Integrated Hot-Compressed Water and Laccase-Mediator Treatments

Subscriber access provided by Yale University Library

Article

Integrated Hot-Compressed Water and Laccase Mediator Treatments of Eucalyptus grandis Fibers: Structural Changes of Fiber and Lignin Jian-Quan Wu, Jia-Long Wen, Tong-Qi Yuan, and Run-Cang Sun J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 31 Jan 2015 Downloaded from http://pubs.acs.org on February 4, 2015

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.

Journal of Agricultural and Food Chemistry 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 38

Journal of Agricultural and Food Chemistry

1

Integrated Hot-Compressed Water and Laccase Mediator Treatments of

2

Eucalyptus grandis Fibers: Structural Changes of Fiber and Lignin

3 4

Jian-Quan Wu, Jia-Long Wen, Tong-Qi Yuan,* and Run-Cang Sun

5 6

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University,

7

Beijing, China

8 9 10 11 12 13 14

* Corresponding author. Tel: +86-10-62336903. Fax: +86-10-62336903. E-mail:

15

(T.-Q.Y.) [email protected].

1

ACS Paragon Plus Environment


16

ABSTRACT

17

Eucalyptus grandis fibers were treated with hot-compressed water (HCW) and

18

laccase mediator to enhance the fiber characteristics and to produce an active lignin

19

substrate for binderless fiberboard production. The composition, morphology and

20

crystallinity index (CrI) analysis of fibers showed that the HCW treatment increased

21

the CrI and lignin content of the treated fibers through partial removal of

22

hemicelluloses. Simultaneously, the HCW treatment produced some granules and

23

holes on the surface of the fibers, which possibly facilitated the accessibility of the

24

laccase mediator. Milled wood lignins and enzymatic hydrolysis lignins isolated from

25

the control and treated fibers were comparatively characterized. A reduction of

26

molecular weight was observed, which indicated that a preferential degradation of

27

lignin occurred after exposure to the laccase mediator. Quantitative 13C, 2D-HSQC

28

and 31P NMR characterization revealed that the integrated treatment resulted in the

29

cleavage of β-O-4' linkages, removal of G' (oxidized α-ketone) substructures, an

30

increase in the S/G ratio and free phenolic hydroxyls.

31 32 33

KEYWORDS: Eucalyptus grandis, hot-compressed water, laccase mediator, milled

34

wood lignin, quantitative 13C, 2D-HSQC, 31P NMR

35

2


Page 2 of 38

Page 3 of 38


36

INTRODUCTION

37

Medium density fiberboard (MDF) is a wood composite product made following one

38

basic principle, whereby solid wood is broken into fibers, the fibers are then mixed with

39

binder, and the mixture is hot-pressed to form a new wood-like structure. By this method,

40

the anisotropy of wood is reduced and a valuable product could be manufactured by small

41

pieces of wood or recycled timber.1 In this process, synthetic adhesives, such as urea

42

formaldehyde (UF) and phenol-formaldehyde (PF), are commonly used to glue the fibers

43

all together to form a wood composite material.2 However, the emission of formaldehyde

44

vapors from the adhesive may cause environmental or health hazards. The new approach

45

for bonding of boards is to use environmentally friendly enzymes, for instance, laccase

46

and peroxidases, for cross-linking and polymerization of wood-based material.3,4

47 48

Technical applications for bonding of wood fibers through activation of surface lignin

49

by enzyme-catalyzed oxidation seem to be promising.5,6 The stable radicals in lignin,

50

which is generated by oxidation, are contributed to the enzyme-catalyzed bonding. It also

51

reported that these radicals can loosening or generate cross-linking of the lignin

52

structure,7-9 as well as produce the adhesive effect during the hot-press process.7,10,11 Both

53

phenolic and non-phenolic aromatic substrates could be modified by the laccase-mediator

54

system treatments with common redox mediators, such as

55

2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS).8 As a matter of fact, it has been

56

confirmed that the internal bonding in dry process boards, which was made from laccase

57

pretreated fibers, was enhanced by the laccase actively.12 The adhesion improvement was

3



58

attributed to the generation of covalent bonds and radical-radical coupling, generating

59

from phenoxy radicals in the laccase incubated fiber lignin. However, the investigation of

60

the detailed changes to lignin structures during the laccase treatment was scarce. Therefore,

61

it is of interest to study this further.

62 63

Generally, since physical and chemical barriers inhibit the accessibility of an enzyme to

64

a substrate, an efficient enzymatic contact of substrate plays an important role in the

65

maximized bio-utilization.13 The extent of contact may be improved by a modification of

66

the surface morphology of the fiber or by a change in internal chemical components.14 It

67

has been reported that during the autohydrolysis process, the more reactive hemicelluloses

68

were solubilized, the lignocellulosic matrix was disrupted, and accordingly a more

69

reactive lignocellulose was generated.15-17 The solubilized components, primarily

70

hemicelluloses and a portion of lignin, were separated from the biomass matrix.

71

Consequently, a redistribution of the original lignin by migration or by dissolved lignin

72

precipitated on the surface results in morphology differences conducive to the coupling

73

reaction.18 Therefore, it is significant to investigate the detailed structural changes of the

74

fiber and lignin during integrated hot-compressed water (HCW) and laccase-mediated

75

treatments.

76 77

The purpose of this study was to understand the structural changes of fiber and lignin

78

during individual HCW and laccase-mediated treatments, as well as the integrated

79

treatment (HCW followed by laccase mediator treatments). Morphologic and chemical

4


Page 4 of 38

Page 5 of 38


80

composition changes of the control and treated fibers, as well as lignin preparations

81

isolated from the corresponding fibers were thoroughly characterized by various advances

82

techniques.

83 84

MATERIALS AND METHODS

85

Materials. Thermo-mechanical pulp (TMP) of E. grandis (5 years old) fibers was

86

supplied by a local fiberboard company in Guangdong Province, China. The choice of

87

TMP of E. grandis fibers was due to it being extensively planted in southern China. The

88

fibers without bark were collected in this study. A fungal laccase (phenoloxidases, EC

89

1.10.3.2) originated from Aspergillus oryzae was purchased from Hubei Yuancheng

90

Pharmaceutical Co., Ltd., China. One unit of laccase activity was defined as the amount of

91

enzyme that oxidized 1 µmol of 2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate

92

(ABTS)/minute, under standard conditions of pH 4.0 and temperature 30 °C, respectively.

