Lignin Alkylation Enhances Enzymatic Hydrolysis ... - ACS Publications

Subscriber access provided by University of Florida | Smathers Libraries

Article

Lignin Alkylation Enhances Enzymatic Hydrolysis of Lignocellulosic Biomass Chenhuan Lai, Maobing Tu, Changlei Xia, Zhiqiang Shi, Shao-Long Sun, Qiang Yong, and Shiyuan Yu Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b02405 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 21, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Energy & Fuels is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

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

Energy & Fuels

1

Lignin Alkylation Enhances Enzymatic Hydrolysis

2

of Lignocellulosic Biomass

3

Chenhuan Lai,†,§ Maobing Tu,*,‡,§ Changlei Xia,‡ Zhiqiang Shi,§ Shaolong Sun,‡,┴ Qiang Yong,†

4

and Shiyuan Yu†

5

†

College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, China

6

‡

Department of Chemical and Environmental Engineering, University of Cincinnati, Cincinnati,

7

Ohio 45221, USA

8

§

9

Auburn, Alabama 36849, USA

Forest Products Laboratory and Center for Bioenergy and Bioproducts, Auburn University,

10

┴

11

Guangzhou, Guangdong 510642, China

12

* Corresponding author

13

Email address: [email protected]

14

Fax: +1 513 556 2259; Tel: +1 513 556 4162

College of Natural Resources and Environment, South China Agricultural University,

15 16 1 ACS Paragon Plus Environment

Energy & Fuels

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

Page 2 of 34

1

ABSTRACT: Understanding the interactions between lignin and cellulase will help better

2

design biomass pretreatment and enzymatic hydrolysis processes. Ethanol organosolv lignins

3

(EOLs) were isolated from organosolv pretreatment of sweetgum (SG) and loblolly pine (LP)

4

under different temperature. The structural changes of EOLs were determined by 13C NMR and

5

Heteronuclear multiple bond correlation (HMBC) NMR to elucidate their positive and/or

6

negative effects on enzymatic hydrolysis. Alkylation of lignin with ethanol resulting in the

7

etherification of Cα was observed in both EOL-SG and EOL-LP. The effect of EOL-SG on

8

enzymatic hydrolysis was correlated with the content of ethoxyl group. The lignin alkylation in

9

EOLs appeared to be reversible at high temperature, resulting in the inverse change of their

10

effects on enzymatic hydrolysis. A strong correlation was observed between the binding affinity

11

of cellulases onto EOLs and their corresponding hydrolysis yields. Lignin alkylation likely

12

reduce the affinity of enzyme onto lignin, which can enhance the enzymatic hydrolysis.

13

KEYWORDS: adsorption, biomass, enzymatic hydrolysis, organosolv lignin, pretreatment

14 15 16 17 18 19 20 2 ACS Paragon Plus Environment

Page 3 of 34

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

Energy & Fuels

1

1. INTRODUCTION

2

Lignocellulosic biomass, as a renewable resource, is the most abundantly available feedstock. It

3

has a great potential to produce biofuels, and valuable chemicals.1-4 However, the biorefinery of

4

lignocelluloses is not cost effective, mainly because producing fermentable sugars from

5

lignocellulosic biomass is costly and inefficient. The influential factors affecting the enzymatic

6

digestibility of lignocelluloses have been studied, such as the presence of lignin and

7

hemicellulose in biomass, the degree of polymerization and crystallinity of cellulose, or

8

accessible surface areas.5 Lignin is a crosslinked polymer with different phenylpropane units in

9

biomass, which cements the celluloses in the plant cell wall, and limits the enzyme accessibility,

10

by physically blocking the enzyme attack and non-productive binding of the cellulase enzyme.6-8

11

It has been widely accepted that the lignin in biomass played a negative role in enzymatic

12

hydrolysis and increased the cellulase cost to achieve the high sugar yields.9

13

To understand the mechanism of lignin inhibition, the physicochemical properties and

14

chemical structures of lignin, which might impact the enzymatic hydrolysis, have been examined,

15

including hydrophobicity, surface charges, phenolic hydroxyl group contents, carbonyl group

16

contents, and degree of condensation.10-13 The characteristics of lignin in pretreated biomass

17

varied considerably with the types of biomass and the pretreatment methods. The composition of

18

three phenol building blocks (p-hydroxyphenyl, guaiacyl, and syringyl) differs in the lignin of

19

softwood, hardwood and herbaceous plants.14 More importantly, lignin structures are altered

20

significantly during the thermochemical pretreatment. A decrease in the percentage of β-O-4

21

bonds, but an increase in the percentage of C-C condensed structures were observed in lignins

22

after pretreatment, which indicated that lignin depolymerization and repolymerization reactions

23

occurred simultaneously.15 It has been reported that the more condensed lignin tended to adsorb 3 ACS Paragon Plus Environment

Energy & Fuels

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

Page 4 of 34

1

more cellulases enzyme.16 The phenolic hydroxyl groups in lignin might be the necessary sites

2

for the enzyme nonproductive adsorption and enzyme inactivation.17, 18 By contrast, the

3

carboxylic groups in lignin could enhance the enzymatic hydrolysis of pretreated lignocellulose,

4

by increasing the electrostatic repulsion to enzymes.19 However, a clear understanding of lignin

5

structure-inhibition/stimulation relationship on enzymatic hydrolysis is lacking.6, 20, 21

6

Organosolv pretreatment is one the promising pretreatment processes for softwood and

7

hardwood, which can enhance the substrates hydrolyzability and produce high purity of lignin.22,

8

23

9

organosolv process.24 During the process, the solubilized organosolv lignin could be precipitated

Cleavage of α-aryl ether and β-aryl ether has been suggested to the major reaction in the

10

out from the spent liquor by adding water.25, 26 Recently, certain EOL has been observed to

11

enhance the enzymatic hydrolysis of cellulosic substrates considerably.13, 27 Effect of EOLs on

12

enzymatic hydrolysis was suggested to be controlled by lignin hydrophobicity and negative zeta

13

potentials.28 Lignin hydrophobicity and zeta potential are further governed by lignin structures

14

and functional groups. However, which lignin structures and functional groups are responsible

15

for EOLs inhibition and/or stimulation has not been fully understood. Furthermore, which

16

reactions are accountable for the positive effect of EOLs from organosolv process has not been

17

explored.

18

Acid catalyzed alkylation of lignin with methanol or ethanol at 30°C has been reported, in

19

which the methylene proton of ethoxyl group was observed at δH 3.4 ppm.29 Liberation of

20

phenolic groups has also been observed in alkylation of spruce lignin.29 Early research also

21

demonstrated that extensive methylation took place in alkylation of lignin with alcoholic

22

hydrochloric acid and suggested the etherification of benzyl alcohol groups or the re-

23

etherification of benzyl ether group was the main reaction under mild conditions (20-30°C) for 4 ACS Paragon Plus Environment

Page 5 of 34

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

Energy & Fuels

1

solvent pretreatment with acids. In the organosolv pretreatment, the condition was much severe

2

(160–190°C). As a result, the reactions could be different and more complicated. The alcohols

3

have been proposed to have a significant effect on acidic delignification processes.30 The lignin

4

condensation reactions predominating under acidic conditions were reduced substantially by the

5

addition of solvents. 2-naphthol has also been found to suppress the lignin condensation in steam

6

explosion pretreatment of aspen,15 in which 2-naphthol was suggested to react with Cα in lignin.

