Structural Characteristics of Lignin Macromolecules from Different


Oct 18, 2017 - Understanding of the fundamental chemistry of lignin macromolecules in lignocellulosic biomass will facilitate the further development ...
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Structural characteristics of lignin macromolecules from different Eucalyptus species Han-Min Wang, Bing Wang, Jia-Long Wen, Tong-Qi Yuan, and Run-Cang Sun ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02970 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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Structural characteristics of lignin macromolecules from different Eucalyptus species Han-Min Wang†, Bing Wang†, Jia-Long Wen†*, Tong-Qi Yuan†, and Run-Cang Sun†* †

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, No.35 Tsinghua East Road, Haidian District, 100083, Beijing, China.

Corresponding Author: Tel.: +86-10-62336903; Fax: +86-10-62336903. *Email addresses: [email protected] (R.-C. Sun); [email protected]

Abstract Understanding of the fundamental chemistry of lignin macromolecules in lignocellulosic biomass will facilitate the further development of renewable biofuel and biomaterial production. In the present study, to thoroughly delineate the detailed structural characteristics of the native lignin in the three major Eucalyptus wood, MWL, CEL, and EHL were sequentially isolated from the diverse Eucalyptus wood. Parallelly, a modified enzyme lignin (DEL) based on double ball-milling and enzymatic hydrolysis was also prepared with super-high yield (102.5-109.5%) from same Eucalyptus wood. The structural characteristics of all the lignin preparations were elaborately investigated by HPAEC, GPC, and NMR techniques (2D-HSQC and

31

P-NMR). Results showed that the

contents of different substructures (β-O-4, β-β, β-5, and β-1) and S/G ratios of lignin macromolecules varied among these Eucalyptus species. As compared to the β-O-4 content in the extracted lignins (MWLs and CELs, 44.5-60.2/100Ar), the residual lignins (EHLs and DELs) exhibited a higher content of β-O-4 linkages (59.1-64.3/100Ar). DEL preparations can be served as “lignin preparation” to develop depolymerization strategy of lignin due to the higher content of β-O-4 linkages and abundant syringyl units. Moreover, the sufficient understanding of lignin will facilitate subsequent processing of these Eucalyptus species for production of biochemicals, bioenergy, and biomaterials.

Keywords: Lignin macromolecules, Structural characteristics, NMR, Eucalyptus species 1 ACS Paragon Plus Environment

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Introduction Lignin, as the most abundant natural biopolymer in the vascular plant cell wall on earth after cellulose and hemicelluloses, is mainly composed of three units: syringyl (S) units with two methoxyl groups, guaiacyl (G) units with one methoxyl group, and p-hydroxyphenyl (H) units without methoxyl groups. The main linkages exist in these three units are aryl ether (β-O-4, α-O-4, etc.) and carbon-carbon (β-5, β-β, etc.) bonds.1, 2 In addition, lignin and carbohydrates (mainly hemicelluloses) are also linked by different chemical linkages, such as phenyl glycosides, benzyl ethers, and γ-esters, forming lignin-carbohydrate complex (LCC).3,

4

In fact, these physical and chemical structural

features of lignin are extremely important for their subsequent value-added application, such as lignin-based green chemicals and biomaterials.5 Although the primary structure of lignin has been well depicted, the detailed molecular structures of the lignin polymer from plants cell wall have not been clearly understood due to the complicated structure and property.1 Therefore, achieving more representative lignin samples from plant cell wall and thoroughly investigating their molecular structural features seem to be vitally significant in current biorefinery. In the past several decades, various efforts have been contributed to isolate lignin from all kinds of plants. Historically, lignin extracted from finely ball-milled wood by the aqueous dioxane (96%) was named as “milled wood lignin” (MWL), which was firstly developed by Björkman.6 Afterwards, enzyme was used to remove the majority of carbohydrates, prior to solvent extraction with aqueous dioxane, resulting in cellulolytic enzyme lignin (CEL).7, 8 It is generally believed that the structural features of CEL are similar to those of MWL,8, 9 whereas CEL is considered to be more representative samples for the structure analysis of native lignin due to its high yield. Next, mild preswelling pretreatment was adopted to obtain a higher yield of CEL.10, 11 In 2003, enzymatic mild acidolysis lignin (EMAL) method, which combined enzymatic hydrolysis and mild acidolysis, was developed by Wu and Argyropoulos.12 Comparing to MWL and CEL, EMAL possesses a higher yield and purity, which can better represent the lignin in the plant cell wall.13 Following the pace of the pioneer, swollen residual enzyme lignin (SREL) with a higher yield (up to 95%, based on Klason lignin) and purity was isolated based on 2 ACS Paragon Plus Environment

