Revealing the topochemistry and structural features of lignin during

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Revealing the topochemistry and structural features of lignin during the growth of Eucalyptus grandis×E.urophylla Wei-Jing Chen, Bao-Cheng Zhao, Yun-Yan Wang, Tong-Qi Yuan, Shuang-Fei Wang, and Run-Cang Sun ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01542 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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ACS Sustainable Chemistry & Engineering

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Revealing the topochemistry and structural features of lignin during

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the growth of Eucalyptus grandis×E.urophylla

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Wei-Jing Chen†, Bao-Cheng Zhao†, Yun-Yan Wang‡, Tong-Qi Yuan†,*, Shuang-Fei Wang§,

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and Run-Cang Sun†,*

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†

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Tsinghua East Road Haidian District, Beijing 100083, China.

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‡

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

Center for Renewable Carbon, Department of Forestry, Wildlife, and Fisheries, University of

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Tennessee Institute of Agriculture, Knoxville, Tennessee 37996, United States.

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§

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Road, Nanning 530000, China.

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

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

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

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ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 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

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Abstract The heterogeneity of the topochemistry and molecular structure of lignin has impeded the

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potential application of lignocellulosic biomass. In this study, confocal Raman microscopy

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(CRM), 2D heteronuclear single-quantum coherence (2D HSQC), and phosphorous magnetic

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resonance spectroscopy (31P NMR) were used to elucidate the topochemistry and structural

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variations of Eucalyptus lignin during the growth. CRM results confirmed that the lignin

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concentration was in the following order: cell corner middle lamella (CCML) > compound

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middle lamella (CML) > S2 regions in cell walls. The growth rate of lignin concentration in

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CCML decreased from 1 to 4 years old, which was contrary to the tendency in S2 regions.

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Swollen residue enzyme lignin (SREL) was isolated as a representative sample to be used in

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the characterization of the structural features of lignin. High yield (83.9–94.2%) and low

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carbohydrate content (5.11–6.25%) were observed in SRELs. An alteration in predominant

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monosaccharide content in the lignin samples between galactose and glucose was found during

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the wood maturation. Moreover, 2D NMR showed that β-O-4′ (60.75‒64.55%) and β-β′

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(8.56‒11.16%) were the main linkages in lignin. The S/G ratio, from 3.12 to 3.67, exhibited an

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increasing trend with growth in SRELs.

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Keywords: lignin topochemistry, structural variation, SREL, confocal Raman microscopy,

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NMR

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Introduction As one of the three principal components of the cell wall, lignin plays a significant and

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complex role in providing rigidity and hydrophobicity to cell walls, as well as protecting plants

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from pathogens that cause infection.1–2 Lignin polymers generally consist of three predominant

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units: p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S).3 However, previous studies have

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found differences in the content, composition, and distribution of lignin among different plant

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species, tissues, cell types, and even wall layers in a single cell.1, 4–7 Owing to radical coupling

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in various sites during lignin polymerization, the resulting polymers constituted a complex

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structure.8–9 Apart from interunit linkages of lignin, linkages between lignin and carbohydrate

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also occur, particularly hemicelluloses, which form the lignin–carbohydrate complex (LCC).

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Generally, phenyl glycosides, esters, and benzyl ethers are regarded as the three major types of

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LCC linkages in wood. 3, 8 Therefore, the heterogeneity of the topochemistry and molecular

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structure of lignin during plant growth is key and crucial to basic research in phytochemistry.

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This quality significantly affects the potential use of plants in the papermaking and production

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of artificial board and cellulosic ethanol, among others.10–11

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To identify regularities in lignin distribution, various techniques were proposed. Traditional

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methods, such as ultraviolet microscopy, bromination or mercurization with energy dispersive

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X-ray analysis, fluorescence microscopy, transmission electron microscopy, and scanning

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electron microscopy12–13 have limitations, which could diminish the accuracy of an experiment.

