Comparative study on the properties of lignin isolated from different pretreated sugarcane bagasse and its inhibitory effects on enzymatic hydrolysis

Journal Pre-proof Comparative study on the properties of lignin isolated from different pretreated sugarcane bagasse and its inhibitory effects on enzymatic hydrolysis

Chao Xu, Fen Liu, Md. Asraful Alam, Huanjun Chen, Yu Zhang, Cuiyi Liang, Huijuan Xu, Shushi Huang, Jingliang Xu, Zhongming Wang PII:

S0141-8130(19)39369-9

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.270

Reference:

BIOMAC 14298

To appear in:

International Journal of Biological Macromolecules

Received date:

16 November 2019

Revised date:

29 December 2019

Accepted date:

31 December 2019

Please cite this article as: C. Xu, F. Liu, M.A. Alam, et al., Comparative study on the properties of lignin isolated from different pretreated sugarcane bagasse and its inhibitory effects on enzymatic hydrolysis, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2019.12.270

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.

© 2020 Published by Elsevier.

Journal Pre-proof

Comparative study on the properties of lignin isolated from different pretreated sugarcane bagasse and its inhibitory effects on enzymatic hydrolysis Chao Xu a, d, e, f, Fen Liu a, d, e, f, Md. Asraful Alam b, Huanjun Chen a, d, e, f, Yu Zhang a*, Cuiyi Liang a, d, e, f, Huijuan Xu a, Shushi Huang c, Jingliang Xu a, b*, Zhongming Wang a

Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 501640, China

b

School of Chemical Engineering, Zhengzhou University, Zhengzhou 50001, China

c

Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Academy

ro

of

a

-p

of Sciences, Nanning 530007, China

CAS Key Laboratory of Renewable Energy, Guangzhou 501640, China

e

Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou

f

na

501640, China

lP

re

d

University of Chinese Academy of Sciences, Beijing 10049, China

Abstract

Jo ur

* Corresponding Author; E-mail: [email protected], Tel: 86-13580316368; [email protected]

Five sugarcane bagasse lignin samples, namely, dilute sulfuric acid (DSAL), sodium hydroxide (SHL), ethanol (EL), hot liquid water (HLWL)-pretreated residual solids, and raw material (cellulolytic enzyme lignin, CEL), were extracted. Comparative studies on the physicochemical properties of isolated lignin, nonproductive adsorption of cellulase by lignin, and its effect on enzymatic hydrolysis was performed. Results showed that the molecular weight and homogeneity of lignin remarkably decreased after pretreatment compared with CEL. Lignin with low negative zeta potential, high phenolic hydroxyl group content and hydrophobicity exhibited

1 / 43

Journal Pre-proof

strong nonproductive adsorption performance to cellulase. This phenomenon was positively correlated with it’s inhibitory effect on enzymatic hydrolysis. Compared with the control (without lignin), the Avicel conversion rate (40 mg lignin /200 mg Avicel) decreased by 10.74%, 9.28%, 8.73%, 4.22%, and 2.80% after digestion of Avicel for 72 h with the presence of EL, SHL, CEL, HLWL, and DSAL, respectively.

na

Keywords

lP

re

-p

ro

of

Graphical abstract

1. Introduction

Jo ur

Sugarcane bagasse; Lignin; Absorption; Cellulase; Enzymatic hydrolysis

Continued large-scale use of fossil fuels adversely affects the environment. Thus, developing alternative sustainable energy resources is necessary to respond to climate change and ensure sustainable energy security [1]. The second-generation biofuel with lignocellulosic biomass as a substrate is highly anticipated to accomplish the target. Sugarcane is the most important sugar-producing crop that belongs to the Gramineae (Poaceae) family, and its production has steadily increased. By 2014, the annual production of sugarcane has reached 1.88 Gt worldwide [2]. Sugarcane bagasse is the main residual from sugarcane generated in sugar manufacturing industry, and its yield is approximately 30% of the weight of sugarcane [3]. Sugarcane bagasse

2 / 43

Journal Pre-proof

has been applied in many fields, such as fertilizers to cultivate plants, feedstock for papermaking, biosorbents for the removal of heavy metal ions, fuel to produce power, building materials etc. [3-5]. In recent years, increasing interest has been attached to sugarcane bagasse due to feasibility to use as an ideal lignocellulosic biomass source of fermentable sugars to produce various bio-products [3, 6]. The advantages of sugarcane bagasse include (1) low cost for collection and transportation and (2) rich in carbohydrates with cellulose and

of

hemicellulose contents of approximately 40%–45% and 25–30%, respectively [3]. Moreover, (3) enzymatic

ro

saccharification is easier to perform in sugarcane bagasse than both of hardwood and softwood [7].

-p

During the biological conversion of lignocellulosic biomass, enzymatic hydrolysis is a crucial step to produce

re

fermentable sugars for microbial fermentation [7]. Generally, it is difficult to directly release fermentable sugars

lP

from natural lignocellulosic biomass due to its tight structure. In order to increase the digestibility of

na

lignocellulose for enhancing enzymatic hydrolysis efficiency, breaking down the strong cell wall by various is performed which is known as pretreatment [1, 7]. Many pretreatment methods can break the barrier structure of

Jo ur

lignocellulosic biomass to boost enzymatic saccharification. Among them, dilute acid [8, 9], alkali [10], organosolv [11], and hot liquid water [5] pretreatments are popular methods due to the advantages of direct catalyzing ability to cellulose and hemicellulose into fermentable sugars, good delignification performance, pretreatment solvent recyclable, and no chemical reagents addition. Previous studies have demonstrated that these pretreatment methods have different mechanisms in enhancing the digestibility of lignocellulosic biomass. For example, HLWL pretreatment mainly removes hemicellulose to reduce the recalcitrance of biomass [5], dilute acid pretreatment can hydrolyze the carbohydrates and a portion of lignin [3], ethanol pretreatment can deconstruct the β-O-4 linkages and ester bonds of lignin, thus cutting its linkages to cellulose and hemicellulose [12], and alkali pretreatment tends to remove lignin and a portion of hemicellulose [4, 13].

3 / 43

Journal Pre-proof

Lignin is the major non-carbohydrate component of sugarcane bagasse with typical content approximately 18%– 25% [14]. Lignin is a three-dimensional cross-linked complex phenylpropanoid polymer composed of three subunits: guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H), connected by C–C and C–O linkages [7, 15]. Lignin has shown great application potential in many fields [16, 17], however, it generally acts as a major

of

component that hinders the enzymatic saccharification of lignocellulosic biomass in the field of biomass energy

ro

[1, 7]. Generally, the lignin content in lignocellulosic biomass exhibits negative correlation with its digestibility [7]. Therefore, many pretreatment methods focus on the removal of lignin components to enhance the enzymatic

-p

hydrolysis efficiency of lignocellulose. Unfortunately, the pursuing of a high lignin removal rate requiring for

re

high inputs of energy, chemicals, or water, which subsequently causing more carbohydrates losses. Therefore,

lP

controlling lignin content in a reasonable range becomes a consensus for researchers. However, the lignin

na

structure is unstable and easy to be deconstructed and/or removed under the pretreatment conditions such as acid, alkali, high temperature and so on, therefore, the physicochemical properties of lignin in the pretreated residue

Jo ur

are often different from those in the raw material [1]. The main mechanisms of inhibitory effect on enzymatic hydrolysis caused by lignin including act as physical barriers and nonproductive adsorption of enzymes [18, 19]. Particularly, studying the mechanism of non-productive adsorption of cellulase on lignin is of great significance in improving the utilization efficiency of cellulase, the reuse of enzymes, and the high solid enzyme hydrolysis of biomass [7, 8, 13]. The adsorption of cellulase in lignin is associated with hydrophobicity, hydrogen bonding, and electrostatic interactions [1, 7, 20], which would reduce the free enzymes content of the hydrolysis system, thereby reducing the enzymatic saccharification efficiency. Some studies reported that the adsorption capacity of lignin is positively correlated with its phenolic hydroxyl groups content [21, 22]. Lignin with less carboxyl groups content exhibits high hydrophobicity, which would cause more serious non-productive adsorption to

4 / 43

Journal Pre-proof

enzymes [20]. In addition to the physicochemical properties of lignin, the differences in its molecular weight also cause remarkably effect of on enzymatic saccharification [25]. Reports about the effect of the ration of different lignin subunits on its adsorption to cellulase are inconsistent. Some studies observed a positive correlation between the adsorption affinity of lignin to cellulase and its S/G ratio [19, 24], whereas others have reported opposite results [15, 25]. Furthermore, the lignin extracted from organosolv-pretreated hardwood can

of

enhance enzymatic hydrolysis, whereas the lignin prepared from organosolv-pretreated softwood has a

ro

remarkable inhibitory effect [26, 27].

