Structural elucidation of lignin macromolecule from abaca during alkaline hydrogen peroxide delignification

Journal Pre-proof Structural elucidation of lignin macromolecule from abaca during alkaline hydrogen peroxide delignification

Cheng-Ye Ma, Han-Min Wang, Jia-Long Wen, Quentin Shi, Shuang-Fei Wang, Tong-Qi Yuan, Run-Cang Sun PII:

S0141-8130(19)36656-5

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.080

Reference:

BIOMAC 14107

To appear in:

International Journal of Biological Macromolecules

Received date:

21 August 2019

Revised date:

5 December 2019

Accepted date:

10 December 2019

Please cite this article as: C.-Y. Ma, H.-M. Wang, J.-L. Wen, et al., Structural elucidation of lignin macromolecule from abaca during alkaline hydrogen peroxide delignification, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/ j.ijbiomac.2019.12.080

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

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Structural Elucidation of Lignin Macromolecule from Abaca during Alkaline Hydrogen Peroxide Delignification Cheng-Ye Ma1, Han-Min Wang1, Jia-Long Wen1, 2*, Quentin Shi3, Shuang-Fei

1

of

Wang4, Tong-Qi Yuan1, 2, Run-Cang Sun1*

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University,

Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing

-p

2

ro

Beijing 100083, China

re

Forestry University, Beijing, 100083, China

Shanghai Dssun New Material Co., Ltd, Shanghai 200223, China

4

College of Light Industry and Food Engineering, Guangxi University, Nanning

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530004, China

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3

*Corresponding author:

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing 100083, China. Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing 100083, China. Tel: +86-10-62336903. Fax: +86-10-62336903. E-mail: [email protected]; [email protected]

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Abstract To maximize the utilization of Abaca lignin in the current biorefinery, structural characteristics of native lignin from Abaca were firstly comprehensively investigated. Parallelly, effective delignification of Abaca was achieved by alkaline hydrogen peroxide (AHP) process, which facilitated the production of specialty paper in

of

industry. The structural changes of lignin macromolecules during the AHP delignification were illustrated by comparing the structural differences of the released

31

P NMR, pyrolysis-GC/MS, and GPC

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analytical methods, such as 2D-HSQC NMR,

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lignin fraction and corresponding native lignin, which were analyzed via the advanced

re

techniques. It was found that Abaca lignin is a HGS-type lignin, which is

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overwhelmingly composed of β-O-4 linkages and abundant hydroxycinnamic acids

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(mainly p-coumaric acid). In addition, partial cleavage of β-O-4 linkages and p-coumarate in lignin occurred during the AHP delignification process. Meanwhile,

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AHP process also led to the elevation of H-type lignin units in AHPL. Considering that β-O-4 bond is vulnerable in the catalytic degradation process of lignin, the lignin with abundant β-O-4 linkages is beneficial to the downstream conversion of lignin into aromatic chemicals.

Keywords: Abaca; lignin isolation; NMR analysis; lignin characterization

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1. Introduction Lignocellulosic biomass (LCB) is mainly composed of cellulose, hemicelluloses, and lignin. These natural macromolecules can be used for the production of biofuels, energy, value-added products and chemicals [1]. However, selective separation or fractionation of these components is still difficult since they are physically entangled

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among them in the plant cell wall. These tight physical binding and chemical linkages between lignin and carbohydrates restrict the isolation of the components in an

ro

unaltered manner [2]. In general, physical and chemical pretreatments result in the

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deconstruction and separation of cell wall components. In terms of chemical structure,

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any pretreatment will inevitably lead to the change or destruction of structural features

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of these macromolecules, especially lignin macromolecular. Up to now, advantages

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and limitations of various pretreatment techniques (i.e., physical, chemical, biological methods and combinations of these methods) have been elaborately reviewed [3]. In