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These methods provide limited information on all major chemical components and mostly lead

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to the disruption of the native-state structure as they require isolation of tissue or chemical

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agent treatment.6–7 Raman microscopy has been widely used in characterizing materials

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because of its noninvasiveness and negligible water signal interference. The technique can also

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provide information on the molecular structure of a material.14–15 The application of this 3



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nondestructive in situ approach in plant cell walls can obtain relatively intact composition and

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structural information for further research.16 Previous studies indicated that confocal Raman

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microscopy (CRM) facilitated the determination of lignin and carbohydrate distribution in the

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plant cell wall, as well as in the identification of variations in the cell wall structure after

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pretreatment. 17–18

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To determine the precise structure of lignin, a representative lignin sample is needed to be

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isolated firstly. The milled wood lignin (MWL) sample proposed by Björkman is considered an

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ideal sample with a native structure.19 However, the yield of MWL is only 25–50%, and

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inevitable destruction of lignin structure was found during the milling procedure.20 Cellulolytic

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enzyme lignin (CEL) was subsequently proposed to maintain a lignin structure as completely

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as possible and to improve the yield.21 Cellulose enzymatic hydrolysis combined with organic

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solvent preswelling was also conducted to enhance the yield ulteriorly.22–23 By isolating lignin

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via enzymolysis and mild acidolysis, Wu et al. developed an efficient procedure and obtained

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lignin sample named as enzymatic mild acidolysis lignin (EMAL).24 However, these methods

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only focus on isolating lignin fractions from the plant cell wall, producing an unsatisfactory

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yield of lignin samples. Swollen residue enzyme lignin (SREL) was proposed as an ideal lignin

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sample.25 In the SREL method, ball-milled wood powder was pretreated with mild alkali and

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subsequent in situ enzymatic hydrolysis, avoiding extraction with a neutral solvent. According

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to the study, the SREL yield reached 95%, which was apparently higher than those of previous

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lignin samples that were isolated using the aforementioned methods. In an early study

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evaluating the influence of NaOH concentration on structural features, 2% NaOH was proven

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to be effective for the preparation of SREL.26

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In determining the molecular structure of lignin, a low yield of degradation fractions was 4


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identified as a major disadvantage of conventional chemical degradation techniques, such as

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nitrobenzene oxidation, permanganate oxidation, thioacidolysis, and derivatization followed by

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reductive cleavage (DFRC).27 As an approach to in situ lignin characterization, solution-state

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NMR has recently become predominant. Two-dimensional heteronuclear single-quantum

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coherence (2D HSQC) NMR spectroscopy can discern the overlapping signals of

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carbohydrates and the side-chain region of lignin.28 Spectra are typically measured using whole

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cell wall dissolution systems with deuterated solvents.29–31 However, ball-milled cell walls

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exhibit poor solubility in the system. Although the acetylated cell wall is more easily dissolved,

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the presence of a large number of residual carbohydrates impedes signal identification.25 These

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challenges call for a more effective pretreatment method by which the structure of lignin can

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be maintained to the greatest extent, and a large amount of carbohydrates can be removed

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simultaneously.

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Eucalyptus is one of the most important and fastest growing sources of fiber.32 In China,

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wide Eucalyptus plantations provide abundant raw materials for the artificial board, pulping

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and papermaking industries to satisfy a large market demand.33–34 Despite previous efforts to

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determine structural changes during wood growth, the yield and the representativeness of

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lignin samples remained unsatisfactory, and the previous study was lack of a micro-scale

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characterization of cell walls with wood maturation.35 In the present study, Eucalyptus grandis

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× E. urophylla at different growth years (1–4 years old) was collected to reveal variations in

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the topochemistry and molecular structure of lignin during the growth. CRM was conducted to

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observe changes in lignin distribution with growth. SREL was isolated for structural

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characterization. Quantitative information was obtained by analyses, including the chemical

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composition, carbohydrate analysis, molecular weight analysis, 2D HSQC spectra, and 31P

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NMR spectra. The current study is expected to help elucidate the heterogeneity of 5



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topochemistry and molecular structure of lignin in the plant cell wall.

