The Nature of Hololignin - ACS Sustainable Chemistry & Engineering

Subscriber access provided by READING UNIV

Article

The nature of hololignin Xianzhi Meng, Yunqiao Pu, Poulomi Sannigrahi, Mi Li, Shilin Cao, and Arthur Jonas Ragauskas ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03285 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 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.

ACS Sustainable Chemistry & Engineering 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 26

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

ACS Sustainable Chemistry & Engineering

The nature of hololignin

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

Xianzhi Meng†, Yunqiao Pu‡, Poulomi Sannigrahi§, Mi Li‡, Shilin Cao∥, Arthur J. Ragauskas*,†,‡,⊥ †

Department of Chemical & Biomolecular Engineering, University of Tennessee Knoxville, Knoxville, TN 37996, United States ‡ Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States § School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, United States ∥College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou 350002, China ⊥ Department of Forestry, Wildlife and Fisheries, Center of Renewable Carbon, The University of Tennessee Knoxville, Institute of Agriculture, Knoxville, TN 37996, United States Corresponding Author * Art J. Ragauskas. Fax: 865-974-7076; Tel: 865-974-2042; E-mail: [email protected].

35 36 1

ACS Paragon Plus Environment


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

37

ABSTRACT

38

Acid chlorite delignification is frequently used to obtain a mixture of cellulose and

39

hemicellulose known as holocellulose from biomass. While a majority of lignin is removed after

40

holocellulose pulping, there appears to be a minor fraction of lignin that is more resistant to acid

41

chlorite treatment and remains in the holocellulose even after repeated delignification treatment.

42

This type of lignin, defined as hololignin, has not been characterized, is likely to contribute to

43

biomass recalcitrance and is clearly of fundamental interest to understand its structural

44

characteristics. In this study, hololignin isolated from poplar holocellulose was characterized

45

with a wide array of techniques including GPC, quantitative 13C, DEPT-13, HSQC, and 31P

46

NMR. The results were compared to those from milled wood lignin, the representative native

47

lignin isolated from poplar. NMR analysis demonstrated a depletion of cinnamyl aldehyde,

48

acetyl group, and decrease of p-hydroxybenzoate structural units in hololignin. An enrichment of

49

condensed structures in hololignin was observed. Hololignin also had a significantly lower

50

molecular weight than milled wood lignin. Finally, hololignin is relatively enriched in guaiacyl

51

units and has a lower S/G ratio, lower β-O-4 ether linkages, lower aliphatic and phenolic

52

hydroxyl group, and higher carboxylic acid group than the MWL.

53

KEYWORDS

54

Acid chlorite, holocellulose pulping, hololignin, delignification, milled wood lignin,

55

INTRODUCTION

56 57

Cellulose, hemicellulose and lignin, the three major components of lignocellulosic biomass are closely associated in the plant cell wall.1 An in-depth understanding of the structure of the 2


Page 2 of 26

Page 3 of 26

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


58

carbohydrates and lignin is necessary to manipulate lignocellulosics for material applications

59

and/or to minimize the recalcitrance of biomass for the biological conversion of bioresources to

60

biofuels.2 Bulk compositional information of these biomolecules can be obtained through

61

hydrolysis or degradation of biomass without separating them from the lignocellulosic matrix.

62

However, in order to gain a better understanding of the structure and morphology of cellulose,

63

hemicellulose and lignin, it is essential to isolate each component from the lignocellulosic matrix

64

for detailed analytical studies.3-5

65

Lignin, a three dimensional cross-linked heteropolymer, accounts for 15-30% of biomass and

66

is responsible for structural integrity and water transport in plants.6 Currently, only less than 5%

67

of lignin is used in low-value commercial applications such as dispersants and surfactants, or as

68

concrete additive, while the remainder is burnt as fuel or discarded as waste.7-10 Therefore, how

69

to engineer lignin based products of increased value, which requires us to develop and optimize

70

efficient lignin isolation process became crucial. Meanwhile, understanding the structural

71

features of lignin isolated from different processes is equally important as their applications are

72

strongly associated with their structure. For example, lignin with aliphatic thiol groups known as

73

kraft lignin is mainly used in heat and electricity generation due to its high sulfur content.11

74

The extraction of lignin from lignocellulosic substrates, known as delignification, has been

75

extensively studied by the pulp and paper industry and can be achieved by a variety of methods

76

including kraft pulping, alkaline peroxide delignification, oxygen delignification, and chlorine

