Structural Features of Alkaline Dioxane Lignin and Residual Lignin

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Structural features of alkaline dioxane lignin and residual lignin from Eucalyptus grandis × E. urophylla Wei-Jing Chen, Bao-Cheng Zhao, Xuefei Cao, TongQi Yuan, Quentin Shi, Shuang-Fei Wang, and Run-Cang Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05760 • Publication Date (Web): 23 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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Journal of Agricultural and Food Chemistry

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Structural features of alkaline dioxane lignin and residual lignin from Eucalyptus

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grandis × E. urophylla

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Wei-Jing Chen†, Bao-Cheng Zhao†,‡, Xue-Fei Cao†, Tong-Qi Yuan*,†, Quentin Shi, § ,

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Shuang-Fei Wang‖, and Run-Cang Sun*,†

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Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University,

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†

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No.35 Tsinghua East Road Haidian District, Beijing 100083, China

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‡

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and Technological Development Zone, Danyang City 212300, China

Power Dekor (JiangSu) Wood Research Co., Ltd. Dare Industrial Park, Economic

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§Jining

Mingsheng New Materials Co., Ltd, Xinglong Industrial Park, Jining 272000,

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China

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‖

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Daxue East Road, Nanning 530000, China

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

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

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Sun).

College of Light Industry and Food Engineering, Guangxi University, No. 100

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ACS Paragon Plus Environment


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ABSTRACT

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In the present study, lignin from eucalyptus was extracted with 80% alkaline

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dioxane (0.05 M NaOH) from ball-milled wood and subsequently fractionated by

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gradient acid precipitation from the filtrate. Meanwhile, the residual lignin was

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prepared by a double enzymatic hydrolysis process. The yield of the lignin extracted

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by alkaline dioxane (LA-2) was 29.5%. The carbohydrate contents and molecular

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weights of the gradient acid precipitated lignin fractions gradually decreased from

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4.90 to 1.36% and from 7770 to 5510 g/mol, respectively, with the decline of the pH

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value from 6 to 2. Results from 2D HSQC NMR and 31P NMR showed that an evident

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reduction of β–O–4′ linkages with the pH value decrease, while the contents of

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aliphatic –OH, phenolic –OH and carboxylic groups displayed an increasing trend.

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Moreover, the residual lignin exhibited the highest molecular weight (11690 g/mol),

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as well as the most abundant β–O–4′ linkages (71.1%) and the highest S/G ratio

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(4.68).

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Keywords: Lignin, Alkaline Dioxane, Gradient acid precipitation, Structural

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characterization, Nuclear magnetic resonance (NMR)

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Introduction

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Lignocellulosic biomass has received widespread attention due to its abundance,

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renewability and availability for being converted into value-added chemicals, energy

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and materials.1 However, cellulose, hemicelluloses and lignin are tightly linked

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together in the plant cell wall, resulting in difficulties in isolation and efficient

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utilization of lignocellulose. Therein, lignin acts as adhesive and contributes to

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intensive biomass recalcitrance. Accordingly, in order to realize the value-added

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applications of lignocellulose, a comprehensive acquisition of chemical composition

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and structural characteristics of lignin is of vital significance.2 Lignin is the most

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abundant natural aromatic polymer and mainly consists of three types of units,

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including guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units, which are

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mainly linked by aryl ether and carbon–carbon (C–C) bonds.3 Although the structure

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of lignin has been extensively investigated for more than one hundred years and the

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main structural features have been studied in many respects, it is neither absolutely

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definable nor determinable due to the complex and heterogeneous structure.4 In

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general, how to isolate lignin from plant cell wall with unaltered structure ought to be

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established prior to analyzing the structural features of lignin macromolecules in the

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plant cell wall. Björkman extracted lignin from ball milled wood with 96% aqueous

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dioxane,5 and the isolated “milled wood lignin” (MWL) is considered to be a model to

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elucidate the native lignin macromolecular structure. Subsequently, other isolation

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methods were put forward, such as cellulolytic enzyme lignin (CEL),6 and enzymatic

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mild acidolysis lignin (EMAL).7 However, although these methods contribute to

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enhancing the yield and purity of lignin to some extent, the milled wood lignin is still



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regarded as the most representative and widely used sample. In our previous works,

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mild alkaline solution was used as a pre-swelling agent before extraction of lignin.8-9

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The NaOH solution proved to be conducive to enhancing the lignin yield in the

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subsequent isolation processes, and had minimum effects on the lignin structure. It

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was deduced that the NaOH solution could loosen the compact structure of cell wall,

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transform the crystal form of cellulose, and remove a proportion of hemicelluloses.10

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Therefore, it is reasonable to speculate that the addition of alkaline solution into

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dioxane could facilitate the extraction of MWL.

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Although MWL is considered as the representative lignin, it is a mixture of variant

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lignin fractions with different molecular weights and structures. Generally, three kinds

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of methods are used for fractionating lignin mixtures, including organic solvents

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precipitation,11,12 ultrafiltration method,13,14 and acid precipitation.15,16 As compared

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with the other two methods, acid precipitation is superior in low cost and convenience.

