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Insights of ethanol organosolv pretreatment on lignin properties of Broussonetia papyrifera Lan Yao, Congxin Chen, Chang Geun Yoo, Xianzhi Meng, Mi Li, Yunqiao Pu, Arthur Jonas Ragauskas, Chengyu Dong, and Haitao Yang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b03290 • Publication Date (Web): 11 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

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ACS Sustainable Chemistry & Engineering

Insights of ethanol organosolv pretreatment on lignin properties of Broussonetia papyrifera

Lan Yao †, ‡,§,1, Congxin Chen †,1, Chang Geun Yoo Yunqiao Pu

∥

, Arthur J. Ragauskas §,

∥

, Xianzhi Meng §, Mi Li

∥

,

∥ ,⊥

, Chengyu Dong #, Haitao Yang *,†,‡

†

Hubei Provincial Key Laboratory of Green Materials for Light Industry, Hubei University of Technology, 28th of Nanli Road, Wuhan 430068, China ‡ Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education/Shandong Province, Qilu University of Technology, Jinan, 250353, China § Department of Chemical and Biomolecular Engineering, University of Tennessee Knoxville, Knoxville, TN 37996-2200, USA ∥ Joint Institute for Biological Sciences, Biosciences Division, Oak Ridge National Laboratory (ORNL), Oak Ridge, TN 37831, USA ⊥ Department of Forestry, Wildlife and Fisheries, Center for Renewable Carbon, University of Tennessee Knoxville, Institute of Agriculture, Knoxville, TN 37996-2200, USA # Department of Civil and Environmental Engineering, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region 1

The two authors contribute the same to the paper. Corresponding Author * Haitao Yang. Tel: +86 18607128211; E-mail address: [email protected].

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ABSTRACT Delignification

of

Broussonetia

papyrifera

during

ethanol

organosolv

pretreatment was studied. Milled wood lignin (MWL), the ethanol organosolv lignin recovered at 5 min (PL5), 10 min (PL10), and 60 min (PL60) during the pretreatment, and the residual lignin (RL60) after the pretreatment were characterized and compared. GPC results demonstrated that the weight average molecular weights (Mw) of the ethanol organosolv lignin fractions increased as the pretreatment time extended from 5 to 60 min. 31P NMR analysis revealed that the content of aliphatic OH decreased in the following order: RL60 < PL60 < PL5 < PL10 < MWL. HSQC NMR results suggested that the contents of syringyl, guaiacyl, p-hydroxybenzoate unit, phenylcoumaran, and resinol in the solubilized lignin fractions were significantly changed during the pretreatment. In addition, a significant portion of β-O-4 inter-unit linkages of lignin was extensively cleaved during the pretreatment, leading to the formation of stilbene structures in lignin samples such as PL60.

KEYWORDS Delignification, Broussonetia papyrifera, ethanol organosolv pretreatment, lignin characterization


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INTRODUCTION Biomass-derived renewable fuels have been proposed as a possible solution for alternating the current petroleum products and reducing their greenhouse gas emission. In particular, production of bioethanol has been highlighted due to its environment-friendly and renewability profile.1 Broussonetia papyrifera, also known as paper mulberry, is a fast-growing tree mainly distributed in Asia and Pacific countries. It has been utilized for many applications. For instance, its inner bark and leaves are used for manufacturing paper and animal feed, respectively. In addition, the roots and fruits are used in traditional Chinese medicines.2 Although the cellulose-rich stem part has a great potential as bioenergy feedstock, its applications are still understudied. Pretreatment is an essential step in the biological conversion process for bioethanol production. It reduces biomass recalcitrance and improves the efficiency of the subsequent biological conversion steps by altering several features of the plant cell wall such as removing and/or redistributing hemicellulose/lignin, disrupting the ultrastructure of cellulose and increasing the accessible surface area of cellulose.3 Among various biomass pretreatment methods, organosolv pretreatment is a well-known delignification method using various organic solvents. It substantially solubilizes lignin, results in a cellulose-rich solid residual with the enhanced reactivity toward enzymes. In a typical acid-catalyzed organosolv pretreatment, delignification is promoted by the cleavage of β-aryl ether bonds via either acidolysis or homolysis.4 The



beneficial effect of organosolv pretreatment on the subsequent enzymatic hydrolysis stage has been illustrated in several studies.5-7 Ethanol, one of the cheapest and readily available green solvents that can be produced from biomass, has been widely applied to organosolv pretreatment,8 due to its low boiling point, ease of recovery, and the ability to generate sulfur-free technical lignins.