7

In a catalytic depolymerization of lignin process (>350°C)under supercritical ethanol, ethanol

8

has been also found to suppress the lignin condensation though O-alkylation and C-alkylation

9

capping reactions.31 It is hypothesized that alkylation of lignin with ethanol occurs in organosolv

10

pretreatment processes, and this alkylation of lignin suppresses lignin condensation and results in

11

positive of effect of EOLs.

12

The primary goal of this study is to investigate the effect of lignin alkylation on enzymatic

13

hydrolysis of cellulosic substrates and to elucidate the structural and functional groups changes

14

in EOLs and their influence on enzymatic hydrolysis. A series of EOLs were produced from

15

organosolv pretreatment of sweetgum and loblolly pine under different temperature (160-200 °C).

16

Effect of pretreatment temperature on the alkylation reactions between ethanol and lignin was

17

examined by 13C, HSQC and HMBC NMR. The physicochemical properties of lignin, including

18

hydrophobicity, surface charge, and molecular weight (Mw), and the binding affinity of lignins

19

with enzymes were examined as well. The stimulatory or inhibitory effects of EOLs on

20

enzymatic hydrolysis of Avicel and pretreated biomass were correlated to the ethoxyl groups in

21

alkylated lignins.

22

5 ACS Paragon Plus Environment

Energy & Fuels

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

1

Page 6 of 34

2. MTERIALS AND MEHTODS

2

2.1. Organosolv Pretreated Biomass Preparation. Sweetgum (Liquidambar styraciflua)

3

wood chips and loblolly pine (Pinus taeda) wood chips with the size of 1.0 × 1.0 cm (L × W)

4

were collected by Forest Products Laboratory at Auburn University. Organosolv pretreated

5

sweetgum (OPSG) was prepared in a 1.0 L Parr batch reactor as described previously.13 Briefly,

6

sweetgum wood chips (80 g) soaked in 25% ethanol solution with 1.0% (w/w) sulfuric acid

7

overnight in a solid to liquid ratio of 1:7. After that, the wood chips with solution were loaded

8

into the reactor and pretreated at 160 °C for 1 h. For organosolv pretreatment of loblolly pine

9

(OPLP), wood chips were pretreated similarly in 75% ethanol solution at 170 °C for 1 h. After

10

pretreatment, the reactor was cooled down in a water bath. The pretreated substrate was collected

11

by filtration, washed with 95% ethanol solution at room temperature three times (1:10 of

12

solid/liquid), and followed by at least three times water washing.

13

2.2. Ethanol Organosolv Lignin Preparation. To prepare EOLs from sweetgum (EOL-SG),

14

four kinds of spent liquor were collected from organosolv pretreatment of sweetgum with 75%

15

ethanol and 1.0% sulfuric acid, at 160, 170, 180, and 190 °C for 1 h, respectively. EOLs were

16

precipitated by adding 3-fold volumes of water into the organosolv spent liquor. The precipitated

17

EOLs were collected by filtration on Whatman No. 1 filter paper and washed three times with

18

warm water. Similarly, four EOLs from loblolly pine (EOL-LP) were collected from organosolv

19

pretreatment of loblolly pine with 75% ethanol and 1.0% sulfuric acid, at 170, 180, 190, and 200

20

°C for 1 h, respectively. The EOL was designated based on the biomass name and pretreatment

21

temperature. For example, EOL from organosolv pretreatment of sweetgum at 160 °C was

22

designated as EOL-SG160.


Page 7 of 34

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

1

Energy & Fuels

2.3. Cellulase Enzymes. Commercial cellulase, Novozym 22C, was provided from

2

Novozymes (Franklinton, NC), and used for enzymatic hydrolysis of Avicel (pure cellulose),

3

OPSG, and OPLP (pretreated substrates). The filter paper enzyme activity of Novozym 22C was

4

100 FPUmL-1, determined by using Whatman NO. 1 filter paper as the substrate;32 and the β-

5

glucosidase activity of Novozym 22C was 343 IUmL-1, determined by using p-nitrophenyl-β-D-

6

glucoside as the substrate.33 Cellulase from Trichoderma reesei ATCC 26921 was purchased

7

from Sigma-Aldrich (St. Louis, MO) and only used for cellulase adsorption isotherm

8

determination.

9

2.4. Enzymatic Hydrolysis of Avicel, OPSG and OPLP with the Addition of Various

10

EOLs. Enzymatic hydrolysis was performed in 50 mL of 50 mM sodium citrate buffer (pH 4.8)

11

at 2% glucan (w/v) with commercial enzyme (Novozym 22C) as previously described.13 In brief,

12

the hydrolysis reaction was performed at 50 °C and 150 rpm for 72 h. The enzyme loading of

13

Novozym 22C was 5.0 FPUg-1 glucan in enzymatic hydrolysis of Avicel and OPSG, but 10.0

14

FPUg-1 glucan in enzymatic hydrolysis of OPLP. To investigate the effects of EOLs on

15

enzymatic hydrolysis, EOLs were added into the enzymatic hydrolysis system prior to the

16

enzyme addition. In detail, 4 gL-1 EOLs were added to a mixture of the substrates and citrate

17

buffer, and then incubated for 1 h at room temperature. After that, the enzyme was added to

18

initiate the enzymatic hydrolysis. To examine the substrate digestibility, the samples were taken

19

from the hydrolysis solution at various time intervals. The glucose and xylose content was

20

determined by HPLC with Aminex HPX-87P column. The hydrolysis yield of the substrates was

21

calculated from the released glucose content, as a percentage of the theoretical sugars available

22

in the substrates. Initial hydrolysis rate was calculated based on the released glucose in the first 3


Energy & Fuels

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

1

h of enzymatic hydrolysis. Enzymatic hydrolysis was carried out in duplicate, and each data

2

point was presented as the average of two replicates.

3

Page 8 of 34

2.5. Enzyme Adsorption Isotherm. To examine the affinity of cellulases on EOL samples,

4

cellulase C2730 (protein content 41 mgmL-1) with low β-glucosidase content was used. Cellulase

5

C2730 was incubated with 2% (w/w) lignin at 4 °C and 150 rpm for 3 h. A range of enzyme

6

concentration was used from 0.01 to 0.4 mgmL-1. After reaching equilibrium, the sample was

7

taken to determine the protein content in the supernatant by Bradford assay as free enzyme

8

content in the enzymatic hydrolysis solution. The adsorbed enzyme was calculated from the

9

difference between the initial enzyme content and the free enzyme content. The classical

10

Langmuir adsorption isotherm (Γ=ΓmaxKC/(1+KC)) was used to fit the cellulase enzyme

11

adsorption on substrates. Where, Γmax is the surface concentration of protein at full coverage

12

(mgg-1 substrate); K is the Langmuir constant (mLmg-1); and C is the free enzyme content in

13

solution (mgmL-1). Moreover, the distribution coefficient (R) can be expressed as R= ΓmaxK.