77

dioxide bleaching.12-14 During these processes, lignin is usually broken down to lower fragments,

78

resulting in changes to its physicochemical features.10 Oxidative biomass delignification methods 3



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

79

rely on a reactive oxidant to oxidize phenolic and/or non-phenolic lignin structures. In oxygen

80

delignification, the reaction between oxygen and lignin under alkali conditions involves an

81

electron transfer from phenolates to oxygen followed by subsequent coupling reactions between

82

the phenoxy radical and oxygen which ultimately fragments aromatic rings.15 The electron transfer

83

reaction to oxygen also leads to the generation of peroxy radical, hydrogen peroxide, and the

84

hydroxyl radical which also oxidatively fragment lignin. Hence, alkaline hydrogen peroxide

85

delignification of lignin has some similarities to oxygen delignification but differences in process

86

conditions frequently differentiates the extent of delignification and oxidative products.16

87

Acid chlorite delignification is one of the most commonly used holocellulose pulping methods

88

to produce holocellulose which is prepared both in academic and industrial labs to characterize

89

cellulosic properties.17 The mixture of acetic acid and sodium chlorite has been shown as an

90

effective reagent to selectively remove lignin from biomass with only trace solubilization of

91

glucan and xylan at moderate temperatures. The mechanism by which chlorite oxidation

92

delignifies biomass is still under debate; however, it is known that this process is based on the

93

oxidation of lignin by chlorine dioxide which is generated from sodium chlorite under acidic

94

condition.18 It has been reported that the aromatic structures with free phenolic hydroxyl groups in

95

lignin can be rapidly degraded to muconic acid and quinone structures.19 It was also reported that

96

chlorine dioxide exerted oxidative attack on both the side chains and aromatic ring of etherified

97

phenolics.20 Kolar et al. reported that the methylene group in allylic position can be oxidized by

98

chlorine dioxide to a carbonyl group.21 Other reactions, such as reduction of methoxyl content and

99

introduction of chlorine into lignin, via ClO2 generated Cl2, have also been reported.20 In addition, 4


Page 4 of 26

Page 5 of 26

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


100

Kumar et al. showed that holocellulose pulping had a detrimental effect on cellulose chain length

101

and molecular weight.22 Hubbell and Ragauskas reported that these detrimental effects on

102

cellulose degree of polymerization are minimized to a great extent by the presence of small

103

amounts of lignin in the holocellulose of poplar.17 These studies found that once the lignin

104

content is reduced to below approximately 1%, further extended holocellulose pulping would

105

lead to the polysaccharides susceptible to degradation by acid hydrolysis and oxidative

106

cleavage.17 Therefore, although acid chlorite delignification can be effectively used to delignify

107

biomass, the lignin content should be monitored throughout the process and not allowed to drop

108

below a certain threshold (i.e., ~1% for poplar).

109

While carbohydrate loss is negligible during mild acid chlorite delignification, there is

110

always a chlorite resistant lignin fraction left in holocellulose even after repeated delignification

111

treatment.23 The chemistry nature of this type of residual lignin, termed as hololignin, is still

112

quite limited to our best knowledge. Therefore, understanding the chemistry of hololignin that

113

remains associated with holocellulose even after repeated acid chlorite treatments would provide

114

insight into the understanding of current oxidizing delignification mechanisms and further lead to

115

improvements in biomass delignification. In this study, hybrid poplar biomass was subjected to

116

acid chlorite delignification and the residual lignin was extracted from holocellulose by refluxing

117

with acidic dioxane following literature procedures.24 The acidic dioxane extracted lignin from

118

holocellulose (hereafter referred to as hololignin) was extensively characterized with NMR

119

spectroscopy and gel permeation chromatography. These results were then compared to those

120

from milled wood lignin (MWL) isolated from the untreated hybrid poplar biomass. The 5



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

121

structure of hololignin was characterized for the first time in this study, aiming to provide

122

detailed insight into the impact of oxidative delignification on the lignin structure and

123

subsequently provide valuable information on the future development of biomass delignification.

124

MATERIALS AND METHODS

125

Biomass feedstock. Hybrid poplar (Populus trichocarpa x deltoides) milled to pass a 0.841

126

mm screen in a Mini Wiley Mill (Thomas Scientific, Swedesboro, NJ) was obtained from Oak

127

Ridge National Laboratory (ORNL), Oak Ridge, TN. Extractives were removed by placing the

128

biomass samples into an extraction thimble in a Soxhlet apparatus (Foss, SoxtecTM 2050). The

129

extraction flask was filled with ethanol/toluene (1:2, v/v) and then refluxed with boiling rate of

130

24 solvent cycles per h for ~8 h.