-p

The structure of lignin is complex and has large differences in different species and is prone to diverse changes

re

under different pretreatment conditions. Furthermore, although the physicochemical properties of lignin have

lP

been proven to significantly affect its non-productive adsorption of cellulases, however, the mechanism is still

na

unclear. Therefore, it is of great significance for the efficient utilization of cellulases to study the relationships between the physicochemical properties of lignin and its influence on the enzymatic hydrolysis efficiency of

Jo ur

cellulose. In this work, lignin extracted from dilute acid, alkali, organosolv, and hot liquid water-pretreated sugarcane bagasse and raw material, respectively. Its physicochemical properties were analyzed by gel permeation chromatography (GPC), Fourier-transform infrared (FTIR) spectroscopy, two-dimensional heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance (2D HSQC NMR), thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS). Subsequently, the cellulase adsorption isotherm on different lignin and the relationship between physicochemical properties of lignin and its cellulase adsorption characteristic were analyzed. Moreover, the effects of different lignins on enzymatic hydrolysis of cellulose were investigated.

5 / 43

Journal Pre-proof

2. Materials and method 2.1 Materials Sugarcane bagasse raw material was supplied by Guangxi Fenghao Sugar Co., Ltd. (Chongzuo, China); milled to 0.18–0.425 mm; and dried to constant weight prior to use. Avicel, protease (from Streptomyces), and cellulase (C2730, from Trichoderma reesei ATCC 26921) were purchased from Sigma-Aldrich (St Louis, MO). Toluene,

of

ethanol, dioxane, sodium hydroxide, and glacial acetic acid were provided by Macklin Co., Ltd. (Shanghai,

-p

2.2. Procedures of different pretreatments

ro

China).

re

Dilute sulfuric acid (DSA) pretreatment was performed in accordance with the method of Gabriel et al. [3].

lP

Sugarcane bagasse raw material and 1.09% DSA mixture (solid/liquid ratio 1:2.8) were reacted at 121 °C for 30

na

min. Sodium hydroxide (SH) pretreatment was conducted in 2% (w/v) SH at 80 °C for 2 h, and the solid/liquid ratio was 1:20 [4]. Ethanol (E) pretreatment was conducted by mixing 60% (v/v) E and raw material at a

Jo ur

solid/liquid ratio of 1:10 and reacting the mixture at 160 °C for 45 min [11]. Hot liquid water (HLW) pretreatment was performed at 180 °C for 30 min with a solid/liquid ratio of 1:20 [5]. After each pretreatment, we collected the solid fraction, washed it with tap water (natural pH), and then dried it at 50 °C until constant weight.

2.3. Lignin preparation Cellulolytic enzyme lignin (CEL) was prepared in accordance with the method of Yoo et al. [19]. First, raw material was kept in Soxhlet-extractor with toluene and ethanol mixture (2:1, v/v) for 8 h. The solid fraction was washed and collected and ball-milled with a PMQW planetary ball mill (Nanjing, China) for 6 h. Second, the

6 / 43

Journal Pre-proof

carbohydrates of sugarcane bagasse were thrice removed by an overloading cellulase (40 FPU/ g dry biomass) hydrolysis. Subsequently, the residual enzymes were removed by 1 U/ mL Pronase (Sigma Chemical Company, USA), this experiment was conducted in 50 mM phosphate buffer (pH 7.4) at 37 °C for 24 h, then the Pronase was deactivated at 90 °C for 2 h. Finally, lignin was extracted by 96% (v/v) dioxane (20 mL/ g biomass) at 50 °C for 48 h, and the CEL was obtained after evaporating and freeze-drying the extracting solution. The

of

DSAL, SHL, EL, and HLWL samples were prepared from DSA, SH, E, and HLW pretreated sugarcane bagasse

-p

2.4. Structural characterization of isolated lignin

ro

by using the same methods described above.

re

The FTIR spectra of different lignins from 4000 cm−1 to 800 cm−1 were obtained by FTIR spectroscopy (Bruker,

lP

Germany). The molecular weight of isolated lignin was measured by GPC (Agilent Technologies, Inc., Santa

na

Clara, CA) using tetrahydrofuran as the mobile phase [19, 28]. Approximately 100 mg of different lignins was dissolved in 1 mL of DMSO-d6, and the sample was filtered. Then, the suitable sample was placed in NMR

Jo ur

tubes for 2D 13C–1H HSQC NMR analysis. A Bruker Avance III 400 MHz spectroscope equipped with a 5 mm broadband observe probe (5 mm BBO 400 MHz W1 with Z-gradient probe, Bruker) was used to measure the cross signals of different lignins at 298 K. The working parameters of Bruker standard pulse sequence (“hsqcetgpsi2”) was as follows: spectral width of 11 ppm in F2 ( 1H) with 2048 data points and 190 ppm in F1 (13C) with 256 data points; 128 scans (NS) and 1 s interscan delay (D1). Vario EL Cube (Elementar, Germany) was applied to analyze the elemental components of isolated lignin. The sample was prepared by weighing 5 mg of lignin and encapsulating it in a tin container. TGA of different lignins was performed using a synchronized thermal analysis gas mass spectrometry system (STA449F5-GC7890-5975MS, Agilent Technologies, Inc., Santa Clara, CA). The temperature was set to 30 °C–600 °C at a heating rate of 10 °C/min. N2 was used as the

7 / 43

Journal Pre-proof

protective gas during this process.

2.5. Assessment of cellulase adsorption isotherm on different isolated lignins Cellulase was added to 10 mL of 50 mm sodium acetate buffer (pH 4.8) to increase the protein content from 0.05 mg mL−1 to 2.0 mg mL−1. Then, 0.2 g of different lignins was loaded to conduct lignin adsorption

of

experiment of cellulase, and the adsorption was performed at 4 °C for 3 h to reach equilibrium [19].

ro

Subsequently, the supernatant was collected by centrifuging the mixture at 12,000 rpm for 10 min to measure the protein content. Enzymatic hydrolysis of Avicel was performed in 0.2 M sodium acetate buffer (pH 4.8) at

-p

150 rpm and 50 °C for 72 h. The working volume was set to 5 mL, and 200 mg (4%, w/v) of Avicel and 10 (or

re

40) mg of different lignin (CEL, DSAL, EL, HLWL, or SHL) mixtures were loaded for enzymatic hydrolysis.

lP

The cellulase (C2730, from Trichoderma reesei ATCC 26921, Sigma-Aldrich, Germany) dosage was 10 FPU/g

Jo ur

2.6. Analysis methods

na

Avicel. The samples were collected at 0, 6, 12, 24, 36, and 48 h for the detection of sugars.

The zeta potential of isolated lignin was measured with a Zetasizer Nano ZS and Mastersizer 2000E (Malvern Instruments, England). The Folin–Ciocalteu method was used to estimate the free phenolic groups of different lignins [29]. Free cellulase protein in the supernatant was determined by Bradford assay method using bovine serum albumin as the protein standard [30]. The difference of cellulase content in the supernatant before and after adsorption experiment was calculated as the adsorbed cellulase on lignin. Glucose was quantified by high-performance liquid chromatography in accordance with a previous study [4]. We also determined the chemical components of isolated lignin following the protocol of the National Renewable Energy Laboratory [31]. Surface hydrophobicity of isolated lignin was estimated using the method described by Huang et al. [32].