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The lignin fractions with different molecular weights can be directly precipitated by

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changing the pH value of the acidic water. Moreover, it was found that the chemical

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structures and the contents of certain functional groups of lignin macromolecules

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varied with the change of lignin molecular weights.17 Accordingly, it is feasible to

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fractionate lignin with descending pH values to avoid co-precipitation, and obtain

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lignin fractions with different chemical structures and properties.

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A direct elucidation of the chemical structure of the lignin left in the residual wood

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meal after extraction of MWL was indispensable for elucidating the structure of the

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whole lignin in the plant cell wall.18 However, due to the poor solubility caused by


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high contents of remnant carbohydrate, the analysis of the residual lignin is restricted

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to methods such as derivatization followed by reductive cleavage (DFRC) or

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solid-state NMR spectroscopy,19,20 which provide limited information.21 A sufficient

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enzymatic hydrolysis of the solid residue is favorable for removing the carbohydrate

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and elevating the yield of lignin.22,23 Consequently, in this study, the wood meal

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residue after the extraction of milled wood lignin was further enzymatic hydrolyzed

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twice to obtain a pure residual lignin sample.

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In this work, the lignin (LA-2) extracted from Eucalyptus grandis × E. urophylla by

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alkaline dioxane solution and precipitated directly by HCl solution (pH 2) was

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compared with the MWL extracted by the classical method.24 In addition, gradient

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acid precipitation was applied to fractionate the lignin mixture to find out the

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structural changes of lignin during the extraction and separation. Moreover, the

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residual lignin obtained by adequate enzymatic hydrolysis was also characterized to

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elucidate the whole lignin structure in the cell wall.

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Materials and Methods

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Materials. Oven-dried wood of 3-year-old Eucalyptus grandis × E. urophylla,

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collected from Guangxi province, China was ground into 40-60 mesh. The powders

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were extracted with toluene/ethanol (2:1, v/v) in a Soxhlet extractor until the liquid

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was colorless. The extractive-free wood meal contained 38.3% cellulose, 16.0%

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hemicelluloses, 25.2% Klason lignin and 5.6% acid-soluble lignin, according to the



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methods given by National Renewable Energy Laboratory (NREL).25 The dewaxed

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sample was further milled in a planetary ball mill (Fritsch GmbH, Idar-Oberstein,

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Germany) equipped with a 500 mL ZrO2 bowl containing mline 133)ixed balls (10

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balls of 2 cm diameter and 25 balls of 1 cm diameter). The milling was conducted for

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5 h (a 10-min interval after every 10-min milling) at 450 rpm. The commercial

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cellulolytic enzyme used in this study was Cellic@CTec2 (100 FPU/mL), which was

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provided by Novozymes (Beijing, China). All chemicals were analytical or reagent

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grade without further purification.

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Isolation of Lignin. Lignin fractions were isolated according to the scheme in

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Figure 1. The 10 g ball-milled wood powder was dispersed in 80% alkaline dioxane

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(0.05 M NaOH) with a solid to liquid ratio of 1:20 (g/mL) at 80 °C, and the whole

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system was stirred for 6 h. The extraction procedure was repeated twice and the final

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solid residue was washed with fresh dioxane until the filtrate was clear. The combined

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supernatants were first adjusted the pH value to nearly 7 with dilute HCl solution,

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followed by concentration with rotary evaporation at reduced pressure. Then the

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concentrated supernatants were precipitated in 10 times the volume of HCl solution

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(pH 6). The precipitates were collected through filtration and freeze-dried to obtain L6

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fraction. After that, the supernatant was concentrated, and then precipitated in 10

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times the volume of acidic water (pH 4) to get the lignin fraction labeled as L4.

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Subsequently, the corresponding supernatant was concentrated with a rotary

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evaporator under reduced pressure, and then precipitated in 10 times the volume of

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acidic water (pH 2) to obtain L2 fraction. Besides, the concentrated extracted liquid


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was also directly dripped into the pH 2 acidic water to obtain LA-2 lignin fraction

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under the same extraction condition. After the extraction of LA-2, the residue was

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enzymatic hydrolyzed at 50 °C for 48 h in a medium of acetate buffer solution (pH

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4.8). The cellulase was added at 50 FPU/g substrate with 5% solid loading. The

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enzymatic hydrolysis procedure was repeated twice and the enzyme-treated residue

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was washed with buffer solution and water to obtain LR fraction.

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Milled wood lignin (MWL) was obtained from the same material (3-year-old

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Eucalyptus grandis × E. urophylla) in an earlier study.24 The MWL was extracted

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with 96% dioxane, further dissolved in 90% acetic acid, and regenerated in water. The

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characterization results of the MWL were referenced directly.