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Materials and Methods Materials. Samples of Eucalyptus grandis × E. urophylla aged 1–4 years old were

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harvested from Guangxi Province, China. The whole stems were debarked, and the middle part

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was sectioned. Prior to use, the raw materials were smashed to 40–60 mesh and then extracted

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with toluene–ethanol (2:1, v/v) in a Soxhlet extractor until the liquid becomes colorless. After

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drying, the dewaxed eucalypt sawdust (30 g, 40–60 mesh) was ball-milled in a planetary ball

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mill (Fritsch GmbH, Idar-Oberstein, Germany) at room temperature under 450 rpm/min for 5 h

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(10 min milling with 10 min interval). The composition of eucalypt wood was analyzed in

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accordance with the standards set by the National Renewable Energy Laboratory.36 These raw

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materials were labeled as R-1, R-2, R-3, and R-4. Commercial cellulase (Cellic® CTec2, 100

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FPU/mL) was provided by Novozymes (Beijing, China). All other chemicals used in this study

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were purchased from Beijing Chemical Works.

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Isolation of swollen residual enzyme lignin. The overall scheme for the isolation of SREL

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is presented in Figure 1. The ball-milled eucalypt wood powder (1 g) aged 1–4 years old was

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first swollen in 2% sodium hydroxide (NaOH) (1:50, g/mL) for 24 h, with stirring, at room

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temperature. After preswelling, the concentration of the whole system was reduced to 1% by

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adding distilled water, and the pH value was adjusted to 4.8 with acetic acid. The reaction

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mixtures were exposed to enzymatic hydrolysis with a loading of 75 FPU cellulase at 48 °C in

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a rotary shaker (150 rpm) for 72 h. Subsequently, the mixture was centrifuged, and the residue

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was repeatedly washed with hot acidic water (pH=2.0, 80 °C) to remove the hydrolyzed

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carbohydrate and the remnant enzyme. The SREL residue was obtained by subjecting again the

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mixture to centrifugation and freeze-drying. The four lignin samples were labeled as SREL-1, 6


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SREL-2, SREL-3, and SREL-4.

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Figure 1.

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Characterization of lignin distribution. Lignin distribution was detected by CRM. Raman

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spectra were obtained using a LabRam Xplora confocal Raman microscope (Horiba Jobin

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Yvon, Longjumeau, France) equipped with a confocal microscope (Olympus X51, Tokyo,

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Japan) and a motorized x–y stage. To detect the Raman light, an air-cooled illuminated charge

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coupled device behind the spectrograph was used. For high spatial resolution, the

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measurements were conducted using an MPlan 100× oil immersion microscope objective

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(Olympus, NA=1.40) and a linear-polarized laser (λ=532 nm). For mapping, the relevant

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parameters were set as previous studies.18 Labspec5 was used for image processing and

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spectral analysis. Spectra were obtained from cell corner middle lamella (CCML) and S2 in the

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fiber.

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Characterization of SREL. Carbohydrate analysis was conducted by high-performance

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anion exchange chromatography (Dionex ICS3000, USA) as described in a previous study.37

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Dried to a constant weight at 105 °C, 5 mg of the lignin sample was first treated with 0.125 mL

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of 72% sulfuric acid for 5 min at room temperature. Ultrapure water was added into the

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solution to dilute H2SO4 into 4%, and the reaction system was transferred into an oven set to

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105 °C for 150 min. Prior to molecular weight analysis, the four lignin samples were acetylated

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as described in a previous study.38 Weight average molecular weight (Mw) and number average

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molecular weight (Mn) were determined by gel permeation chromatography (GPC, Agilent

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1200, USA) by using an ultraviolet detector at 240 nm. GPC was conducted on a PLgel 10 mm

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Mixed-B 7.5 mm i.d. column, as described in a previous study.39 2D HSQC NMR spectra were

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obtained on a Bruker AVIII 400 MHz spectrometer (Germany) at 25 °C. Up to 20 mg of the 7



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lignin sample was dissolved in 0.5 mL DMSO-d6. Data acquisition and analysis were