9-11

From the perspective of biorefinery, lignin is a key feedstock component

that needs to be valorized in order to accomplish a total biomass utilization.12 As a prerequisite to identifing the viable opportunities for lignin valorization, the structural features of lignin solubilized during the pretreatment and retained in the pretreated residue need to be fully understood. Lignin is an important byproduct to improve the economic feasibility of cellulosic ethanol.13 It is well known that organosolv pretreatment dissolves a significant fraction of lignins in the biomass, and these lignins are known as organosolv lignin (OSL). They show great potential in multiple applications. For instance, esterification of OSL with anhydrides could obtain polycaprolactone (PCL) with the enhanced miscibility.14 The antioxidant property of OSL has also been studied extensively. 15,16 While many studies have reported structural characterization of OSL,17-19 most of these studies were either conducted on the basis of comparison of the pretreated and untreated biomass with a fixed pretreatment severity or only focused on the structural information of one individual lignin characteristic. Observation of lignin modification during the organosolv pretreatment cannot only provide insights into the mechanism of lignin solubilization but also provide guidelines on how to utilize these lignins to provide


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value-added products. Therefore, it is of vital importance to illustrate the structural characteristics changes of OSL during organosolv pretreatment in order to improve lignin valorization. In this study, structural changes of lignin during ethanol organosolv pretreatment of Broussonetia papyrifera were studied. This was achieved by recovering and characterizing lignins at different time periods of ethanol organosolv pretreatment. Previous studies indicated that delignification effectively occurred during the first few minutes of ethanol organosolv pretreatment.20,21 Our preliminary studies have found that 60 min was the optimal pretreatment time in terms of glucan digestibility. Hence, 5, 10 and 60 minutes were chosen as the pretreatment time in the present study. Residual lignin after pretreatment was also isolated and characterized for understanding its physicochemical properties. Ethanol-soluble lignins and residual lignin were investigated using FT-IR, GPC,

31

P and HSQC NMR techniques. The structural

changes of lignin during the ethanol organosolv pretreatment can provide valuable information to elucidate the pretreatment delignification process, and more importantly, to develop strategies for the valorization of organosolv lignin.

MATERIALS AND METHODS Materials. Samples of Broussonetia papyrifera were harvested from Enshi, Hubei province, China in 2013. All the chemicals used in this research were purchased from Fisher Scientific (USA). The chemical composition of the extractive-free Broussonetia papyrifera analyzed by NREL procedure is 13.28 % of Klason lignin, 39.88 % of



glucan, 28.28 % of xylan and 2.13 % of ash.22 Ethanol organosolv pretreatment. Broussonetia papyrifera was debarked, Wiley-milled (screen set to 2 mm) and soxhlet-extracted by toluene/ethanol (2:1, v/v) for 8 h. The extractive-free materials were air-dried and loaded into a 4560 Parr 1L pressure reactor (Parr Instrument Company) and pretreated under the following conditions: 50% ethanol/water (v/v) solution and 0.9% sulfuric acid (wt.%) with 10 wt. % solid loading (dry basis). The pretreatment was conducted at 160 ± 2 °C for 5, 10 and 60 min (±0.5 min) individually. The pretreated slurry was filtered to collect the liquid and solid residue. The solid was washed using 200 mL of ethanol/water (1:1, v/v) 3 times followed by washing with an excess amount of deionized water until the effluent became neutral. The residual solid was air-dried at room temperature for 24 h. Lignin isolation and purification. The solubilized lignin fractions during the ethanol organosolv pretreatment were isolated and purified as described in Fig. 1. The crude lignin was obtained from the liquid fractions by rotary-evaporating at 30 °C under reduced pressure and freeze-drying. This crude lignin was further purified according to the purification method of milled wood lignin (MWL).23 In brief, the lignin was dissolved in acetic acid-water (9:1, v/v) and precipitated in deionized water. The recovered solid was dried, dissolved in 1,2-dichloroethane/ethanol (2:1, v/v) and precipitated in diethyl ether. The precipitated lignins (PL5, PL10 and PL60 for 5 min, 10 min and 60 min, respectively) were recovered by centrifugation, washed with petroleum ether, dried under vacuum at 40 °C for overnight, and finally stored in a desiccator over P2O5 until further analysis.


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The residual lignin (RL60) was isolated by enzymatic hydrolysis (100 FPU/g substrate) of 60 min ethanol organosolv pretreated Broussonetia papyrifera for 48 h. During the hydrolysis, fresh buffer and cellulase was used every 24 h. The recovered lignin-enriched residues were extracted by 85% dioxane (98.6 mg of 37% hydrochloric acid were mixed with 100 ml of dioxane-water mixture) for 2 h and washed with acidic 85% dioxane.24 The collected liquid through the extraction and washing processes was neutralized with anhydrous sodium carbonate. The purified residual lignin was precipitated in distilled water with a dilute HCl solution (pH=2), followed by centrifugation and freeze-drying.