8 / 43

Journal Pre-proof

3. Results and discussion 3.1. Components and molecular weight analysis of isolated lignins The purity of various samples were evaluated by analyzing their chemical components, and the results are presented in Table 1. More than 93% (w/v) of total lignin content and less than 2% (w/v) of glucan and xylan

of

were obtained in each isolated lignin, suggesting a high purity level. Higher lignin content was achieved in

ro

pretreated preparations than that of CEL possibly because the breakdown of the lignin–carbohydrate complex during pretreatment helps to enhance the digestibility of carbohydrates [33]. The number average molecular

-p

weight (Mn), weight average molecular weight (Mw), and polydispersity index (PDI, Mw/Mn) of different

re

lignin samples are described in Table 1. Compared with those of CEL, both Mn and Mw of pretreated lignin

lP

were reduced, whereas the PDI increased, implying that the pretreatment decreased the uniformity of residual

na

lignin in sugarcane bagasse in terms of molecular weight. For different isolated lignins, the Mn of DSAL was the lowest, followed by SHL, HLWL, EL, and CEL. The PDI of CEL, DSAL, SHL, EL, and HLWL were 1.35,

Jo ur

2.61, 2.03, 1.51, and 1.85, respectively. The difference in the changes of molecular weight of isolated lignins indicated that each pretreatment could have different lignin removal and/or depolymerization mechanisms [19]. Please insert Table 1 here

3.2. Structural characterization of isolated lignin by FTIR analysis The functional groups and bonds of isolated lignins were analyzed by FTIR spectroscopy (Fig. 1), and their relative peak intensities are listed in Table 2. The assignments of major signals were referenced with previous publications [19, 34, 35]. The peak at 3450 cm−1 represents the hydroxyl group, and the peak intensity of each pretreated lignin at this position was reduced compared with that of CEL. This result indicated that all

9 / 43

Journal Pre-proof

pretreatment methods showed the ability to remove hydroxyl groups. The C-H stretching and vibration peaks were observed at 2924, 2852, 1458, and 834 cm−1. These peaks in DSAL and SHL were notably lower than that of CEL, suggesting that both DSAL and SHL pretreatment could significantly break the methyl and/or methylene groups. The unconjugated carbonyl group appeared at 1704 cm−1. Its relative peak intensity hardly changed in EL, but it was significantly reduced in other pretreated lignins compared with CEL. The peaks at

of

1602, 1510, and 1423 cm−1 are attributed to aromatic ring vibration, and the decrease of relative intensity of these peaks in residual lignins indicated that the aromatic ring structures suffered destruction after different

ro

pretreatments. The C–O vibrations in syringyl (S) and guaiacyl (G) rings were assigned at 1330 and 1264 cm −1,

-p

respectively. The relative intensity of these peaks decreased in all pretreated lignins compared with CEL,

re

suggested that the S and G subunits suffered destruction during pretreatment. For pretreated lignin samples, the

lP

relative peak intensity of C–O in DSAL was the lowest, whereas it was the highest in EL. The peak at 1126 cm−1

na

represents the carbon stretching of cellulose contaminants, and the relative peak intensity in the pretreated lignin sample was lower than that of CEL, indicating high purity. This result was consistent with the results of

Jo ur

chemical component analysis of isolated lignins. Moreover, the low peak at 1033 cm −1 in pretreated lignins than CEL demonstrated the removal of C–O–C during different pretreatments. Please insert Fig. 1 here

Please insert Table 2 here

3.3. Structural characterization of isolated lignin by 2D HSQC NMR spectrum analysis In order to obtain deeper understanding the effect of four pretreatment method on the lignin structures, 2D HSQC NMR analysis was performed. Aliphatic regions spectra of different lignins are presented Fig. 2, and

10 / 43

Journal Pre-proof

main 13C–1H cross signals are assigned in Table 3 [36-39]. The predominant signal in all isolated lignins appeared at δC/δH 56.26/3.72 ppm, represents the methoxyl. The α position of β-aryl ether (β-O-4) assigned at δC/δH 72.44/4.91 ppm, signals observed at δC/δH 84.03/4.39 ppm and δC/δH 86.69/4.12 ppm were ascribed to the Cβ-Hβ in β-O-4 linked to G- and S-subunit. All of these signals were disappeared in DSAL and SHL, indicating that the β-O-4 linkage was violently cleaved during dilute sulphuric acid and sodium hydroxide pretreatment process. These peaks could be clearly monitored in EL, implying that ethanol pretreatment can preserve the

of

β-aryl ether. The peak for resinol (β-β) was observed at δC/δH 53.75/3.13, this peak could not be detected in any

ro

pretreated lignin samples. The β-5 linkage was appeared at δC/δH 83.63/4.95 ppm, the signal of this peak could

-p

only be detected in CEL and EL. The peaks for C2-H2, C3-H3 and C4-H4 linkages in β-D-Xylopyranoside (X)

re

were monitored at δC/δH 72.90/3.05 ppm, δC/δH 73.81/3.25 ppm and δC/δH 75.53/3.52 ppm, respectively. All of

lP

these signals were disappeared in DSAL and SHL, only X4 was monitored in HLWL, indicating that the

na

linkages hemicellulose and lignin were remarkably hydrolyzed with acid, alkali and hydrothermal pretreatment,

Jo ur

which are confirmed with the results of chemical components analysis in Table 1. Please insert Fig. 2 here

Aromatic regions spectra of isolated lignins are described in Fig. 3. The peaks for S 2,6 and oxidized S2,6 (S′2,6) were observed at δC/δH 103.96/6.72 and δC/δH 107.23/7.40, the absence of S′2,6 in pretreated lignins suggested the destruction of oxidized (Cα=O) phenolic syringyl units during different pretreatment procedures. The peaks for G2, G5, and G6 units were appeared at δC/δH 11.53/6.99, δC/δH 116.23/6.77, and 119.63/6.80, respectively. The noise level of these peaks in pretreated lignin were obviously reduced compared with CEL. Among them, C5-H5 was the predominant linkage, the signals for C6-H6 were disappeared both in DSAL and SHL. The signal at δC/δH 128.24/7.18 was remarkably lower in pretreated lignins than in CEL, demonstrating the severely

11 / 43

Journal Pre-proof

deconstruction of H2,6 with different pretreatment. In addition, the signals at δC/δH 122.6/7.15, δC/δH 144.7/7.50, δC/δH 130.5/7.35, and δC/δH 113.8/6.29 were ascribed to FA6, pCAα+FAα, pCA2,6, and PCAβ (FA, ferulate substructures; pCA, p-coumaroylated substructures). Volume integration of the signal of FA in pretreated lignins was reduced at varying degrees, the signals for FAα even disappeared in DSAL. FA residues are important cross-linkers between lignin and arabinoxylan polymer chains, and between arabinoxylans [40]. This

of

observation can be attribute to the destruction of lignin–hemicellulose linkages during pretreatment process,

ro

which are in line with the chemical analysis data (Table 1).