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performed using the Bruker Topspin 2.1 software. Signal assignment in 2D HSQC spectra was

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based on an earlier study.31 31P NMR spectroscopy of the lignin samples was conducted as

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previously reported.25, 40–41

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Results and discussion

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Chemical Composition. The chemical composition of the eucalypt wood at different

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growth years (1–4 years old) is shown in Table 1. The Klason lignin (KL) content in the 1-

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year-old wood was 22.98% and increased to 25.08% and 25.17% in the 2-year-old wood and 3-

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year-old wood, respectively. The KL content of the 4-year-old wood decreased to 22.41%, the

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lowest in all of the wood samples. By contrast, the acid-soluble lignin (ASL) content showed

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no distinct changes in the wood samples aged 1 to 4 years old (5.53–5.74%). The cellulose

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content showed an increasing trend during the growth, although the changes were small.

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Meanwhile, the hemicelluloses exhibited no large variations in the samples. In previous

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studies, no significant and consistent differences in chemical composition were found between

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juvenile and mature wood .42–43

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Table 1.

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Confocal Raman microscopy. The Raman images and spectra of lignin distribution in the

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fiber cell wall of the middle sections of the eucalypt wood are shown in Figure 2. According to

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previous studies, the peak at 1599 cm-1 was attributed to symmetric aryl ring stretching, which

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was the most representative contribution of lignin. The other peak at 1654 cm-1 was the feature

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attributed to coniferaldehyde and coniferyl alcohol units.44 In the current study, the Raman

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images were obtained by integrating the band region of 1547–1707 cm-1.

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Figure 2. 8


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As shown in Figure 2(a), lignin concentrations can be reflected by varying intensities in

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morphologically distinct cell wall regions. For all samples, the highest concentration of lignin

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was found in the CCML, followed by that in the compound middle lamella and then in the

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secondary walls. The lignin concentrations in CCML in a single cell clearly varied, which was

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attributed to the heterogeneity of lignin distribution.17 The color changes in the Raman images

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indicate that lignin concentration distinctly increased with wood maturity. However, more

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details could be obtained from the Raman spectra, as shown in Figures 2(b) and 2(c). In

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general, both the cell corners and secondary walls showed increasing trends in lignin

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concentration during the growth, and the maximal integral of CCML was more than twice that

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of S2, as shown in R-4. However, the rate of increase in lignin concentration varied markedly

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between CCML and S2. In CCML regions, the growth rate of the 2-year-old wood sample (R-

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2) markedly increased; the growth rates of R-3 and R-4 decreased, but the concentrations were

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almost identical. By contrast, in the S2 regions, the lignin concentrations were initially nearly

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equal between R-1 and R-2, and the growth rate of concentration markedly increased in R-3

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and R-4. These findings indicated that initial lignification occurred in the cell corners and then

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spread toward the cell walls, which was consistent with earlier studies.4, 45 Tobimastu et al.

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used fluorescence-tagged monolignols and obtained similar observations in Pinus radiate in

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which the probe fluorescence spread to the compound middle lamella and then to the

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secondary walls as the cells matured.45

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Yield and carbohydrate analysis of the SRELs. In previous studies, the SREL yields were

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distinctly higher than the CEL yields.25–26 Similarly, the SREL yields (as shown in Table 2) in

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this study reached 83.9–94.2% of the total lignin of different eucalypt wood samples (1–4

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years old). Transformation from cellulose I to cellulose II caused by preswelling of aqueous 9



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NaOH was proved in an earlier study.26 During the transformation, the initial parallel chain

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crystal structures changed into antiparallel chains,46 and the hydrogen bond network underwent

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reorganization. Some changes were observed in cellulose II, including the weakening of van

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der Waals interaction and reduction in crystallinity. Moreover, the higher surface area and

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porous volume than those of cellulose I47–48 positively contributed to the high yield of

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enzymatic hydrolysis after the ball-milled powder was preswollen in NaOH. Meanwhile,

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thorough hydrolysis led to relatively low (5.11–6.25%) carbohydrate content in the prepared

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SREL samples, particularly the 1-year-old lignin sample (SREL-1).