93

The cellulolytic enzyme was Celluclast 1.5 L (Novozymes, filter paper activity, 100

94

FPU/g), containing hemicellulase activities. All chemicals used as received were of

95

analytical or reagent grade.

96 97

Treatment of the Fibers. The E. grandis fibers were extracted with toluene/ethanol

98

(2:1, v/v) in a Soxhlet apparatus for 6 h. The extracted fibers without exposure to any

99

other processes were regarded as the control fibers (F1). For the HCW treatment, 25 g of

100

extracted fibers (dry weight, dw) was soaked in 750 mL deionized water in a

101

stainless-steel reactor with a magnetic stirrer. This reactor was sealed with a steel cover

5



102

and autoclaved at the 170 °C for 1 h. After the HCW treatment, the mixtures were cooled

103

to about 30 °C and then the treated fibers were filtrated with a Buchner funnel. The treated

104

fibers were washed repeatedly with deionized water until the filtrate was neutral and then

105

dried for 24 h at 40 °C in a drying oven. The resulting dry samples were named

106

hot-compressed water treated fibers (F2). For the laccase-mediated treatment, the extracted

107

fibers were treated with laccase in an aqueous suspension at 5% consistency with a dosage

108

of 24 U/g fiber (dw) and with ABTS 1.5% (dw, w/w) at the saturated air bubbling

109

atmosphere. They were held at approximately 25 °C and pH 4.0 for 2 h. After the

110

laccase-mediated treatment, the mixtures were filtered and the treated fibers were dried at

111

40 °C for 24 h. These samples were considered as the laccase-mediated fibers (F3).

112

Integrated fibers (F4) were obtained by the same HCW treatment used to prepare the F2

113

samples followed by the same laccase-mediated treatment used to prepare the F3 samples.

114

It should be mentioned that all the treatments were carried out in duplicate.

115 116

Isolation of MWL. Milled wood lignin (MWL) was prepared from the fibers according

117

to the method of Björkman.19 Approximately 15 g of fibers were milled (5 h) in a Fritsch

118

planetary ball mill (Germany) according to a previous literature.20 The ball-milled sample

119

was extracted with dioxane/water solution (96:4, v/v) for 24 h. The mixture was filtered

120

and then the filtrate was concentrated to ca. 30 mL. The dissolved lignin was isolated by

121

precipitating the concentrated liquor into 10 volumes of acidified water (pH 2.0) adjusted

122

by 6 M HCl. The precipitated MWL was obtained by first centrifugation, then washed

123

with same acidified water and finally freeze-dried. The four fiber samples (F1, F2, F3 and

6


Page 6 of 38

Page 7 of 38


124

F4) underwent the same processes, and the four resulting lignin samples were titled as L1,

125

L2, L3 and L4, respectively.

126 127

Preparation of EHL. Enzymatic hydrolysis lignin (EHL) was isolated as described by

128

Wen et al.20 The ball-milled sample (0.3 g) was subjected to enzymatic hydrolysis (100

129

FPU cellulase/g substrate) at 2% solid loading with 50 mM NaOAc buffer (pH 4.8). The

130

mixed slurry was incubated in a rotary shaker (150 rpm) at 50 °C for 48 h. After the

131

enzymatic hydrolysis treatment, the residue (named as EHL) was obtained by centrifuged

132

and thoroughly washed with 70 °C water, and then freeze-dried. No further purification

133

was conducted to preserve all structural features of the isolated EHL. The four fiber

134

samples (F1, F2, F3 and F4) underwent the same processes, and the four resulting lignin

135

samples were named EHL1, EHL2, EHL3 and ELL4, respectively. The freeze-dried sample

136

(30 mg) was suspended in DMSO-d6 (0.5 mL) and the mixture was ultrasonic processed at

137

45 °C for 5 h to complete dissolution.

138 139

Characterization of the Fibers. The composition of the four fiber samples was

140

determined by the National Renewable Energy Laboratory (NREL) protocol using a

141

two-step acid hydrolysis method.21 FT-IR spectra of the fibers were collected in the range

142

of 4000 to 700 cm-1 at 4 cm-1 resolution and 128 scans per sample.20 Scanning electron

143

microscopy images were carried out with a Hitachi S-3400N II (Hitachi, Tokyo, Japan)

144

instrument at 15 kV. For each sample, five different surface areas in average were imaged

145

on each single piece by the functionalized SEM tip. Ten sample pieces were imaged by

7



146

different amplification factors and 150 recognition images were randomly selected for the

147

analysis. The crystallinity index of fiber samples was measured using an XRD-6000

148

instrument (Shimadzu, Japan) with a Cu Kα radiation source (λ=0.154 nm) at 40 kV and 30

149

mA. Samples were scanned from 5 ° to 40 ° (2θ) at a speed of 2 ° /min.

150 151

Characterization of the Lignins. The carbohydrate moieties associated with the four

152

lignin samples were determined by hydrolysis with dilute sulfuric acid according to a

153

previous report.22 The weight-average (Mw) and number-average (Mn) molecular weights

154

of the acetylated lignin preparations were determined by GPC with an ultraviolet detector

155

(UV) at 280 nm. The column used was a 300 mm×7.5 mm i.d., 10um, PL-gel 10 um

156

mixed-B, with a 50 mm × 7.5 mm i.d. guard column of the same material (Agilent

157

Technologies, Scotland, UK). For the quantitative 13C NMR experiments, 140 mg of lignin

158

was dissolved in 0.5 mL of DMSO-d6, and 20 µL of chromium (III) acetylacetonate (0.01

159

M) was added as a relaxation agent to reduce the relaxation delay. For the quantitative

160

2D-HSQC spectra, approximately 90 mg of lignin was dissolved in 0.5 mL of DMSO-d6.