Figure 1. Pretreatment and lignin extraction process. Fourier transform infrared (FT-IR) analysis. Structural characteristics of lignin fractions were determined by FT-IR spectroscopy (Spectrum One FTIR system, Perkin Elmer, Wellesley, MA), employing 64 scans with 2 cm-1 resolution from 4000 to 500 cm-1. Lignin molecular weight distribution analysis. Gel permeation chromatography (GPC) analysis was conducted to determine the molecular weights of each lignin



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fraction. Prior to the analysis, lignin samples were acetylated according to a previously described method.25 GPC was performed on an Agilent 1200 HPLC system (Agilent Technologies, Inc., Santa Clara, CA) equipped with Waters Styragel columns (HR1, HR4, and HR5; Waters Corporation, Milford, MA). Nuclear magnetic resonance (NMR) analysis. About 50 mg of lignin samples in DMSO-d6 (0.4 mL) was characterized by two-dimensional (2D) 1H–13C heteronuclear single quantum coherence (HSQC) NMR conducted at 298 K with a Bruker Advance III 400-MHz spectroscopy utilizing a 5-mm Broadband Observe probe (5-mm BBO 400MHz W1 with Z-gradient probe, Bruker). A Bruker standard pulse sequence (‘hsqcetgpsi2') was used, and the spectral width was 11 ppm in F2 (1H) with 2,048 data points and 190 ppm in F1 (13C) with 256 data points (96 scans and 1 s interscan delay). 31

P NMR spectra were acquired after phosphitylation of each lignin fraction with

TMDP

(2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane),

following

the

literature.26 The conditions for 31P NMR spectra were as follows: a 90° pulse angle, 25s pulse delay, and 256 transients at room temperature. Bruker’s TopSpin 3.5 software was used for processing the lignin spectra data.

RESULTS AND DISCUSSIONS Lignin removal during ethanol organosolv pretreatment. As our previous studies reported, ethanol organosolv pretreatment showed effective delignification with woody biomass.27,28 The pretreatment yield and lignin removal of ethanol organosolv pretreated Broussonetia papyrifera at different pretreatment times are presented in


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Table 1. These results indicated that during the first 5 min, 40.29% of lignin was dissolved in the pretreatment liquor, which accounted for more than half of the whole delignification process. About 60.77% of lignin was removed after 10 min of the pretreatment, and this delignification yield increased to 71.50 % by extending the pretreatment time to 60 min. These results indicate that the majority of lignin was solubilized in the first 5 min of pretreatment.

Delignification during the last 50 min

(between 10 to 60 min) was not as efficient as the first 10 min since the easier removed fraction of lignin might already dissolve in the first 10 min. 29

Table1. Pretreatment yield and lignin removal of ethanol organosolv pretreated Broussonetia papyrifera at different times Klason lignin of Pretreatment time

Solid remaining (%)

residual biomass

Lignin removal (%)

(min)

(%) 0

100.00

13.28

-

5

53.55

14.81

40.29

10

51.32

10.15

60.77

60

42.87

8.83

71.50

Structural characterization of lignin by FTIR. FTIR is one of the most common techniques to analyze the structural characteristics of lignin. Fig. 2 presents the FTIR spectra of the three organosolv lignin fractions as well as the residue lignin



and MWL. FTIR adsorption bands from lignin were assigned based on the published studies.30,31 Strong signals at 3420 and 2937 cm-1 were ascribed to the hydroxyl bond (O-H) stretching and C-H stretching vibrations, respectively. Signals assigned to aromatic skeletal vibrations at 1590, 1510 and 1420 cm-1 were also observed.31 The band at 1460 cm-1 was attributed to the C-H asymmetric deformation with aromatic ring vibration. The signals at the wavenumbers of 1330 and 1120 cm-1 were assigned to syringyl units, and those at around 1262 cm-1 belonged to guaiacyl units. The absence of signals at 1168 cm-1 and low intensities of a signal at 833 cm−1 (C-H out of plane in position 2 and 6 of S, all positions of H) also confirmed that the lignin in Broussonetia papyrifera was GS-type.30,32 The bands at 1034 and 833 cm-1 were attributed to aromatic C-H in-plane deformation vibrations and C-H out-of-plane stretching, respectively.