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Characterization of Lignin. The analysis of the carbohydrate moieties associated

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with the isolated lignin fractions was conducted by hydrolysis with dilute sulfuric acid

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according to the previous literature.24 The weight-average (Mw) and number-average

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(Mn) molecular weights of the acetylated lignin precipitations were determined by gel

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permeation chromatography (GPC) on a 1200 instrument (Agilent Corporations,

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Scotland, U.K.) with an ultraviolet (UV) detector. The column used was a 300

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mm×7.5 mm i.d., 10um, PL-gel 10 um 156 mixed-B, with a 50 mm × 7.5 mm i.d.

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guard column of the same material.26 The method of acetylation was the same as

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previously used.26 All the acetylated lignin samples completely dissolved in THF. The

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2D HSQC NMR and

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spectrometer (Bruker, Karlsruhe, Germany) at 25 °C. For the 2D HSQC NMR, about

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40 mg of sample was dissolved in 0.5 mL of DMSO–d6. The 31P NMR spectroscopy

31P

NMR spectra were recorded on an AVIII 400 MHz



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of the lignin samples was conducted as previously reported.8, 27 All the lignin samples

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showed good solubility before the 2D HSQC NMR and the 31P NMR analyses.

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Results and Discussion

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Alkaline Dioxane Lignin. MWL has been regarded as a structural model of native

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lignin, however, the low yield of MWL is a notable drawback. In order to improve the

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yield of lignin, 80% alkaline dioxane containing 0.05 M NaOH was applied to extract

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lignin in the present study. The yield of LA-2 was 29.5% (Table 1), which was higher

153

than that of MWL extracted from the same raw material (8.0%).24 The sugar analysis

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showed that the carbohydrate contents of LA-2 was 3.24%, lower than that of MWL

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(5.5%).24 The primary detectable monosaccharides were xylose, glucose, and

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galactose. The relative content of xylose was significantly lower in the LA-2 in

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comparison with that of MWL. This suggests that a part of hemicelluloses could be

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dissolved in the present extraction condition. Meanwhile, LCC bonds were not

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observed in the 2D HSQC spectra of the alkaline dioxane lignin (Figure 3) as

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compared with that of MWL obtained in the earlier study24, indicating that the

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alkaline dioxane treatment could cleave LCC linkages to some extent and obtain

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lignin sample with higher purity. However, some other interactions between

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carbohydrate and lignin might also contribute to the remnant carbohydrates.

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According to the previous study in which NaOH was used as a pre-swelling agent,10 it

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was found that NaOH resulted in a transformation of cellulose crystal form and was

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conducive to change the morphology of ball-milled cell wall from rigid and compact


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to a looser one. In consequence, the effects of NaOH in the extraction process of this

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study could be the destruction of the cell wall and the removal of hemicelluloses,

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which facilitated the lignin to dissolve out of the cell wall, and enhanced the yield and

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purity of the lignin sample.

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As shown in Table 2, the molecular weight of LA-2 was 6990 g/mol, which was

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higher than that of the MWL (5630 g/mol), while the polydispersity index (PDI)

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values of the LA-2 and the MWL were close.24 Moreover, from the calculation results

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of 2D HSQC NMR (Table 3), the LA-2 contained higher amounts of β–O–4′ ether

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linkages (64.6%) than that of the MWL (53.0%).24 These results indicated that the

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LA-2 contained more intact structure as compared with that of MWL. This could be

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attributed to that the addition of NaOH accelerated the dissolving of large fractions of

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lignin. The higher S/G ratio indicated that the S-type lignin units were more

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susceptible to the alkaline condition. Therefore, the extraction of lignin with alkaline

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dioxane was an ideal way to obtain a lignin sample in high yield and purity, which

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also maintained more β–O–4′ ether linkages as compared with the traditional

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Björkman MWL.

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Fractionation of Alkaline Dioxane Lignin by Gradient Acid Precipitation. It

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has been demonstrated that the changes of lignin structure were inevitable during the

185

isolation, especially the milling process.20, 28 Generally, the lignin obtained by direct

186

acid precipitation is a complex mixture which limits elucidation of the structural

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features comprehensively. In the present study, for the first time, different lignin



188

fractions were obtained by the gradient acid precipitation of the filtrate after the

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alkaline dioxane treatment. The lignin fractions (L6, L4, and L2) were expected to

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reveal the whole structure of LA-2.

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Lignin fractions precipitated successively at various pH values (pH 6, 4, and 2)

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were labeled as L6, L4, and L2, respectively. As shown in Table 1, the yield of LA-2

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approximated the sum of the yields of L6, L4, and L2, suggesting that the lignin yield

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was not affected by gradient acid precipitation. As for the carbohydrate contents of

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the three lignin fractions, there was an evident declining trend with the pH decrease.