161

Functional groups of the lignin samples were determined by 31P NMR spectra according to

162

previous publications.20,24 The detailed NMR analysis operation conditions have been

163

given previously.20,23,24

164 165

RESULTS AND DISCUSSION

166

Chemical Composition of the Fibers. It should be mentioned that the OH groups in

167

hemicelluloses and cellulose are mainly attributed to the hygroscopicity and dimensional

8


Page 8 of 38

Page 9 of 38


168

changes of fiberboard. From the perspective of fiberboard performance standards, it is

169

desired to simultaneously minimize the water-absorbing capacity and maximize the

170

strength of the product as much as possible. A feasible solution for this challenge is to

171

remove the hemicelluloses but keep the cellulose. In the present study, HCW treatment at

172

a lower temperature of 170 °C for 1 h was performed to partially remove hemicelluloses

173

and boost the fiberboard performance. The selection of the operational parameters

174

employed in this work was based on our previous research and other published work.25,26

175

The elevate of pretreatment temperature from 110 to 170 °C for 1 h and 170 to 230 °C for

176

0.5 h resulted a notable increase of the degradation and solubilization of hemicelluloses.

177

However, the dissolving rate of the hemicelluloses tended to be gently in the range of 170

178

to 230 °C for 0.5 h.25 The similar fractionation of hemicelluloses could be achieved in the

179

experiments performed under the mildest conditions assayed (175 °C, 1 h) according to

180

the report of Romaní etc.26 In addition, considering of the energy consumption and the

181

cost, the experimental condition of 170 °C for 1 h was chosen.

182 183

As shown in Table 1, the yield of F3 (97.5%) was relatively very high, whereas, the

184

yields of F2 (67.2%) and F4 (62.8%) were decreased greatly due to the HCW treatments.

185

The content of arabinose, galactose, mannose and xylose, which are representative of

186

hemicellulosic fractions, was significantly affected by the HCW treatment. A reduction of

187

their total contents from 18.51% in the control fiber (F1) to 12.66% in the HCW fiber (F2)

188

was observed. In particular, the xylose presented a maximal decrease of 30.00%. It has

189

been reported that the HCW treatment can cause a significant reduction of the proportion

9



190

of hemicelluloses.27 However, the content of glucose, the main constituent of cellulose,

191

was relatively stable in this study, possibly explained by the less intensive HCW treatment

192

conditions used. Furthermore, the lignin content slightly increased in all of the treated

193

samples. This result could be explained by the fact that the hemicelluloses content was

194

reduced and the relative content of lignin was increased proportionally. The CrI of F3

195

increased just 1.50% compared to the control sample, and F1 was inferior to that of F2

196

(4.63%). It was assumed that the increase of CrI could be ascribed to the partial removal

197

of the amorphous portion, mainly hemicelluloses and to some extent of lignin. These

198

results indicated that the HCW and laccase-mediated treatments could enhance the

199

mechanical properties of fiber by increasing the CrI.

200

201

Surface Morphology of the Fibers. Scanning electron microscope images of the fibers

202

revealed a rather conspicuous structure on the surface of the fibers (Figure 1). The F1 was

203

observed to be relatively smooth, exhibiting a homogeneous surface that was covered by a

204

thin layer of non-cellulosic material. This smooth layer was probably due to the melted

205

lignin spread on the surface of the fiber. It was logical to suppose that the lignin of the

206

middle lamella was plasticized at a high temperature above its glass-transition-point

207

during the thermo-mechanical pulping process and then spread over the nearby fiber

208

surfaces. When the temperature cooled down, a surface coating was likely formed.28

209

Fernando et al.29 also reported a crust on the surface of the unbleached TMP fibers.

210

However, it was obviously observed that the cellulose microfibril aggregates and granules

211

on the surface of F3 showed a rough and wrinkled mark. Simultaneously, it could be

10


Page 10 of 38

Page 11 of 38


212

noticed that a large number of holes, which would enhance the accessibility of the enzyme,

213

were present on the surface of F2 and F4 but not in the other two samples. The

214

phenomenon could be ascribed to the hydronium ion (H3O+) initially causing

215

depolymerization of xylose and cleavage of the acetyl group. At a high temperature, H3O+

216

is originated from water autoionization, causing hydrolysis and deacetylation of

217

hemicelluloses. Meanwhile, H3O+ is reproduced from the released acetic acid with the

218

further degradation of hemicelluloses. Since the abundance of side-groups and the less

219

uniform structure of the hemicelluloses would be easily solubilised and hydrolyzed in

220

water,16,17,30,31 the formation of holes may have been promoted. The “granular” materials

221

on the surface of F4 after the integrated treatment were due to the deposition of lignin,

222

which was in line with previous reports.32,33 Through the analysis of the SEM and AFM

223

images, Kristensen et al.34 reported that the hydrothermal pretreatment could induce the

224

formation and migration of spherical lignin deposits onto the surface of fibers. The

225

incremental content of deposited lignin on the surface of the fibers increased the effect of

226

the laccase-mediated treatment.

227 228

FT-IR Analysis of the Fibers. Figure 2 shows the FT-IR spectra of the four fiber

229

samples with the bands assigned according to previous literature results.4,6,35 The O-H

230

stretching vibration in OH groups was observed as a wide absorption band at 3347cm-1,

231

and the band at 2903 cm-1 was attributed to the C-H asymmetric vibrations in methyl and

232

methylene groups. The signals at 1599, 1508, and 1421 cm-1 were originated from the

233

aromatic skeletal vibrations and C-H deformation. The symmetric stretching of C−O−C

11



234

presented at 1037 cm-1 was due to the presence of cellulose and hemicelluloses. Some

235

different absorption peaks were clearly distinguished in the four FT-IR spectra. An evident

236

signal at 1736 cm-1 could be found in the spectra of F1 and F3, which was originated from

237

the carbonyl stretching in unconjugated ketone, carbonyl, and ester groups.22 The intensity

238

of this signal was decreased in the spectra of F2 and F4. The same trend was observed

239

regarding the intensity of the band at 1651 cm-1 (carbonyl stretching in conjugated

240

ketones). As mentioned above, the hydronium ions (H3O+) could cause depolymerization

241

of hemicelluloses and cleavage of the acetyl group, which could decrease the content of

242

carbonyl groups. Furthermore, the deposition of lignin on the surface of the fibers, which

243

contains minor amounts of carbonyl groups as compared to hemicelluloses, may reduce

244

the signals of carbonyl groups.