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Figure 2. FT-IR spectra of lignin samples. The relative intensities of various cross peaks, calculated as the ratio of the intensity of the signal to that at 1510 cm−1,31 are shown in Table 2. Lower intensities of the aromatic ring in the organosolv lignin fractions comparing to MWL indicated that some extent of lignin degradation occurred during the ethanol organosolv pretreatment. It was also found that the content of total hydroxyl groups was increased as the pretreatment time extended, suggesting that more hydroxyl groups were formed due to the acid catalyzed cleavage of various ether linkages with the increase of pretreatment time.33 Furthermore, similar observations were also found for signals at 2937 cm-1 from C-H stretching, 1730 cm-1 from unconjugated carbonyl group and 1330 cm-1 from C-O vibration of syringyl. These decreased signal intensities with the increased pretreatment time indicated that lignin degradation is a function of the reaction time.



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Table2. Signal assignment and relative intensities in FTIR spectra of lignin samples. Assignment 1 2 3 4 5 6 7 8 9 10 11 12 13

Hydroxyl group C-H stretching Unconjugated carbonyl group Aromatic ring Aromatic ring C-H deformation Aromatic ring C-O vibration of syringyl Guaiacyl C-O units C-O vibration of guaiacyl Aromatic C-H deformation in syringyl C-O-C stretching Aromatic C-H deformation out of plane

Wavenumber cm-1

MWL

PL5

PL10

PL60

RL60

3420

1.40

0.74

0.89

0.95

0.93

2937

0.82

0.68

0.69

0.79

0.87

1730

0.51

0.31

0.33

0.46

0.84

1590 1510

0.91 1.00

0.82 1.00

0.81 1.00

0.88 1.00

0.92 1.00

1460

1.01

0.94

0.94

0.97

1.02

1420

0.90

0.82

0.83

0.90

0.93

1330

0.82

0.79

0.80

0.88

0.93

1262

1.00

0.92

0.91

0.92

0.98

1225

1.10

0.97

0.97

0.99

1.06

1120

1.37

1.08

1.07

1.06

1.10

1034

1.12

0.88

0.87

0.92

0.99

833

0.34

0.34

0.33

0.40

0.47

Note. The relative intensity was calculated as the ratio of the intensity of the signal to

the intensity of the band at 1,510 cm−1. Molecular weight distributions of lignin by GPC. The molecular weight analysis of lignin reflects the variations in depolymerization/repolymerization reactions occurring during the ethanol organosolv pretreatment. The results of weight-average (Mw), number-average molecular weights (Mn) and polydispersity of lignin samples are


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summarized in Table 3. The Mw of MWL from untreated Broussonetia papyrifera, PL5, PL10, PL60, and RL60 were 12581, 8567, 9229, 13882 and 23027 g/mol, respectively. Both PL60 and RL60 had higher Mw compared with MWL. It is possibly due to the lignin repolymerization during ethanol organosolv pretreatment. A previous study reported that lignin inter-unit linkages were extensively cleaved during organosolv pretreatment and lignin degradation was a function of the reaction time.34 It is well known that both depolymerization and repolymerization of lignin occurs during an acid-catalyzed organosolv pretreatment process. 31 The increased Mw of the organosolv lignin (PLs) with pretreatment time indicated that some repolymerization reactions probably occurred. The polydispersity of the dissolved lignin fractions ( PL10 (0.54 mmol/g) > PL60 (0.47 mmol/g) > RL60 (0.32 mmol/g). For p-hydroxybenzoate and carboxylic acid hydroxyl groups, there were no significant differences in the different lignin fractions. The results indicated that due to the acid-catalyzed cleavage of various ether linkages, more phenolic hydroxyl groups were formed during the pretreatment. The decrease of aliphatic OH group could be due to the loss of the γ-methylol group as formaldehyde and the OH groups on Cα,β to form stilbene structures.20 More phenolic OH groups is a desired feature for potentially using ethanol organosolv lignin as antioxidant, was maximized in PL10 for this study.


16

which

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7 OH (mmol/g lignin)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6

MWL

5

PL5

4

PL10

3

PL60

2

RL60

1 0

Figure. 3 31P-NMR spectra analysis results of lignin fractions from Broussonetia papyrifera.

2D NMR analysis. HSQC NMR analysis was employed to acquire additional structural information of the lignin fractions from the ethanol organosolv pretreatment of Broussonetia papyrifera. The HSQC spectra were shown in the Fig. S1. The aliphatic and aromatic spectral data for each lignin fraction were analyzed according to the literature. 36-38 The aromatic region offers important information about lignin monolignol compositions. The cross peaks for S2/6 was present at δc/δH 104.6/6.67 ppm. The C-H correlations from guaiacyl subunits were observed at 111.9/6.91, 115.7/6.93 and 119.7/6.75 ppm for G2, G5, and G6, respectively. Furthermore, cross signals from p-hydroxyphenyl benzoate unit were clearly found at δc/δH 116.2/6.76 ppm (PB3/5) and δc/δH 130.5/7.42 ppm (PB2/6). The semi-quantitative analysis of HSQC NMR spectra is shown in Table 4. The relative contents of lignin S units were similar in PL5, PL10,