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addition, how different biomass pretreatment processes alter chemical composition and change cell wall structure has been comprehensively investigated [3, 4]. Generally, biomass pretreatment is the key procedure of the biorefinery. It undoubtedly affects the fate of the individual lignocellulose constituents and further downstream valorisation of these constituents, especially for lignin. Based on the current practice of biorefinery, these factors will affect the final utilization of lignin in different areas, such as the content of β-O-4 bonds, the amounts of different hydroxyl contents, S/G/H ratio, chemical reactivity, molecular weight, etc. Recently, researchers

have

found

that

available 3

aromatic

hydroxyl

groups

in

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high-molecular-weight lignin were critical for the successful preparation of carbon fibre [5, 6]. Additionally, the content of β-O-4 linkage in lignin is a key to achieve monomer production during catalytic degradation process [7]. Traditionally, the current pulping industries produce pulp for different industrial use and also release a lot of industrial lignin as waste. The main lignin varieties are kraft lignin, alkali lignin, and lignosulfonates. In detail, the largest source of lignin,

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kraft lignin is highly condensed and contains a low amount of residual β-O-4 bonds. It

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also incorporates sulfur in the form of thiol groups, which can complicate downstream

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valorisation [4]. Sulfite pulping is the second most important chemical pulping

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process, lignosulfonates are typically water-soluble and highly degraded and have a

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higher sulfur content (4-8 wt%) compared to kraft lignin. The third traditional pulping

na

process is soda pulping, soda lignin is typically characterised by a low β-O-4 content, and can be isolated through precipitation. Nevertheless, the complex structures of

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lignin and wide range of molecular weights undoubtedly limited their further value-added applications.

Lignin is an amorphous and aromatic polymer, comprising of different basic compositions and common linkages, such as β-O-4, α-O-4, β-β, β-5, β-1, 5-5, 4-O-5, and dibenzodioxocin (DBDO) linkages, depending on different lignocellulosic species [8, 9]. The intrinsic structural complexity of lignin macromolecule in the plant cell wall makes it difficult to be efficiently isolated lignin polymers from LCB. Although the primary structures and linkages of lignin have been well depicted, the structural changes of lignin macromolecule during different pretreatments have not been 4

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completely elucidated [8-10]. Additionally, the unclear understanding of the chemical reactivity of the isolated lignin will also make it more difficult to achieve value-added applications of lignin. Based on these considerations, a comprehensive elucidation of structural features and chemical reactivity of lignin during the pretreatment process is of vital importance to meet the overall goals of biorefinery.

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The double enzymatic lignin (DEL) recently developed has proven to be a representative native lignin to acquire panoramic insights of lignin structures in

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different feedstock [11, 12]. Among these pretreatment methods, alkaline hydrogen

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peroxide (AHP) pretreatment has been considered as a promising and eco-friendly

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chemical pretreatment to remove lignin from biomass [13, 14]. Furthermore, AHP has

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been successfully applied to isolate hemicelluloses and lignin from herbaceous

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biomass under mild conditions [15]. Abaca (Musa textilis), also called ‘‘Manila hemp’’, is a plant belonged to the Musaceae family, which is similar to the banana tree

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but having inedible fruit and stalk that provides high-quality fibres [16]. Traditionally, Abaca has been used to make fishing nets, banknotes, cigarettes, baby napkins, toilet paper, machine filters, hospital textiles, and tea bags, etc [16]. In the 1970s, soda and alkaline pulping of Abaca was firstly performed to produce pulp [17]. In the1980s, Abaca pulp was obtained by cooking and bleaching with alkali and hydrogen peroxide, respectively [18]. Subsequently, the effects of soda pulping of Abaca on the yield, kappa, viscosity, breaking length, stretch, tear index of pulp as well as paper sheets were performed [16]. Recently, bleaching of soda pulp of Abaca with peracetic acid was reported [19]. However, structural characterization and structural changes of 5

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lignin from Abaca during the pulping and bleaching process have not been investigated until now. In addition, the molecular characteristics of native lignin from Abaca have not been addressed. Although the detailed and accurate structure of lignin macromolecule in the plant is still in exploration, improvements in methods for isolating native lignin and identifying lignin structures in spectroscopic techniques

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have enabled researchers to elucidate the structural changes of lignin during different pretreatment and delignification processes, which is important for the lignin chemistry

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and value-added applications of lignin.