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Table 2.

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As for the composition of monosaccharides, glucose, galactose, and xylose were identified

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as the dominating monosaccharides. The high glucose content was related to the small amount

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of stubborn unhydrolyzed cellulose. The abundant xylose could be attributed to the potential

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existence of the lignin–carbohydrate complex (LCC). The rather large quantity of galactose

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probably contributed to the reinforcement of enzymatic hydrolysis. As compared with those in

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our previous study, the dosage of enzyme in the current study was increased from 50 FPU g-1

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to 75 FPU g-1, and the time of enzymatic digestion was extended from 48 h to 72 h.25 Thus, the

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changes in enzymatic hydrolysis conditions facilitated a more thorough breakage of LCC

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linkages. Du et al. observed a considerable increase in galactose after LCC enzymatic

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treatment.49 Zhao et al. also found an increase in galactose content after the second enzymatic

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hydrolysis to isolate lignin residue.35 Moreover, as compared with the glucose contents, the

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galactose contents in SREL-1 and SREL-2 were higher. However, the opposite result was

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observed in SREL-3 and SREL-4; i.e., glucose was the predominant monosaccharide in these

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two lignin samples. In general, the glucose content gradually increased and replaced galactose

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as the primary monosaccharide in SRELs. This change in predominant monosaccharides was 10


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not observed in other types of lignin samples (MWL, CEL, EHR) in the previous study.35

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However, the result is similar to that obtained in the study by Rencoret et al. in which galactans

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occurred in significant quantities in the youngest eucalypt wood sample and decreased during

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maturation.50 According to Rencoret et al., the change in galactans may be associated with

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pectin contents in cell walls.50 Therefore, the variation in glucose and galactose contents in the

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present study could be related to carbohydrate biosynthesis during growth of eucalypt wood.

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Molecular weights of SRELs. The average molecular weights (Mw and Mn) and the

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polydispersity indices (PDI, Mw/Mn) of SREL samples calculated from GPC curves are listed

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in Table 3. Generally, no obvious distinctions and variations in molecular weights were

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reflected among the SREL samples isolated from eucalypt wood with different ages. The

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SREL fractions exhibited relatively narrow molecular weight distributions, with Mw/Mn

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ranging from 1.66 to 2.04. The high molecular weights of lignin were closely related to the

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isolation method used. As compared with those of MWL and CEL in previous studies,25,35 the

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molecular weights of SRELs in the present study were considerably higher, suggesting a more

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intact structure obtained using the isolation method proposed by Wen et al.25 However, a

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condensation of lignin may be happened during the alkaline swelling stage, which also could

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attribute to the relative high molecular weight of lignin in the present study. MWL was mainly

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isolated from the middle lamella, whereas CEL was from the secondary wall S2 region.51 After

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NaOH preswelling and enzyme digestion, SREL could be thoroughly isolated from the cell

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walls, which was considered a more representative sample of the whole lignin.

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Table 3.

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2D HSQC spectra analysis. The SREL samples isolated from different growth periods of Eucalyptus were analyzed by 2D HSQC NMR to obtain a detailed chemical structure. The 11



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signals in the spectra indicated main structural characteristics, including basic components and

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diverse substructures. The 2D HSQC spectra of the eucalypt wood are presented in Figure 3.