and PL60, which were lower than that in MWL and RL60. The relative amounts of lignin G units in the organosolv lignin fractions were higher than that in MWL and RL60, indicating that more G units were removed than S units during acid-catalyzed ethanol pretreatment. As a result, the S/G ratio of RL60 was much higher than the other lignin fractions. Most of the PB units were progressively removed during the pretreatment process. Thus only a small quantity of PB unit remained in the RL60. In addition, a signal at δC/δH 127.9/7.01 ppm attributed to the Cα/β/Hα/β of the stilbenes units was observed only in PL60. 39 Similar observations of the stilbenes formation during organosolv pretreatments were also reported in previous studies. 10,19,20 In the aliphatic region of the lignin HSQC NMR spectra, the predominant signals indicated the presence of the methoxyl and major interunit linkages including β-O-4' aryl ether (A), phenylcoumaran (B) and resinol (C). The C–H correlations in β-O-4' substructure were observed from α, β and γ positions at around δc/δH 72.4/4.84 ppm, δc/δH 86.4/4.05 ppm (β position of β-O-4' linked to S unit) and δc/δH 84.1/4.24 ppm (β position of β-O-4' linked to a G unit), 60.3/3.56 ppm, respectively. Phenylcoumaran was well resolved by C–H correlations at δc/δH 87.5/5.38, δc/δH 53.5/3.45 and δc/δH 63.2/3.68 ppm from Cα–Hα, Cβ–Hβ and Cγ–Hγ, respectively. The presence of resinol substructure was confirmed by Cα–Hα correlations at around δc/δH 85.5/4.63 ppm, Cβ– Hβ correlations at around 54.0/3.03 ppm, Cγ–Hγ correlations at around δc/δH 71.0/3.99 ppm and δc/δH 71.6/4.15 ppm. The existence of a signal at δc/δH 64.2/3.30 ppm in all solubilized lignin fractions is ascribed to the methylene in α-ethoxylated β-O-4 (A’),20 suggesting the occurrence of α-ethoxylation during the pretreatment process. The


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cleavage of interunit linkages during ethanol organosolv pretreatment was observed clearly in this study. The content of total lignin inter-unit linkages including β-O-4, β-β, and β-5 linkages over total lignin aromatic subunits (Ar %) in the RL60 was 54.5 % which was lower than MWL (83.6 %). The HSQC analysis of MWL revealed that β-O-4' was the main lignin inter-unit linkages in Broussonetia papyrifera, followed by β-β (11.6 %) and β-5 (4.1 %). The content of total lignin inter-unit linkages over total lignin aromatic subunits (Ar %) in PL5, PL10 and PL60 were 65.7 %, 66.9 %, and 51.6 %, respectively. During the pretreatment process, the aryl ether linkages in PL5, PL10, and PL60 were 50.5 %, 51.1 % and 36.7 % of the total lignin aromatic subunits (Ar %), respectively. These results are consistent with the results in the literature,4 which indicated that the cleavage of β-aryl ether linkages occurred mostly in the early stage of organosolv pretreatment. Although the content of β-O-4' linkage was significantly reduced after the pretreatment, some linkages remained in the RL60. The relative contents of phenylcoumaran and resinol units were increased in organosolv lignin fractions with the increased pretreatment time. Overall, it was found that β-O-4' linkages were significantly cleaved during the ethanol organosolv pretreatment process, in particular, in the first 10 min. More lignin G units were removed than lignin S units during ethanol organosolv pretreatment.

Table4.Quantitative information of three lignin samples in the HSQC spectra. Lignin substructure

MWL %a

PL5

%b

%a

PL10 %b

%a

%b


PL60 %a

%b

RL60 %a

%b


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S

50.4

-

42.5

-

45.7

-

44.8

-

66.7

-

G

49.6

-

57.5

-

54.3

-

55.2

-

33.3

-

PB

4.9

-

4.0

-

4.1

-

3.5

-

1.7

-

S/G

1.0

0.7

-

0.8

-

0.8

-

2.0

β-O-4

70.5

84.3

50.5

76.8

51.1

76.9

36.7

71.0

41.0

75.2

phenylcoumaran

3.4

4.1

3.7

5.6

4.7

7.0

4.7

9.1

2.7

4.9

resinols

9.7

11.6

11.5

17.6

10.7

16.1

10.2

19.9

10.8

19.9

stilbenes

0

0

0

0

0

0

1.3

2.4

0

0

a

Amount of specific functional group was expressed as a percentage of S+G.

