.L.; Xue, B.L.; Sun, R.C. Recent Advances in Characterization of

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Lignin Polymer by Solution-State Nuclear Magnetic Resonance (NMR) Methodology.

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Materials 2013, 6 (1), 359-391.

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(23) Murciano Martínez, P.; Punt, A. M.; Kabel, M. A.; Gruppen, H., Deconstruction of

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lignin linked p-coumarates, ferulates and xylan by NaOH enhances the enzymatic conversion

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of glucan. Bioresource Technol. 2016, 216, 44-51. 20


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(24) Lawoko, M.; Henriksson, G.; Gellerstedt, G. New method for quantitative

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preparation of lignin-carbohydrate complex from unbleached softwood kraft pulp:

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Lignin-polysaccharide networks I. Holzforschung 2003, 57 (1), 69-74.

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(25) Sun, R.; Fang, J.; Tomkinson, J.; Geng, Z.; Liu, J., Fractional isolation,

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physico-chemical characterization and homogeneous esterification of hemicelluloses from

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fast-growing poplar wood. Carbohyd Polym. 2001, 44 (1), 29-39.

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(26) You, T.T.; Zhang, L.M.; Zhou, S.K.; Xu, F., Structural elucidation of

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lignin–carbohydrate complex (LCC) preparations and lignin from Arundo donax Linn. Ind.

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Crop Prod. 2015, 71, 65-74.

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(27) Xiao, L.P.; Shi, Z.J.; Xu, F.; Sun, R.C. Characterization of Lignins Isolated with

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Alkaline Ethanol from the Hydrothermal Pretreated Tamarix ramosissima.Bioenerg. Res.

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2013, 6 (2), 519-532.

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(28) Hendriks, A.; Zeeman, G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresource technol. 2009, 100, 10-18. (29) Faix, O., Classification of lignins from different botanical origins by FT-IR spectroscopy. Holzforschung 1991, 45 (1), 21-28. (30) Faix, O., Condensation indices of lignins determined by FTIR-spectroscopy. Eur J. Wood Wood Prod. 1991, 49 (9), 356-356. (31) Faix, O.; et al. Fourier transform infrared spectroscopy. In Methods in lignin

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chemistry; Lin, S. Y., Dence, C. W. Eds. Springer-verlag: Berlin Heidelberg, New York.,

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1992.

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(32) Yang, S.; Yuan, T.Q.; Sun, R.C. Structural Elucidation of Whole Lignin in Cell 21



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Walls of Triploid of Populus tomentosa Carr. ACS Sustain. Chem. Eng. 2016, 4 (3),

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1006-1015.

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(33) Terashima, N.; Ralph, S. A.; Landucci, L. L. New facile syntheses of monolignol

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glucosides; p-glucocoumaryl alcohol, coniferin and syringin. Holzforschung 1996, 50 (2),

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151-155.

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(34) Cetinkol, Ö. P.; Dibble, D. C.; Cheng, G.; Kent, M. S.; Knierim, B.; Auer, M.;

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Wemmer, D. E.; Pelton, J. G.; Melnichenko, Y. B.; Ralph, J. Understanding the impact of

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ionic liquid pretreatment on eucalyptus. Biofuels. 2010, 1(1), 33-46.

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(35) Balakshin, M. Y.; Capanema, E. A.; Chang, H.M. MWL fraction with a high

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concentration of lignin-carbohydrate linkages: Isolation and 2D NMR spectroscopic analysis.

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Holzforschung 2007, 61(1), 1-7.

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(36) Yelle, D. J.; Ralph, J.; Frihart, C. R. Characterization of nonderivatized plant cell

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walls using high-resolution solution-state NMR spectroscopy. Magn. Reson. Chem. 2008, 46

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(6), 508-517.

461 462 463 464

(37) Kim, H.; Ralph, J. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d(6)/pyridine-d(5). Org. Biomol. Chem. 2010, 8 (3), 576-591. (38) Hu, Z.; Yeh, T.F.; Chang, H.M.; Matsumoto, Y.; Kadla, J. F.Elucidation of the structure of cellulolytic enzyme lignin. Holzforschung 2006, 60 (4), 389-397.

465 466

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FIGURECAPTIONS

467 468

Figure 1. Scheme for isolation of milled wood lignin (MWL), lignin-carbohydrate

469

complex-rich fraction (LCC-AcOH), cellulolytic enzyme lignin (CEL) and enzymatic

470

hydrolysis residue (EHR) from Eucalyptus grandis×E.urophylla.

471 472

Figure 2. Main lignin-carbohydrate complex (LCC) linkages and substructures of lignin:

473

(PhGlc) phenyl glycoside; (Est) γ-ester; (BE) benzyl ether; (A) β-O-4′ linkages; (B) resionl

474

substructures formed by β-β′,α-O-γ′and γ-O-α′ linkages; (C) phenylcoumarance structures

475

formed by β-5′ and α-O-4′ linkages; (D) spirodienone structures formed by β-1′ and α-O-α′

476

linkages; (I) p-hydroxycinnamyl alcohol end groups; (S) syringyl units; (S′) oxidized syringyl

477

units; and (G) guaiacyl units.