-p

Please insert Fig. 3 here

lP

re

Please insert Table 3 here

na

The relative contents of S, H, and G subunits are summarized in Table 4. Compared with G-units, more S-units were deconstructed by DSA, SH, and HLW pretreatment, thus decreasing the S/G ratio in the DSAL, SHL, and

Jo ur

HLWL samples. Nevertheless, the ratio of S/G in EL was the same as that in CEL. In particular, the S/G ratios of DSAL, SHL, HLWL, EL, and CEL were 0.43, 0.18, 0.24, 0.43, and 0.39, respectively. The detected H-unit in pretreated residual lignins was significantly decreased compared with that in CEL. In particular, the H-unit decreased by 16.97%, 12.36%, 3.96%, and 7.56% in DSAL, SHL, EL, and HLWL, respectively. The content of S-unit in residual lignin slightly changed after pretreatment with HLW, whereas the H-unit suffered more serious destruction than the G-unit. Please insert Table 4 here

3.4. Surface properties of lignin preparations 12 / 43

Journal Pre-proof

The elemental compositions of different lignins are presented in Table 5. Oxygen and carbon were the predominant elements (more than 90%) of lignin samples. Compared with CEL, the carbon element content of pretreated lignins was increased. According to the results of component analysis, this phenomenon might be related to the high purity of lignin samples extracted from the pretreated residues. The O/C ratios of DSAL, SHL, EL, and HLWL were 0.34, 0.39, 0.52, and 0.42, which were significantly lower than that of CEL (0.48) (except EL). The oxygen of lignin mainly exists in the forms of free or esterified hydroxyl groups and free or

ro

of

esterified carboxyl groups [42], as well as methoxy and β-O-4 linkages. The decrease in the relative content of oxygen also provided the evidence of alteration of these groups in lignin by pretreatment. The detection of

-p

nitrogen is related to the presence of proteins in the lignin samples. In addition, the protein sources mainly

re

include plant protein of sugarcane bagasse itself and enzyme proteins added during the process of lignin

lP

extraction. The results of elemental analysis showed that the nitrogen content in different samples was extremely

na

low, implying that almost no protein was present in the isolated lignins. Significant differences were observed in the contents of carbon, hydrogen, and oxygen in different lignin samples. This result suggested that the structure

Jo ur

of residual lignin suffered diverse modification after pretreatments.

The phenolic group (Ph-OH) content and zeta potential of isolated lignins are presented in Table 5. The Ph-OH contents of CEL, DSAL, SHL, EL, and HLWL were 0.50, 0.52, 0.43, 0.54, and 0.26 mmol/g, respectively. The Ph-OH groups in EL were similar to those of CEL, whereas the Ph-OH groups in other pretreated lignins increased by 14%–48% compared with those of CEL. This result could be attributed to the cleavage of ether bonds during pretreatment. The functional groups based on the C1s peaks were measured by XPS analysis (Table 5). The quantities of C–C/C–H bonds (284.6 eV) on the surface of CEL and DSAL were 71.58% and 63.66%, respectively, which were lower than that of SHL (74.09%), EL (73.88%), and HLWL (73.98%). The

13 / 43

Journal Pre-proof

abundance of C–O bonds (286.6 eV) for CEL, SHL, EL, and HLWL was 20.49%, 18.06%, 18.34%, and 18.17%, and a remarkably increase was observed in DSAL (29.84%) compared with that in CEL. Compared with CEL, the monitored carbon atoms that bonded with carbonyl or noncarbonyl oxygen (O–C=O, 289 eV) were similar in SHL, EL, and HLWL, whereas they decreased by 18.16% in DSAL.

of

Please insert Table 5 here

ro

3.5. Characterization of thermostability and thermogravimetric properties in lignin Lignin thermo stability was reported to be affected by the composition and content of its functional groups,

-p

branch chains, and condensation degree [43]. TGA results of isolated lignins are presented in Fig. 4. The

re

thermogravimetric performance of lignin preparations is shown in Fig. 4(a), and the weight of all lignin samples

lP

did not change when the temperature was below 150 °C, indicating a limited content of volatile components in

na

different lignins [43]. The temperature for 50% weight loss of CEL, DSAL, SHL, EL, and HLWL occurred at 363 °C, 418 °C, 399 °C, 367 °C, and 407 °C, respectively. The low carbohydrate content and/or high

Jo ur

condensation degree of lignin was reported to increase its thermal stability, therefore enabling it a higher 50% weight loss temperature [44]. The 50% weight loss temperature of pretreated lignin was obviously higher than that of CEL, indicating that the carbohydrate content of pretreated lignin was lower than that of CEL. This result has been verified from the compositional analysis and structural characterization of lignin. Meanwhile, all pretreatments may have led to lignin condensation. The derivative thermogravimetric (DTG) curves of isolated lignins are illustrated in Fig. 4(b). As the temperature increased from 80 °C to 600 °C, a main peak appeared at 355 °C at the DTG curves of all isolated lignins, whereas another peak was observed at 255 °C of the DTG curve of CEL and EL, corresponding to decomposition rates of 0.35 and 0.37%* °C−1. The cleavage of ether linkages (β-O-4) and evaporation of monomer phenol (such as p-coumaric acid) were expected to occur at

14 / 43

Journal Pre-proof

200 °C–350 °C [45]. The DTG curves of CEL and EL showed a main peak in this temperature range, indicating a high content of ether bonds and monomeric phenol. This outcome was consistent with the results of FTIR and 2D HSQC NMR analysis. The main peak of the DTG curve at 355 °C for each lignin could be attributed to the degradation of C–C bonds and aromatic rings [46]. The decomposition rates for CEL, DSAL, SHL, EL, and HLWL were 0.37, 0.33, 0.42, 0.36, and 0.38%* °C−1, respectively. The volatile products of lignin were reported

of

to produce demethoxylation and/or re-condensation reactions between 400 °C and 600 °C [46, 47]. High O/C

ro

ratio in lignin favors the gasification reaction. Otherwise, it tends to carbonize. The residual amount of different lignin samples at 600 °C exhibited the following order: DSAL > HLWL > SHL > EL > CEL. The elemental

-p

composition of isolated lignin samples obtained from TGA results was consistent with elemental analysis

re

results.

na

lP

Please insert Fig. 4 here

3.6. Adsorption isotherms of cellulase by isolated lignin and its effect on enzymatic hydrolysis

Jo ur

Langmuir adsorption isotherms of different isolated lignin samples are presented in Fig. 5, and the related parameters are presented in Table 6. The maximum adsorption capacity (Γ max) of cellulase on lignin refers to the maximum amount of enzyme that can be adsorbed by per unit mass of lignin, reflecting the amount of cellulase adsorbed on lignin during enzymatic hydrolysis to some extent. Γ max of HLWL (30.96 mg cellulase/g lignin) was much lower than that of CEL (40.65 mg cellulase/g lignin), whereas it increased by 65.45%, 16.03%, and 8.5% for DSAL, EL, and SHL, respectively. The adsorption affinity (K) of different lignin samples to cellulase were in the order of EL > CEL > SHL > HLWL > DSAL, which did not show a significant correlation with the maximum adsorption capacity of lignin preparations. Nevertheless, neither the maximum adsorption capacity nor the adsorption affinity can accurately evaluate the adsorption characteristics of cellulase on lignin. Binding

15 / 43

Journal Pre-proof

strength (R, R = Γmax × K) was introduced to estimate the comprehensive effects of maximum adsorption capacity and adsorption affinity of lignin on its adsorption to cellulase [21]. In general, the binding strength reflects the chance of cellulase adsorbed by lignin [19, 42]. The binding strengths of DSAL, SHL, EL, HLWL, and CEL were 108.70, 163.93, 256.41, 125.00, and 156.25 L mg −1, respectively.

of

Please insert Fig. 5 here

ro

Please insert Table 6 here

-p

The effect of adding 5 mg of different lignin samples on the enzymatic hydrolysis of Avicel is illustrated in Fig.

re

6(a). Compared with control (Avicel only), the lignin samples of CEL, DSAL, and HLWL did not cause notable

lP

differences in the digestibility of Avicel–lignin mixture, whereas the glucose yield of Avicel decreased by 7.56%

na

and 5.59% with the addition of EL and SHL. The addition of 20 mg of isolated lignins distinctly decreased the glucose yield at 72 h hydrolysis (Fig. 6(b)). Avicel digestibility decreased by 10.74% and 9.28% with the

Jo ur

addition of EL and SHL, respectively, showing much stronger inhibitory effect on enzymatic hydrolysis than other lignins. The presence of CEL, HLWL, and DSAL reduced the glucose yield in the mixtures by 8.73%, 4.22%, and 2.80%, respectively.