245 246

Yield and Carbohydrate Content in the Isolated MWL Samples. The yields and

247

carbohydrate contents of the isolated MWL samples are given in Table 2. The yield of L1,

248

L2, L3 and L4 was 9.8%, 11.3%, 9.4% and 11.9%, respectively. MWL could act as a

249

representative of native lignin,36,37 but its yield is limited and extensively rely upon

250

milling time and the nature of the lignocellulosic materials.38 Although all MWL samples

251

obtained have been purified, some carbohydrates still associated with the MWL samples.

252

As shown in Table 2, glucose and galactose were the primary sugars, while the amounts of

253

arabinose, rhamnose and xylose were slight. However, the glucose content decreased to

254

0.25% and 0.62% after the HCW and laccase-mediated processes, respectively.

255

Furthermore, the xylose disappeared in L2 and L4, suggesting that most of the xylose that

12


Page 12 of 38

Page 13 of 38


256

was attached to lignin was removed after the HCW treatment. This observation indicated

257

that the HCW treatment was an effective method to remove hemicelluloses.

258 259

Molecular Weight Distributions of the Four MWL Samples. As can be seen from

260

Table 3, the molecular weights of these four MWL samples were ranged from 4030-4790

261

g/mol. As compared to the Mw of L1, the other three lignin samples exhibited a decreasing

262

tendency. Specifically, the Mw of L4 after the integrated treatment was 4030 g/mol. The

263

results revealed that the integrated treatment reduced the molecular weight of L4, which

264

was related to the depolymerization of lignin.39 The difference in the carbohydrates

265

contents could also be explained for this phenomenon.23,36 In addition, the four lignin

266

samples presented a relatively low polydispersity (Mw/Mn < 2). Furthermore, after a

267

relatively short incubation with laccase mediator, more activated sites and stable lignin

268

fragments were obtained. Cleavage of linkages was the main reaction and as a result

269

phenolic monomers were formed.40

270 271

Quantitative 13C NMR Spectra of the Four MWL Samples. The quantitative 13C

272

NMR spectra of the four MWL samples are presented in Figure 3. The amount of specific

273

substructures was calculated based on an aromatic ring (Ar).23,24 The detailed assignments

274

of the signals in the spectra have been given in a previous literature.24 The spectra of the

275

MWL samples were similar to each other with the exception of several peaks. The low

276

carbohydrate contents were verified by the diminished signals between 90 and 102 ppm,

277

which was also confirmed by the aforementioned sugar analysis, and suggested that the

13



278

lignin-carbohydrate complex linkages were partly cleaved after the treatments. The peak at

279

163.2 ppm originated from carboxyl groups was visible in L1 and L3, whereas its intensity

280

was decreased in L2 and L4 due to the HCW treatment. The aryl ether bond (β-O-4', δ

281

61.3-58.0) and the methoxy group (OMe, δ 58.0-54.0) regions were applied to calculate

282

the quantitative information of the structural features. The value of OMe was decreased

283

from 1.89/Ar in L1 to 1.76/Ar (L2) after HCW treatment and to 1.74/Ar (L3) after laccase

284

mediator treatment, which was mainly as a result of the demethoxylation reactions that

285

occurred in the G and/or S units. The content of β-O-4' substructures was reduced after the

286

treatments, and there was an especially marked decline from 0.60 to 0.50/Ar after the

287

integrated treatment. It has been demonstrated that the predominant reactions in lignin

288

were fragmentation rather than polymerization. This fact was also confirmed by 2D NMR

289

integration results.

290 291

Quantitative 31P NMR Spectra of the Four MWL Samples. The reactivity of lignin is

292

mainly defined by the OH groups, especially by the free phenolic OH groups.41 31P NMR

293

spectroscopy is an ideal technique for quantitating the major OH groups in lignin, such as

294

aliphatic OH, condensed and uncondensed phenolic OH, and carboxylic acids groups.23,42

295

The 31P NMR spectra of L1 to L4 are shown in Figure 4 and the contents of different OH

296

groups are given in Table 4. The amounts of non-condensed syringyl OH were calculated

297

to be 0.38, 0.51, 0.38, and 0.66 mmol/g for L1, L2, L3, and L4, respectively. These results

298

suggested that the HCW treatment could induce an augment of the non-condensed

299

syringyl OH, revealing the split of β-O-4' linkages. The content of non-condensed guaiacyl

14


Page 14 of 38

Page 15 of 38


300

OH was gradually decreased after the HCW and laccase mediator treatments; however

301

there was no obvious difference of the condensed guaiacyl OH content among these

302

samples. This implied that parts of the guaiacyl-type lignin units were degraded and

303

possibly released into the hydrolysate.43 The amount of carboxylic groups in L2, L3 and L4

304

were elevated, up to 0.92, 0.43 and 1.03 mmol/g, respectively, as compared to L1 (0.22

305

mmol/g). This increment was likely attributable to the oxidation of lignin side-chains

306

during the HCW and laccase mediator treatments.5

307 308

2D-HSQC NMR Spectra of the Four MWL Samples. The four MWL samples were

309

investigated by 2D-HSQC NMR techniques to reveal their detailed structures. The

310

corresponding spectra are exhibited in Figure 5 and the main substructures are described

311

in Figure 7. As shown in Figure 5, the prominent correlations observed in the side-chain

312

region (δC/δH 50-90/2.5-6.0) of all the four spectra were the β-O-4' ether linkages

313

(substructure A). Specifically, the correlations at δC/δH 71.7/4.85, δC/δH

314

83.4-85.9/4.10-4.30 and δC/δH 59.8/3.39-3.69 belong to the Cα-Hα, Cβ-Hβ and Cγ-Hγ

315

linkages of the β-O-4' ether substructures, respectively. β-β' (resinol, B) and β-5'

316

(phenylcoumaran, C) linkages could also be observed. Strong signals for resinol

317

substructures B were detected at δC/δH 84.9/4.66 (Cα-Hα) and δC/δH 53.5/3.07 (Cβ-Hβ). The

318

correlations located at δC/δH 71.0/3.80 and δC/δH 70.9/4.18 were ascribed to its γ-position.