14

2.6. EOLs Surface Hydrophobicity Determination. The surface hydrophobicity of EOLs

15

was estimated by the Rose Bengal (hydrophobic dye) adsorption method.13, 34 To mimic the

16

enzymatic hydrolysis conditions, Rose Bengal was mixed with EOL at pH 4.8, 50 °C, and 150

17

rpm for 2 h. The concentration of Rose Bengal was fixed at 40 mgL-1, while the concentration of

18

EOL was in a range of 2–10 gL-1. The hydrophobic dye, Rose Bengal, could be adsorbed on the

19

lignin surface through hydrophobic interaction. The free dye content in the supernatant was

20

determined by measuring the absorbance at 543 nm using a UV-Vis spectrometer. The content of

21

adsorbed dye was calculated from the difference between the initial dye content and the free dye

22

content. The partition quotient (PQ) was calculated from the ratio of the adsorbed dye over the


Page 9 of 34

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

Energy & Fuels

1

free dye. The obtained PQ was plotted against the corresponding lignin content. The slope from

2

the linear plot was defined as the surface hydrophobicity of lignin (Lg-1).

3

2.7. EOLs Surface Charge Determination. The surface charges of EOLs were determined

4

by the potentiometric titration.20 Briefly, lignin samples (120 mg) were firstly dissolved in 10.0 g

5

NaOH solution (0.1 M), then the solution was acidified with 3.0 g 1.0 M HCl and stirred for 10

6

min. After that, 30.0 g of 0.1 M NaOH was added to the solution to neutralize the acid and to re-

7

dissolve the lignin. The sample solution was titrated by 0.1 M HCl with the automatic titrator

8

(AUT-701, DKK-TOA) from pH 12 to 2. Blank solution (without lignin) was prepared and

9

titrated as well. The surface charges (mM g-1) on the lignin under pH 4.8 were calculated as

10

following: Q= (Vblank－Vsample) ×M/W, where Q is the surface charge (mM g-1), Vsample and Vblank

11

are the titration volume consumed by lignin sample solution and blank solution at pH 4.8

12

respectively, M is the mole concentration of HCl and W is the weight of lignin samples.

13

2.8. Acetylation of Ethanol Organosolv Lignin. The acetylation of EOLs (0.5 g) was

14

conducted in 6 mL pyridine-acetic anhydride (1:1, v/v) at room temperature, and stirred for 72 h.

15

The solution was added dropwise to 120 mL ice-cold water containing 1 mL concentrated HCl.

16

The precipitated acetylated lignin was extensively washed by water, and then collected on

17

Whatman No. 1 filter paper. The resultant acetylated lignin was air-dried in fume hood, and used

18

for lignin molecular weight determination and 1H NMR analysis.

19

2.9. EOLs Molecular Weight Determination. The acetylated EOLs were used in the

20

determination of their weight-average molecular weights (Mw) by gel permeation

21

chromatography (GPC) using an HPLC system equipped with three 4.6 mm × 300 mm Styragel

22

columns (HR5E, HR4, and HR2) (Waters, Milford, MA) in tandem, and a refractive index 9 ACS Paragon Plus Environment

Energy & Fuels

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

1

detector. The acetylated EOLs (1 mg) were dissolved in 1 mL HPLC-grade tetrahydrofuran

2

(THF), and 50 µL of sample injection volume. THF was used as eluent at a flow rate of 1

3

mLmin-1 operated at 35°C. The columns were calibrated with polystyrene standards.

4

Page 10 of 34

2.10. 1H NMR Analysis of EOLs. Functional groups (phenolic hydroxyl, aliphatic hydroxyl,

5

and methoxyl) of acetylated EOLs were estimated using 1H-NMR on a Bruker 400/600

6

spectrometer. Lignin acetate (50 mg) and p-nitrobenzaldehyde (NBA, 5 mg as internal standard)

7

were dissolved in 0.5 mL of CDCl3.35 The contents of functional groups were calculated from the

8

integration ratios of the protons of functional groups to the protons of the internal standard.

9

2.11. 13C NMR Analysis of EOLs. Quantitative 13C NMR employed a 90º pulse width, a 1.2

10

s acquisition time and a 1.7 s relaxation delay. Chromium (III) acetylacetonate (0.01 M) was

11

added to the solution to provide complete relaxation of all nuclei. Non-acetylated EOLs (120

12

mg) were dissolved in dimethyl sulfoxide (DMSO-d6). A total of 20000 scans per sample were

13

collected.

14

2.12. 2D HMBC NMR Analysis of EOLs. Lignin samples (80 mg) were dissolved in 0.5

15

mL of DMSO-d6. The experiments were carried out at room temperature on a 400 MHz or 600

16

MHz Bruker Avance NMR spectrometer equipped with a cryoprobe. Matrices of 2048 × 256

17

data points were collected. The recycle delay was 1.5 sec with number of scans of 128. The

18

HMBC spectra were recorded via heteronuclear zero and double quantum coherence experiment

19

(HMBCgplpndqf sequence).

20

2.13. 2D HSQC NMR Analysis of EOLs. EOLs (60 mg) were dissolved in 0.5 mL of

21

deuterated dimethyl sulfoxide (DMSO-d). HSQC spectra were recorded on a Bruker 600 MHz


Page 11 of 34

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

Energy & Fuels

1

Bruker Spectrometer using the pulse program “hsqcetgp” as described previously.28 The DMSO

2

solvent peak was used as an internal reference (δC 39.52, δH 2.50 ppm).

3

3. RESULTS AND DISCUSSION

4

3.1. Effects of EOLs on Enzymatic Hydrolysis of Avicel and Pretreated Substrates. To

5

examine the characteristic effects of EOLs, EOL-SG and EOL-LP lignins were added into the

6

enzymatic hydrolysis of Avicel, OPSG and OPLP (Figure 1). Four EOL-SG lignins showed

7

stimulatory effects on enzymatic hydrolysis of Avicel, OPSG and OPLP. EOL-SG lignins from

8

the lower pretreatment temperature resulted in better hydrolysis yields. By contrast, EOL-LP170

9

and 180 showed inhibitory effects on hydrolysis yields; EOL-LP190 and 200 showed stimulatory

10

effects. It was unexpected that higher pretreatment temperature resulted in the positive effects of

11

EOL-LP lignins on enzymatic hydrolysis.

12

Specifically, the addition of EOL-SG160, 170, 180 and 190 increased the 72 h hydrolysis

13

yield of Avicel by 5–8% (Figure1a). These four lignins also enhanced the 72 h hydrolysis yield

14

of OPSG by 10–36% (Figure 1b), and improved the 72 h hydrolysis yield of OPLP by 7–32%

15

(Figure 1c). This indicates that EOL-SG enhanced enzymatic hydrolysis of pretreated biomass

16

better than pure cellulose, which is probably due to the residual lignin (11%) in OPSG and

17

OPLP. Remarkably, it was observed that EOL-SG from lower pretreatment temperature showed

18

much higher stimulation than those from higher temperature. For instance, EOL-SG160

19

increased the 72 h hydrolysis yield of OPSG by 36%, while EOL-SG190 increased it only by

20

12%. It is most likely that the physicochemical properties (such as hydrophobicity, functional

21

groups and surface charge) of EOL-SG changed at high temperature.