131

Holocellulose preparation. Holocellulose was prepared from the extractive-free biomass

132

according to literature procedures by exposure to NaCIO2 (1.30 g/1.00 g lignocellulosic dry

133

biomass) in acetic acid (10% by dry weight of biomass) at 70 oC for 1 h. The process was

134

repeated to ensure maximum lignin removal. The samples were then filtrated and rinsed with an

135

excess of DI water.

136

Carbohydrate and lignin Composition. The chemical composition of feedstock and

137

holocellulose pulp was determined according to a NREL analytical procedure.25 Carbohydrate

138

content was measured by a high-performance anion exchange chromatography with pulsed

139

amperometric detection using Dionex ICS-3000 (Dionex Corp., USA) with an conductivity

140

detector, a CarboPac PA1 column (2 × 250 mm, Dionex), a guard CarboPac PA1 column (2 × 50

141

mm, Dionex), a PC 10 pneumatic controller, and a AS40 automated sampler. 0.20 M and 0.40 M 6


Page 6 of 26

Page 7 of 26

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


142

NaOH was used as the eluent and post-column rinsing effluent, respectively. Calibration was

143

performed with standard solutions of glucose, xylose, arabinose, mannose and galactose, and

144

fucose was used as an internal standard.

145

Isolation of dioxane lignin. Holocellulose was refluxed with dioxane and HCl (9:1, v/v)

146

under nitrogen for 4 h. The sample was then filtered and subjected to two rounds of extraction

147

with dioxane for 24 h, and the combined aliquots were neutralized, filtered and concentrated

148

under reduced pressure. The precipitated lignin was washed with water and then freeze-dried.

149

Isolation of milled wood lignin. Milled wood lignin (MWL) was isolated from the poplar

150

according to a modified literature.26 Briefly, extractive free poplar was placed in a porcelain ball

151

mill jar, along with porcelain grinding media and ground for 5 days in a rotary ball mill. Lignin

152

was extracted from the ground wood with dioxane/water (9:1, v/v). The crude MWL was then

153

purified using a series of extraction steps with 90% acetic acid, 1,2-dichloroehtane/ethanol (2:1,

154

v/v), diethyl ether and petroleum ether. MWL was then dried overnight in a vacuum oven at 40

155

o

C prior to NMR and GPC analysis.

156

Lignin molecular weight distribution analysis. The lignin molecular weight distribution

157

analysis was performed with gel permeation chromatography (GPC) after acetylation. Dry lignin

158

(~25 mg) was added into a mixture of acetic anhydride/pyridine (1:1, v/v, 2.00 mL) and stirred at

159

room temperature for 24 h. Ethanol (25.00 mL) was added to the reaction mixture, left for 30

160

min and removed with a rotary evaporator. The addition and removal of ethanol was repeated

161

until all traces of acetic acid were removed from the sample. The residue was dissolved in

162

chloroform (2.00 mL) and precipitated with diethyl ether (100.00 mL). The precipitate was 7



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

163

centrifuged, washed with diethyl ether and dried under vacuum prior to GPC analysis. The

164

molecular weight distributions of the acetylated lignin samples were analyzed on a PSS-Polymer

165

Standards Service (Warwick, RI, USA) GPC SECurity 1200 system featuring Agilent HPLC

166

1200 components equipped with four Waters Styragel columns (HR1, HR2, HR4 and HR6) and

167

an UV detector (270 nm). Tetrahydrofuran was used as the mobile phase and the flow rate was

168

1.0 mL/min. Data collection and processing were performed using Polymer Standards Service

169

WinGPC Unity software (Build 6807). Standard narrow polystyrene samples were used for

170

calibration.