319

Phenylcoumaran substructures C presented relatively low levels, as shown by the

320

correlations at δC/δH 86.97/5.46, 53.3/3.46 and 62.5/3.72 corresponding to Cα-Hα, Cβ-Hβ

321

and Cγ-Hγ, respectively.

15



322 323

In the aromatic region (δC/δH 100-135/5.5-8.5), the predominance correlations of S and

324

G units could be observed in all the spectra. The C2,6-H2,6 correlation for the normal S-type

325

lignin units was found as a prominent signal at δC/δH 104.2/6.69, whereas the

326

corresponding correlation in Cα-oxidized S′ units was shifted to δC/δH 106.3/7.21. The

327

C2-H2, C5-H5, and C6-H6 correlations for the G units were identified at δC/δH 111.2/6.99,

328

114.5/6.82 and 118.8/6.79, respectively. The correlation for the C2-H2 in oxidized α-ketone

329

G′ units (δC/δH 110.1/7.47) was only detected in the spectra of L1 and L3. Moreover, some

330

condensed lignin structures could also be detected at S2/6, especially for L2 and L4, but the

331

formed mechanism was still unrevealed.44

332 333

The absolute percentage of lignin substructures in the four MWL samples are listed in

334

Table 5. The primary substructures were estimated to be β-O-4' aryl ether linkages, ranged

335

from 46.0% to 50.3%. The secondary substructures were detected to be β-β' linkages

336

(13.6-14.6%). The β-5' linkages was present in lower proportions (1.9-3.8%). It could be

337

found that the percentage of the β-O-4' aryl ether linkages (A) decreased to 46.0% in L4.

338

Three different reactions have been considered to take place with the lignin polymers

339

during the incubation period with laccase mediator: (i) oxidation; (ii) partial

340

polymerization with perhaps concomitant degradation and possible repolymerization; and

341

(iii) cleavage of lignin carbohydrate bonds.3,5,6, 9,11 According to the present data, it could

342

be concluded that a preferential degradation of lignin was occurred after incubated with

343

laccase mediator. As can be seen form Table 5, the S/G ratio of L1 was calculated to be

16


Page 16 of 38

Page 17 of 38


344

1.80. However, for L2 isolated from HCW treated fibers and L4 isolated from the

345

integrated treated fibers the S/G ratio was estimated to be 3.03 and 3.58, respectively. The

346

increased S/G ratios of L2 and L4 further confirmed the preferential degradation or removal

347

of the G-type lignin for the treated fibers during the hydrothermal pretreatment.45 Exactly,

348

the correlations of the G' units (oxidized G units with a Cα-ketone) were disappeared in

349

the corresponding spectra of L2 and L4, which could only be seen at lower contour levels.

350

In addition, the S/G ratio in L3 (1.78) isolated from the laccase mediator treated fiber

351

exhibited a slight reduction, which was also in line with the previous literatures.46

352 353

2D-HSQC NMR Spectra of the Four EHL Samples. To further verify the structural

354

transformations of lignin in the fibers, the EHL samples were isolated to make a

355

comparison with the MWL samples. Undoubtedly, almost all lignin fractions in the

356

materials could be obtained as EHL samples, which were more representative than the

357

MWL samples. To preserve all structural features of the isolated EHL, no further

358

purification was conducted. As shown in Figure 6, the four EHL samples exhibited clearly

359

distinguishable spectra, which were similar to those of the corresponding MWL samples.

360

The primary structural features of lignin, including S, G, and H units as well as various

361

interunit linkages, such as β-O-4', β-β', and β-5' linkages, can be clearly identified in the

362

2D-HSQC spectrum of EHL.

363 364

In the side chain region, however, the correlations at δC/δH 86.7/5.43, which were

365

originated from the Cα-Hα of the phenylcoumaran C (β-5') substructures, could only be

17



366

found in the spectra of EHL1 and EHL3, but still be observed at lower contour levels in

367

other two samples. In the aromatic region, the cross-signals from S, G, and H units could

368

be clearly identified. The C2-H2 correlations in the G′ units (δC/δH 110.1/7.47) were

369

disappeared in all the EHL samples. Whereas, the C2,6-H2,6 correlations from H units were

370

clearly observed at δC/δH 127.8-128.7/7.23, which were disappeared in all the MWL

371

samples. This phenomenon could be ascribed to MWL including a higher proportion of

372

middle lamella material, containing few H units, and the enzymatic treatment removing

373

the most of the carbohydrates, preserving the inner H units.

374 375

The absolute percentages of β-O-4', β-β', and β-5' linkages as well as S/G ratios of EHL

376

samples are also listed Table 5. Obviously, the contents of β-O-4' linkages and the S/G

377

ratios presented similar features to the corresponding MWL samples. The downtrend of

378

the content of β-β' linkages was observed after the hot-compressed water, laccase-mediator

379

system, and integrated treatments. However, the content of β-5' was very low, even not

380

detected in the EHL1 and EHL4 samples. In general, the MWL and EHL samples display

381

great similarity and either could be considered as a good representative of native lignin.

18


Page 18 of 38

Page 19 of 38


382 383

ACKNOWLEDGMENTS We are grateful for the financial support of this research from the National Natural

384

Science Foundation of China (31400296, 31430092 and 31110103902) and Beijing

385

Municipal Commission of Education (20131002201).

386 387 388 389

REFERENCES (1) Hüttermann, A.; Mai, C.; Kharazipour, A. Modification of lignin for the production of new compounded materials. Appl. Microbiol Biot. 2001, 55, 387-394.