Energy & Fuels

Glucose yield / %

60

80

(a) EOL-SG/160 EOL-SG/170 EOL-SG/180 EOL-SG/190

60

40

20

0 12

24

36

48

60

20

(c) EOL-SG160 EOL-SG170 EOL-SG180 EOL-SG190

40

20

0

0

12

24

Time / h

36

48

60

72

0

12

24

36

48

60

72

Time / h

Time / h

Figure 1. Effects of EOL-SG on enzymatic hydrolysis of Avicel (a), OPSG (b), and OPLP (c). Avicel Avicel+ Avicel+ Avicel+ Avicel+

60

80

(a) EOL-LP170 EOL-LP180 EOL-LP190 EOL-LP200

OPSG OPSG+ OPSG+ OPSG+ OPSG+

60

40

20

0

80

(b) EOL-LP170 EOL-LP180 EOL-LP190 EOL-LP200

40

20

12

24

36

48

Time / h

60

72

40

20

0

0 0

(c)

OPLP OPLP+ EOL-LP170 OPLP+ EOL-LP180 OPLP+ EOL-LP190 OPLP+ EOL-LP200

60 Glucose yield / %

80

3

OPLP OPLP+ OPLP+ OPLP+ OPLP+

60

40

72

Glucose yield / %

2

80

(b) EOL-SG160 EOL-SG170 EOL-SG180 EOL-SG190

0

0

1

OPSG OPSG+ OPSG+ OPSG+ OPSG+

Glucose yield / %

Avicel Avicel+ Avicel+ Avicel+ Avicel+

Glucose yield / %

80

Glucose yield / %

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

Page 12 of 34

0

12

24

36

48

60

72

Time / h

0

12

24

36

48

60

72

Time / h

4

Figure 2. Effects of EOL-LP on enzymatic hydrolysis of Avicel (a), OPSG (b), and OPLP (c).

5

By contrast, EOL-LP lignins (EOL-LP170, 180, 190, and 200) changed the 72 h hydrolysis

6

yields of Avicel from 60.5 (control) to 50.4, 58.6, 61.8, and 63.8%, respectively (Figure 2a).

7

Similarly, EOL-LP170 and 180 decreased the 72 h hydrolysis yields of OPSG and OPLP, but

8

EOL-LP190 and P200 increased those of OPSG and OPLP (Figure 2b and Figure 2c).

9

Unexpectedly, the higher pretreatment temperature (190 - 200 °C) changed the negative effects

10

of EOL-LP on enzymatic hydrolysis to be positive. This suggested that the higher temperature in

11

organosolv process changed the lignin structures and potentially increased the carboxylic acid

12

and ester functional groups in EOL-LP lignins resulting in positive effects (Tables S2 in

13

Supporting Information).

14

Similar results have been reported on the effects of wood extractives on enzymatic hydrolysis

15

of Avicel,36 in which it was observed that that the phenolic extractives from birch wood

16

increased the glucose yield by 7% and the phenolic extractives from pine wood decreased it by 12 ACS Paragon Plus Environment

Page 13 of 34

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

Energy & Fuels

1

9%. It was suggested that these phenolic extractives are composed of lignin oligomers created by

2

steam explosion.36 They proposed that wood extractives can prevent nonproductive binding of

3

cellulase enzymes on to cellulose surface. Likewise, lignosulfonate has also been reported to

4

improve the 72 h hydrolysis yield of pretreated poplar by 26%.37 It was suggested that

5

lignosulfonate could reduce the nonproductive binding between enzymes and residual lignin via

6

electrostatic repulsion from the negative charged lignosulfonate groups. In this study, as the

7

pretreatment temperature increased, the positive effects of EOL-SG decreased, whereas the

8

negative effects of EOL-LP reduced. We believe that cellulase enzymes would adsorb onto

9

EOLs with two binding modes (tight binding and weak binding). The tight binding would result

10

in irreversible nonproductive binding, and the weak binding would stabilize cellulase enzyme

11

activity and reduce nonproductive binding between enzymes and cellulose/residual lignins. The

12

net effect of EOLs depends on the collective influence of tight binding and weak binding. These

13

two binding modes most likely are controlled by lignin hydrophobicity, surface charge and

14

functional groups.

15

3.2. Effects of Pretreatment Temperature on Lignin Structural Changes Revealed by

16

Quantitative 13C NMR. To understand the chemical structures of EOLs, 13C NMR and 2D

17

HMBC NMR were used to characterize EOL-SG and EOL-LP lignins.


Energy & Fuels

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

Page 14 of 34

1 2

Figure3. Quantitative

3

SG190.

4

13

C NMR spectra of EOL-SG160, EOL-SG170, EOL-SG180 and EOL-

13

C NMR spectra showed a distinct methyl (CH3) peak at 15.4 ppm in EOL-SG lignins,

5

which was not observed in original lignin (MWL-SG) (Figure 3).A corresponding methylene

6

group (-OCH2CH3) was also observed at δc 63.9 ppm. It is possible that an ethoxyl group was

7

introduced on lignin through etherification of benzyl alcohol group (Scheme 1). A similar peak

8

has been reported on isolated lignin from Miscanthus by ethanol organosolv process under reflux

9

condition previously by HSQC-TOCSY NMR.38 Unexpectedly, higher pretreatment temperature

10

results in lower ethoxyl group in EOL-SG. The amount of ethoxyl related methyl carbon (per Ar)

11

decreased gradually in EOL-SG (Figure 3 and Table S1 in Supporting Information). The 14 ACS Paragon Plus Environment

Page 15 of 34

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

Energy & Fuels

1

ethoxyl group gave a HSQC correlation with a methylene group at δC/δH 63.9/3.36 ppm, the peak

2

signal also gradually decreased with the increase of pretreatment temperature. We believe that

3

the newly formed ether bonds were cleaved under the higher temperature, which decreased the

4

content of ethoxyl groups in EOL-SG. This suggested this etherification reaction was reversible

5

and higher temperature results in lower etherification. The decrease of ethoxyl group was

6

coincident with the decrease of stimulatory effect of EOL-SG on enzymatic hydrolysis. It is

7

possible that ethanol alkylation suppresses lignin condensation. C5 and Cα condensation has been

8

observed in lignin model compounds.39 In this case, the Cα was etherified with ethanol and

9

would not be available for further condensation reactions. Similar observation has been reported

10

in organosolv pulping, in which it was suggested that lignin condensation under acidic condition

11

was significantly hindered by the addition of alcohols.30

12

Meanwhile, it was also observed the content of etherified S3,5 at 152 ppm decreased

13

gradually and non-etherified S3,5 and G3 at 147 ppm increased correspondingly at higher

14

temperature (Table S1 in Supporting Information). The increase of the signal of non-etherified

15

S3,5 and G3 in 13C NMR spectra indicated the formation of new phenolic hydroxyl group in EOL-

16

SG170, 180 and 190. This was consistent with the 1H NMR analysis result (Table 1), which

17

showed the increasing pretreatment temperature increased the content of phenolic hydroxyl

18

group by 44, 77, and 84% in EOL-SG170, 180 and 190 respectively, as comparing to EOL-

19

SG160.