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To extend and maximize the downstream valorisation possibilities of the lignin

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stream, the structural changes of Abaca lignin during alkaline hydrogen peroxide

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(AHP) delignification were comprehensively investigated in the present study. In

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addition, the isolated lignin fractions (DEL and AHPL) from Abaca were firstly elaborately characterized by means of the state-of-the-art analytical methods, such as 31

P NMR, pyrolysis-GC/MS, and gel permeation chromatography

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2D-HSQC NMR,

(GPC) as well as FT-IR spectrascopy techniques. In short, the results obtained will facilitate the understanding of the lignin structure and structural changes of lignin in the plant cell wall of Abaca and then achieve an efficient delignification as well as value-added utilization of the isolated lignin polymers.

2. Materials and Methods 2.1 Materials

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Abaca (Musa textilis) was provided from Fengyuan Paper Co., Ltd. in Hebei province, China. The compositional analysis of Abaca was measured according to the National Renewable Energy Laboratory (NREL) method [20]. The raw material was smashed into powder (40-60 mesh) by a laboratory pulverizer and dried in oven at 60 o

C for 24 h. The sample (40-60 mesh) was extracted with toluene/ethanol (2:1, v/v) in

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a Soxhlet extractor for 6 h. The chemical components of the plant cell wall of Abaca were listed as below, cellulose 52.50%, hemicelluloses 21.48%, lignin 18.61%, and

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extractives 5.19%. For the delignification process, the pretreatment liquor of Abaca

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was provided by Fengyuan Paper Co., Ltd. (Hebei, China), which is a diluted and

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wine-colored transparency liquid and obtained after alkaline hydrogen peroxide (AHP)

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pretreatment (20 wt% NaOH and 10 wt% H2O2 based on the dried feedstock,

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solid/liquid ratio of 1:20 (g/mL), 105 oC cooking temperature, and 3 h holding time). All the chemical reagents used in this study were analytical grade and purchased from

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Macklin Chemical Co., Ltd. (Shanghai, China). The cellulase (Cellic@ CTec2, 100 FPU/mL) was provided from Novozymes (Beijing, China).

2.2 Fractionation of alkaline hydrogen peroxide lignin (AHPL) The pretreated liquor (200 ml) was concentrated to about 20 ml under reduced pressure. Subsequently, the concentrated liquor was added dropwise into the ethanol (60 ml, kept at pH 6) under continuous stirring to precipitate the dissociated hemicelluloses. After removing precipitated carbohydrates, the lignin-containing supernatants were obtained and re-concentrated by vacuum distillation, and then 7

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slowly pouring it into plenty of acidic water (pH 2.0, 200 ml) to precipitate the AHPL. After washing with acidic water and freeze-drying, the purified AHPL was recovered.

2.3 Preparation of double enzymatic lignin (DEL) To delineate the structural characteristics of the native lignin in the raw material,

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double enzymatic lignin (DEL) from Abaca was prepared. The detailed preparation process was according to the previous publications [11, 12]. As shown in Fig. S1, the

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ball-milled powder (5 g) was mixed with the desired amounts of sodium acetate

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buffer (pH 4.8) with a solid-to-liquid ratio of 1:20 (g/mL) and cellulase (35 FPU/g

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substrate). Then the mixture was incubated at 50 oC in a rotary shaker with a

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rotational velocity of 150 rpm for 48 h. Next, the mixture was centrifuged and the

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residue was washed thoroughly with sodium acetate buffer (pH 4.8) to remove the hydrolyzed carbohydrates, and then freeze-dried. Finally, the dried residual solid was

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repeatedly subjected to ball-milling for 2 h and enzymatic hydrolysis again as above-mentioned processes. After washing with acidic water (pH 2.0) and freeze-drying, DEL sample with the yield of 95.7% (without carbohydrates) from Abaca was obtained. To increase the solubility of the lignin in tetrahydrofuran (THF) for the determination of molecular weights by GPC technique, the acetylation of lignin was performed as previously [11]. All experiments in this study were conducted in duplicate and the results reported were the average values.