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mild NaOH preswollen and in-situ enzymatic hydrolysis from most of hardwood species.14 Generally, MWL and CEL are considered to be more representative native lignin as compared to residual enzyme lignin (REL) after CEL extraction, because REL is not easily dissolved in DMSO-d6, due to the existence of stubborn cellulose, which restricts the direct structural characterization of lignin macromolecules by NMR technique.15 In fact, some of the carbohydrates remaining in the REL can be hydrolyzed by appropriate pretreatment. To obtain the structural features of REL in the plant cell wall, the further enzymatic hydrolysis of REL (the “enzymatic hydrolysis of REL” also labeled as EHREL or EHL) was also applied to eliminate residual carbohydrates.16 Furthermore, it was reported that ball-milling process can facilitate the decrystallization of crystalline structure of celluloses and improve the accessibility of cellulase to celluloses.17 Thus, a modified enzyme lignin based on double ball-milling and enzymatic hydrolysis (Double enzymatic lignin, DEL) was presented in this study, which could significantly improve the yield and maintain structural integrity of the native lignin in the plant cell wall. As an important strategic tree species for the development of fast-growing and high-yielding forests, Eucalyptus has become an indispensable fast-growing hardwood species in the south of China (with an area of about 3.6 million hectares), which is considered to be the second biggest high yield raw material for pulping and related fields in China. Up to 2016, the timber production of Eucalyptus achieved more than 30 million cubic meters, which accounted for 27% of total production in China. Previously, the lignin from Eucalyptus globulus was characterized by composition and structure.18-22 In addition, the chemical structures of MWLs extracted from Eucalyptus globulus, Eucalyptus nitens, Eucalyptus maidenii, Eucalyptus grandis, and Eucalyptus dunnii were comparably investigated.23, 24 However, the conventional MWL method used in those studies was less representative for fully understanding of chemical structures of lignin macromolecules due to the low yield. In the present study, to better understand the structural characteristics of lignin macromolecules in these Eucalyptus species (Ecamaldulensis, Eucalyptus grandis and Eucalyptus urophydis) that widely grown in China, a modified MWL, CEL and EHL preparations were successively isolated from different Eucalyptus species. As a parallel experiment, DEL samples with super-high yield were also isolated from the same 3 ACS Paragon Plus Environment

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feedstock, respectively. The obtained lignin preparations were comprehensively investigated

by

high-performance

anion-exchange

chromatography

two-dimensional heteronuclear single-quantum coherence (2D-HSQC) NMR,

(HPAEC), 31

P-NMR

spectroscopy, and gel permeation chromatography (GPC). It is believed that better understanding of lignin structures in these Eucalyptus species will facilitate the development of a more efficient deconstruction strategy in the current biofinery chemistry.

Materials and Methods Materials Eucalyptus wood was collected from 4 year-old trees of Ecamaldulensis, Eucalyptus grandis and Eucalyptus urophydis, harvested from Guangxi Province, China. The raw materials were smashed into sawdust (40–60 mesh), dried in an oven at 60 oC, and then extracted with ethanol/benzene (1:2, v/v) using a soxhlet extractor for 6 h. Finally, the extractive-free Eucalyptus wood was air-dried and subjected to mill in a planetary ball mill (Fritsch GmbH, Idar-Oberstein, Germany) at a fixed frequency of 450 rpm for 5 h according to a previous literature.25 To avoid high temperature destroying the subtle molecular structure of lignin, the ball-milling procedure was set a 10 min interval after every 10 min of milling. All chemicals used were purchased from Sigma Chemical Co. (Beijing, China), except for cellulase. Commercial cellulase (Cellic® CTec2, 100 FPU/mL) was kindly provided from Novozymes, Beijing, China.

Fractionation of lignin fractions The overall scheme for the isolation of MWL, CEL, and EHL is shown in Figure 1. The ball-milled wood powder (20 g) was suspended in 80% dioxane with a solid-to-liquid ratio of 1:20 (g/mL) at 50 °C in the dark for 12 h under stirring. The mixture was filtered and then the residue was washed with the same solvents until the filtrate was clear. The above procedure was repeated twice. All the supernatants were concentrated under reduced pressure and then slowly dropped into 3 volumes of 95% ethanol to precipitate the hemicelluloses, which were separated by filtration. The mixed filtrates were concentrated again, and then precipitated in acidic water (pH=2, adjust by aqueous HCl). 4 ACS Paragon Plus Environment

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After centrifugation and freeze-drying, the MWLs was obtained. Herein, MWL-c, MWL-g, and MWL-u represent the MWL sample isolated from Ecamaldulensis, Eucalyptus grandis and Eucalyptus urophydis, respectively. The residues obtained after MWL extraction were then subjected CEL extraction. The residues were suspended in an acetate buffer solution (pH=4.8) with a concentration of 5% solid loading. After that, cellulase was added to the suspension with 35 FPU/g substrate, and the mixture was incubated at 50 °C in a rotary shaker for 48 h. After enzymatic hydrolysis, the mixture was separated by centrifugation, and the hydrolyzed residue was washed with buffer solution and deionized water repeatedly. Finally, the freeze-dried residue was extracted with 80% aqueous dioxane to obtain CEL fractions, and the detailed extraction process was same to the aforementioned procedure of MWL. The obtained CEL fractions were named as CEL-c, CEL-g, and CEL-u, respectively. The final residues after CEL isolation were further subjected to the above-mentioned enzymatic treatment. After enzymatic hydrolysis, the mixture was centrifuged and the enzymatic hydrolysis lignin (EHL) was thoroughly washed with buffer solution and deionized water, and then freeze-dried to obtain EHL fractions, which were labeled as EHL-c, EHL-g, and EHL-u, respectively.