20 21


Energy & Fuels

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

Page 16 of 34

1

Table 1. Determination of functional groups in EOL-SG and EOL-LP by 1H NMR analysis

2

using p-nitrobenzaldehyde as internal standard [mM g-1] Lignin

Phenolic hydroxyl

Aliphatic hydroxyl

Methoxyl

EOL-SG160

2.75

5.27

11.75

EOL-SG170

3.96

3.98

11.83

EOL-SG180

4.88

2.77

11.10

EOL-SG190

5.07

2.68

10.43

EOL-LP170

2.91

5.74

8.99

EOL-LP180

3.59

5.48

8.74

EOL-LP190

3.53

4.87

7.14

EOL-LP200

3.60

4.37

6.14

3 HO HO

MeO

HO HC

O OMe OMe O

HO H3C

O

+

H

-H2O MeO

MeO

OMe MeO

O

O

H+, CH3CH2OH

OMe O

MeO

OMe MeO

OMe O

4 5 6 7

Scheme 1. Alkylation of lignin with ethanol resulting in the etherification of Cα (reversible reaction). For EOL-LP, the similar etherification of lignin substructure was observed based on 13C

8

NMR (Figure S1 in Supporting Information). As the pretreatment temperature increased, the

9

amount of ethoxyl related methyl group in EOL-LP decreased gradually (Table S2 in Supporting

10

Information). The cleavage of the newly formed ether bonds were not significant for softwood

11

lignin at higher temperature, and the etherified ethoxyl groups in loblolly pine appeared to be

12

more stable than those in sweetgum. Unexpectedly, higher temperature resulted an increased

13

signal at 14.7 ppm in EOL-LP. Meanwhile, the signals of carboxylic acid/ester groups at 178– 16 ACS Paragon Plus Environment

Page 17 of 34

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

Energy & Fuels

1

168 ppm in EOL-LP increased gradually with the increase of temperature (Table S2 in

2

Supporting Information). The aliphatic esters probably were formed between ethanol and

3

aliphatic acid groups. Similar results have been reported in catalytic depolymerization of lignin

4

with supercritical ethanol, in which the formation of alkyl ester was observed in HSQC NMR

5

and esterification took place between ethanol and lignin side chains.31 The aliphatic acid groups

6

were most likely generated from the oxidation of the propyl side chain. The substructure

7

etherified aliphatic ester was also confirmed by HMBC NMR in our study (Figure S2 in

8

Supporting Information). The C-H of methyl group correlation was observed at δC/δH 14.4/1.2

9

ppm in HMBC NMR. The C of methyl group can see the H of methylene at δH 4.05 ppm. The

10

corresponding C in methylene at δC 60.2 ppm, gives further HMBC correlation to C=O at 173

11

ppm. The ester gave a HSQC correlation with a methylene group at δC/δH 60.2/4.05 ppm (Figure

12

4). Similar correlation has been observed in the residual lignin of a catalytic reaction,31 in which

13

they suggested the esterification can also suppress the lignin condensation. In this study, this

14

cross signal increased gradually with the increase of temperature. This probably can explain why

15

the EOL-LP from higher temperature became stimulatory, because more esterification took place

16

and inhibited the lignin condensation. The chemical shifts of both ethyl carbons in lignin ester

17

agreed well with those of model carboxylic esters.40 Similar aliphatic ethyl esters reaction

18

products have been identified in the reaction of lignin and ethanol under toluene sulfonic acid

19

catalyzation.41 Meanwhile, the content of etherified G3,4 at 149 ppm decreased slightly and non-

20

etherified G3,4 at 147 ppm increased correspondingly at higher temperature (Table S2 in

21

Supporting Information). This indicated that higher temperature increased the content of

22

phenolic hydroxyl group in EOL-LP slightly, which was confirmed by 1H NMR analysis.


Energy & Fuels

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

1

Compared to the EOL-SG, the phenolic hydroxyl groups in EOL-LP did not increase much as

2

the temperature increased (Table 1).

Page 18 of 34

3

The aromatic region (160–103 ppm) and higher aliphatic region (89–58 ppm) in 13C NMR

4

spectra were analyzed as well. The increase of temperature increased the content of condensed

5

aromatics in both EOL-SG and EOL-LP, which contributed to the decrease of the content of

6

protonated aromatics. It was observed that the condensed aromatics in both EOL-SG and EOL-

7

LP increased with the increase of temperature (Table S1 and Table S2 in Supporting

8

Information). This indicated that the condensation reaction in EOLs took place in higher

9

temperature of organosolv process. A similar observation has been reported for isolated lignins

10

from hydrothermal pretreatment of aspen, in which the condensed aromatics also increased after

11

pretreated at 170 °C.42 It should be noted that this condensation reaction was significantly

12

suppressed by lignin alkylation and esterification in organosolv processes.

13

The propyl side chain related aliphatic region in 13C NMR can be separated into three areas

14

(89–78, 73–71, and 61–58 ppm), which was mainly identified as Cβ, Cα and Cγ areas,

15

respectively in β-O-4 linkage substructures. Organosolv process breaks the interunit linkages of

16

α-aryl-ether and β-aryl-ether bonds in both EOL-SG and EOL-LP. An increase of temperature in

17

the organosolv process resulted in a significant decrease of Cβ, Cα, and Cγ areas. This agrees with

18

previous reports that cleavage of β-O-4 linkage is the main mechanism for the organosolv

19

process.24 It was also confirmed by the increase of the signals (130–145 ppm) of non-etherified

20

G3 and S3,5 groups as the pretreatment temperature increased.

21

The HSQC spectra of EOL-SG and EOL-LP from different pretreatment temperature was

22

compared in Figure 4. The inter-unit linkages of β-aryl-ether (β-O-4, A), resinol (β-β, B), 18 ACS Paragon Plus Environment

Page 19 of 34

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

Energy & Fuels

1

phenylcoumaran (β-5, C) were identified by their cross-peaks (Figure 4 and Table S3). HSQC

2

NMR spectra showed that an α-ethoxylated β-O-4ʹ substructure (Aʹ) was formed in both EOL-

3

SG and EOL-LP. The methylene group of Aʹα-OCH2CH3 was observed at δC/δH 63.9/3.36 ppm.

4

The correlation of Cα-Hα in Aʹ was observed at δC/δH 80.1/4.57 ppm. As the temperature

5

increased, the cross signals of Aα andAʹα decreased significantly and became not visible in EOL-

6

SG180 and EOL-SG190 (Figure 4a). This suggested that most of β-O-4 linkage was cleaved,

7

including the newly formed β-O-4ʹ in Aʹ. The cross signals of Bγ did not change at higher

8

temperature. This indicated resinol was stable in organosolv processes. The HSQC spectra

9

showed the cross signals of Aʹα-OEt decreased gradually as the temperature increased, which 13

10

agreed well with the observations in the

C NMR spectra. For EOL-LP, one cross signal

11

increased dramatically in HSQC at δC/δH 60.2/4.05 ppm (Figure 4b). This agrees well with the

12

observed correlation of methylene group in ethyl esters in lignin depolymeration.41

13


Energy & Fuels

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

Page 20 of 34

1 2

(a) HSQC NMR spectra of EOL-SG lignins (side chain region)


Page 21 of 34

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

Energy & Fuels

1 2 3

(b) HSQC NMR spectra of EOL-LP lignins (side chain region) Figure 4. HSQC NMR spectra of EOL-SG and EOL-LP lignins.