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This result suggests that the remnant carbohydrate is more liable to be co-precipitated

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with lignin at a higher pH value, which agreed with the previous study.15

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With the decrease of the pH value, the precipitated lignin fractions exhibited

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different molecular weights. As shown in Table 2, the molecular weights of the three

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lignin fractions were 7770, 6800, and 5510 g/mol, respectively. This illustrated that

201

the lignin molecular weights gradually declined with the decrease of the pH value,

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which was consistent with the previous studies.15,29 This trend during the gradient

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precipitation corresponded to the change of the carbohydrate contents, suggesting that

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the hemicelluloses and lignin fractions with large molecular weight were prone to be

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precipitated at higher pH value, while the remnant carbohydrate and small lignin

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fractions co-precipitated at lower pH value. It is known that the lignin fractions with

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higher molecular weights usually have the larger particle size. The larger the lignin

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colloid particles in alkaline solution, the stronger the Van der Waal’s attractive forces

209

were, and thus the larger lignin particles were favored to coagulate first in the acid


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precipitation.15,16 The molecular weight of LA-2 approximated that of L4 and it was

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almost equal to the average molecular weights of L6, L4, and L2. The GPC

212

chromatograms displayed in Figure 2 also revealed the same result. Combined with

213

the GPC chromatograms and the decreasing molecular weights, it was shown that

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there barely existed lignin fractions of very low molecular weight, and the destruction

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and depolymerization of the lignin macromolecule were not very extensive during the

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ball-milling and extraction processes. The polydispersity index values (PDI) of the

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lignin fractions, ranged from 1.54–1.58, did not show significant variations, and they

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were approximate to those of the LA-2 (1.56) and MWL (1.59). This suggested that the

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alkaline dioxane extraction did not affect the polydispersity of the lignin as compared

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with that of the traditional MWL. Meanwhile, similar PDI values were observed

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among these lignin fractions obtained by gradient precipitation, indicating the

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homogeneity of the lignin.

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Since 2D HSQC NMR can provide important compositional and structural

224

information of lignin, it has been widely used to analyze the lignin.21,30,31 In the

225

present study, 2D HSQC NMR was also applied to further elucidate the lignin

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fractions. The side-chain region (δC/δH 50–90/2.5–6.0) and aromatic region (δC/δH

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100–125/5.5–8.0) of the 2D HSQC spectra are shown in Figures 3 and 4, respectively.

228

In the side-chain region, prominent signals including methoxy group, β–O–4′ (A), β–β′

229

(resinol, B), β–5′ (phenylcoumaran, C), and β–1′ (spirodienone, D) are ascribed

230

according to previous results.32 The signals at δC/δH 61.4/4.10, which are assigned to

231

the Cγ–Hγ correlations for p-hydroxycinnamyl alcohol end groups (I) were also



232

observed. Meanwhile, signals of some carbohydrate were detected in the side chain

233

regions, such as C5–H5 correlations in β-D-xylopyranoside (X5), which were found at

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δC/δH 62.3/3.40 and 3.72. However, the signals of Cα–Hα correlations in the

235

substructure D were not detected at the same contour levels in the side-chain region of

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L6 fraction, but found in the L2 and L4 fractions. The signals of the S-type and G-type

237

units were observed in the aromatic region, while the signals of H-type units were not

238

detected. This suggested that the lignin preparations of Eucalyptus grandis ×

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E.urophylla fractionated by the alkaline dioxane and gradient acid precipitation were

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typical G–S type lignins. The Cα-oxidized C2,6–C2,6 correlations of S units were

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observed at δC/δH 106.2/7.23 and 7.07, suggesting that some oxidation reactions

242

during the isolation process occurred in α-position of the lignin unit.

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Table 3 displays the semi-quantitative HSQC calculation results of the main lignin

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substructures and S/G ratios of the lignin fractions, referring to previous

245

calculations.32,33 There showed a slight decrease of the contents of the β–O–4′

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linkages from L6 to L2, and the amounts of the resinols and phenylcoumarans in the L6

247

fraction were higher than those of the L4 and L2 fractions. Associated with the results

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of the molecular weights of these lignin fractions, it was found that the L6 fraction,

249

with the highest molecular weight, also contained the most abundant aryl–ether

250

linkages and carbon–carbon (C–C) bonds. The amounts of β–O–4′, β–β′, β–5′, and

251

β–1′ linkages decreased along with the decline of the molecular weight, except a

252

slight fluctuation of the contents of β–5′ linkages. The decline of the amounts of

253

linkages in the L6, L4, and L2 together with the variations of the molecular weights of


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the three samples as stated above, it could be concluded that lignin precipitated at

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higher pH value maintained more intact structure.

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The S/G ratio is an important parameter to elucidate the chemical structure of lignin

257

macromolecule. The S/G ratios of the L6, L4, and L2 fractions were 1.95, 2.17, and

258

2.20, respectively (Table 3), showing an increasing trend with the decrease of

259

molecular weights of the lignin fractions.