Please insert Fig. 6 here

The surface hydrophobicity of different lignins are presented in Table 7. Similar hydrophobicity was observed in DSAL, HLWL, and CEL compared with CEL. SH pretreatment reduced the hydrophobicity by more than 50%, whereas E pretreatment increased it by 33%, which indicated that the dielectric constant of lignin was differentially changed after pretreatment with SH and E pretreatment. A positive correlation between the

16 / 43

Journal Pre-proof

hydrophobicity of lignin and its inhibitory effect on the hydrolysis efficiency of Avicel enzymes was observed. Some previous studied have reported that the hydrophobicity of pretreated biomass was negative corrected with its enzymatic hydrolysis efficiency [8, 9], which was persistent with the results of this study. Electrostatic force is one of the main factors leading to non-productive adsorption of cellulase by lignin [18]. Zeta potential on the surface of lignin determines its interaction strength with enzymes, in general, the cellulases possess electric

of

charges, so they can be adsorbed/ or repelled by the opposite / or same electric charges on the lignin surface [21].

ro

The zeta potentials of CEL, DSAL, SHL, EL, and HLWL were -11.22, -18.44, -9.79, -9.31, and -12.04 mV, respectively.

re

-p

Please insert Table 7 here

lP

All the lignin preparations used in this study exhibited an inhibitory effect on enzymatic hydrolysis, and this

na

effect was remarkably affected by their physicochemical properties. Studies have found that the physicochemical properties of lignin, such as molecular weight, phenolic group content, zeta potential, and

Jo ur

hydrophobicity, affect its nonproductive adsorption of cellulase [1, 7, 18, 19, 48]. Hydrophobic interactions happen between the aromatic amino acid residues of enzymes and hydrophobic patches on the surface of lignin, which is the main mechanism leading non-productive adsorption between cellulase and lignin. Therefore, the hydrophobic structural of lignin was responsible for its non-productive association with enzymes during enzymatic hydrolysis process [1, 40, 49]. Organosolv lignins from softwood exhibited higher hydrophobicity than from hard wood, DA and HLW pretreatment can cause the degradation products of carbohydrates to form pseudo lignin covering the surface of biomass, which adversely affects the enzymatic hydrolysis of cellulose [26, 27]. Functional groups of lignin such as hydroxyl and carboxyl and amino acid residues of enzymes are charged in an aqueous solution, thereby generating electrostatic interactions. Lignin has a characteristically negative

17 / 43

Journal Pre-proof

charge [50], while the pH of the solution has a remarkably effect on the surface charge of the enzymes. When the pH of the solution is less than the isoelectric point of the enzyme, the enzyme has a positive charge, and conversely, the enzyme has a negative charge, such as the electrical point of β-glucosidase is 5.7–6.4, while the isoelectric points of EG and CBH are less than 5.0, so the electrostatic interaction between EG and CBH and lignin during enzymatic hydrolysis (pH 4.8-5.0) is smaller than that of β-glucosidase [25, 51]. In addition,

of

functional groups containing hydrogen atoms in lignin (especially phenolic hydroxyls of lignin) and enzymes

ro

may cause hydrogen bonding between them. Studies have proven that the addition of surfactants can be reduce the nonproductive adsorption of lignin on cellulase by forming hydrogen bonds with phenolic hydroxyls of

-p

lignin [9, 52]. In general, the lignin that suffered severe destruction will result in small molecular weight and

re

therefore has high specific surface area, therefore providing more enzyme adsorption sites, and causing more

lP

serious adsorption to cellulase. Many studies have reported that the ratio of S/G in lignin is related to its effect

na

on cellulase hydrolysis, unfortunately, these findings are still controversial [15, 19, 25]. The results of this study indicated that the S/G ratio of lignin has no significant correlation with its adsorption to cellulase. A negative

Jo ur

correlation was showed between the molecular weight of lignin and its maximum adsorption capacity. The binding strength of isolated lignin to cellulase was positively affected by the combined effects of its phenolic hydroxyl content, zeta potential, and hydrophobicity. Compared with CEL, HLW and DSA pretreatment attenuated the inhibitory effect of lignin on the digestibility of Avicel, while SH and E pretreatment leads to enhanced inhibition of enzymatic hydrolysis by lignin.

4. Conclusion Relationships between the physicochemical properties of five lignin samples and their effects on enzymatic hydrolysis were studied. Compared with the CEL, a remarkable reduction of molecular weight and S/G ratio

18 / 43

Journal Pre-proof

was observed in pretreated lignin preparations, while their thermal stability were enhanced. The adsorption of cellulase by lignin has no obvious relationship with the above properties of lignin, but exhibited positive correlations with its zeta potential, hydrophobicity, and phenolic hydroxyl content. The digestibility of Avicel was decreased with the presence of all kinds lignins, and their inhibitory effect was as follows: EL > SHL >

of

CEL > HLWL > DSAL.

-p

The authors declare that they have no competing interests.

ro

Conflicts of interest

re

Acknowledgments

lP

This research was financially supported by the National Key Research and Development Program China (grant

na

number 2016YFB0601004), the National Key Research and Development Program China (grant number 2018YFB1501701), the Bureau of International Cooperation, Chinese Academy of Sciences (grant number

2017A010105018).

Jo ur

182344KYSB20160056), and the Science & Technology Project of Guangdong Province (grant number

References [1]

X. Li, Y. Zheng, Lignin-enzyme interaction: Mechanism, mitigation approach, modeling, and research prospects, Biotechnol. Adv. 35 (2017) 466-489. https://doi.org/10.1016/j.biotechadv.2017.03.010.

[2]

FAOSTAT, 2017. FAO Statistical Database. http://faostat3.fao.org (cited 2017 May 22).

[3]

G.J. Betancur, N. Pereira, Sugarcane bagasse as feedstock for second generation ethanol production: Part I: Diluted acid pretreatment optimization, Electron. J. Biotech. 13 (2011) 1-9.

19 / 43

Journal Pre-proof

https://doi.org/10.2225/vol13-issue3-fulltext-3. [4]

Y.Y. Liu, Y. Zhang, J.L. Xu, Y.M. Sun, Z.H. Yuan, J. Xie, Consolidated bioprocess for bioethanol production with alkali-pretreated sugarcane bagasse, Appl. Energ. 157 (2015) 517-522. https://doi.org/10.1016/j.apenergy.2015.05.004.

[5]

H.D. Zhang, S.H. Xu, S.B. Wu, Enhancement of enzymatic saccharification of sugarcane bagasse by liquid

of

hot water pretreatment, Bioresour. Technol. 143 (2013) 391-396.

[6]

ro

https://doi.org/10.1016/j.biortech.2013.05.103.

S. Al Arni, Extraction and isolation methods for lignin separation from sugarcane bagasse: A review, Ind.

A.C. dos Santos, E. Ximenes, Y. Kim, M.R. Ladisch, Lignin–Enzyme Interactions in the Hydrolysis of

re

[7]

-p

Crop. Prod. 115 (2018) 330-339. https://doi.org/ 10.1016/j.indcrop.2018.02.012.

lP

Lignocellulosic Biomass, Trends. Biotechnol. 37 (2018) 518-531.

[8]

na

https://doi.org/10.1016/j.tibtech.2018.10.010.

C.X. Huang, W.Q. Lin, C.H. Lai, X. Li, Y.C. Jin, Q. Yong, Coupling the post-extraction process to remove

Jo ur

residual lignin and alter the recalcitrant structures for improving the enzymatic digestibility of acid-pretreated bamboo residues, Bioresour. Technol. 285 (2019) 121355. https://doi.org/10.1016/j.biortech.2019.121355. [9]

W.Q. Lin, D.F. Chen, Q. Yong, C.X. Huang, S.L. Huang, Improving enzymatic hydrolysis of acid-pretreated bamboo residues using amphiphilic surfactant derived from dehydroabietic acid, Bioresour. Technol. 293 (2019) 122055. https://doi.org/10.1016/j.biortech.2019.122055.