2.4 Characterization of lignin macromolecules 8

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FT-IR spectra of lignin preparations were obtained using a Thermo Scientific Nicolet iN10 FT-IR microscope (Thermo Nicolet Corp., Madison, WI, USA) equipped with liquid nitrogen cooled MCT detector. The dried lignin samples were ground and pelletized using BaF2, and their spectra were recorded in the range from 4000 to 700 cm-1 at 4 cm-1 resolution and 128 scans per sample. The fingerprint

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region was baseline corrected between 1900 and 750 cm-1. The carbohydrates analysis of lignin samples was carried out by the H2SO4 hydrolysis and determined by

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high-performance anion-exchange chromatography (Dionex ICS3000) as previously

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reported [21, 22]. The weight-average (Mw) and number-average (Mn) molecular

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weights of the lignin polymers were measured by gel permeation chromatography

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(GPC, Agilent 1200, USA) with an UV detector at 240 nm on a PL-gel 10 μm

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Mixed-B 7.5 mm ID column, corrected with PL polystyrene standards (1390, 4830, 9970, 29150, 69650 g/mol).

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NMR spectra of lignin samples were recorded on a Bruker AVIII 400 MHz spectrometer at 25 oC in DMSO-d6. The quantitative 2D-HSQC and

31

P NMR

experiments were performed according to the previous literatures [23-26], which can quantitatively reflect the content of chemical functional groups (carboxyl, aliphatic hydroxyl, and phenolic hydroxyl groups). Py-GC/MS was performed with a CDS Pyroprobe 5200HP pyrolyzer (Chemical Data Systems) connected to a Perkin Elmer GC/MS apparatus (Clarus 560) equipped with an Elite-35MS capillary column (30×0.25 mm i.d., 0.25 μm film thickness). Compounds were identified by comparing their mass spectra with the NIST library. The syringyl/guaiacyl (S/G) ratio was 9

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calculated by dividing the sum of peak areas from syringyl units by the sum of the peak areas of guaiacyl derivatives of the selected markers [26].

3. Results and Discussion 3.1 Carbohydrate contents of the lignin preparations and their molecular weight

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distributions To further characterize the composition and content of the associated

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carbohydrates in the lignin fractions, the carbohydrates analysis of double enzymatic

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lignin (DEL) and alkaline hydrogen peroxide lignin (AHPL) was performed (Table 1).

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It was clearly observed that DEL contained a small quantity of associated

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carbohydrates (10.72%, w/w dry lignin), while AHPL only contained trace of

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carbohydrates (1.26%, w/w dry lignin). After examining the detailed composition of the associated carbohydrates, it was found that glucose was the major sugar in DEL,

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the higher content of glucose (6.90%) is ascribed to the small amount of unhydrolyzable cellulose in DEL. The lower amount of carbohydrates in AHPL suggested

that

the

AHP

treatment

facilitated

the

cleavage

of

specific

lignin-carbohydrate complex and dissolution of cleaved lignin fractions in alkaline media. The values of average molecular weights (Mw and Mn) and polydispersity indexes (PDI) of lignins can reveal the structural changes of lignin during the AHP process. As shown in Fig. 1, the Mw of DEL (17330 g/mol) is significantly higher than that (4390 g/mol) of AHPL. The relatively higher Mw of DEL was possibly due to its 10

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carbohydrates content (10.72%). It has been reported that the carbohydrate associated with lignin increases the hydrodynamic volume of lignin macromolecule and therefore increases the apparent molecular weight of the lignin when molecular weight is determined by GPC technique. Another reason for the low Mw of AHPL is presumed to partial alkaline hydrolysis of β-O-4 ether linkages during the AHP

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process, which will be confirmed in the subsequent NMR section.

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3.2 FT-IR Spectra

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To further investigate the structural differences between the DEL and AHPL,

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FT-IR spectra of the lignin fractions were investigated. Fig. 2 exhibits the FT-IR

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spectra of AHPL and DEL and the main assignments in the FT-IR spectra of these

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lignin fractions are assigned in Table S1 according to previous literatures [27, 28]. As illustrated in Fig. 2, the absorption bands located at 3410-3430 cm-1 are attributed to

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the aliphatic OH group stretching, which is a broad and strong absorption band. Besides, these bands in the spectra of DEL are higher than those of AHPL due to the high content of carbohydrates in DEL. The absorbance located at 2940 cm-1 and 2850 cm-1 is corresponding to the stretching vibration of C-H in the methyl and methylene groups, respectively. These weakened bands in AHPL further verified that methyl and methylene-rich carbohydrates have been mostly removed. The bands at 1600, 1508, 1420 and 1267 cm-1 are assigned to aromatic skeletal vibrations, and the C-H deformations in -CH3 and -CH2 with aromatic ring vibration at 1460 cm-1. The band at 1326 cm-1 (syringyl and condensed guaiacyl units) in the AHPL was decreased, 11

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suggesting that the AHP treatment probably led to the demethoxylation reactions of the lignin macromolecule. However, this deduction needs to be further confirmed by NMR techniques. In short, the significant differences in the FT-IR spectra was reflected in the weakened bands located at 1738 cm-1 (C=O in ester groups) in the spectrum of AHPL.