478 479

Figure 3. Side-chain region in 2D HSQC NMR of milled wood lignin (MWL),

480

lignin-carbohydrate complex-rich fraction (LCC-AcOH), cellulolytic enzyme lignin (CEL)

481

and enzymatic hydrolysis residue (EHR) fractions isolated from Eucalyptus

482

grandis×E.urophylla at different growth stages.

483 484

Figure 4. Aromatic region in 2D HSQC NMR of milled wood lignin (MWL),

485

lignin-carbohydrate complex-rich fraction (LCC-AcOH), cellulolytic enzyme lignin (CEL)

486

and enzymatic hydrolysis residue (EHR) fractions isolated from Eucalyptus

487

grandis×E.urophylla at different growth stages.

488

23



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Page 24 of 33

489

TABLES

490 491

Table 1. Chemical composition (%) of the extractive-free Eucalypt. Sample 2-year-old 3-year-old 4-year-old

Cellulose 38.4±1.2 41.4±1.4 44.2±1.7

Hemicelluloses 15.4±0.7 17.0±0.8 18.3±0.8

Klason lignin 27.6±0.3 27.9±0.7 29.2±0.9

Acid-solublelignin 4.4±0.1 4.9±0.2 4.5±0.2

492 493

24


Ash 0.54±0.02 0.44±0.02 0.43±0.02

Page 25 of 33

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494 495

Table 2. Yield and carbohydrate content of lignin and LCC fractions. Sample

Yielda(%)

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 (%) 5.7±0.28 5.5±0.26 3.9±0.19 8.9±0.44 11.6±0.57 10.5±0.52 4.4±0.22 6.2±0.31 5.4±0.26 4.0±0.20 4.4±0.22 4.7±0.23

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

Relative carbohydrate content (%) Gal Glc Xyl Man 5.3 8.6 68.7 N.D 4.9 5.2 70.8 N.D 4.1 8.1 72.7 N.D 5.1 22.3 57.7 N.D 4.1 14.0 63.8 N.D 3.0 16.2 64.8 N.D 19.0 29.5 28.2 4.3 23.7 26.5 27.2 3.3 19.0 29.2 29.2 3.8 21.2 14.1 40.4 4.3 28.2 8.0 36.5 2.5 20.1 9.6 42.1 5.4

496

a

497

Xyl = xylose, Man = mannose, GlcA = glucuronic acid, N.D = not detected.

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

Based on Klason lignin of dewaxed wood. Ara = arabinose, Gal = galacose, Glc = glucose,

498

25



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Page 26 of 33

499 500

Table 3. Weight-average (Mw) and number-average (Mn) and Mw/Mn of lignin and LCC

501

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±30 5630±50 5890±40 2790±20 2660±25 2580±30 8500±60 8070±70 7920±40 8440±55 6680±40 6830±50

Mn 3480±20 3540±25 3690±20 2490±25 2370±30 2320±30 4810±40 4420±50 4360±25 3540±35 2540±20 2480±30

502 503

26


Mw/Mn 1.58 1.59 1.60 1.12 1.12 1.11 1.77 1.83 1.82 2.38 2.63 2.75

Page 27 of 33

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504 505

Table 4. Quantification of LCC and lignin substructures from lignin and LCC fractions by

506

2D-HSQC (%, based on 100Ar)

507

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 8.9 122 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.

508

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509 510

Table 5 Quantification of the MWL and CEL fractions by quantitative 31P-NMR analysis

511

(mmol/g)

Guaiacyl OH Ca NCb MWL-2 3.95 0.23 0.09 0.38 MWL-3 5.01 0.40 0.11 0.46 MWL-4 4.46 0.34 0.09 0.47 CEL-2 5.24 0.27 0.06 0.28 CEL-3 3.95 0.38 0.09 0.24 CEL-4 5.54 0.35 0.05 0.30 a b 512 C, condensed. 5-substitued lignin; NC, non-condensed. Samples

Aliphatic OH

Syringyl OH

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

513

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FIGURE GRAPHICS

515 516

Figure 1

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518 519

520

521

522 523 524

Figure 2

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525

526 527 528

Figure 3

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530

531 532

Figure 4 32


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533

For Table of Contents Use Only 534

Structural variation of lignin and lignin-carbohydrate complex in Eucalyptus grandis ×

535

E.urophylla during its growth process

536

Bao-Cheng Zhao†, Bo-Yang Chen†, Sheng Yang†, Tong-Qi Yuan†,*, Adam Charlton‡, and

537

Run-Cang Sun†,*

538

539

Unveiling the structural variation of lignin and lignin-carbohydrate complex in plant cell wall 540

With the growth of Eucalypt.

33


Structural Variation of Lignin and Lignin ... - ACS Publications

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