Preparation of DELs To delineate the structural features of lignin macromolecules in these Eucalyptus species, double enzymatic lignin (DEL) in the diverse Eucalyptus wood were isolated, and the detailed procedure is presented in Figure 2. Typically ball-milled of Eucalyptus wood powder (5 g) was suspended 100 mL acetate buffer (pH = 4.8) with loading of 2.5 mL of cellulase (100 FPU/mL). The reaction mixture was incubated at 50 oC in a rotary shaker (150 rpm) for 48 h. After that, the mixture was centrifuged and the residual solid was washed extensively with acetate buffer (pH=4.8) and hot water, and then freeze-dried. The dried residual solid was further subjected to ball-milling process for 2 h and enzymatic hydrolysis as above-mentioned procedures. After washing and freeze-drying, the DEL fractions (named as DEL-c, DEL-g, and DEL-u, respectively) were obtained.

Lignin acetylation 5 ACS Paragon Plus Environment

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To improve the solubility of the lignin macromolecules in tetrahydrofuran for GPC analysis, the acetylation of lignin was performed as previously.26 In detail, 20 mg of lignin fractions were completely dissolved in DMSO/N-methylimidazole (2:1, v/v, 0.9 mL), after continually shaking for 24 h at room temperature in the dark, the acetic anhydride (0.6 mL) was added to the mixture and maintained for 2 h. Finally, the solution was dropped slowly into ice water (pH=2, 100 mL) to precipitate the acetylated lignin samples. After centrifugation and freeze-drying, all the acetylated lignin (MWLs, CELs, EHLs, and DELs) were obtained.

Structure elucidation of lignin preparations Carbohydrate analysis of lignin fractions was conducted by hydrolysis with dilute sulfuric acid and analyzed by high-performance anion-exchange chromatography (HPAEC) as previously.25, 27 The weight-average (Mw) and number-average (Mn) molecular weights of all the acetylated lignin samples were determined by gel permeation chromatography (GPC, Agilent 1200, and USA). The GPC system was equipped with UV detector at 240 nm on a PL-gel 10 μm Mixed-B 7.5 mm ID column, which was calibrated with monodisperse PL polystyrene standards (1390, 4830, 9970, 29150, 69650 g/mol). 4 mg acetylated lignin sample was dissolved in 2 mL of tetrahydrofuran (THF), and then a 10 μL solution was injected. The column was operated at ambient temperature and eluted with THF at a flow rate of 1 mL min-1. Carbohydrate analysis and GPC experiments were performed in duplicate, and the data reported were the average values. For quantitative 2D-HSQC spectra, about 50 mg of lignin was dissolved in 0.5 mL of DMSO-d6 (99.8% D),24,

28, 29

the Bruker standard pulse program hsqcetgp was used for 2D-HSQC

experiments.25, 30, 31 The spectral widths were 5000 Hz and 20000 Hz for the 1H- and 13

C-dimensions, respectively. The number of collected complex points was 1024 for

1

H-dimension with a recycle delay of 1.5 s. The number of transients was 64, and 256 time

increments were always recorded in the

13

C-dimension. Prior to Fourier transformation,

the data matrixes were zero filled up to 1024 points in the 13C-dimension. Data processing was performed using standard Bruker Topspin-NMR software (Topspin 2.1). A quantitative analysis of the intensities of the 2D-HSQC cross-signal was performed 6 ACS Paragon Plus Environment

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according to the following formula: I(C9) units = 0.5I(S2,6) + I(G2)

(1)

Where I(S2,6) is the integration of S2,6, including S and S', I(G2) is the integral value of G2. I(C9) represents the integral value of the aromatic ring. According to the internal standard (I(C9)), the amount of I(X) could be obtained by the following formula: I(X) = I(X)/I(C9) /100Ar

(2)

Where I(X) is the integral value of the α-position of A (β-O-4), B (β-β), and C (β-5), the integration should be in the same contour level. In the aromatic region, C2,6-H2,6 correlations from S units and C2-H2 correlation from G units were used to estimate the S/G ratio of lignin. The S/G ratio could be obtained by the following formula: S/G = 0.5I(S2,6)/I(G2)

(3)

For quantitative 31P NMR, 20 mg lignin was dissolved in 500 μL of anhydrous pyridine and deuterated chloroform (1.6:1, v/v) under stirring.32 This was followed by the addition 100 μL of cyclohexanol (10.85 mg/mL in anhydrous pyridine and deuterated chloroform 1.6:1, v/v) as an internal standard and 100 μL of Chromium (III) acetylacetonate solution (5 mg/mL in anhydrous pyridine and deuterated chloroform 1.6:1, v/v) as relaxation reagent. After that, the mixture was reacted with 100 μL of phosphitylating reagent (2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholate, TMDP) for about 10 min and was transferred into a 5 mm NMR tube for subsequent 31P NMR analysis.30, 33