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The C=O groups are mainly originated from ester groups in lignin (esterfied p-coumaric acid, pCE) rather than carbohydrate because that only lower contents of

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carbohydrates (especially trace amount of xylan) were detected in DEL and AHPL.

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Furthermore, the absorption peak (1167 cm-1) was assigned to HGS lignin based on

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the classification system in a publication [28], indicating that both AHPL and DEL

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from Abaca were belonged to HGS-type lignin.

3.3 2D-HSQC spectra of the lignin

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The 2D-HSQC NMR technique can supply detailed information on the structural features of the lignin macromolecules, such as chemical composition (S/G ratio), chemical linkages and their quantitative results [29]. To further reveal the structural changes of DEL and AHPL, 2D-HSQC spectra were used for the elaborated analysis of the lignin fractions. The different regions, aromatic region (δC/δH 100-124/6.1-7.6) and side-chain (δC/δH 49-95/2.6-5.6), and the major identified substructures are shown in Fig. 3 and quantified in Table 2. In the side-chain region of 2D-HSQC spectra of lignin fractions (AHPL and DEL), the main structures (depicted in Fig. 3) could be obviously differentiated 12

Journal Pre-proof according to previous publications [10, 30, 31]. The methoxyl groups (-OCH3, δC/δH 55.7/3.70) and β-aryl-ether (β-O-4) linkages were the most dominating substructures in the Abaca lignin fractions. Additionally, γ-acylated lignin (A′γ) structure was also found in the DEL, which was in agreement with a previous report [32]. The detailed assignments of these substructures are listed in Table S2. By interpreting the

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2D-HSQC spectra, it can be found that all of the lignin fractions presented similar spectral patterns apart from tiny differences. For instance, the 2D-HSQC spectra of

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DEL displayed the typical spectrum of native lignin from grass species, which is in

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line with the FT-IR spectra [11]. The appearance of A′γ in the spectrum of DEL

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suggested the existence of acylated β-O-4 linkages at the γ-position [33]. By contrast,

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the disappeared A′γ in the spectrum of AHPL suggested that the AHP delignification

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could effectively destroy the γ-acylated substructure in lignin. Interestingly, it was noted that the signal for Aβ(S) in AHPL is stronger than that in DEL, further indicating

linkages.

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that most of β-O-4 linkages in DEL were existed in the form of acylated β-O-4

In the aromatic/unsaturated region of 2D-HSQC spectra of lignin fractions (AHPL and DEL), the signals from the syringyl (S) and guaiacyl (G) units were distinctly observed [32, 34]. In detail, the S units showed a visibly signal for the C2,6-H2,6 correlations at δC/δH 103.5/6.66, and the lower signal of the Cα-oxidized S-units (S′) appeared at δC/δH 106.3/7.32. Besides, there are three distinct cross-signals of G units: C2-H2 (δC/δH 110.8/6.97), C5-H5 (δC/δH 114.5/6.70 and 115.1/6.95), and C6-H6 (δC/δH 119.0/6.78). Moreover, the H units were also 13

Journal Pre-proof distinguished at δC/δH 127.0/6.95 in the spectra of DEL and AHPL. Simultaneously, the existence of pCE could be also revealed by the correlations, such as C7-H7 correlation at δC/δH 144.4/7.39, C8-H8 correlation at δC/δH 113.6/6.25, and C2,6-H2,6 correlations at δC/δH 129.9/7.45. The content of G units in the AHPL were lower than that in DEL, while the amount of H units was elevated to 28% in the AHPL as

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compared to that (4%) in DEL, suggesting that the AHP process likely resulted in the demethoxylation of G and S-type lignin (Table 2). Additionally, DEL contained a

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large amount of p-coumarate (pCE), which is a pensile unit in the gramineous lignin

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process removed the pCE to some extent.