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The main structures are also quantified in Table 4 according to a previous study.35

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A large amount of useful information regarding the interunit linkages of lignin could be

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obtained from the side chain region of the spectra. The prominent signals clearly corresponded

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to β-O-4′ ether units (A), resinols (B), and phenylcoumarans (C), which were assigned

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according to previous studies.29–30, 52 In addition, a small quantity of spirodienones (D) was

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detected in some samples. The signal located at δC/δH 61.2/4.05, which is assigned to the Cγ–Hγ

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correlation of p-hydroxycinnamyl alcohol end groups (I), was also observed. The Cα–Hα

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correlations in the β-O-4′ linkages were observed at δC/δH 71.8/4.82, and the Cβ–Hβ

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correlations were detected at δC/δH 83.4/4.28 and 85.9/4.09 for the substructures consisting of

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G and S units, respectively. Cγ–Hγ correlations of β-O-4′ were found at δC/δH 59.9/3.3–3.8 but

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partly overlapped with the signal of the remnant xylan (X5). For the resinol substructures (β-

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β′), correlations of Cα–Hα, Cβ–Hβ, and Cγ–Hγ were found at δC/δH 84.9/4.64, 53.4/3.05, and

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71.2/3.80–4.17, respectively. Moreover, Cα–Hα correlations in phenylcoumaran substructures

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(β-5′) in SREL-2 and SREL-3 and spirodienone substructures (β-1′) occurred in small

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quantities in SREL-1, SREL-3, and SREL-4.

281

Figure 3.

282

Figure 4.

283 284

In addition to the substructure linkages of lignin, signals of polysaccharides were also

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observed in this area. Signals of C5–H5 correlation in β-D-xylopyranoside (X5) were found at

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δC/δH 62.5/3.38 in all SREL samples. A trace amount of C3-H3 correlation in β-D-

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xylopyranoside was detected in SREL-1 and SREL-2. Apart from these signals, traces of 12


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benzyl ether LCC structure were found in SREL-3. Overall, signals of polysaccharides were

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relatively weak, which corresponded to the results of the carbohydrate analysis. In the aromatic

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region of the HSQC spectra, signals of S and G units could be clearly distinguished.

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Meanwhile, an H unit was also observed at δC/δH 127.7/7.2 in all SREL samples, occurring in

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small quantities, except that in SREL-2. The S units showed a prominent signal at δC/δH

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103.8/6.69, while the Cα–oxidized S′ appeared at δC/δH 106.3/7.32. The G units, including C2–

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H2, C5–H5, and C6–H6 correlations, were observed at δC/δH 110.9/6.95, 114.8/6.72–6.94, and

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118.7/6.76, respectively.

296 297

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

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100 aromatic units and as a percentage of the total side chains) calculated from the 2D HSQC

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spectra of the SREL samples are presented in Table 4 with reference to a previous study.35 The

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β-O-4′ linkage was distinctly the most predominant substructure in SRELs, with 60.75–64.55%

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content; the β-O-4′ content initially increased and then decreased slightly. The second

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abundant linkage was β-β′, with 8.56% to 11.16% content, followed by β-1′ in smaller amounts

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(0.09–0.94%). An earlier study on lignin biosynthesis indicated that β-O-4′ coupling was the

304

only essential pathway available for either monolignol to couple with an S unit to which was

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attributed the higher β-ether structure in high-S lignins.3

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Table 4.

307 308

The S/G ratio exhibited a distinct increasing tendency with wood growth among the SREL

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samples, which was consistent with a relevant study.50 This finding confirmed to the lignin

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depositing sequence explained earlier.53 During lignification in cell walls, H units are deposited

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first, followed by G units and finally, S units.54 Moreover, G units continued to be incorporated

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from the early to late stages of xylem differentiation, while the S units were deposited mainly 13



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during the middle and late stages. Owing to later lignification, fibers contained fewer G units

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but more S units.1 Studies on wood anatomical properties showed that the content of juvenile

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wood decreased with the extension of the rotation age; in addition, juvenile wood has a shorter

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fiber length and thinner cell wall, as compared with mature wood.42 H units were detected in

317

most SREL samples with low levels, except for SREL-2. The content of H units exhibited no

318

distinct regularity attributable to the premier deposition of H units that rendered itself difficult

319

to isolate. However, in comparison with CEL prepared from the same materials where H-units

320

were not detected,25 SRELs, with a high yield and a low carbohydrate content, can still be

321

assumed as a more ideal sample to characterize the whole lignin in the plant cell walls.