390

(2) Liu, C.; Zhang, Y.; Wang, S.; Meng, Y.; Hosseinaei, O. Micromechanical properties of the

391

interphase in cellulose nanofiber-reinforced phenol formaldehyde bondlines. BioResources 2014, 9,

392

5529-5541.

393

(3) Euring, M.; Trojanowski, J.; Horstmann, M.; Kharazipour, A. Studies of enzymatic

394

oxidation of TMP-fibers and lignin model compounds by a Laccase-Mediator-System using

395

different 14C and 13C techniques. Wood Sci. Technol. 2012, 46, 699-708.

396 397

(4) Nasir, M.; Gupta, A.; Beg, M. D. H.; Chua, G. K.; Laccase application in medium density fibreboard to prepare a bio-composite. RSC Adv. 2014, 4, 11520-11527.

398

(5) Euring, M.; Trojanowski, J.; Kharazipour, A. Laccase-mediator catalazed modification of

399

wood fibers: studies on the reaction mechanism and making of medium-density fiberboard. Forest

400

Prod. J. 2013, 63, 54-60.

401

(6) Nasir, M.; Gupta, A.; Beg, M. D. H.; Chua, G. K.; Kumar, A. Fabrication of medium density

402

fibreboard from enzyme treated rubber wood (Hevea brasiliensis) fibre and modified organosolv

403

lignin. Int. J. Adhes. Adhes. 2013, 44, 99-104.

404

(7) Euring, M.; Ruhl, M.; Ritter, N.; Kues, U.; Kharazipour, A. Laccase mediator systems for 19



405

eco-frendly production of medium-density fiberboard (MDF) on a pilot scale: Physicoochemical

406

analysis of the reaction mechanism. J. Biotechnol. 2011, 6(s1), 1253-1261.

407

(8) Shiraishi, T.; Sannami, Y.; Kamitakahara, H.; Takano, T. Comparison of a series of laccase

408

mediators in the electro-oxidation reaction of nonphenolic lignin model compounds. Electroi. Acta.

409

2013, 106, 440-446.

410

(9) West, M. A.; Hickson, A. C.; Mattinen, M. L.; Lloyd-Jones, G. Evaluating lignins as enzyme

411

subatrates: insight and methodological recommendations from a study of laccase-catalyzed

412

lignin polymerization. BioResources 2014, 9, 2782-2796.

413 414

(10) Widsten, P.; Kandelbauer, A. Adhesion improvement of lignocellulosic products by enzymatic pre-treatment. Biotechnol. Adv. 2008, 26, 379-386.

415

(11) Felby, C.; Thygesen, L. G.; Sanadi, A.; Barsberg, S.; Native lignin for bonding of fiber

416

board-evaluation of bonding mechanisms in boards made from laccase-treated fibers of beech

417

(Fagus sylvatica). Ind. Crop. Prod. 2004, 20, 181-189.

418

(12) Alvarez, C.; Rojano, B.; Almaza, O.; Rojas, O. J.; Ganan, P. Self-bonding boards from

419

plantain fiber bundles after enzymatic treatment: adhesion improvement of lignocellulosic

420

products by enzymatic pre-treatment. J. Polym. Environ. 2011, 19, 182-188.

421 422 423

(13) Hu, F.; Jung, S.; Ragauskas, A. Impact of pseudolignin versus dilute acid-pretreated lignin on enzymatic hydrolysis of cellulose. ACS Sustainable Chem. Eng. 2012, 1, 62-65. (14) Liu, J.; Hu, H. R.; Xu, J. F.; Wen, Y. B.; Optimizing enzymatic pretreatment of recycled

424

fiber to improve its draining ability using response surface methodology. BioResources 2012, 7,

425

2121-2140.

426

(15) Runge, T.; Wipperfurth, P.; Zhang, C. H. Improving biomass combustion quality using a

20


Page 20 of 38

Page 21 of 38


427

liquid hotwater treatment. Biofuels 2013, 4, 73-83.

428

(16) Ma, X. J.; Yang, X. F.; Zheng, X.; Lin, L.; Chen, L. H.; Huang, L. L.; Cao, S. L.

429

Degradation and dissolution of hemicelluloses during bamboo hydrothermal pretreatment.

430

Bioresour. Technol. 2014, 161, 215-220.

431 432 433

(17) Xu, J.; Thomsen, M. H.; Thomsen, A. B. Feasibility of hydrothermal pretreatment on Maize Silage for bioethanol production. Appl. Biochem. Biotech. 2010, 162, 33-42. (18) Solala, L.; Antikainen, T.; Reza, M.; Johansson, L. S.; Hughes, M.; Vuorinen, T. Spruce

434

fiber properties after high-temperature thermomechanical pulping (HT-TMP). Holzforschung 2014,

435

68, 195-201.

436 437 438 439 440

(19) Björkman, A. Isolation of lignin from finely divided wood with neutral solvents. Nature 1954, 174, 1057-1058. (20) Wen, J. L.; Xue, B. L.; Xu, F.; Sun R. C. Unveiling the structural heterogeneity of bamboo lignin by in situ HSQC NMR technique. Bioenerg. Res. 2012, 5, 886-903. (21) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D.

441

Laboratory Analytical Procedure (LAP): determination of structural carbohydrates and lignin in

442

biomass. Technical Report: NREL/TP-510-42618, 2008. National Renewable Energy Laboratory,

443

Golden. Co, USA.

444

(22) Yuan, T. Q.; Xu, F.; He, J.; Sun, R. C. Structural and physico-chemical characterization of

445

hemicelluloses from ultrasound-assisted extractions of partially delignified fast-growing poplar

446

wood through organic solvent and alkaline solutions. Biotechnol. Adv. 2010, 28, 583-593.

447

(23) Yuan, T. Q.; Sun, S. N.; Xu, F.; Sun, R. C. Characterization of lignin structures and

448

lignin–carbohydrate complex (LCC) linkages by quantitative 13C and 2D HSQC NMR

21



449

spectroscopy. J. Agric. Food Chem. 2011, 59, 10604-10614.