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[35]. By contrast, the weakened signals of pCE in AHPL suggested that the AHP

lP

As can be seen from the Table 2, the relative abundances of different linkages in

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the lignin fractions were quantified according to the calculation method described in the previous literatures [32, 33, 36]. It was observed that the relative content of the

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β-O-4 aryl ether units (A) in AHPL (45.54/100Ar, Ar=aromatic) was lower than in DEL (64.16/100 Ar), confirming that the β-O-4 linkages in lignin were partially cleaved during the AHP process. Accordingly, it was found that syringyl/guaiacyl (S/G) ratio of DEL and AHPL was 4.94 and 4.14, respectively. In general, the plentiful S-type units in hardwood or grass lignin are positively related to the content of β-O-4 linkages in the lignin. In short, the abundant β-O-4 linkages in these lignin fractions are beneficial to the downstream conversion of lignin into aromatic molecules.

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Journal Pre-proof 3.4 31P NMR spectra of the lignin Quantitative

31

P NMR technique is a valid method to evaluate the multifarious

functional groups in different lignin fractions [24, 25, 37]. In this study,

31

P NMR

spectra of lignin are shown in Fig. S2 and the integral ranges for different OH groups are listed in Table S3. The contents of the various OH groups are illustrated in Fig. 4.

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As shown in Fig. 4, the content of phenolic OH groups (G and S-type OH) in DEL is close to that in AHPL, suggesting that the structural features of AHPL are similar to

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those of DEL. Simultaneously, the content of aliphatic OH in AHPL was calculated to

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be 2.26 mmol/g, which was lower than that (2.66 mmol/g) of DEL, implying that

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some aliphatic OH groups were oxidized during the AHP process. It was noted that

lP

the content of carboxylic group in AHPL was markedly increased, implying that a

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large amount of esterified p-coumaric acid in the raw material was transformed into free p-coumaric acid during AHPL preparation. Meanwhile, part oxidization also

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occurred during the delignification process of AHP, which also leads to the increase of COOH in AHPL. Furthermore, the low content of phenolic OH groups in AHPL suggested that the released phenolic OH groups from partial cleavage of β-O-4 linkages were further oxidized into other structures (e.g., quinone) [32].

3.5 Pyrolysis-gas chromatography/mass spectrometry The Py-GC/MS chromatograms of AHPL and DEL are shown in Fig. 5. The identities and relative abundances of the released compounds are listed in Table S4. Among them, syringyl (S) and guaiacyl-type (G) phenols were obviously identified 15

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(Fig. S3). Only a minor quantity of phenol-type compounds from p-hydroxyphenyl (H) units could be detected. The most important compounds identified were phenol (peak 1), phenol, 4-ethyl (peak 4), 1,2-benzenediol, 3-methoxy (peak 7), phenol, 2,6-dimethoxy (peak 10), benzaldehyde, 4-hydroxy-3,5-dimethoxy (peak 13), phenol, 2,6-dimethoxy-4-(2-propenyl) (peak 17).

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Py-GC/MS has been successfully used to analyze the relative S/G ratio of lignin [38]. The syringyl/guaiacyl (S/G) ratios of the lignins obtained from DEL and AHPL

ro

are shown in Table S4. In all those samples, the G-type and S-type degradation

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products were released in different amounts, with an S/G ratio ranging from AHPL

re

(1.78) to DEL (5.77). For example, DEL showed a higher S/G ratio as compared to

lP

the corresponding AHPL, which was in line with the 2D-HSQC results

na

aforementioned. The lower S/G ratio of AHPL (1.78) suggested that a part of S-type lignin was degraded and removed during AHP process [26, 36]. Another aspect is that

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although the S/G ratio of the lignins obtained from Py-GC/MS is different with the S/G ratio acquired by 2D-HSQC NMR results aforementioned, the overall trend is consistent. The differences of S/G ratios were probably related to the demethoxylation induced by the pyrolysis process. It was indicated that demethoxylation of S units easily occurred than guaiacyl units during pyrolysis process, thus resulting in the formation of more guaiacyl-type degradation products and then influencing the syringyl/guaiacyl ratio [39].