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Quantitative 31P NMR is an important technique to characterize the lignin structure,

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which can be applied to investigate the functional groups of the lignin fractions. The

262

31P

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are displayed in Figure 5 and Table 4, respectively. It shows that the amounts of

264

aliphatic hydroxyl, total phenolic hydroxyl and carboxyl groups increased with the

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decline of the pH value of the acid water. This result confirmed the 2D HSQC NMR

266

that the linkages of the lignin fractions with lower molecular weight were

267

preferentially cleaved, thus exposing more dissociated hydroxyl groups. The carboxyl

268

groups, which were induced during the isolating process, were found in the L6, L4 and

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L2 lignin samples with increasing contents. The results were also founded in some

270

other related studies that lignin fractions obtained at lower pH are more

271

oxidized.15,34,38 It is reported that the phenolic hydroxyl and carboxyl groups provide

272

negative charges to the lignin colloids in the alkaline medium, and the electrostatic

273

repulsions keep the solution stable. When the pH value of the whole system becomes

274

acidic, the introduction of the H+ will impair the balance of the solution, change the

275

zeta potential of the lignin colloids, and thus result in the precipitation of lignin.15,16 In

NMR spectra and the counts of the functional groups of L6, L4, and L2 fractions



276

this study, by means of 31P NMR, the changes of the phenolic hydroxyl and carboxyl

277

groups were in accord with related researches,15,16,35 and demonstrated the mechanism

278

of the fractionation through gradient acidic precipitation. The more exposed S-type

279

phenolic OH groups made the S units more prone to be precipitated at lower pH value,

280

which resulted in the increase of S/G ratios of lignin samples with the decline of the

281

pH values. Condensations among lignin units could occur as a side reactions mainly

282

caused by ball-milling process.36 Nevertheless, in the present study, the contents of

283

the condensed hydroxyl groups were low and in line with that of classical MWL.24

284

This indicated that the obtained lignin fractions did not present much condensed

285

structure.

286

Residual Lignin. Due to only a part of the lignin being extracted by the alkaline

287

dioxane treatment, it could not represent the whole lignin structural information in the

288

plant cell wall. Therefore, the residual wood meal after extraction by alkaline dioxane

289

was subjected to enzymatic hydrolysis twice to remove as much carbohydrate as

290

possible. The obtained residual lignin (LR) was also characterized to realize a full

291

elucidation of the chemical structure of the lignin in eucalyptus.

292

The yield of LR was 74.9%, and the sum of the yields of L6, L4, L2 and LR reach

293

nearly 100%. However, the carbohydrate could not be removed thoroughly by the

294

enzymatic hydrolysis due to inhibition of the complex structure of the cell wall.

295

Hence there was still 5.92% of carbohydrate remaining in the LR fraction. As

296

compared with the lignin fractions obtained by the gradient acid precipitation, it was

297

found that the LR fraction had the highest molecular weight (11690 g/mol) (Table 2),


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which was similar to that of the cellulolytic enzyme lignin obtained in earlier work

299

(10060 g/mol).8

300

Although a proportion of the carbohydrates still remained, the LR also performed

301

good solubility in DMSO-d6. The spectra (Figures 3 and 4) and calculation results

302

(Table 3) of 2D HSQC NMR showed that the content of β–O–4′ linkages in LR was

303

as high as 71.1%, the β–β′ linkage was 7.1%, while the β–5′ and the β–1′ were not

304

detected. The S/G ratio of the LR was 4.68, indicating that the LR contained more

305

S-type units as compared with the acid precipitated lignin fractions. This could be

306

attributed to that the LR was most likely originated from the secondary wall of plant

307

cell, in which the lignin are consisted of more syringyl units.39–41 Therefore, the

308

abundant content of β–O–4′ ether linkages detected in the LR sample could be

309

explained by the fact that the β–O–4′ coupling was the most possible pathway

310

available for monolignols to couple with the syringyl units.37 The content of the

311

syrigyl hydroxyl groups detected by

312

was a lignin fraction with abundant S-type units, while the lower amounts of carboxyl

313

groups could imply milder effects caused by the isolating process.

31P

NMR (Table 4) also confirmed that the LR

314

In the present study, a combination of alkaline dioxane extraction and gradient acid

315

precipitation was applied to fractionate lignin from eucalyptus, and the residual lignin

316

obtained by adequate enzymatic hydrolysis was also characterized to achieve a

317

comprehensive elucidation of the lignin structure of eucalyptus. As compared with

318

traditional extraction method with dioxane, the results indicated that the extraction

319

with alkaline dioxane could elevate the yield of lignin, and resulted in minimal



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320

structural variations. By means of the gradient acid precipitation, different lignin

321

fractions were separated. The contents of the inter-unit linkages and the typical

322

functional groups of the lignin fractions indicated that depolymerization of lignin

323

macromolecular structure existed during the alkaline dioxane extraction. However, the

324

molecular

325

depolymerization of the lignin during the isolation processes were not dramatic. The

326

carbohydrate in the residual lignin was significantly removed by double enzymatic

327

hydrolysis so that the solution-state NMR could be applied to reveal the detailed

328

structures. The results showed that the residual lignin had the highest contents of

329

β–O–4′ linkages and S/G ratio as compared with the precipitated lignin samples.

weights

of

the

fractions

illustrated

that

the

destruction

and

330

331

Acknowledgements

332

This work was financially supported by the National Key R&D Program of China

333

(2017YFB0307903), the National Natural Science Foundation of China (31430092),

334

and the Fundamental Research Funds for the Central Universities (2015ZCQ-CL-02).