4

3.3. Effects of Pretreatment Temperature on Hydrophobicity, Surface Charge and

5

Molecular Size of EOLs. To examine the changes on physicochemical properties of EOLs in

6

the organosolv process, surface charge, molecular weight and hydrophobicity of EOL-SG and

7

EOL-LP lignins were determined (Table 2). As pretreatment temperature increased, the surface

8

charges of EOL-LP lignins increased by 11-55%, as compared to EOL-LP170, which could be

9

attributed to the increase on carboxylic acid groups as shown in 13C NMR analysis (Table S2 in

10

Supporting Information). The increasing negative charge in lignins could come from the more

11

side chain oxidized under the higher temperature. The more negatively charged groups in EOL-

12

LP could result in the stronger electrostatic repulsion between enzyme and lignins, thus reduced

13

non-productive binding and enhanced enzymatic hydrolysis. Compared to EOL-LP, the surface 21 ACS Paragon Plus Environment

Energy & Fuels

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

Page 22 of 34

1

charges of EOL-SG first decreased and then increased. GPC analysis indicated that the Mn, Mw,

2

and Mw/Mn (degree of polydispersity) decreased in both EOL-SG and EOL-LP, as the

3

pretreatment temperature increased. The decrease on molecular weight was mainly due to the

4

cleavage of ether linkages of the lignin, which was observed in 13C NMR spectra (Table S1 and

5

Table S2 in Supporting Information). A similar result was reported that increasing temperature

6

resulted in extensive cleavage of ether linkages of EOL from poplar.26, 43As for the

7

hydrophobicity of lignins, the increasing temperature seemed decrease the hydrophobicity in

8

both EOL-SG and EOL-LP. EOL-SG160 showed the highest hydrophobicity, and EOL-LP170

9

showed the highest hydrophobicity. The increase of negative charge could decrease the enzyme

10

nonproductive binding due to the electrostatic repulsion. The decrease of hydrophobicity in EOL

11

could decrease the hydrophobic interaction between enzyme and lignin. However, the net effect

12

of lignin inhibition or stimulation depends on the combined influence of hydrophobicity and

13

surface charge.28

14

Table 2. Hydrophobicity, surface charges, and molecular weight of organosolv lignins. Lignin

Surface charge at pH 4.8

Mw

Hydrophobicity

[mM g-1]

[g M-1]

[L g-1]

EOL-SG160

-0.542

5457

0.56

EOL-SG170

-0.458

3039

0.23

EOL-SG180

-0.583

2013

0.45

EOL-SG190

-0.583

1881

0.33

EOL-LP170

-0.375

5210

1.07

EOL-LP180

-0.417

3532

0.47


Page 23 of 34

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

Energy & Fuels

EOL-LP190

-0.583

2920

0.49

EOL-LP200

-0.583

2855

0.27

1 2

3.4. Effects of Pretreatment Temperatures on EOLs Affinity to Enzyme. To assess the

3

cellulases affinity to EOLs, Langmuir adsorption isotherms were determined (Table 3). The

4

results showed that EOL-SG had lower affinity to cellulases than EOL-LP. The Langmuir

5

constant (K) from EOL-LP was two or three fold of that from EOL-SG. The distribution

6

coefficient (R) from EOL-LP was 10–15 folds higher than that from EOL-SG. Specifically for

7

EOL-SG, the higher pretreatment temperature resulted in the higher binding affinity between

8

lignin and enzyme based on the Langmuir constant (K) and distribution coefficient (R).

9

Table 3. Langmuir adsorption isotherm parameters from enzyme adsorption on lignins. Lignins

Гmax [mg g-1]

K [mL mg-1]

R [L g-1]

EOL-SG160

1.05

7.21

0.008

EOL-SG170

2.66

8.82

0.023

EOL-SG180

4.51

9.90

0.045

EOL-SG190

5.59

10.18

0.057

EOL-LP170

6.40

20.73

0.139

EOL-LP180

5.81

20.96

0.122

EOL-LP190

5.49

21.17

0.116

EOL-LP200

5.17

18.90

0.098

10


Energy & Fuels

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

1

Page 24 of 34

On the contrary for EOL-LP, the higher pretreatment temperature resulted in the lower

2

binding affinity based on R value. A good correlation (r2>0.70) was found between R values and

3

the amount of surface charge of EOL-LP. This indicated the increase in surface charge could

4

increase the electrostatic repulsion between enzyme and lignin, thus reduced the binding affinity

5

of high-temperature EOL-LP to enzyme.

6

The R value has been considered as the binding strength between enzyme and lignin, which

7

was related to enzyme adsorption and desorption. A lower R value of EOL-SG resulted in the

8

higher positive effect on enzymatic hydrolysis. A strong negative correlation (r2>0.97) was

9

observed between R value of EOL-SG lignins and their corresponding 72 h hydrolysis yields of

10

OPSG and OPLP (Figure 5). This indicated that weaker binding from EOL-SG lignin could

11

stimulate enzymatic hydrolysis.

12

For EOL-LP, a similar correlation (r2>0.85) was found between R value and their

13

corresponding 72 h hydrolysis yields of OPSG and OPLP (Figure 5). Higher temperature in

14

pretreatment typically resulted in higher R value for lignin.42 However in this study, the higher

15

temperature results in lower R value for EOL-LP. It was most likely because the esterification

16

took place at higher temperature and effectively suppressed the lignin condensation and reduced

17

the hydrophobicity of EOL-LP lignins.


Page 25 of 34

70

72 h Glucose yield (%)

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

Energy & Fuels

60

50

Avicel OPSG OPLP Left: EOL-SG Right: EOL-LP

40

30

0.00

1

0.04

0.08

0.12

0.16

Distribution coefficient (R)

2

Figure 5. Correlation between distribution coefficient (R) and 72 h hydrolysis yield of Avicel,

3

OPSG and OPLP.

4

3.5. The Potential Effects of Lignin Alkylation in Organosolv Pretreatment Process.

5

Quantitative 13C NMR suggested that an alkylation of sweetgum lignin with ethanol in the

6

organosolv process resulted in the etherification of the propyl side chain. As mentioned earlier,

7

this remarkable change was observed in the lower aliphatic region (~15 ppm) for EOLs. A signal

8

of 15.4/15.7 ppm was assigned to a methyl carbon in an ethoxyl group, which has been

9

suggested to be due to a nucleophilic addition reaction between ethanol and benzylic position in

10

β-aryl ether substructure of lignin.38, 44 As the pretreatment temperature increased, the amount of

11

this ethoxyl group in EOL-SG gradually decreased (Table S1 in Supporting Information). More

12

importantly, a strong negative correlation (r2=0.90) was observed between the affinity of enzyme

13

on EOL-SG (R value) and the content of ethoxyl group in EOL-SG. This indicated that the

14

decrease of ethoxyl group content in EOL-SG probably increased the affinity of enzyme to

15

lignins, and thus reduced their stimulatory effects. Moreover, the higher temperature in

16

organosolv resulted in the less etherification of lignin. This has a potential application in the 25 ACS Paragon Plus Environment

Energy & Fuels

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

Page 26 of 34

1

biorefinery if higher temperatures or severity are not necessary for hardwood biomass.

2

Quantitative 13C NMR and HMBC NMR indicated that an alkylation of loblolly pine lignin with

3

ethanol at higher temperature (>190 °C) resulted in the esterification of aliphatic acid side chain,

4

which changed the inhibitory effects of EOL-LP lignins to become positive.