450

(24) Wen, J. L.; Sun, S. L.; Yuan, T. Q.; Xu, F.; Sun, R. C. Structural elucidation of lignin

451

polymers of Eucalyptus chips during organosolv pretreatment and extended delignification. J.

452

Agric. Food Chem. 2013, 61, 11067-11075.

453

(25) Sun, S. L.; Wen, J. L.; Ma, M. G.; Sun, R. C. Structural elucidation of sorghum lignins

454

from an integrated biorefinery process based on hydrothermal and alkaline treatments. J. Agric.

455

Food Chem. 2014, 62, 8120-8128.

456 457 458

(26) Romaní, A.; Garrote, G.; López, F.; Parajó, J. C. Eucalyptus globulus wood fractionation by autohydrolysis and organosolv delignification. Bioresour. Technol. 2011, 102, 5896-5904. (27) López-Linares, J. C.; Romero, I.; Cara, C.; Ruiz, E.; Castro, E.; Moya, M. Experimental

459

study on ethanol production from hydrothermal pretreated rapeseed straw by simultaneous

460

saccharification and fermentation. J. Chem. Technol. Biotechnol. 2014, 89, 104-110.

461

(28) Heinemann, S.; Wang, S, X.; Peltonen. J.; Kleen, M. Characterization of fiber wall surface

462

structure of chemically modified TMP fibers from Norway spruce. Nord. Pulp. Pap. Res. J. 2011,

463

26, 21-30.

464

(29) Fermando, D.; Muhic, D.; Engstrand, P.; Daniel, G. Fundamental understanding of pulp

465

property development under different thermo-mechanical pulp refining condition as observed by a

466

new Simons' staining method and SEM observation of the ultrastructure of surfaces.

467

Holzforschung 2011, 65, 777-786.

468

(30) Dogaris, I.; Karapati, S.; Mamma, D.; Kalogeris, E.; Kekos, D. Hydrothermal processing

469

and enzymatic hydrolysis of sorghum bagasse for fermentable carbohydrates production.

470

Bioresour.Technol. 2009, 100, 6543-6549.

22


Page 22 of 38

Page 23 of 38


471 472 473

(31) Kim, T. H.; Lee, Y. Y. Fractionation of corn stover by hot-water and aqueous ammonia treatment. Bioresour. Technol. 2006, 97, 224-232. (32) Ruiz, H. A.; Rodríguez-Jasso, R. M.; Fernandes, B.D.; Vicente, A. A..; Teixeira, J. A.

474

Hydrothermal processing, as an alternative for upgrading agriculture residues and marine biomass

475

according to the biorefinery concept: A review. Renew. Sust. Energ. Rev. 2013, 21, 35-51.

476

(33) Zhang, M.M.; Chen, G. J.; Kumar, R.; Xu, B. Q. Mapping out the strutural changes of

477

natural and pretreated plant cell wall surfaces by atomic force microscopy single molecular

478

recognition imaging. Biotechnol. Biofuels. 2013, 6, 147.

479 480 481 482 483 484 485 486 487

(34) Kristensen, J. B.; Thygesen, L. G.; Felby, C.; Jørgensen, H.; Elder, T. Cell-wall structural changes in wheat straw pretreated for bioethnol production. Biotechnol. Biofuels. 2008, 1, 5. (35) Wang, S. H.; Liu, J. L.; Li, F.; Dai, J. M. ; Jia, H. S.; Xu, B. S. Study on converting cotton pulp fiber into carbonaceous microspheres. Fiber Polym. 2014, 15, 286-290. (36) Yuan, T. Q.; Sun, S. N.; Xu, F.; Sun, R. C. Structural characterization of lignin from triploid of Populus tomentosa Carr. J. Agric. Food Chem. 2011, 59, 6605-6615. (37) Wen, J. L.; Xue, B. L.; Xu, F.; Sun, R. C.; Pinkertc, A. Unmasking the structural features and property of lignin from bamboo. Ind. Crops. Prod. 2013, 42, 332-343. (38) Rencoret, J.; Marques, G.; Gutiérrez, A.; Nieto, L.; Santoe, J. L.; Jiménez-Barbero, J.;

488

Martínez, A. T.; del Río, J. C. HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and

489

Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state.

490

Holzforschung 2009, 63, 691-698.

491 492

(39) Yelle, D. J.; Kaparaju, P.; Hunt, C. G..; Hirth, K.; Kim, H.; Ralph, J.; Felby, C. Two-dimensional NMR evidence for cleavage of lignin and xylan substituents in wheat straw

23



493

through hydrothermal pretreatment and enzymatic hydrolysis. Bioenergy. Res. 2013, 6, 211-221.

494

(40) Trajano, H. L.; Engle, N. L.; Foston, M.; Ragauskas, A. J.; Tschaplinski, T. J.; Wyman, C.

495

E. The fate of lignin during hydrothermal pretreatment. Biotechnol. Biofuels. 2013.

496

(41) Akim, L. G.; Argyropoulos, D. S.; Jouanin, L.; Leplé, J.-C.; Pilate, G.; Pollet, B.; Lapierre,

497

C. Quantitative 31P NMR spectroscopy of lignins from transgenic poplars. Holzforschung 2001, 55,

498

386-390.

499 500 501

(42) Argyropoulos, D. S. 31P NMR in wood chemistry: A review of recent progress. Res. Chem. Intermed. 1995, 21, 373-395. (43) Nicolas, B.; Roland, E.-H.; Mounir, C.; Mathieu, P.; Stéphane, D.; Philippe, G.

502

Investigation of the chemical modifications of beech wood lignin during heat treatment. Polym

503

Degrad Stabil. 2010, 95, 1721-1726.

504

(44) Torr, K. M.; Love, K. T.; Cetinkol, Ö. P.; Donaldson, L. A.; George, A.; Holmes, B. M.;

505

Simmons, B. A. The impact of ionic liquid pretreatment on the chemistry and enzymatic

506

digestibility of Pinus radiata compression wood. Green Chem. 2012, 14, 778-787.

507

(45) Xiao, L. P.; Shi, Z. J.; Xu, F.; Sun, R. C. Characterization of lignins isolated with alkaline

508

ethanol from the hydrothermal pretreated Tamarix ramosissima. Bioenerg. Res. 2013, 6, 519-532.