Page 17 of 33


335

References

336

1.

337

Bogel‐Łukasik, R.; Andreaus, J.; Pereira Ramos, L., Current pretreatment

338

technologies for the development of cellulosic ethanol and biorefineries.

339

ChemSusChem 2015, 8, 3366-3390.

340

2.

341

does plant cell wall nanoscale architecture correlate with enzymatic digestibility?

342

Science 2012, 338, 1055-1060.

343

3.

344

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

345

oxidative coupling of 4-hydroxyphenyl-propanoids. Phytochemistry Reviews 2004, 3,

346

29-60.

347

4.

348

lignins via definitive lignin models and NMR. Biomacromolecules 2016, 17,

349

1906-1920.

350

5.

351

Nature 1954, 174, 1057-1058.

352

6.

353

enzymes. Tappi 1957, 40, 553-558.

354

7.

355

and purity. J. Pulp Pap. Sci. 2003, 29, 235-240.

356

8.

Silveira, M. H. L.; Morais, A. R. C.; da Costa Lopes, A. M.; Olekszyszen, D. N.;

Ding, S. Y.; Liu, Y. S.; Zeng, Y.; Himmel, M. E.; Baker, J. O.; Bayer, E. A., How

Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.; Marita, J. M.;

Yue, F.; Lu, F.; Ralph, S.; Ralph, J., Identification of 4–O–5 units in softwood

Björkman, A., Isolation of lignin from finely divided wood with neutral solvents.

Pew, J. C., Properties of powdered wood and isolation of lignin by cellulytic

Wu, S.; Argyropoulos, D., An improved method for isolating lignin in high yield

Wen, J. L.; Sun, S. L.; Yuan, T. Q.; Sun, R. C., Structural elucidation of whole



357

lignin from Eucalyptus based on preswelling and enzymatic hydrolysis. Green Chem.

358

2015, 17, 1589-1596.

359

9.

360

Revealing the topochemistry and structural features of lignin during the growth of

361

Eucalyptus grandis × E. urophylla. ACS Sustainable Chem. Eng. 2018.

362

10. Chen, W. J.; Yang, S.; Zhang, Y.; Wang, Y. Y.; Yuan, T. Q.; Sun, R. C., Effect of

363

alkaline preswelling on the structure of lignins from Eucalyptus. Sci. Rep. 2017, 7.

364

11. Cui, C.; Sun, R.; Argyropoulos, D. S., Fractional precipitation of softwood Kraft

365

lignin: isolation of narrow fractions common to a variety of lignins. ACS Sustainable

366

Chem. Eng. 2014, 2, 959-968.

367

12. Li, M. F.; Sun, S. N.; Xu, F.; Sun, R. C., Sequential solvent fractionation of

368

heterogeneous bamboo organosolv lignin for value-added application. Sep. Purif.

369

Technol. 2012, 101, 18-25.

370

13. Toledano, A.; García, A.; Mondragon, I.; Labidi, J., Lignin separation and

371

fractionation by ultrafiltration. Sep. Purif. Technol. 2010, 71, 38-43.

372

14. Toledano, A.; Serrano, L.; Garcia, A.; Mondragon, I.; Labidi, J., Comparative

373

study of lignin fractionation by ultrafiltration and selective precipitation. Chem. Eng.

374

J. 2010, 157, 93-99.

375

15. Santos, P. S. B. d.; Erdocia, X.; Gatto, D. A.; Labidi, J., Characterisation of Kraft

376

lignin separated by gradient acid precipitation. Ind. Crops Prod. 2014, 55, 149-154.

377

16. Wang, G.; Chen, H., Fractionation of alkali-extracted lignin from steam-exploded

378

stalk by gradient acid precipitation. Sep. Purif. Technol. 2013, 105, 98-105.

Chen, W. J.; Zhao, B. C.; Wang, Y. Y.; Yuan, T. Q.; Wang, S. F.; Sun, R. C.,


Page 18 of 33

Page 19 of 33


379

17. Tolbert, A.; Akinosho, H.; Khunsupat, R.; Naskar, A. K.; Ragauskas, A. J.,

380

Characterization and analysis of the molecular weight of lignin for biorefining studies.

381

Biofuel Bioprod Bior 2015, 8, 836-856.

382

18. Furuno, H.; Takano, T.; Hirosawa, S.; Kamitakahara, H.; Nakatsubo, F.,

383

Chemical structure elucidation of total lignins in woods. Part II: Analysis of a fraction

384

of residual wood left after MWL isolation and solubilized in lithium

385

chloride/N,N-dimethylacetamide. Holzforschung 2006, 60, 653-658.

386

19. Holtman, K. M.; Chen, N.; Chappell, M. A.; Kadla, J. F.; Xu, L.; Mao, J.,

387

Chemical structure and heterogeneity differences of two lignins from loblolly pine as

388

investigated by advanced solid-state NMR spectroscopy. J. Agric. Food Chem. 2010,

389

58, 9882-9892.