Results and discussion Chemical composition of Eucalyptus species The chemical composition of the diverse Eucalyptus species is shown in Table 1. The Klason lignin content of E. camaldulensis (21.4%) and E. urophydis (21.0%) was lower than that of E. grandis (23.8%), which exactly accorded with the variation of the acid-soluble lignin content in the three Eucalyptus species. The high lignin content of E.grandis suggested that the lignification degree of this Eucalyptus was slightly higher than those of other species at the same age. As shown in Table 1, the content of cellulose (glucan) was 41.65%, 40.04%, and 41.01% in E. camaldulensis, E. grandis, and E. 7 ACS Paragon Plus Environment

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urophydis, respectively. In addition, the content of hemicelluloses (Rhamnose, arabinan, galactan, xylan, and glucuronic acid) in E. camaldulensis

(17.15%) and E. urophydis

(17.56%) was lower than that of E. grandis (18.20%), which was consistent with the diversity of the relevant lignin (Klason and acid-soluble lignin) contents. In short, understanding the chemical composition of the three Eucalyptus wood is precondition for further deconstruction of these Eucalyptus wood into single component and other value-added products.

Yields and carbohydrate content of lignin preparations MWL is traditionally considered as a representative lignin to understand the structural characteristics of lignin macromolecules in the plant cell wall. However, the low yield of MWL implied that it only represents a part of lignin fragments in the plant cell wall. It was found that the yield of native lignin extracted with 80% aqueous dioxane is higher than that extracted with 96% aqueous dioxane.23,

34

Based on these considerations, 80%

aqueous dioxane was used to extract MWL sample in this study. However, the MWL samples extracted from diverse Eucalyptus wood still present the yields ranged from 13.8 to 15.8 % (based on Klason lignin of Eucalyptus). Subsequently, the modified CEL preparations were consecutively extracted from the residues after MWL extraction and the corresponding yields ranged from 7.2 to 11.8 % (Table 2). By contrast, the yields of EHL and DEL preparations were 78.2-80.8 % and 102.5-109.5 %, respectively. The ultrahigh yields of EHLs and DELs are related to the fact that these lignin preparations are residual lignin after enzymatic hydrolysis, rather than extracted lignin, such as MWL and CEL. In the present study, the entire lignin in the plant cell wall of Eucalyptus was divided into three lignin preparations (MWL, CEL and EHL). Although these lignin fractions cannot exhibit panoramic structural features of lignin in different Eucalyptus species, they can better delineate the structural characteristics of lignin fractions from morphologically distinct regions (cell corner middle lamella, compound middle lamella, secondary wall) of plant cell wall in Eucalyptus. By contrast, the entire lignin from plant cell wall of Eucalyptus can be isolated via DEL method, which can better depict the entire structural characteristics of lignin macromolecules of these Eucalyptus species. 8 ACS Paragon Plus Environment

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To evaluate the composition and content of carbohydrate in the lignin preparations, the carbohydrate analysis of all the lignin preparations was performed (Table 2). It was observed that the contents of the total carbohydrates in the MWL and CEL fractions ranged from 6.80 to 9.31%. In detail, the MWL and CEL fractions contained a large percentage of xylose among the total sugars and uronic acids, implying that xylans were the predominant carbohydrates both in MWL and CEL fractions.25 Similarly, the amounts of associated carbohydrates in the EHL samples ranged from 11.44 to 14.04%, suggesting that EHL samples are also associated with small amount of xylans, in addition to some stubborn cellulose. As compared to EHL fractions, the DEL preparations from Eucalyptus wood contained relatively lower carbohydrates, which can be also reflected from the few correlated signals of carbohydrates in 2D-HSQC spectra of DELs. In general, low amount of carbohydrates in lignin is beneficial to structural elucidation of lignin macromolecules. Therefore, the DEL fraction with less carbohydrate can be regarded as a representative lignin for deep understanding the structural characteristics of lignin from these Eucalyptus species.

Molecular weight distributions The weight-average (Mw) and number-average (Mn) molecular weights of all the acetylated lignin samples obtained from Eucalyptus wood were measured by GPC and calculated from the GPC curves (relative values related to polystyrene), and the corresponding polydispersity indexs (PDI, Mw/Mn) were also given in Table 3. For MWL, the Mw of MWL-u was 6910 g/mol, which was the lowest molecular weight in the three MWL samples. For CEL fractions, it was found that the Mw of CEL-u (7230 g/mol) was apparently lower than those of CEL-c (8650 g/mol) and CEL-g (8770 g/mol). By contrast, the corresponding Mw of EHL samples showed no obvious fluctuation, ranging from 10810 to 11670 g/mol. In short, the molecular weight of these lignin fractions increased in the order of MWL < CEL < EHL. The highest Mw of EHL is probably attributable to the existence of stubborn cellulose and other associated carbohydrates.35 In addition, a high β-O-4 content also led to a high Mw of EHL. 36 Similarly, the DEL samples isolated from different Eucalyptus species all exhibited relatively high molecular weight (8130-8790 9 ACS Paragon Plus Environment

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g/mol) and narrow polydispersity index (Mw/Mn, 1.32-1.35). In detail, the Mw of DEL-g (8760 g/mol) and DEL-u (8790 g/mol) was slightly higher than that of DEL-c (8130 g/mol). Additionally, the narrow polydispersity of DEL suggested that the DEL is relatively more homogeneous lignin macromolecules among these lignin preparations. Considering the super-high yield, relatively lower carbohydrate content, and higher molecular weight as well as the narrow polydispersity of lignin macromolecules, it was concluded that DEL can be served as an ideal sample for the structural elucidation of lignin macromolecules in different Eucalyptus species.