5

4. CONCLUSIONS

6

The structural changes of EOLs in organosolv processes and their positive/negative effects on

7

enzymatic hydrolysis were investigated. Quantitative 13C NMR was used to characterize the

8

effects of pretreatment temperature on structural changes of EOLs. It was observed ethanol

9

etherification of propyl side chain occurred in hardwood lignin (EOL-SG) and this etherification

10

is a reversible reaction. Remarkably, as organosolv pretreatment temperature increased, the

11

positive effects of EOL-SG from sweetgum decreased. On the contrary, the negative effects of

12

EOL-LP from loblolly pine changed to become positive at higher temperature. It was proposed

13

that this change was associated with the ethanol esterification of aliphatic acid side chain of

14

lignin substructure at higher temperature in softwood lignin. We believe that lignin etherification

15

and esterification suppress lignin condensation reactions and resulted in lignins low affinity to

16

enzyme, which in turn enhanced the enzymatic hydrolysis by reducing the non-productive

17

binding between residual lignin and cellulases.

18

ASSOCIATED CONTENT

19

Supporting Information

20

Table S1. Summary of chemical groups in EOL-SG lignins by quantitative 13C NMR analysis.

21

Table S2. Summary of chemical groups in EOL-LP lignins by quantitative 13C NMR analysis.

22

Table S3. Assignments of 13C-1H cross-signals in 2D-HSQC spectra of lignins. Figure S1. 26 ACS Paragon Plus Environment

Page 27 of 34

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

Energy & Fuels

1

Quantitative 13C NMR spectra of EOL-LP170, EOL-LP180, EOL-LP190 and EOL- LP200.

2

Figure S2. HMBC NMR spectra of EOL-LP200 lignin. Figure S3. HSQC NMR spectra of

3

EOL-SG lignins. Figure S4. HSQC NMR spectra of EOL-LP lignins.

4

AUTHOR INFORMATION

5

Corresponding Author

6

* E-mail:[email protected]

7

Notes

8

The authors declare no competing financial interest.

9

ACKNOWLEDGMENT

10

The study was supported in part by grants from Alabama Agricultural Experimental Station, Sun

11

Grant and USDA NIFA (Grant NO. 2011-68005-30410). Lai’s study was also partially supported

12

by Natural Science Foundation of Jiangsu Province for youth (BK20150874) and National

13

Natural Science Foundation for youth (31600463).

14 15 16 17 18 19 20 21 22 27 ACS Paragon Plus Environment

Energy & Fuels

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

1

Page 28 of 34

REFERENCES

2 3

(1) Bhutto, A. W.; Qureshi, K.; Harijan, K.; Zahedi, G.; Bahadori, A. Strategies for the

4

consolidation of biologically mediated events in the conversion of pre-treated

5

lignocellulose into ethanol. RSC Adv.2014, 4, 3392-3412.

6

(2) Himmel, M. E.; Ding, S.-Y.; Johnson, D. K.; Adney, W. S.; Nimlos, M. R.; Brady, J. W.;

7

Foust, T. D. Biomass recalcitrance: engineering plants and enzymes for biofuels

8

production. Science 2007, 315, 804-807.

9

(3) Chundawat, S. P.; Beckham, G. T.; Himmel, M. E.; Dale, B. E. Deconstruction of

10

lignocellulosic biomass to fuels and chemicals. Annu.Rev.Chem.Biomol.Eng.2011, 2,

11

121-145.

12 13 14 15

(4) Luterbacher, J.; Alonso, D. M.; Dumesic, J. Targeted chemical upgrading of lignocellulosic biomass to platform molecules. Green Chem.2014, 16, 4816-4838. (5) Mansfield, S. D.; Mooney, C.; Saddler, J. N. Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol. Progr.1999, 15, 804-816.

16

(6) Mooney, C. A.; Mansfield, S. D.; Touhy, M. G.; Saddler, J. N. The effect of initial pore

17

volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresour.

18

Technol.1998, 64, 113-119.

19

(7) Kumar, L.; Arantes, V.; Chandra, R.; Saddler, J. The lignin present in steam pretreated

20

softwood binds enzymes and limits cellulose accessibility. Bioresour. Technol.2012, 103,

21

201-208.


Page 29 of 34

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

Energy & Fuels

1

(8) Rahikainen, J. L.; Evans, J. D.; Mikander, S.; Kalliola, A.; Puranen, T.; Tamminen, T.;

2

Marjamaa, K.; Kruus, K. Cellulase-lignin interactions—The role of carbohydrate-binding

3

module and pH in non-productive binding. Enzyme Microb. Technol.2013, 53, 315-321.

4

(9) Zeng, Y.; Zhao, S.; Yang, S.; Ding, S.-Y. Lignin plays a negative role in the biochemical

5

process for producing lignocellulosic biofuels. Curr.Opin.Biotechnol.2014, 27, 38-45.

6

(10)

Pereira, A.; Hoeger, I. C.; Ferrer, A.; Rencoret, J.; del Rio, J. C.; Kruus, K.;

7

Rahikainen, J.; Kellock, M.; Gutierrez, A.; Rojas, O. J. Lignin films from spruce,

8

eucalyptus, and wheat straw studied with electroacoustic and optical sensors: Effect of

9

composition and electrostatic screening on enzyme binding. Biomacromolecules 2017,

10 11

18, 1322-1332. (11)

Pielhop, T.; Larrazábal, G. O.; Studer, M. H.; Brethauer, S.; Seidel, C.-M.; von

12

Rohr, P. R. Lignin repolymerisation in spruce autohydrolysis pretreatment increases

13

cellulase deactivation. Green Chem.2015, 17, 3521-3532.

14

(12)

Yu, Z.; Gwak, K. S.; Treasure, T.; Jameel, H.; Chang, H. m.; Park, S. Effect of

15

lignin chemistry on the enzymatic hydrolysis of woody biomass. ChemSusChem 2014, 7,

16

1942-1950.

17

(13)

Lai, C.; Tu, M.; Shi, Z.; Zheng, K.; Olmos, L. G.; Yu, S. Contrasting effects of

18

hardwood and softwood organosolv lignins on enzymatic hydrolysis of lignocellulose.

19

Bioresour. Technol.2014, 163, 320-327.

20

(14)

Campbell, M. M.; Sederoff, R. R. Variation in Lignin Content and Composition

21

(Mechanisms of Control and Implications for the Genetic Improvement of Plants). Plant

22

Physiol.1996, 110, 3.


Energy & Fuels

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

1

(15)

Page 30 of 34

Li, J.; Henriksson, G.; Gellerstedt, G. Lignin depolymerization/repolymerization

2

and its critical role for delignification of aspen wood by steam explosion. Bioresour.

3

Technol. 2007, 98, 3061-3068.

4

(16)

Nakagame, S.; Chandra, R. P.; Kadla, J. F.; Saddler, J. N. The isolation,

5

characterization and effect of lignin isolated from steam pretreated Douglas-fir on the

6

enzymatic hydrolysis of cellulose. Bioresour. Technol.2011, 102, 4507-4517.

7 8 9

(17)

Pan, X. Role of functional groups in lignin inhibition of enzymatic hydrolysis of

cellulose to glucose. J. Biobased Mater.Bioenergy 2008, 2, 25-32. (18)

Sewalt, V.; Glasser, W.; Beauchemin, K. Lignin impact on fiber degradation. 3.