b

Amount of specific functional group was expressed as a fraction of β-O-4 + β-5 + β-β

The aforementioned results suggest that characteristics of lignin vary depending on the reaction conditions, and this information can be a clue to developing lignin valorization strategies. For example, PL10 exhibiting the most phenolic OH groups would be an attractive material for polyurethanes synthesis, whereas PL60 containing stilbenes is applicable to the future approaches for plant disease resistance and human health. 40 CONCLUSION FT-IR, GPC and NMR techniques elucidated the structural characteristics of different lignin fractions formed during ethanol organosolv pretreatment of Broussonetia papyrifera techniques. Deconstruction of lignin was observed during ethanol organosolv pretreatment, causing different structural characteristics in recovered lignin fractions. Among the lignin fractions, PL60 had the highest weight


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average molecular weight. PL10 showed the highest aliphatic OH and phenolic hydroxyls content. Different contents of S unit, G unit, PB unit, β-O-4 and phenylcoumaran were observed in each removed lignin fractions. Stilbenes units were formed during the pretreatment process but only presented in PL60. Further lignin valorization strategies can vary depending on the structural properties of lignins generated at different pretreatment time. AUTHOR INFORMATION Corresponding Author *

Haitao Yang. Tel: +86 18607128211; E-mail address: [email protected].

Author Contributions The manuscript was prepared through contributions of all authors. All authors have approved the final version of the manuscript. Notes The authors declare that they have no competing financial interests. ACKNOWLEDGMENTS The authors are grateful for the support by the National Natural Science Foundation of China (No. 31500496), China Scholarship Council (No. 2011842330 and No. 201508420257), Key project of Hubei Provincial Department of Education (No. D20161402) and Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education of China (NO. KF-201719 and NO. KF201611)

REFERENCES



(1) Ragauskas, A.; Williams, C.; Davison, B.; Britovsek, G.; Cairney, J.; Eckert, C.; Frederick, W.; Hallett, J.; Leak, D.; Liotta, C.; Mielenz, J.; Murphy, R.; Templer, R.; Tschaplinski, T. The Path Forward for Biofuels and Biomaterials. Science 2006, 311, 484-489, DOI 10.1126/science.1114736. (2) Zheng, Z; Cheng, K; Chao, J.; Wu, J.; Wang, M. Tyrosinase inhibitors from paper mulberry (Broussonetia papyrifera). Food Chem. 2008, 106, 529-535, DOI 10.1016/j.foodchem.2007.06.037. (3) Pu, Y.; Zhang, D.; Singh, P.; Ragauskas, A. The new forestry biofuels sector. Biofuels Bioprod. Biorefin. 2008, 2, 58-73, DOI 10.1002/bbb.48. (4) Kangas, H.; Liitiä, T.; Rovio, S.; Ohra-aho, T.; Heikkinen, H.; Tamminen, T.; Poppius-Levlin, K. Characterization of dissolved lignins from acetic acid Lignofibre (LGF) organosolv pulping and discussion of its delignification mechanisms. Holzforschung 2015, 69, 247-256, DOI 10.1515/hf-2014-0070. (5) Jang, S.; Kim, H.; Jeong, H.; Kim, J.; Yeo, H.; Choi, I. Effect of ethanol organosolv pretreatment factors on enzymatic digestibility and ethanol organosolv lignin structure from Liriodendron tulipifera in specific combined severity factors. Renew. Energ. 2016, 87, 599-606, DOI 10.1016/j.renene.2015.10.045. (6) Pan, X.; Gilkes, N.; Kadla, J.; Pye, K.; Saka, S.; Gregg, D.; Ehara, K.; Xie, D.; Lam, D.; Saddler, J. Bioconversion of hybrid poplar to ethanol and co-products using an organosolv fractionation process: optimization of process yields. Biotechnol. Bioeng. 2006, 94, 851–861. DOI 10.1002/bit.20905. (7) Pan, X.; Xie, D.; Yu, W.; Lam, D.; Saddler, N. Pretreatment of lodgepole pine killed by mountain pine beetle using the ethanol organosolv process: fractionation and process optimization. Ind. Eng. Chem. Res. 2007, 46, 2609–2617, DOI 10.1021/ie061576l. (8) Arato, C.; Pye, K.; Gjennestad, G. The Lignol Approach to Biorefining of Woody Biomass to Produce Ethanol and Chemicals. Appl. Biochem. Biotech.2005, 123,(1-3) ,– 882, DOI 10.1385/ABAB:123:1-3:0871. (9) Zhao, X.;Cheng, K.; Liu, D. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Appl. Microbiol. Biotechnol. 2009, 82, 815–827, DOI 10.1007/s00253-009-1883-1. (10) Hallac, B.; Pu, Y.; Ragauskas, J. Chemical Transformations of Buddleja davidii Lignin during Ethanol Organosolv Pretreatment. Energ Fuel 2010, 24, 2723–2732, DOI 10.1021/ef901556u. (11) Zhang, Z.; Harrison, D.; Rackemann, W.; Doherty, O.; O'Hara, M. Organosolv Pretreatment of Plant Biomass for Enhanced Enzymatic Saccharification. Green Chem. 2012, 47 (12), 360-381, DOI 10.1039/C5GC02034D. (12) Kautto, J.; Realff, M. J.; Ragauskas, A. J.; Kassi, T. Economic analysis of an organosolv process for bioethanol production. BioResources 2014, 9 (4) 6041-6072, DOI 10.15376/biores.9.4.6041-6072. (13) Ragauskas, A.; Beckham, G.; Biddy, M.; Chandra, R.; Chen, F.; Davis, M.; et al. Lignin valorization: improving lignin processing in the biorefinery. Science 2014, 344, 709–718, DOI 10.1126/science.1246843. (14) Teramoto, Y.; Lee, S.; Endo, T. Molecular composite of lignin: Miscibility and