2D-HSQC NMR analysis The 2D-HSQC NMR spectra can provide detailed information of the structural characteristics of the lignin polymer,37,

38

including substructures (linkages) and S/G

ratios.29 To investigate the composition and detailed chemical structures of the entire lignin fractions from different Eucalyptus species, all the lignin preparations (MWLs, CELs, EHLs, and DELs) were elaborately analyzed by the 2D-HSQC NMR technique. The side-chain (δC/δH 49−95/2.6−5.6) and aromatic (δC/δH 100−124/6.1−7.6) regions of the 2D-HSQC NMR spectra of the lignin fractions are shown in Figures 3, 4, and 5. The main identified substructures are also depicted in Figures 3 and 4.14, 24, 28 In the side-chain regions of the 2D-HSQC spectra of lignin preparations (MWLs, CELs, EHLs, and DELs) from different Eucalyptus species, aside from the conspicuous methoxyl groups (OMe), the substructures, such as β-O-4 aryl ethers (A), resinols (B), phenylcoumarans (C), and spirodienone substructures (D), could be assigned according to previous publications.39, 40 The detailed assignments of these substructures are listed in Table S1. From the 2D-HSQC spectra, it was found that all the lignin fractions exhibited similar spectral patterns except for tiny differences. For example, the signal intensity of carbohydrates in 2D-HSQC spectra of DEL (Figure 5) was relatively weak as compared to that of EHL, which was in agreement with the aforementioned carbohydrate analysis. By contrast, the DEL displayed a typical spectrum of native lignin, such as MWL and CEL. Briefly, common substructures were clearly displayed in the 2D-HSQC spectrum of DEL. Moreover, a small signal located at δC/δH 61.3/4.08 was assigned to the Cγ-Hγ 10 ACS Paragon Plus Environment

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correlation of p-hydroxycinnamyl alcohol end groups (I). In the aromatic region, syringyl (S) and guaiacyl (G) units were clearly distinguished. C2,6–H2,6 correlations in S-type unit were defined at δC/δH 103.8/6.68, while the signals for the Cα-oxidized S-units (S′) were observed at δC/δH 106.3/7.21. Interestingly, G2 position showed two correlated signals at δC/δH 108.8/6.92 and 110.9/6.96, respectively. Based on existing knowledge in lignocellulose chemistry, the two signals were tentatively assigned to phenolic type (G2′) and non-phenolic type (G2) units, respectively (Figure S2). To better explain the different chemical shifts of G2, the detailed reasons for the different chemical shifts are listed in the section of supporting materials. The relative abundances of linkages in the Eucalyptus lignin macromolecules were calculated based on the quantitative method.29 As shown in Table 4, the relative content of β-O-4 aryl ether units (A) in CEL-g (60.2/100Ar) was higher than that (47.2/100Ar) of MWL-g, which was in accord with the corresponding S/G ratios (3.2 vs 1.6). Theoretically, abundant S-type precursor is beneficial to the formation of β-O-4 linkage. Thus, a higher content of β-O-4 linkage positively related to the S/G ratio of the lignin macromolecules.41 The changes of S/G ratio in the MWL-g, CEL-g, and EHL-g preparations basically confirmed it. In fact, the relatively higher content of β-O-4 in CEL fraction is mostly attributed to the fact that MWL is probably originated from middle lamella of plant cell wall, while the subsequent CEL fractions are mainly originated from secondary wall of plant cell wall.42 That is, the initial MWL fraction is composed of lignin fragments with relatively low S/G ratios as compared to those of CEL fraction. Similarly, previous study found that MWL from E. grandis exhibited a low S/G ratio (1.7, based on 2D-HSQC).24 Besides, the ball-milling process may partly destroy β-O-4 linkage, which resulted in the decreased content of β-O-4 linkages in MWL. Similarly, the increased S/G ratios in the CEL fractions were in agreement with that reported in a recent publication, in which G-type lignin preferentially located in the middle lamella in contrast to S-type lignin.43 By contrast, it was observed that there was no difference in S/G ratios of CELs and EHLs preparations from these Eucalyptus wood, suggesting that CELs and EHLs were probably originated from same region of plant cell wall, such as S2 layer. As a residual lignin, the high-yield (range from 78.2 to 80.8 %, based on the Klason lignin) of EHL samples with 11 ACS Paragon Plus Environment