171

Lignin NMR characterization. All NMR experiments were performed on a Bruker

172

Avance-III 400 MHz spectrometer operating at a frequency of 100.59 MHz. For quantitative 13C

173

NMR, lignin (70.0 mg) was dissolved in DMSO-d6 (0.5 ml) with slight heating and stirring with

174

a micro stir bar. The 13C spectra were acquired at 50 oC in order to reduce viscosity. An

175

inverse-gated decoupling pulse sequence was used to avoid nuclear Overhauser effect (NOE) and

176

10, 000 scans were collected with a pulse delay of 12 s. NMR experiments termed distortionless

177

enhancement by polarization transfer (DEPT) were used to distinguish between primary,

178

secondary and tertiary carbon atoms in lignin. The DEPT NMR spectra were acquired using a

179

135° pulse angle, 3-s pulse delay and 8192 scans. A standard Bruker heteronuclear single

180

quantum coherence pulse sequence (hsqcetgp) was used on a BBO probe for the

181

two-dimensional 13C-1H HSQC NMR spectra with the following conditions: 13 ppm spectra

182

width in F2 (1H) dimension with 1024 data points and 210 ppm spectra width in F1 (13C)

183

dimension with 256 data points; a 1.5 s pulse delay, a 90o pulse, a JC-H of 145 Hz, and 32 scans. 8


Page 8 of 26

Page 9 of 26

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


184

The DMSO solvent peak around 39.5 ppm (C) and 2.50 ppm (H) was used for chemical shifts

185

calibration. 31P NMR spectra were acquired after dissolving lignin (~25 mg) in a pyridine/CDCl3

186

(1.5:1.0, v/v) solution and derivatizing with TMDP (75 µL). Chromium acetylacetonate and

187

endo-N-hydroxy-5-norbornene-2,3-dicarboximide (NHND) were also added into the solution as

188

relaxation agent and internal standard, respectively. Quantitative 31P NMR spectra were acquired

189

using an inverse-gated decoupling (Waltz-16) pulse sequence with a 25 second pulse delay and

190

128 scans. 13C, DEPT, HSQC, and 31P NMR data were processed using TopSpin 2.1 (Bruker

191

BioSpin).

192

Error Analysis. Error bars shown in each figure represent standard error which is the standard

193

deviation of the sampling distribution of a statistic, defined as the standard deviation divided by

194

the square root of the sample size – three independent assays unless otherwise specified.

195

RESULTS AND DISCUSSION

196

Carbohydrate and Lignin Composition. Figure 1 shows the glucan, xylan, and Klason

197

lignin contents in raw feedstock and holocellulose of poplar. Holocellulose pulping removes

198

almost no cellulose and hemicellulose sugars, and the Klason lignin content is decreased by 81%

199

after holocellulose pulping of poplar. The hololignin extraction yield based on the initial Klason

200

lignin content in holocellulose is around 75%. The compositional analysis results obtained in this

201

study are in agreement with other data reported for poplar holocellulose in literature.27

9



60

Glucan

Xylan

Lignin

50 Weight percentage (%)

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

40 30 20 10 0 Raw feedstock

Holocellulose

202 203

Figure 1. Glucan, xylan, and lignin content of raw feedstock and holocellulose of poplar.

204

Lignin molecular weight distribution. The number-average molecular weight (Mn) and

205

weight-average molecular weight (Mw) of the lignin fractions including milled wood lignin

206

(MWL) and hololignin were determined by gel permeation chromatography (GPC). The poplar

207

hololignin demonstrates a much lower molecular weight than those for MWL by an order of

208

magnitude (Figure 2). For example, the Mw and Mn of MWL decreased by 76% and 68% after

209

holocellulose pulping, respectively. In addition, the hololignin shows a lower PDI (3.1)

210

compared to that of MWL (3.8), indicating that the lignin residues after holocellulose pulping

211

become more uniform in terms of molecular weight distribution. This is in agreement with other

212

data reported that residual lignin in pulp after oxygen delignification mainly contains fragments

213

with smaller molecular weights which were also more uniformly distributed.28

10


Page 10 of 26

Page 11 of 26

14000

MWL

Hololignin

12000 Molecular weight (g/mol)

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


10000 8000 6000 4000 2000 0 Mn (g/mol)

Mw (g/mol)

214 215

Figure 2. Molecular weight distribution analysis of acetylated poplar milled wood lignin and

216

hololignin as determined by gel permeation chromatography Lignin 13C NMR analysis. The 13C NMR spectra data of poplar MWL and hololignin are

217 218

shown in Figure 3 and the 13C NMR quantitative analysis results are presented in Table 1. Signal

219

assignments and quantitative analysis of these lignin samples were performed following

220

literature reports.29-31 The aromatic region of lignin 13C NMR spectrum can be usually divided

221

into three regions of interest: unsubstituted aromatic (δ 123 – 106 ppm), carbon-substituted