Page 20 of 23

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


complex formation of organosolv lignin and its acetates with synthetic polymers containing vinyl pyrrolidone and/or vinyl acetate units. J. Appl. Polym. Sci. 2012,125, 2063-2070, DOI 10.1002/app.36294. (15)García, A.; Spigno, G.; Labidi, J. Antioxidant and biocide behavior of lignin fractions from apple tree pruning residues. Ind Crop Prod. 2017,104, 242–252, DOI 10.1016/j.indcrop.2017.04.063. (16) Sadeghifar, H.; Wells, T.; Le, R.; Sadeghifar, F.; Yuan, J.; Ragauskas, A. Fractionation of Organosolv Lignin Using Acetone: Water and Properties of the Obtained Fractions. ACS Sustainable Chem. Eng. 2017, 5, 580-587, DOI 10.1021/acssuschemeng.6b01955. (17) Hu, G.; Cateto, C.; Pu, Y.; Samuel, R.; Ragauskas A. Structural Characterization of Switchgrass Lignin after Ethanol Organosolv Pretreatment. Energ Fuel 2013, 26 (1), 740-745, DOI 10.1021/ef201477p. (18) Bai, Y.; Xiao, L.; Shi, Z.; Sun, R. Structural Variation of Bamboo Lignin before and after Ethanol Organosolv Pretreatment. Int. J. Mol. Sci. 2013, 14, 21394-21413, DOI 10.3390/ijms141121394. (19) Zhu, M; Wen, J; Su, Y; Wei, Q; Sun, R. Effect of structural changes of lignin during the autohydrolysis and organosolv pretreatment on Eucommia ulmoides Oliver for an effective enzymatic hydrolysis. Bioresour. Technol. 2015, 185, 378–385, DOI 10.1016/j.biortech.2015.02.061. (20) Guo, Y.; Zhou, J.; Wen, J.; Sun, G.; Sun, Y. Structural transformations of triploid of Populus tomentosa Carr. Lignin during auto-catalyzed ethanol organosolv pretreatment. Ind Crop Prod 2015, 76, 522–529, DOI 10.1016/j.indcrop.2015.06.020. (21) Jang, S.; Kim, H.; Jeong, H.; Kim, J.; Yeo, H.; Choi, I. Effect of ethanol organosolv pretreatment factors on enzymatic digestibility and ethanol organosolv lignin structure from Liriodendron tulipifera in specific combined severity factors. Renew Energ 2016, 87, 599-606, DOI 10.1016/j.renene.2015.10.045. (22) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker D. Determination of structural carbohydrates and lignin in biomass. 2008. Technical Report No. TP-510–42618. Golden, CO: National Renewable Energy Laboratory. (23) Björkman, A. Studies on finely divided wood. Part 1.-Extraction of lignin with neutral solvent. Sven.Papperstidn 1956, 59, 477-485. (24) Wu, S.;Argyropoulos, D. An improved method for isolating lignin in high yield and purity. J Pulp. Pap. Sci. 2003, 29, 235-240. (25) Kumar, R.; Hu,F.; Hubbell, A.; Ragauskas, A.; Wyman,C. Comparison of laboratory delignification methods, their selectivity, and impacts on physiochemical characteristics of cellulosic biomass. Bioresour. Technol. 2013, 130, 372-381, DOI 10.1016/j.biortech.2012.12.028. (26) Pu, Y.; Cao, S.; Ragauskas, A. Application of quantitative 31P NMR in biomass lignin and biofuel precursors characterization. Energy Environ. Sci. 2011, 4, 3154-3166, DOI 10.1039/c1ee01201k. (27) Yoo, C.; Li, M.; Meng, X.; Pu, Y.; Ragauskas, A. Effects of organosolv and ammonia pretreatments on lignin properties and its inhibition for enzymatic hydrolysis. Green Chem. 2017, 19(8), 2006-2016, DOI 10.1039/C6GC03627A.