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the high content of β-O-4 (range from 60.6 to 63.6/100Ar) suggested that most of the lignin still remained in the residue after MWL and CEL extraction. Moreover, the highest S/G ratio in EHL-g implied that more S-type lignin units may exist in S2 layer of the E. grandis wood. In this study, DEL preparations were isolated with ultrahigh yield (range from 102.5 to 109.5 %, based on the Klason lignin) to delineate the structural differences of the native lignin macromolecules from different Eucalyptus wood. As shown in Table 4, the S/G ratios of DELs were found to be lower than those of corresponding CELs and EHLs. As compared to S/G ratios of CELs, the decreased S/G ratios of DELs were mostly related to the fact that the DELs and CELs are originated from different morphological regions in the plant cell wall, respectively. As aforementioned, CELs are mostly originated from secondary cell wall (S2), in which the syringyl (S) unit is abundant as compared to guaiacyl (G) unit in hardwood and grass species.13, 34 Therefore, DELs isolated from the plant cell wall (S2, CCML, and CML) can better represent the entire structural characteristics of lignin macromolecules. Besides the S/G ratios, the relative contents of linkages (β-O-4, β-β, β-5, and β-1) in DELs are important parameters for thoroughly investigating the structural features of entire lignin macromolecules. For example, the β-O-4 content in DEL-g was higher than those of DEL-c and DEL-u, which was in agreement with a higher S/G ratio in DEL-g. In general, the structures of lignin, whether derived from raw biomass, a pretreatment, or cellulosic ethanol production, influence its suitability for future processing.44 Usually, it is expected that lignin with high S/G ratios or high β-O-4 contents that facilitate subsequent biorefinery process.45 In addition, the starting lignin with a high proportion of β-O-4 linkages will also be beneficial to subsequent depolymerization and upgrading of lignin.46, 47 Therefore, DEL can be served as an ideal lignin fraction to develop depolymerization strategy for residual lignin (i.e. enzyme lignin) after enzymatic saccharification during the bioethanol production. Moreover, it was found that the DEL from Eucalyptus grandis (DEL-g) presented the highest content of β-O-4 linkage (64.3/100Ar) and abundant S/G ratio (3.1). These characteristics suggested that Eucalyptus grandis is a promising feedstock in biorefinery process.45 12 ACS Paragon Plus Environment

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31P-NMR

spectral analysis

To further compare the differences of functional groups in the lignin preparations, quantitative

31

P-NMR spectra of all the MWLs, CELs, EHLs, and DELs samples from

diverse Eucalyptus wood were performed. Quantification was carried out via peak integration with cyclohexanol as internal standard. The spectra of the lignin fractions are shown in Figure S1 and the corresponding quantitative results are listed in Table 5. Remarkably, it was found that MWL fractions contained more phenolic OH groups (G and S-type units) than the corresponding CEL preparations. The high content of phenolic OH groups in MWL samples is directly related to the ball milling process, which often results in a slight cleavage of β-O-4 linkages of lignin and release of more phenolic OH groups,13 which can be also revealed by the aforementioned 2D-HSQC quantitative results of β-O-4 linkages. However, the content of syringyl OH groups was lower than that of guaiacyl OH groups in MWL fractions, implying that the majority of syringyl OH groups in lignin existed in the form of β-O-4 linkages, and only a small amount of dissociative syringyl OH groups could be detected by the

31

P-NMR technique. That is, the cleavage of β-O-4

linkages during the ball-milling process mainly occurred at G-type lignin units. This fact was also reflected from the 2D-HSQC spectra of MWL, in which the signal for phenolic G units (G2′) confirmed this speculation. In fact, a previous study demonstrated that the MWL is mainly originated from middle lamella of plant cell wall, in which the content of G-type unit is higher than that in secondary wall.43 Correspondingly, the molecular weight and S/G ratios of MWLs were also lower than those of CELs, implying that the MWL fractions extracted from the Eucalyptus species had less etherified lignin units owing to the cleavage of β-O-4 structures. In addition, the content of COOH in MWL (0.12 to 0.16 mmol/g) preparations was apparently higher than that of CEL samples (0.06 to 0.08 mmol/g). The high content of COOH implied that oxidation reaction occurred in the ball-milling process. Moreover, it was also noted that the content of condensed G-type phenolic OH groups in MWL fractions was higher (0.16-0.28 mmol/g) than that (0.03-0.06 mmol/g) in other lignin samples (CELs, EHLs, and DELs). Furthermore, it was observed that MWL-c and MWL-g contained lower phenolic OH groups (G and S-type units) than that of MWL-u, suggesting that the lower Mw of MWL-u is related to the high 13 ACS Paragon Plus Environment

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content phenolic OH group. In fact, the quantification from

31

P-NMR is based on weight

of lignin (mmol/g lignin). If the Mw of lignin is low, the mole number of lignin becomes larger. Therefore, the 31P-NMR data are actually related to the molecular weight of lignin. With regarding to the different contents of OH groups in EHLs and DELs, it was found that EHL and DEL preparations from diverse Eucalyptus showed no obvious changes except for different contents of COOH. The highest content of COOH in DEL preparations among these lignin fractions is mostly related to the oxidation of aliphatic OH groups during double ball-milling processes, as also revealed by the decreased aliphatic OH groups in DELs. Moreover, fewer amounts of phenolic OH groups in the DEL of Eucalyptus grandis suggested that the lignin is rich in β-O-4 linkages (Table 4). To present a visualized cognition of lignin structures in different Eucalyptus species, the possible schematic diagrams (Figure 6 and Figure S3) can be obtained based on the quantitative data of S/G ratios and relative contents of different linkages in DEL samples. In short, the better understanding of lignin structures can facilitate the subsequent deconstruction of these Eucalyptus species into main components, such as lignin, hemicelluloses, and cellulose.