[10]

Y.Y. Liu, J.L. Xu, Y. Zhang, C.Y. Liang, M.C. He, Z.H. Yuan, J. Xie, Reinforced alkali-pretreatment for enhancing enzymatic hydrolysis of sugarcane bagasse, Fuel. Process. Technol. 143 (2016) 1-6. https://doi.org/10.1016/j.fuproc.2015.11.004.

20 / 43

Journal Pre-proof

[11]

Y.C. Park, T.H. Kim, J.S. Kim, Effect of organosolv pretreatment on mechanically pretreated biomass by use of concentrated ethanol as the solvent, Biotechnol. Bioproc. E. 22 (2017) 431-439. https://doi.org/10.1007/s12257-017-0088-1.

[12]

X.B. Zhao, K.K. Cheng, D.H. Liu, Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis, Appl. Microbiol. Biot. 82 (2009) 815-827. https://doi.org/10.1007/s00253-009-1883-1. C. Xu, J. Zhang, Y. Zhang, Y. Guo, H.J. Xu, J.L. Xu, Z.M. Wang, Enhancement of high-solids enzymatic

of

[13]

ro

hydrolysis efficiency of alkali pretreated sugarcane bagasse at low cellulase dosage by fed-batch strategy

https://doi.org/10.1016/j.biortech.2019.121993.

C. Krishnan, L.D. Sousa, M.J. Jin, L.P. Chang, B.E. Dale, Alkali-based AFEX pretreatment for the

re

[14]

-p

based on optimized accessory enzymes and additives. Bioresour. Technol. 292 (2019) 121993.

[15]

na

https://doi.org/10.1002/bit.22824.

lP

conversion of sugarcane bagasse and cane leaf residues to ethanol, Biotechnol. Bioeng. 107 (2010) 441-450.

F.F. Guo, W.J. Shi, W. Sun, X.Z. Li, F.F. Wang, J. Zhao, Y.B. Qu, Differences in the adsorption of

Jo ur

enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism, Biotechnol. Biofuels. 7 (2014) 38. https://doi.org/10.1186/1754-6834-7-38. [16]

M. Culebras, H. Geaney, A. Beaucamp, P. Upadhyaya, E. Dalton, K.M. Ryan, M.N. Collins, Bio-derived carbon nanofibres from lignin as high-performance Li-Ion anode materials. Chemsuschem. (2019). https://doi.org/10.1002/cssc.201901562

[17]

M. Pishnamazi, J. Iqbal, S. Shirazian, G.M. Walker, M.N. Collins, Effect of lignin on the release rate of acetylsalicylic acid tablets, Int. J. Biol. Macromol. 124 (2019) 354-359. https://doi.org/10.1016/j.ijbiomac.2018.11.136

[18]

Q. Yang, X.J. Pan, Correlation between lignin physicochemical properties and inhibition to enzymatic

21 / 43

Journal Pre-proof

hydrolysis of cellulose, Biotechnol. Bioeng. 113 (2016) 1213-1224. https://doi.org/10.1002/bit.25903. [19]

C.G. Yoo, M. Li, X.Z. Meng, Y.Q. Pu, A.J. Ragauskas, Effects of organosolv and ammonia pretreatments on lignin properties and its inhibition for enzymatic hydrolysis, Green. Chem. 19 (2017) 2006-2016. https://doi.org/10.1039/c6gc03627a.

[20]

S. Nakagame, R.P. Chandra, J.F. Kadla, J.N. Saddler, Enhancing the enzymatic hydrolysis of

[21]

ro

Bioeng. 108 (2011) 538-548. https://doi.org/10.1002/bit.22981.

of

lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin, Biotechnol.

S.L. Sun, Y. Huang, R.C. Sun, M.B. Tu, The strong association of condensed phenolic moieties in isolated

-p

lignins with their inhibition of enzymatic hydrolysis, Green. Chem. 18 (2016) 4276-4286.

Z.Y. Yu, K.S. Gwak, T. Treasure, H. Jameel, H.M. Chang, S. Park, Effect of lignin chemistry on the

lP

[22]

re

https://doi.org/10.1039/c6gc00685j.

na

enzymatic hydrolysis of woody biomass, Chemsuschem. 7 (2014) 1942-1950. https://doi.org/10.1002/cssc.201400042.

Y. Li, B.K. Qi, J.Q. Luo, Y.H.Wan, Effect of alkali lignins with different molecular weights from alkali

Jo ur

[23]

pretreated rice straw hydrolyzate on enzymatic hydrolysis, Bioresour. Technol. 200 (2016) 272-278. https://doi.org/10.1016/j.biortech.2015.10.038. [24]

L.P. Tan, W. Sun, X.Z. Li, J. Zhao, Y.B. Qu, Y.M. Choo, S.K. Loh, Bisulfite pretreatment changes the structure and properties of oil palm empty fruit bunch to improve enzymatic hydrolysis and bioethanol production, Biotechnol. J. 10 (2015) 915-925. https://doi.org/10.1002/biot.201400733.

[25]

J.K. Ko, E. Ximenes, Y. Kim, M.R. Ladisch, Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods, Biotechnol. Bioeng. 112 (2015) 447-456. https://doi.org/ 10.1002/bit.25359.

[26]

C.H. Lai, M.B. Tu, M. Li, S.Y. Yu, Remarkable solvent and extractable lignin effects on enzymatic

22 / 43

Journal Pre-proof

digestibility of organosolv pretreated hardwood, Bioresour. Technol. 156 (2014) 92-99. https://doi.org/10.1016/j.biortech.2014.01.030. [27]

C.H. Lai, M.B. Tu, Z.Q. Shi, K. Zheng, L.G. Olmos, S.Y. Yu, Contrasting effects of hardwood and softwood organosolv lignins on enzymatic hydrolysis of lignocellulose, Bioresour. Technol. 163 (2014) 320-327. https://doi.org/10.1016/j.biortech.2014.04.065. R. Kumar, F. Hu, C.A. Hubbell, A.J. Ragauskas, C.E. Wyman, Comparison of laboratory delignification

of

[28]

ro

methods, their selectivity, and impacts on physiochemical characteristics of cellulosic biomass, Bioresour. Technol. 130 (2013) 372-381. https://doi.org/10.1016/j.biortech.2012.12.028. E.A. Ainsworth, K.M. Gillespie, Estimation of total phenolic content and other oxidation substrates in

-p

[29]

M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein

na

[30]

lP

https://doi.org/10.1038/nprot.2007.102.

re

plant tissues using Folin-Ciocalteu reagent, Nat. Protoc. 2 (2007) 875-877.

utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254.

[31]

Jo ur

https://doi.org/10.1016/0003-2697(76)90527-3. A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, D. Crocker, Determination of structural carbohydrates and lignin in biomass, NREL, 2011. https://www.nrel.gov/docs/gen/fy13/42618.pdf. [32]

Y. Huang, S.L. Sun, C. Huang, Q. Yong, T. Elder, M.B. Tu, Stimulation and inhibition of enzymatic hydrolysis by organosolv lignins as determined by zeta potential and hydrophobicity, Biotechnol. Biofuel. 10 (2017) 162. https://doi.org/10.1186/s13068-017-0853-6.

[33]

H.D. Zhang, S.B. Wu, J. Xie, Evaluation of the effects of isolated lignin on enzymatic hydrolysis of cellulose, Enzyme. Microb. Technol. 101 (2017) 44-50. https://doi.org/10.1016/j.enzmictec.2017.03.001.

23 / 43

Journal Pre-proof

[34]

O. Faix, Classification of lignins from different botanical origins by FT-IR spectroscopy, Holzforschung. 45 (1991) 21-27. https://doi.org/10.1515/hfsg.1991.45.s1.21.