322 323

31

P NMR spectra analysis. To further evaluate the functional groups of the lignin samples,

324

the four SREL samples were analyzed by quantitative 31P NMR. The spectra are presented in

325

Figure 5, and the corresponding results are listed in Table 5. On the basis of the calculated

326

results, no apparent variation tendency during wood maturation was observed. Regardless, the

327

amounts of aliphatic OH in the SREL samples were lower than that in CEL isolated from

328

eucalypt in a previous study; this difference indicated lower carbohydrate contents in SREL.25

329

G-OH content was higher than S-OH content in the SREL samples, which was consistent with

330

earlier research on hardwood.55 This finding suggested that most S-type units were involved in

331

the formation of β-O-4′ linkages, and only a small amount of S-OH was detected. The

332

carboxylic group in SREL was probably attributed to the release of glucuronic acid resulting

333

from the cleavage of LCC linkages during alkaline treatment.25

334

Figure 5.

335

Table 5.

336

14


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337


With consideration of the challenges impeding the study of lignin topochemistry and

338

molecular structure, SREL, which was obtained by NaOH preswelling followed by enzymatic

339

hydrolysis, was used as a representative sample to undergo characterization, given its high

340

yield but low carbohydrate content. In addition, CRM, 2D HSQC NMR, and 31P NMR were

341

employed to synthetically analyze the lignin distribution and structural variations of Eucalyptus

342

grandis×E.urophylla during the growth.

343

Figure 6.

344 345

Spectra of CRM showed that the order of lignin concentration was as follows: CCML >

346

CML > S2 regions in the cell wall. Despite an increasing trend in lignin concentration along

347

with growth in both CCML and S2, the rate markedly decreased in CCML from 1-year-old to

348

4-year-old wood; by contrast, an opposite regularity was observed in S2 regions. The higher

349

galactose content in SREL was attributed to reinforcement of enzymatic hydrolysis. The

350

monosaccharide compositional variations in SRELs, particularly the changes in galactose and

351

glucose contents, could be correlated to pectin and carbohydrate biosynthesis during the

352

growth; however, further intensive studies have to be conducted. By NMR analysis, β-O-4′ was

353

identified as the most abundant linkage, followed by β-β′. An increasing tendency in the S/G

354

ratio during wood maturation was observed, which was related to the sequence of lignification

355

in fiber cells. Moreover, H units were detected in the majority of SREL samples, indicating a

356

more intact lignin structure. On the basis of the results of the characterization of SRELs,

357

particularly with respect to the molecular weight, 2D HSQC spectra, and 31P NMR spectra, a

358

possible panorama of the whole lignin structure in the Eucalyptus grandis × E. urophylla cell

359

wall was proposed, as shown in Figure 6.

360 361 15



362 363 364

ACKNOWLEDGMENTS This work was financially supported by the National Key R&D Program of China (2017YFB 0307903) and the National Natural Science Foundation of China (31430092 and 31400296).

365

16


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23



519

Figure Captions

520 521

Figure 1. Scheme for the isolation of swollen residue enzyme lignin (SREL) from Eucalyptus

522

grandis × E. urophylla

523 524

Figure 2. (a) Raman images (approximately 29 µm×26 µm) of lignin distribution in the cell

525

walls of eucalypt wood (1–4 years old)

526

(b) Average Raman spectra obtained from the CCML of eucalypt wood cell walls

527

(c) Average Raman spectra obtained from the S regions of eucalypt wood cell walls

528 529

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

530

eucalypt wood with different growth years (1–4 years old), as well as identified substructures

531

(A) β-O-4′ linkages; (B) resinol substructures formed by β-β′, α-O-γ′, and γ-O-α′ linkages; (C)

532

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

533

formed by β-1′ and α-O-α′ linkages; (I) p-hydroxycinnamyl alcohol end groups

534 535

Figure 4. Aromatic region in 2D HSQC NMR spectra of SREL samples isolated from eucalypt

536

wood of different growth years (1–4 years old), as well as the identified substructures. (S)