Conclusions In summary, MWL, CEL, and EHL samples were sequentially isolated from the diverse Eucalyptus species under mild conditions, which can better delineate the structural characteristics of lignin macromolecules from different morphologically distinct regions of plant cell wall in Eucalyptus species. It was found that MWL fractions contained more phenolic OH groups (1.17-1.58 mmol/g) than those (0.46-0.69 mmol/g) of CEL and EHL preparations, which was directly related to the ball milling process and cleavage of β-O-4 linkages. By contrast, the DEL preparations with ultrahigh yield (102.5-109.5%) can better depict the entire structural differences of lignin macromolecules among these Eucalyptus species. Results showed that DEL samples from these Eucalyptus species are syringyl-rich lignin macromolecules (S/G > 2.8), having more abundant β-O-4 linkages (59.1-64.3/100Ar), which can be served as an ideal “lignin preparation” to develop depolymerization strategy for current enzyme lignin originated from the bioethanol 14 ACS Paragon Plus Environment

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production. Moreover, it was found that the Eucalyptus grandis was a more promising feedstock among these Eucalyptus species in biorefinery process due to the highest content of β-O-4 linkage (64.3/100Ar) and abundant syringyl units in the corresponding double enzyme lignin (DEL). Based on these characteristics, it can be concluded that these Eucalyptus species are prospective biomass for the production of bio-based materials and chemicals in green sustainable biorefinery process. In short, the sufficient understanding the structural characteristics of lignin macromolecules in lignocellulose will facilitate subsequent deconstruction of the biomass in an advisable approach.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The related Figures and Tables for detailed introduction of this study. (PDF)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (R.-C. Sun); [email protected] Tel.: +86-10-62336903. Fax: +86-10-62336903. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors are extremely grateful to financial support from the National Natural Science Foundation of China for the key projects (31430092), Fundamental Research Funds for the

Central

Universities

2015ZCQ-CL-02,

and

Beijing

(Z161100005016041).

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Key

Lab.

Project

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Figure Captions and Tables

Figure 1. Isolation scheme of milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHL) from Eucalyptus wood. Figure 2. Isolation procedure of double enzymatic lignin (DEL) from different Eucalyptus species. Figure 3. Side-chain region in 2D-HSQC NMR spectra of MWLs, CELs, and EHLs isolated from Eucalyptus wood. Figure 4. Aromatic region in 2D-HSQC NMR spectra of MWLs, CELs, and EHLs, as well as the identified substructures. Figure 5. Side-chain and aromatic region in the 2D-HSQC NMR spectra of the DELs isolated from Eucalyptus wood. Figure 6. Potential structural diagram of DEL in plant cell wall of Eucalyptus wood (Eucalyptus grandis) based on the quantitative data of DELs (Carb: carbonhydrate). Table 1. Chemical composition of three Eucalyptus wood. Table 2. Yields and carbohydrate content of lignin fractions Table 3. Weight-average molecular weight (Mw), number-average (Mn) molecular weight, and polydispersity (Mw/Mn) of lignin fractions. Table 4. Quantification of the lignin fractions by quantitative 2D-HSQC NMR method. Table 5. Quantification of the lignin fractions by quantitative 31P-NMR method (mmol/g).

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Figure 1. Isolation scheme of milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHL) from Eucalyptus wood.

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Figure 2. Isolation procedure of double enzymatic lignin (DEL) from different Eucalyptus species.

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Figure 3. Side-chain region in 2D-HSQC NMR spectra of MWLs, CELs, and EHLs isolated from Eucalyptus wood.

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Figure 4. Aromatic region in 2D-HSQC NMR spectra of MWLs, CELs, and EHLs, as well as the identified substructures.

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Figure 5. Side-chain and aromatic region in the 2D-HSQC NMR spectra of the DELs isolated from Eucalyptus wood.

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Figure 6. Potential structural diagram of DEL in plant cell wall of Eucalyptus wood (Eucalyptus grandis) based on the quantitative data of DELs (Carb: carbohydrate).

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Table 1 Chemical composition of three Eucalyptus wood. Sample E. camaldulensis E. grandis E. urophydis a b

Rhamnan 0.45 0.44 0.48

Arabinan 0.25 0.27 0.26

Substrate composition (w/w, %) Glua Galacan Glucan Xylan 1.55 41.65 14.38 0.52 0.98 40.04 15.82 0.69 1.57 41.01 14.66 0.59

KL , Klason lignin (i.e., acid insoluble lignin). ASL, acid soluble lignin.