10

Reversal of inhibition of enzymatic hydrolysis by chemical modification of lignin and by

11

additives. J. Agric. Food Chem.1997, 45, 1823-1828.

12

(19)

Nakagame, S.; Chandra, R. P.; Kadla, J. F.; Saddler, J. N. Enhancing the

13

enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content

14

of the associated lignin. Biotechnol.Bioeng.2011, 108, 538-548.

15

(20)

Rahikainen, J. L.; Martin-Sampedro, R.; Heikkinen, H.; Rovio, S.; Marjamaa, K.;

16

Tamminen, T.; Rojas, O. J.; Kruus, K. Inhibitory effect of lignin during cellulose

17

bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption.

18

Bioresour. Technol.2013, 133, 270-278.

19

(21)

Selig, M. J.; Viamajala, S.; Decker, S. R.; Tucker, M. P.; Himmel, M. E.; Vinzant,

20

T. B. Deposition of lignin droplets produced during dilute acid pretreatment of maize

21

stems retards enzymatic hydrolysis of cellulose. Biotechnol.Progr.2007, 23, 1333-1339.


Page 31 of 34

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

1

Energy & Fuels

(22)

Kim, D. E.; Pan, X. J. Preliminary study on converting hybrid poplar to high-

2

value chemicals and lignin using organosolv ethanol process. Ind. Eng. Chem. Res.2010,

3

49, 12156-12163.

4

(23)

Pan, X. J.; Arato, C.; Gilkes, N.; Gregg, D.; Mabee, W.; Pye, K.; Xiao, Z. Z.;

5

Zhang, X.; Saddler, J. Biorefining of softwoods using ethanol organosolv pulping:

6

Preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-

7

products. Biotechnol.Bioeng.2005, 90, (4), 473-481.

8 9 10 11 12

(24)

McDonough, T. J. The chemistry of organosolv delignification. Tappi J.1992, 76,

186-193. (25)

Zhao, X.; Cheng, K.; Liu, D. Organosolv pretreatment of lignocellulosic biomass

for enzymatic hydrolysis. Appl.Microbiol.Biotechnol.2009, 82, 815. (26)

Pan, X.; Gilkes, N.; Kadla, J.; Pye, K.; Saka, S.; Gregg, D.; Ehara, K.; Xie, D.;

13

Lam, D.; Saddler, J. Bioconversion of hybrid poplar to ethanol and co‐products using an

14

organosolv

15

Biotechnol.Bioeng.2006, 94, 851-861.

16

(27)

fractionation

process:

Optimization

of

process

yields.

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

17

effects on enzymatic digestibility of organosolv pretreated hardwood. Bioresour. Technol.

18

2014, 156, 92-99.

19

(28)

Huang, Y.; Sun, S. L.; Huang, C.; Yong, Q.; Elder, T.; Tu, M. B. Stimulation and

20

inhibition of enzymatic hydrolysis by organosolv lignins as determined by zeta potential

21

and hydrophobicity. Biotechnol. Biofuels 2017, 10.


Energy & Fuels

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

1

(29)

Page 32 of 34

Adler, E.; Brunow, G.; Lundquist, K. Investigation of the acid-catalyzed

2

alkylation of lignins by means of NMRspectroscopic methods. Holzforschung 1987, 41,

3

199-207.

4 5 6

(30)

Lai, Y. Z.; Mun, S. P. The chemical Aspects of acidic delignification processes .1.

Role of aryl-ether hydrolysis in aspen. Holzforschung 1994, 48, 203-206. (31)

Huang, X. M.; Koranyi, T. I.; Boot, M. D.; Hensen, E. J. M. Ethanol as capping

7

agent and formaldehyde scavenger for efficient depolymerization of lignin to aromatics.

8

Green Chem.2015, 17, 4941-4950.

9 10 11 12 13

(32)

Adney, B.; Baker, J. Measurement of cellulase activities. Laboratory analytical

procedure 1996, 6, 1996. (33)

Wood, T. M.; Bhat, K. M., Methods for measuring cellulase activities. Methods in

enzymology 1988, 160, 87-112. (34)

Gessner, A.; Waicz, R.; Lieske, A.; Paulke, B.-R.; Mäder, K.; Müller, R.

14

Nanoparticles with decreasing surface hydrophobicities: influence on plasma protein

15

adsorption. Int.J. Pharm.2000, 196, 245-249.

16 17 18

(35)

Pan, X.; Saddler, J. N. Effect of replacing polyol by organosolv and kraft lignin

on the property and structure of rigid polyurethane foam. Biotechnol.Biofuels2013, 6, 12. (36)

Leskinen, T.; Salas, C.; Kelley, S. S.; Argyropoulos, D. S. Wood Extractives

19

Promote Cellulase Activity on Cellulosic Substrates. Biomacromolecules 2015, 16, 3226-

20

3234.

21 22

(37)

Wang, Z. J.; Zhu, J. Y.; Fu, Y. J.; Qin, M. H.; Shao, Z. Y.; Jiang, J. G.; Yang, F.,

Lignosulfonate-mediated cellulase adsorption: enhanced enzymatic saccharification of


Page 33 of 34

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

Energy & Fuels

1

lignocellulose through weakening nonproductive binding to lignin. Biotechnol. Biofuels

2

2013, 6.

3

(38)

Bauer, S.; Sorek, H.; Mitchell, V. D.; Ibáñez, A. B.; Wemmer, D. E.

4

Characterization of Miscanthus giganteus lignin isolated by ethanol organosolv process

5

under reflux condition. J. Agric.Food Chem.2012, 60, 8203-8212.

6

(39)

Barkhau, R. A.; Malcolm, E. W.; Dimmel, D. R. Insoluble lignin models (4) -

7

condensation-reactions of a polymer-bound guaiacylpropanol model. J. Wood Chem.

8

Technol. 1990, 10, 269-291.

9 10 11

(40)

Couperus, P. A.; Clague, A. D. H.; Vandongen, J. P. C. M. C-13 Chemical-shifts

of some model carboxylic-acids and esters. Org. Magn. Resonance 1978, 11,590-597. (41)

Mbotchak, L.; Le Morvan, C.; Duong, K. L.; Rousseau, B.; Tessier, M.; Fradet,

12

A. Purification, structural characterization, and modification of organosolv wheat straw

13

lignin. J. Agric.Food Chem.2015, 63, 5178-5188.

14

(42)

Sun, S. L.; Huang, Y.; Sun, R. C.; Tu, M. B. The strong association of condensed

15

phenolic moieties in isolated lignins with their inhibition of enzymatic hydrolysis. Green

16

Chem.2016, 18, 4276-4286.

17

(43)

El Hage, R.; Brosse, N.; Sannigrahi, P.; Ragauskas, A. Effects of process severity

18

on

19

Polym.Degrad.Stab.2010, 95, 997-1003.

20

the

(44)

chemical

structure

of

Miscanthus

ethanol

organosolv

lignin.

Balakshin, M. Y.; Capanema, E. A. Comprehensive structural analysis of

21

biorefinery lignins with a quantitative

22

87199.

13

C NMR approach. RSC Adv.2015, 5, 87187-

23 33 ACS Paragon Plus Environment

Energy & Fuels

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

1

Page 34 of 34

TOC Figure

2

3 4


Lignin Alkylation Enhances Enzymatic Hydrolysis ... - ACS Publications

Recommend Documents