[35]

L. Yao, C.G. Yoo, X.Z. Meng, M. Li, Y.Q. Pu, A.J. Ragauskas, H.T. Yang, A structured understanding of cellobiohydrolase I binding to poplar lignin fractions after dilute acid pretreatment, Biotechnol. Biofuel. 11 (2018) 96. https://doi.org/10.1186/s13068-018-1087-y. C.X. Huang, J. He, L.T. Du, D.Y. Min, Q. Yong, Structural characterization of the lignins from the green

of

[36]

ro

and yellow bamboo of bamboo culm (Phyllostachys pubescens), J. Wood. Chem. Technol. 36 (2016) 157-172. https://doi.org/10.1080/02773813.2015.1104544.

C.X. Huang, X.C. Wang, C. Liang, X. Jiang, G. Yang, J. Xu, Q. Yong, A sustainable process for procuring

-p

[37]

re

biologically active fractions of high-purity xylooligosaccharides and water-soluble lignin from Moso

C.X. Huang, Y. Su, J.H. Shi, C. Yuan, S.C. Zhai, Q. Yong, Revealing the effects of centuries of ageing on

na

[38]

lP

bamboo prehydrolyzate, Biotechnol. Biofuels, 12 (2019) 189. https://doi.org/10.1186/s13068-019-1527-3.

the chemical structural features of lignin in archaeological fir woods, New. J. Chem. 43 (2019) 3520-3528.

[39]

Jo ur

https://doi.org/10.1039/c9nj00026g.

C. Xu,; J. Zhang, Y. Zhang, Y. Guo, H. Xu, C. Liang, Z. Wang, J. Xu, Lignin prepared from different alkaline pretreated sugarcane bagasse and its effect on enzymatic hydrolysis. Int. J. Biol. Macromol. 141 (2019) 484-492. https://doi.org/10.1016/j.ijbiomac.2019.08.263.

[40]

J. He, C.X. Huang, C.H. Lai, C. X. Li, Q. Yong, Elucidation of structure-inhibition relationship of monosaccharides derived pseudo-lignin in enzymatic hydrolysis, Ind. Crop. Prod. 113 (2018) 368-375. https://doi.org/10.1016/j.indcrop.2018.01.046.

[41]

B.Y. Chen, B.C. Zhao, M.F. Li, R.C. Sun, Characterization of lignins isolated with alkali from the hydrothermal or dilute-acid pretreated rapeseed straw during bioethanol production, Int. J. Biol. Macromol.

24 / 43

Journal Pre-proof

106 (2018) 885-892. https://doi.org/10.1016/j.ijbiomac.2017.08.090. [42]

W.J. Ying, Z.J. Shi, H.Y. Yang, G.F. Xu, Z.F. Zheng, J. Yang, Effect of alkaline lignin modification on cellulase–lignin interactions and enzymatic saccharification yield, Biotechnol. Biofuels. 11 (2018) 214. https://doi.org/10.1186/s13068-018-1217-6.

[43]

L. Moghaddam, Z.Y. Zhang, R.M. Wellard, J.P. Bartley, I.M. O'Hara, W.O.S. Doherty, Characterisation of

of

lignins isolated from sugarcane bagasse pretreated with acidified ethylene glycol and ionic liquids,

[44]

ro

Biomass. Bioenerg. 70 (2014) 498-512. https://doi.org/10.1016/j.biombioe.2014.07.030. J.K. Ko, Y. Kim, E. Ximenes, M.R. Ladisch, Effect of liquid hot water pretreatment severity on properties

-p

of hardwood lignin and enzymatic hydrolysis of cellulose, Biotechnol. Bioeng. 112 (2015) 252-262.

re

https://doi.org/10.1002/bit.25349.

lP

[45] T. Faravelli, A. Frassoldati, G. Migliavacca, E. Ranzi, Detailed kinetic modeling of the thermal

na

degradation of lignins, Biomass. Bioenerg. 34 (2010) 290-301. https://doi.org/10.1016/j.biombioe.2009.10.018.

Jo ur

[46] T.T. You, X. Li, R.Z. Wang, X.M. Zhang, F. Xu, Effects of synergistic fungal pretreatment on structure and thermal properties of lignin from corncob, Bioresour. Technol. 272 (2019) 123-129. https://doi.org/10.1016/j.biortech.2018.09.145. [47]

J.L. Wen, B.L. Xue, F. Xu, R.C. Sun, A. Pinkert, Unmasking the structural features and property of lignin from bamboo, Ind. Crop. Prod. 42 (2013) 332-343. 10. https://doi.org/1016/j.indcrop.2012.05.041.

[48]

Y.F. Li, Z.P. Sun, X.Y. Ge, J.H. Zhang, Effects of lignin and surfactant on adsorption and hydrolysis of cellulases on cellulose, Biotechnol. Biofuels. 9 (2016) 20. https://doi.org/10.1186/s13068-016-0434-0.

[49]

F. Hu, S. Jung, A. Ragauskas, Impact of pseudolignin versus dilute acid-pretreated lignin on enzymatic hydrolysis of cellulose, ACS. Sustain. Chem. Eng. 1 (2013) 62-65. https://doi.org/10.1021/sc300032J.

25 / 43

Journal Pre-proof

[50]

D. Dong, A.L. Fricke, B.M. Moudgil, H. Johnson, Electrokinetic study of kraft lignin, Tappi. J. 79 (1996) 191-197.

[51]

C. Qin, K. Clarke, K. Li, Interactive forces between lignin and cellulase as determined by atomic force microscopy, Biotechnol. Biofuels. 7 (2014) 65. https://doi.org/10.1186/1754-6834-7-65. Y. Zhang, X. Xu, Y. Zhang, J. Li, Effect of adding surfactant for transforming lignocellulose into

of

fermentable sugars during biocatalysing, Biotechnol. Bioprocess. Eng. 16 (2011) 930–936.

na

lP

re

-p

ro

https://doi.org/10.1007/s12257-011-0138-z

Jo ur

[52]

26 / 43

Journal Pre-proof

Table 1 Chemical components and GPC analysis of lignin fractions Total lignin (%)

Ash (%)

Glucan (%)

Xylan (%)

Mn

Mw

PDI

CEL

93.73 ± 0.63

1.27 ± 0.21

1.61 ± 0.11

1.96 ± 0.10

6030

8114

1.35

DSAL

97.29 ± 0.62

0.98 ± 0.12

0.22 ± 0.01

0.69 ± 0.05

2142

5591

2.61

SHL

96.17 ± 0.40

0.71 ± 0.06

0.35 ± 0.04

0.19 ± 0.04

3268

6636

2.03

EL

95.05 ± 0.40

0.92 ± 0.06

0.59 ± 0.06

0.72 ± 0.04

4181

6441

1.51

HLWL

95.70 ± 0.46

0.42 ± 0.04

0.78 ± 0.06

1.11 ± 0.06

6557

1.85

of

Samples

Jo ur

na

lP

re

-p

ro

3540

27 / 43

Journal Pre-proof

Wavenumber (cm−1)

CEL

DSAL

SHL

EL

HLWL

O–H

3450

1.00

0.69

0.67

0.73

0.72

C–H stretching

2924

1.00

0.83

0.82

0.84

0.90

C–H stretching

2852

1.00

0.89

0.88

0.92

0.94

C=O in unconjugated ketone

1704

1.00

0.66

0.73

0.86

0.75

Aromatic ring

1602

1.00

0.46

0.61

0.80

0.69

Aromatic ring

1510

1.00

0.49

0.66

0.72

0.61

C–H deformation

1458

1.00

0.59

0.72

0.81

0.74

Aromatic ring

1423

1.00

0.68

0.78

0.88

0.79

C–O vibration in syringyl

1330

1.00

0.65

0.77

0.87

0.79

C–O vibration in guaiacyl

1264

1.00

0.53

0.67

0.83

0.72

1.00

0.69

0.87

0.95

0.95

1033

1.00

0.72

0.78

1.18

0.98

834

1.00

0.81

0.89

1.01

0.95

C–O–C stretching C–H deformation

ro

-p

lP

na

Jo ur

Aromatic C–H deformation in 1126 syringyl

of

Assignment

re

Table 2 Assignments of FTIR spectra and their peak relative intensity in lignin samples

Footnotes: Relative intensity = FT-IR detection result of specific peak of lignin / FT-IR detection result of corresponding peak in CEL [19].