(28) Tolbert, A.; Yoo, C.; Ragauskas, A. Understanding the Changes to Biomass Surface Characteristics after Ammonia and Organosolv Pretreatments using ToF-SIMS. ChemPlusChem 2017, 82 (5), 1-6, DOI 10.1002/cplu.201700138. (29) Oliet, M.; Rodríguez, F.; Santos, A.;Gilarranz, A.; García-Ochoa, F.; Tijero, J. Organosolv Delignification of Eucalyptus globulus: Kinetic Study of Autocatalyzed Ethanol Pulping. Ind. Eng. Chem. Res. 2000, 39(1), 34-39, DOI 10.1021/ie9905005. (30) Faix, O. Classification of lignins from different botanical origins by FT-IR spectroscopy. Holzforschung 1991, 45, 21-27, DOI 10.1515/hfsg.1991.45.s1.21. (31) Guo, F.; Shi, W.; Sun, W.; Li, X.; Wang, F.; Zhao, J.; Qu, Y. Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism. Biotechnol. Biofuels 2014, 7, 38-47, DOI 10.1186/1754-6834-7-38. (32) Wen, J.; Xue, B.; Xu, F.; Sun, R.; Pinkert A. Unmasking the structural features and property of lignin from bamboo. Ind. Crop. Prod. 2013, 42,332–343, DOI 10.1016/j.indcrop.2012.05.041. (33) Pan, X.; Kadla, J. F.; Ehara, K.; Gilkes, N.; Saddler, J. N. Organosolv Ethanol Lignin from Hybrid Poplar as a Radical Scavenger: Relationship between Lignin Structure, Extraction Conditions, and Antioxidant Activity. J. Agric. Food Chem. 2006, 54, 5806-5813, DOI 10.1021/jf0605392. (34) Hage, R.; Brosse, N.; Sannigrahi, P.;Ragauskas, A. Effects of process severity on the chemical structure of Miscanthus ethanol organosolv lignin. Polym. Degrad. Stabil. 2010, 95, 997-1003, DOI 10.1016/j.polymdegradstab.2010.03.012. (35) Pan, X.; Arato, C.; Gilkes, N.; Gregg, D.; Mabee, W.; Pye, K.; Xiao, Z.; Zhang, X.; Saddler, J. Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products. Biotechnol. Bioeng. 2005, 90, 473-481, DOI 10.1002/bit.20453 . (36) Cao, S.; Pu, Y.; Studer, M.; Wyman, C.; Ragauskas, A. Chemical transformations of Populus trichocarpa during dilute acid pretreatment. RSC Advances 2012, 2, 10925-10936, DOI 10.1039/c2ra22045h. (37) Li, H.; Pu, Y.; Kumar, R.; Ragauskas, A.; Wyman, C. Investigation of Lignin Deposition on Cellulose During Hydrothermal Pretreatment, Its Effect on Cellulose Hydrolysis, and Underlying Mechanisms. Biotechnol. Bioeng. 2014, 3, 485-492, DOI 10.1002/bit.25108. (38) Wang, K.; Yang, H.; Yao, X.; Xu, F.; Sun, R. Structural transformation of hemicelluloses and lignin from triploid poplar during acid-pretreatment based biorefinery process. Bioresour. Technol. 2012, 116, 99-106, DOI 10.1016/j.biortech.2012.04.028. (39) Wen, J.; Xue, B.; Sun, S.; Sun, R. Quantitative structural characterization and thermal properties of birch lignins after auto-catalyzed organosolv pretreatment and enzymatic hydrolysis. J. Chem. Technol. Biotechnol. 2013, 88, 1663–1671, DOI 10.1002/jctb.4017. (40) Chong, J.; Poutaraud, A.; Hugueney, P. Metabolism, and roles of stilbenes in plants. Plant Sci. 2009, 177, 143–155, DOI 10.1016/j.plantsci.2009.05.012.


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TABLE OF CONTENT

SYNOPSIS Lignin dissolved at different time during ethanol organosolv pretreatment, which were charaterized by FTIR, GPC and NMR, promote a promising approach for lignin valorization.


Insights of Ethanol Organosolv Pretreatment on ... - ACS Publications

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