3.6 Structural changes of lignin macromolecule during the AHP process 16

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Under the current AHP process, the natural lignin macromolecules have been partly deconstructed, as revealed by the decreased molecular weight and the partly cleaved β-O-4 linkages. In fact, the changes of molecular weight are not only related to the disassembly of the macromolecules, but also dependent on the amount of carbohydrates in these lignin macromolecules. In addition, AHP process led to the

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occurrence of demethoxylation, as revealed by the elevated H-type lignin units. Simultaneously, oxidation of aliphatic OH group also resulted in the significant

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increase of COOH groups in AHPL. Moreover, the esterified p-Coumaric acid (pCE),

-p

which is attached to γ-position of lignin molecules, was mostly removed during the

re

AHP process. In short, the structural features of lignin obtained from AHP process are

lP

close to those of native lignin from same feedstock. The single and abundant β-O-4

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linkages in the AHPL suggested that this kind of lignin feedstock has a broad application in the catalytic degradation of lignin. Based on these results, the proposed

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AHP delignification mechanism is shown in Fig. 6.

4. Conclusion

In this study, native lignin (i.e. DEL) from Abaca was firstly comprehensively investigated. It was found that Abaca lignin is composed of p-hydroxyphenyl, guaiacyl, and syringyl-type units as well as abundant p-coumaric acid. Remarkably, the lignin macromolecule is overwhelmingly connected by β-O-4 linkages. The fundamental chemistry of the AHP delignification was illustrated by comparing the structural changes of DEL and AHPL. It was observed that partly cleaved β-O-4 17

Journal Pre-proof linkages facilitated the AHPL fractionation. In short, the dominant β-O-4 linkages in both DEL and AHPL suggested that these lignin fractions are beneficial to the downstream conversion of lignin into aromatic chemicals.

Notes

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The authors declare no competing financial interest. Acknowledgments

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This work was supported by Beijing Forestry University Outstanding Young Talent

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Cultivation Project [2019JQ03006, 2019JQ03005], National Natural Science

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Foundation of China [31430092, 31872698], Open Foundation of Guangxi Key

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na

lP

Laboratory of Clean Pulp & Papermaking and Pollution Control [KF201714].

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Figure Captions

Fig. 1. Molecular weight distribution of the DEL and AHPL lignins. Fig. 2. FT-IR spectra of DEL and AHPL lignins. Fig. 3. 2D-HSQC spectra of the lignins and identified main structures. Fig. 4. Quantification of the lignin fractions by quantitative

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P-NMR spectroscopy.

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(aC = condensed 5-substitued lignin, bNC = non-condensed)

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Fig. 5. Py-GC/MS chromatogram of the lignin samples.

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Fig. 6. Proposed processes of chemical structure evolution of Abaca lignin during

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AHP process

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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Fig. 6

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Journal Pre-proof Table 1. The carbohydrates analysis of the lignin preparations (w/w, %) DEL

AHPL

Total carbohydrates

10.72

1.26

Arabinose

0.53

0.03

Galactose

1.42

0.11

Glucose

6.90

0.91

Mannose

1.37

0.07

Glucuronic acid

0.47

0.13

Galacturonic acid

0.03

0.01

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Sample

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Table 2. Quantification of lignin fractions by quantitative 2D-HSQC NMR spectroscopy Sample

β-O-4a

pCEa

S/Gb

S/G/H

DEL

64.16

17.76

4.94

79/16/5

AHPL

45.54

7.77

4.14

58/14/28

Result expressed per 100 Ar based on quantitative 2D-HSQC spectra. 0.5I(S2,6)

S/G ratio obtained by the equation: S/G =

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I(G2)

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Highlights

1. Structural features of lignin from Abaca were firstly comprehensively investigated 2. Effective delignification was implemented by alkaline hydrogen peroxide (AHP) process 3. Structural changes of lignin during the AHP process were elaborately illustrated

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4. Abaca lignin is primarily composed of β-O-4 and abundant hydroxycinnamic

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acids

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5. Partly cleaved β-O-4 and LCC linkages are responsible for the effective

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delignification

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