390

20. Ikeda, T.; Holtman, K.; Kadla, J. F.; Chang, H. M.; Jameel, H., Studies on the

391

effect of ball milling on lignin structure using a modified DFRC method. J. Agric.

392

Food Chem. 2002, 50, 129-135.

393

21. Kim, H.; Ralph, J.; Akiyama, T., Solution-state 2D NMR of ball-milled plant cell

394

wall gels in DMSO-d6. BioEnerg. Res. 2008, 1, 56-66.

395

22. Chen, T. Y.; Wang, B.; Wu, Y. Y.; Wen, J. L.; Liu, C. F.; Yuan, T. Q.; Sun, R. C.,

396

Structural variations of lignin macromolecule from different growth years of Triploid

397

of Populus tomentosa Carr. Int. J. Biol. Macromol. 2017, 101, 747-757.

398

23. Jaaskelainen, A. S.; Sun, Y.; Argyropoulos, D. S.; Tamminen, T.; Hortling, B.,

399

The effect of isolation method on the chemical structure of residual lignin. Wood Sci.

400

Technol. 2003, 37, 91-102.



Page 20 of 33

401

24. Zhao, B. C.; Chen, B. Y.; Yang, S.; Yuan, T. Q.; Charlton, A.; Sun, R. C.,

402

Structural variation of lignin and lignin–carbohydrate complex in Eucalyptus

403

grandis× E. urophylla during its growth process. ACS Sustainable Chem. Eng. 2016,

404

5, 1113-1122.

405

25. Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker,

406

D., Determination of structural carbohydrates and lignin in biomass. Laboratory

407

analytical procedure 2008.

408

26. Zhao, B. C.; Xu, J. D.; Chen, B. Y.; Cao, X. F.; Yuan, T.-Q.; Wang, S. F.;

409

Charlton, A.; Sun, R. C., Selective precipitation and characterization of

410

lignin–carbohydrate complexes (LCCs) from Eucalyptus. Planta 2018, 1-11.

411

27. Granata,

412

2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane, a reagent for the accurate

413

determination of the uncondensed and condensed phenolic moieties in lignins. J.

414

Agric. Food Chem. 1995, 43, 1538-1544.

415

28. Fujimoto, A.; Matsumoto, Y.; Chang, H. M.; Meshitsuka, G., Quantitative

416

evaluation of milling effects on lignin structure during the isolation process of milled

417

wood lignin. J. Wood Sci. 2005, 51, 89-91.

418

29. Sun, R. C.; Tomkinson, J.; Bolton, J., Effects of precipitation pH on the

419

physico-chemical properties of the lignins isolated from the black liquor of oil palm

420

empty fruit bunch fibre pulping. Polym. Degrad. and Stabil. 1999, 63, 195-200.

421

30. Kim, H.; Ralph, J., Solution-state 2D NMR of ball-milled plant cell wall gels in

422

DMSO-d(6)/pyridine-d(5). Org. Biomol. Chem. 2010, 8, 576-591.

A.;

Argyopoulos,


D.

S.,

Page 21 of 33


423

31. Kim, H.; Ralph, J., A gel-state 2D-NMR method for plant cell wall profiling and

424

analysis: a model study with the amorphous cellulose and xylan from ball-milled

425

cotton linters. RSC Adv. 2014, 4, 7549-7560.

426

32. Wen, J. L.; Sun, S. L.; Xue, B. L.; Sun, R. C., Recent advances in characterization

427

of lignin polymer by solution-state nuclear magnetic resonance (NMR) methodology.

428

Materials 2013, 6, 359-391.

429

33. Zhang, L.; Gellerstedt, G., Quantitative 2D HSQC NMR determination of

430

polymer structures by selecting suitable internal standard references. Magn. Reson.

431

Chem. 2007, 45, 37-45.

432

34. García, A.; Toledano, A.; Serrano, L.; Egüés, I.; González, M.; Marín, F.; Labidi,

433

J., Characterization of lignins obtained by selective precipitation. Sep. Purif. Technol.

434

2009, 68, 193-198.

435

35. Mörck, R.; Yoshida, H.; Kringstad, K. P.; Hatakeyama, H., Fractionation of kraft

436

lignin by successive extraction with organic solvents. 1. Functional groups

437

(13)C-NMR-spectra and molecular weight distributions. Holzforschung 1986, 40.

438

36. Guerra, A.; Filpponen, I.; Lucia, L. A.; Saquing, C.; Baumberger, S.;

439

Argyropoulos, D. S., Toward a better understanding of the lignin isolation process

440

from wood. J. Agric. Food Chem. 2006, 54, 5939-5947.

441

37. Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.; Marita, J. M.;

442

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

443

oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem. Rev. 2004, 3, 29-60.

444

38. Lourençon, T. V.; Hansel, F. A.; da Silva, T. A.; Ramos, L. P.; de Muniz, G. I.;



445

Magalhães, W. L., Hardwood and softwood kraft lignins fractionation by simple

446

sequential acid precipitation. Sep. Purif. Technol. 2015,.154, 82-88.