28 / 43

Journal Pre-proof

Table 3 Main 13C–1H cross signals in the HSQC spectrum of different lignin preparations δC/δH (ppm)

Assignment

Bβ

53.75/3.13

Cβ-Hβ in β-β resinol substructures (B)

OMe

56.26/3.72

C-H in methoxyls

Aγ

59.40/3.53

Cγ-Hγ in β-O-4 substructures (A)

A′γ

63.53/4.38

Cγ-Hγ in Cγ-acetylated β-O-4 substructures (A′)

Cγ

63.71/3.72

Cγ-Hγ in phenylcoumaran substructures (C)

Aα

72.44/4.91

Cα-Hα in β-O-4 substructures (A)

X2

72.90/3.05

C2-H2 in β-D-Xylopyranoside (X)

X3

73.81/3.25

C3-H3 in β-D-Xylopyranoside (X)

X4

75.53/3.52

C4-H4 in β-D-Xylopyranoside (X)

B′α

83.63/4.95

Cα-Hα in β-β (B′, tetrahydrofuran)

Aβ(G)

84.03/4.39

Cβ-Hβ in β-O-4 substructures linked to G (A)

Aβ(S)

86.69/4.12

Cβ-Hβ in β-O-4 substructures linked to S (A)

S2,6

103.96/6.72

C2,6-H2,6 in etherified syringyl units (S)

S′2,6

107.23/7.40

C2,6-H2,6 in oxidized (Cα= O) phenolic syringyl units (S′)

G2

111.53/6.99

C2-H2 in guaiacyl units (G)

G5

116.23/6.77

C5-H5 in guaiacyl units (G)

G6

119.63/6.80

C6-H6 in guaiacyl units (G)

FA6

122.6/7.15

C6-H6 in ferulate units (FA)

H2, 6

128.24/7.18

C2,6-H2,6 in H units (H)

pCA2,6

130.5/7.35

C2,6-H2,6 in p-coumaroylated substructures (pCA)

Jo ur

na

lP

re

-p

ro

of

Labels

29 / 43

Journal Pre-proof

144.7/7.50

Cα-Hα in ferulate units (FA)

pCAα

144.7/7.50

Cα-Hα in p-coumaroylated substructures (pCA)

PCAβ

113.8/6.29

Cβ-Hβ in p-coumarate (PCA)

Jo ur

na

lP

re

-p

ro

of

FAα

30 / 43

Journal Pre-proof

Table 4 Components of S, G, H, and S/G ratio of isolated lignins Relative content (%) S/G ratio b

Samples p-Hydroxyphenyl (H)a

CEL

24.00

55.58

20.42

0.43

DSAL

14.66

81.90

3.45

0.18

SHL

17.74

74.19

8.06

0.24

EL

25.32

58.23

16.46

HLWL

24.29

62.86

12.86

of

Guaiacyl (G)a

ro

0.43 0.39

S, H and G expressed as the integration domain of S2, 6, H2,6 and G2. b S/G ratio calculated by the formula: S/G

-p

a

Syringyl (S)a

Jo ur

na

lP

re

ratio = 0.5 IS2, 6/IG2 [36, 41].

31 / 43

Journal Pre-proof

Table 5 Elemental composition and phenolic group content in isolated lignin samples and their functional groups at peaks of C1s Element composition (%) Samples H

O

Surface chemical groups (%)

O/C

groups

C–C/C–H

C–O (286.6, O–C=O

ratio

(mmol/g)

(284.6, eV)

eV)

(289, eV)

N

CEL

61.37

6.69

31.85

0.09

0.52

0.50 ± 0.01

71.58

20.49

7.93

DSAL

69.52

6.72

23.68

0.08

0.34

0.52 ± 0.02

of

C

Phenolic

29.84

6.49

SHL

65.79

6.29

27.81

0.11

0.42

0.43 ± 0.02

74.09

18.06

7.85

EL

62.96

7.05

29.92

0.07

0.48

0.60 ± 0.02

73.88

18.34

7.78

HLWL

67.12

6.52

26.30

0.06

0.39

0.26 ± 0.02

73.98

18.17

7.85

Jo ur

na

lP

re

-p

ro

63.66

32 / 43

Journal Pre-proof

Table 6 Langmuir adsorption isotherm parameters from enzyme adsorption on lignin samples SHL

EL

HLWL

CEL

Γmax (mg g−1)

61.73

40.49

43.29

30.96

37.31

K (mL mg−1)

1.76

4.05

5.92

4.04

4.19

R (L mg−1)

108.70

163.93

256.41

125.00

156.25

R2

0.97

0.96

0.97

0.97

0.97

Jo ur

na

lP

re

-p

ro

of

DSAL

33 / 43

Journal Pre-proof

Table 7 Zeta potential and hydrophobicity in isolated lignin samples DSAL

SHL

EL

HLWL

Zeta potential (−mV) 11.22 ± 0.94 18.44 ± 0.70 9.79 ± 0.28

9.31 ± 0.51

12.04 ± 0.37 11.22 ± 0.94

Hydrophobicity (L/g) 0.92 ± 0.11

1.21 ± 0.02

0.96 ± 0.01

0.90 ± 0.01

0.44 ± 0.01

CEL

0.92 ± 0.11

na

lP

re

-p

ro

of

CEL

Jo ur

Samples

34 / 43

Journal Pre-proof

Figure Legend: Fig. 1. FTIR analysis of isolated lignins. Fig. 2. Aliphatic regions of 2D HSQC NMR spectra from different lignin samples. A/A′, β-O-4 linkages; B/B′, resinol substructures (β-β); C, phenylcoumaran substructures (β-5). Fig. 3. Aromatic region of 2D HSQC NMR spectra from isolated lignins. H, p-hydroxyphenyl units; G, guaiacyl

of

units; S, syringyl units; lignin units, FA, ferulate substructures; pCA, p-coumaroylated substructures.

ro

Fig. 4. TGA (A) and DTG (B) curves of different lignin preparations. Fig. 5. Cellulase adsorption isotherm on different lignin residues.

-p

Fig. 6. Effect of lignin residues on enzymatic hydrolysis of Avicel. (a) 200 mg Avicel + 10 mg lignin; (b) 200

Jo ur

na

lP

re

mg Avicel + 40 mg lignin.

35 / 43

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Fig. 1.

36 / 43

re

-p

ro

of

Journal Pre-proof

Jo ur

na

lP

Fig. 2.

37 / 43

re

-p

ro

of

Journal Pre-proof

Jo ur

na

lP

Fig. 3.

38 / 43

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Fig. 4.

39 / 43

Jo ur

na

lP

re

-p

Fig. 5.

ro

of

Journal Pre-proof

40 / 43

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Fig. 6.

41 / 43

Journal Pre-proof

Authors Any author who has contributed to this paper is listed as a co-author, and all authors of this paper have no objection to this.

Conflicts of interest

ro

of

The authors declare that they have no competing interests.

Acknowledgments

-p

This research was financially supported by the National Key Research and Development Program China (grant

re

number 2016YFB0601004), the National Key Research and Development Program China (grant number

lP

2018YFB1501701), the Bureau of International Cooperation, Chinese Academy of Sciences (grant number

Jo ur

2017A010105018).

na

182344KYSB20160056), and the Science & Technology Project of Guangdong Province (grant number

42 / 43

Journal Pre-proof

Highlights

Analyzed the physicochemical properties of lignin samples.



Lignin structure altered differentially at different pretreatment conditions.



Lignin with high hydrophobicity and zeta potential causes more adsorption.



The digestibility of cellulose was inhibited at the presence of lignin.

Jo ur

na

lP

re

-p

ro

of



43 / 43