537

syringyl; (S′) oxidized syringyl; (G) guaiacyl, and (H) p-hydroxyphenyl units

538 539

Figure 5. 31P NMR spectra of different SREL samples isolated from eucalypt wood of

540

different growth years (1–4 years old)

541 542

Figure 6. Potential structural diagram (based on SREL quantitative data) of SREL in the plant

543

cell wall of eucalypt wood 24


Page 24 of 36

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544 545

Table 1. Chemical composition of eucalypt wood aged 1–4 years old

Lignin (%) Hemicelluloses Samples

546

a

547

b

Cellulose (%)

Other (%) (%)

KL

a

ASL

Total

b

R-1

36.68

18.06

22.98

5.53

28.51

16.75

R-2

37.27

18.36

25.08

5.74

30.82

13.55

R-3

38.28

15.95

25.17

5.60

30.77

15.00

R-4

40.50

16.89

22.41

5.73

28.14

13.55

Klason lignin. Acid-soluble lignin.

25



Page 26 of 36

Table 2. Yield and carbohydrate content of lignin samples isolated from eucalypt wood aged 1–4 years old

Total

Carbohydrate Content (%)

Yield a carbohydrate

Sample (%)

Rhab

Arab

Galb

Glub

Xylb

Manb

GluAb

GalAb

(%)

a

b

SREL-1

83.9

5.11

0.26

0.27

1.44

1.15

0.71

0.77

0.51

0.02

SREL-2

84.8

6.13

0.24

0.35

1.96

1.77

0.77

0.67

0.36

0.03

SREL-3

86.9

6.14

0.21

0.31

1.58

2.21

0.82

0.67

0.33

0.03

SREL-4

94.2

6.25

0.24

0.33

1.70

2.08

0.82

0.78

0.42

0.02

The yield of lignin was calculated based on the total lignin of different eucalypt wood (1–4 years). Rha=rhamnose; Ara=arabinose; Gal=galactose; Glu=glucose; Xyl=xylose; Man=mannose; GluA=glucuronic acid; GalA=galacturonic acid.

26


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Table 3. Weight average molecular weights (Mw), number average molecular weights (Mn), and polydispersity indices (Mw/Mn) of lignin fractions

Sample

Mw

Mn

Mw/Mn

SREL-1

18250

8940

2.04

SREL-2

16120

8620

1.86

SREL-3

16960

8820

1.92

SREL-4

12410

7460

1.66

27



Page 28 of 36

Table 4. Quantification of lignin fractions from eucalypt wood aged 1–4 years old by 2D HSQC NMR spectroscopy

Samples

β-O-4′

β-β′

β-1′

β-5′

S/G

H

SREL-1

60.75 a

11.16

0.09

N.D.

3.12 b

0.86

SREL-2

64.55

9.04

0.94

1.23

3.23

N.D. c

SREL-3

64.27

10.95

N.D.

2.41

3.31

1.18

SREL-4

63.04

8.56

0.31

N.D.

3.67

1.02

a

Results expressed per 100 Ar based on quantitative 2D HSQC spectra.

b

S/G ratio obtained using the equation S/G ratio = 0.5I(S2,6)/I (G2).

c

N.D., not detected.

28


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Table 5. Quantification of different OH groups in lignin fractions by quantitative 31P NMR spectrocopy (mmol/g) G-type phenolic OH

Carboxylic

Total

group

phenolic OH

0.29

0.09

0.47

0.02

0.17

0.10

0.34

0.20

0.03

0.19

0.13

0.42

0.15

0.02

0.18

0.10

0.35

Aliphatic

S-type

OH

phenolic OH

SREL-1

3.80

0.15

0.03

SREL-2

3.87

0.15

SREL-3

4.73

SREL-4

3.97

Samples

a

C, condensed.

b

NC, noncondensed.

Ca

NC b

29



Figure 1.

30


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Figure 2.

31



Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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For Table of Contents Use Only

Elucidation of the topochemical and molecular structural variations of Eucalyptus lignin will benefit for a biorefinery.

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Revealing the topochemistry and structural features of lignin during

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