447

39. Hu, Z.; Yeh, T. F.; Chang, H. M.; Matsumoto, Y.; Kadla, J. F., Elucidation of the

448

structure of cellulolytic enzyme lignin. Holzforschung 2006, 60, 389-397.

449

40. Zhou, C.; Li, Q.; Chiang, V. L.; Lucia, L. A.; Griffis, D. P., Chemical and spatial

450

differentiation of syringyl and guaiacyl lignins in poplar wood via time-of-flight

451

secondary ion mass spectrometry. Anal. Chem. 2011, 83, 7020-7026.

452

41. Wang, H. M.; Wang, B.; Wen, J. L.; Yuan, T. Q.; Sun, R. C., Structural

453

characteristics of lignin macromolecules from different Eucalyptus species. ACS

454

Sustainable Chem. Eng. 2017, 5, 11618-11627.


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Page 23 of 33


455

FIGURE CAPTIONS

456

Figure 1. Scheme for the isolation of lignin samples from Eucalyptus grandis × E.

457

urophylla.

458

Figure 2. Molecular weight distibutions of the LA-2, L2, L4, and L6 samples.

459

Figure 3. Side-chain region in 2D HSQC NMR spectra of the isolated lignin samples.

460

Figure 4. Aromatic region in 2D HSQC NMR spectra of the isolated lignin samples.

461

Figure 5. 31P-NMR spectra of the L2, L4, L6 and LR fractions



Page 24 of 33

TABLES

462 463

Table 1

464

Yields and Carbohydrate Contents of Lignin Preparations. Relative carbohydrate content (%)

Yielda

Carbohydrate

(%)

contentb (%)

Arac

Gal

Glc

Xyl

Uro

MWLd

9.2

5.0

3.0

4.9

5.2

70.8

16.1

LA-2

29.5

2.9

6.8

27.4

33.1

32.8

NDe

L6

8.5

4.4

2.3

10.4

45.8

35.0

6.5

L4

12.1

1.5

13.0

51.7

ND

35.3

ND

L2

10.9

1.2

21.1

32.4

ND

46.5

ND

LR

74.9

4.8

9.9

25.9

38.7

16.6

8.9

Samples

465

a Based

466

b

467

c

468

(Glucuronic acid and Galaturonic acid).

469

d The

470

e

on Klason lignin of dewaxed wood.

Carbohydrate associated with lignin. Ara: arabinose, Gal: galactose, Glc: glucose, Xyl: xylose, Uro: uronic acid,

data was cited directly from the ref.24.

ND, not detected.


Page 25 of 33


471

Table 2

472

Weight-average Molecular Weight (Mw), Number-average (Mn) Molecular Weight,

473

and Polydispersity Index Value (Mw/Mn) of the Lignin Preparations.

474

a The

Samples

Mw

Mn

Mw/Mn

MWLa

5630

3540

1.59

LA-2

6990

4480

1.56

L6

7770

5040

1.54

L4

6800

4380

1.55

L2

5510

3490

1.58

LR

11690

8490

1.38

data was cited directly from the ref.24.



Page 26 of 33

475

Table 3

476

Semi-quantitative HSQC Analysis of Lignin Substructures and Linkages. Samples

β-O-4a

β-βa

β-5a

β-1a

S/G

MWLb

53.0

14.1

1.9

2.8

2.55

LA-2

64.6

12.9

2.4

0.8

3.02

L6

61.0

17.6

3.9

NDc

1.95

L4

60.0

13.7

1.7

1.4

2.17

L2

57.0

12.9

1.9

0.2

2.20

LR

71.1

7.1

ND

ND

4.68

477

a The

478

b

The data was cited directly from the ref.24.

479

c

ND, not detected.

values were presented on the basis of per 100 aromatic units.


Page 27 of 33


480

Table 4

481

Quantitation of the L6, L4, L2 and LR Fractions by Quantitative 31P-NMR Analysis.

Samples

Aliphatic OH

Syringyl OH

Guaiacyl OH Ca

NCb

Carboxyl group

Total phenolic OH

(mmol/g) MWLc

5.01

0.40

0.11

0.46

0.31

0.96

L6

3.91

0.38

0.06

0.34

0.14

0.78

L4

6.41

0.52

0.10

0.49

0.19

1.10

L2

7.06

0.54

0.10

0.50

0.31

1.14

LR

4.72

0.58

0.12

0.30

0.10

1.00

482

a C,

483

b NC,

484

c

condensed. noncondensed.

The data was cited directly from the ref.24.



FIGURE GRAPHICS

485

Figure 1.


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Page 29 of 33

486


Figure 2.



487

Figure 3.


Page 30 of 33

Page 31 of 33

488


Figure 4.



489

Figure 5.


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490


TABLE OF CONTENTS GRAPHICS


Structural Features of Alkaline Dioxane Lignin and Residual Lignin

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