509

(46) Ibarra, D.; Chavez, M. I.; Rencoret, J.; del Río, J. C.; Gutierrez, A.; Romero, J.; Camarero,

510

S.; Martínez, M. J.; Jiménez-Barbero, J.; Martinez, A. T. Structural modification of eucalypt pulp

511

lignin in a totally chlorine-free bleaching sequence including a laccase-mediator

512

stage. Holzforschung 2007, 61, 634-646.

24


Page 24 of 38

Page 25 of 38


FIGURE CAPTIONS

Figure 1. SEM micrographs of the control and treated fibers: control fibers (F1), hot-compressed water treated fibers (F2), laccase mediator treated fibers (F3), and integrated treated fibers (F4).

Figure 2. FT-IR spectra of the control and treated fibers.

Figure 3. Quantitative 31C NMR spectra of the four MWL samples.

Figure 4. Quantitative 31P NMR spectra of the four MWL samples.

Figure 5. 2D-HSQC NMR spectra of the four MWL samples.

Figure 6. 2D-HSQC NMR spectra of the four EHL samples.

Figure 7. Main classical substructures, involving different side-chain linkages and aromatic units identified by 2D-NMR of MWL: (A) β-O-4' aryl ether linkages with a free -OH at the γ-carbon; (B) resinol substructures formed by β-β', α-O-γ', and γ-O-α' linkages; (C) phenylcoumaran substructures formed by β-5' and α-O-4' linkages; (I) p-hydroxycinnamyl alcohol end groups; (S) syringyl units; (S') oxidized syringyl units with a Cα-ketone; (G) guaiacyl units; (G') oxidized guaiacyl units with a Cα-ketone; (H) p-hydroxyphenyl units.

25



Page 26 of 38

TABLES Table 1. Compositional, Yield and Crystallinity Index of the Control and Treated E. grandis Fibers

sample F1 F2 F3 F4

carbohydratea content (%) Ara Gal Glc Man Xyl 0.21 1.47 48.74 0.77 16.06 0.04 0.68 50.97 0.71 11.23 0.08 0.91 47.96 0.64 14.47 NDc 0.52 50.22 0.71 14.94

AILb 28.68 33.63 29.33 31.41

ASL 1.21 0.83 0.94 0.32

Yield (%) 100 67.2 97.5 62.8

CrI (%) 37.29 41.92 38.79 41.28

a

Ara, arabinose; Gal, galactose; Glc, glucose; Man, mannose; Xyl, xylose.

b

AIL, acid insoluble lignin; ASL, acid soluble lignin; O, other components; CrI, Crystallinity Index.

c

ND, Not detected.

26


Page 27 of 38


Table 2. The Yield and Carbohydrate Contents of the Four MWL Samples

sample L1 L2 L3 L4

yield (%) with without total sugar b sugars sugars content (%) 9.8 9.5 3.11 11.3 11.1 1.12 9.4 9.2 1.78 11.9 11.8 1.01

carbohydratea content (%) Rha Ara Gal Glc Xyl 0.17 0.06 0.09 0.03

0.02 0.30 0.18 0.63

0.78 0.51 0.47 0.22

1.29 0.85 0.25 NDc 0.62 0.42 0.13 ND

a

Rha, rhamnose; Ara, arabinose; Gal, galactose; Glc, glucose; Xyl, xylose.

b

Based on the dry mass of Klason lignin (%, w/w).

c

ND, Not detected.

27



Table 3. Weight-Average (Mw) and Number-Average (Mn) Molecular Weights and Polydispersity (Mw/Mn) of the Four MWL Samples

Mw Mn Mw/Mn

L1 4790 2930 1.64

lignin sample L2 L3 4340 4370 2590 2670 1.68 1.64

L4 4030 2200 1.83

28


Page 28 of 38

Page 29 of 38


Table 4. Functional Groups of the Four MWL Lignins as Determined by Quantitative 31

a

P-NMR Method (Millimoles per Gram)

lignin

aliphtic OH

L1 L2 L3 L4

3.66 4.29 4.12 4.15

syringyl OH Ca NCa 0.10 0.38 0.12 0.51 0.07 0.38 0.12 0.66

guaiacyl OH C NC 0.16 0.53 0.15 0.30 0.12 0.47 0.15 0.31

carboxylic total group phenolic OH 0.22 1.17 0.92 1.08 0.43 1.04 1.03 1.24

Abbreviation: C, condensed; NC, non-condensed.

29



Table 5. Quantitation of the Four MWL and EHL Samples by 2D-HSQC NMR Method sample L1 L2 L3 L4 EHL1 EHL2 EHL3 EHL4

β-O-4'a 47.6 50.3 46.7 46.0 52.41 57.19 49.46 46.93

β-β' 13.6 14.6 14.3 14.3 11.29 10.60 10.12 8.64

β-5' 2.9 1.9 3.8 2.7 NDc 0.91 0.68 ND

S/Gb 1.80 3.03 1.78 3.58 2.32 3.65 2.23 3.73

a

Results expressed per 100 Ar based on quantitative 2D-HSQC spectra.

b

S/G ratio obtained by the equation: S/G ratio = 0.5IS2,6/IG2.

c

ND, Not detected.

30


Page 30 of 38

Page 31 of 38


Figures

F1

F2

F3

F4

Figure 1.

31



Page 32 of 38

817

F3 F2

4000

3500

3000

1736 1651 1599 1508 1366 1324 1234 1109 1037 902

2903

1421

F1

3347

Transmittance (%)

F4

2500

2000 -1

Wavenumbers (cm )

Figure 2.

32


1500

1000

Page 33 of 38


Figure 3.

33



Figure 4.

34


Page 34 of 38

Page 35 of 38


Figure 5.

35



Figure 6.

36


Page 36 of 38

Page 37 of 38


Figure 7.

37



Table of Contents Graphic

38


Page 38 of 38

Integrated Hot-Compressed Water and Laccase-Mediator Treatments

Recommend Documents