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KLa 21.40 23.80 21.00

ASLb 4.80 5.15 4.75

Total (%) 85.00 87.19 84.32

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Table 2 Yields and carbohydrate content of lignin fractions Yieldsa Carbohydrate content (%) b (%) samples Rha Ara Gal Glu Xyl Man Glua Total c MWL-c 15.0 0.23 0.36 0.76 0.51 5.03 N.D. 2.17 9.06 MWL-g 15.8 0.35 0.33 0.54 0.45 6.23 N.D. 1.41 9.31 MWL-u 13.8 0.18 0.24 0.59 0.43 4.26 N.D. 2.00 7.70 CEL-c 7.2 0.14 0.12 0.86 1.72 2.65 0.33 0.98 6.80 CEL-g 8.7 0.19 0.17 0.60 2.22 2.60 0.41 0.95 7.14 CEL-u 11.8 0.21 0.14 1.01 2.13 2.89 0.51 1.23 8.12 EHL-c 80.8 0.33 0.33 1.79 2.16 5.45 0.54 3.44 14.04 EHL-g 78.2 0.41 0.34 1.27 1.95 4.09 0.58 2.80 11.44 EHL-u 79.0 0.42 0.33 1.80 1.69 4.39 0.58 3.05 12.26 DEL-c 105.6 0.25 0.27 1.59 1.03 3.20 0.56 2.55 9.45 DEL-g 102.5 0.31 0.26 0.97 1.18 2.94 0.55 2.09 8.30 DEL-u 109.5 0.35 0.30 1.70 1.10 3.25 0.66 2.60 9.96 a The yields of lignin are based on the Klason lignin of different eucalyptus species. b Rha = rhamnose, Ara = arabinose, Gal = galactose, Glu = glucose, Xyl = xylose, Man = mannose, Glua = glucuronic acid c N.D. = not detected.

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Table 3 Weight-average molecular weight (Mw), number-average (Mn) molecular weight, and polydispersity (Mw/Mn) of lignin fractions. Samples MWL-c MWL-g MWL-u CEL-c CEL-g CEL-u EHL-c EHL-g EHL-u DEL-c DEL-g DEL-u

Mw 8230 8810 6910 8650 8770 7230 11670 10810 11280 8130 8760 8790

Mn 5050 5250 4610 6390 6540 5580 8060 7880 8200 6180 6480 6540

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Mw/Mn 1.63 1.68 1.50 1.35 1.34 1.30 1.45 1.37 1.38 1.32 1.35 1.34

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Table 4 Quantification of the lignin fractions by quantitative 2D-HSQC NMR method. β-O-4 β-β β-5 β-1 S/Gb MWL-c 45.2a 12.0 2.7 Trc 1.5 MWL-g 47.2 10.5 2.4 Tr 1.6 MWL-u 44.5 10.7 2.0 Tr 1.4 CEL-c 58.1 13.0 2.1 0.9 2.8 CEL-g 60.2 12.2 1.1 1.0 3.2 CEL-u 59.2 13.2 1.9 Tr 2.9 EHL-c 63.6 13.4 2.3 1.1 2.9 EHL-g 63.3 13.0 0.7 Tr 3.3 EHL-u 60.6 13.1 1.4 Tr 2.8 DEL-c 60.8 11.7 2.6 0.9 2.8 DEL-g 64.3 12.5 1.8 2.0 3.1 DEL-u 58.1 10.3 0.7 1.7 2.9 a Results expressed per 100 Ar based on quantitative 2D-HSQC spectra b S/G ratio obtained by the this equation: S/G ratio = 0.5I (S2,6) / I (G2). c Tr, trace.

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Table 5 Quantification of the lignin fractions by quantitative 31P-NMR method (mmol/g). Sample

Aliphatic OH

Syringyl OH

Guaiacyl OH a C NCb 0.16 0.69 0.23 0.69 0.28 0.86 0.03 0.25 0.06 0.29 0.05 0.20 0.04 0.17 0.05 0.21 0.04 0.16 0.04 0.16 0.04 0.14 0.04 0.15

MWL-c 4.51 0.32 MWL-g 3.72 0.34 MWL-u 4.02 0.44 CEL-c 4.27 0.22 CEL-g 4.54 0.34 CEL-u 3.51 0.32 EHL-c 5.09 0.29 EHL-g 4.62 0.34 EHL-u 3.67 0.26 DEL-c 2.80 0.25 DEL-g 2.52 0.22 DEL-u 2.80 0.25 a C, condensed. 5-substitued lignin b NC, non-condensed.

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Carboxylic Group 0.15 0.12 0.16 0.08 0.06 0.07 0.13 0.15 0.12 0.26 0.23 0.21

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TOC/Abstract graphic

Synopsis Understanding the lignin structures will facilitate sustainable process of the Eucalyptus species for producing biochemicals, bioenergy, and biomaterials.

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246x153mm (96 x 96 DPI)

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