222

aromatic (δ 140 – 123 ppm), and oxygenated aromatic (δ 156 – 140 ppm) regions. The

223

oxygenated aromatic region (CAr-O) which is the most predominant among the aromatic signals,

224

primarily contains C3 and C4 carbons of guaiacyl (G) units and C3, C4 and C5 carbons of syringyl

225

(S) units on the lignin aromatic ring. The carbon-substituted aromatic region (CAr-C) mostly 11



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

226

consists of C1 carbons plus any ring carbons involved in crosslinking, such as 5-5 or β-5

227

substructures present in lignin.32 The unsubstituted/pronated aromatic region (CAr-H) usually

228

comprises the C2, C5, C6 carbons in G units and C2, C6 carbons in S units. As shown in Figure 3,

229

the 13C NMR spectra of hololignin and MWL exhibit significant differences in signal intensity

230

within both aliphatic and aromatic regions. The most notable differences observed are the

231

substantial decrease in the signal intensity of aromatic carbons associated with S units for

232

hololignin. For example, the signal peaked around 153 ppm attributing to the C3/5 in etherified S

233

units is significantly decreased after holocellulose pulping. The signals of cinnamyl aldehyde

234

(CγHO) at the chemical shift of ~193 ppm and CH3/carbonyl carbon in acetyl group around

235

~20/169 ppm are depleted in hololignin compared to MWL, which is not unusual considering the

236

oxidative acidic conditions employed in chlorite holopulping. The reduction of methoxy group

237

around 56 ppm could be attributed to the lignin oxidation by chlorine dioxide, as it has been

238

shown that vanillin could be converted to β-formyl muconic acid monomethyl ester along with

239

quinoidal and other products.20 The small broad peak centered at 174 ppm in hololignin could be

240

attributed to the presence of carboxylic acid groups which are most likely due to chlorine dioxide

241

oxidation of lignin components during holocellulose pulping.

12


Page 12 of 26

Page 13 of 26

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


Hololignin

ppm OMe

CAr-O

DMSO CAr-C

Cγ HO

MWL

Cβ, Cα, Cγ

CAr-H

CH3 in acetyl

COOR

ppm

242 243

Figure 3. Quantitative 13C NMR spectra of poplar hololignin and MWL dissolved in DMSO-d6.

244

Ar: aromatic; OMe: methoxyl; DMSO; dimethyl sulfoxide. Other vital structural information on lignin that can be gained from quantitative 13C NMR

245 246

analysis is shown in Table 1. A well resolved signal at ~162 ppm is attributed to the aromatic C4

247

in the p-hydroxybenzoate (PB) group. Only MWL has signals in this region, and its content is

248

calculated as 0.01 per aromatic ring while hololignin shows almost no PB structure remained

249

after chlorite delignification. An increase of degree of condensation is also found in hololignin.

250

The S/G ratio of lignin was calculated based on the number of carbons/aromatic ring in C2,6 of S

251

units and C2 of G units.32, 33 The S content of hololignin is much lower than that of MWL in

252

poplar, which in turn results in a large decrease in its S/G ratio. It indicates that S units are

13



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 26

253

probably more reactive and appear to be more easily removed during the acid chlorite

254

delignification process.

255

Table 1. Structural characteristics of poplar MWL and holocellulose lignin calculated from

256

quantitative 13C NMR data. δ (ppm)

Assignment

# per aromatic ring MLW

Hololignin

168 - 64

Conjugated COOR

0.14

0.22

156 - 140

Oxygenated aromatic C

2.03

2.57

140 - 123

C-substituted aromatic C

1.67

1.25

123 - 106

Unsubstituted aromatic C

2.27

2.20

S:G

0.99

0.24

Degree of condensation

0.73

0.80

257 258

To further understand the structure of poplar MWL and hololignin, the samples were analyzed

259

using DEPT-135 13C NMR which only shows carbons attached to protons. The spectral

260

assignments are annotated on the spectra based on literature references in Figure 4.34, 35 The

261

guaiacyl unit shows three aromatic carbons from 6-, 5-, and 2-position CH groups at 118.5, 114.5,

262

and 111.2 ppm, and syringyl unit shows two aromatic carbons from 6- and 2-position CH groups

263

at 103.9 ppm. The DEPT NMR spectra confirm the reduction in S units as seen by the substantial

264

decrease of the s-2/6 peak at 103.2 ppm associated with only weak signals (especially G6 signal) 14


Page 15 of 26

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


265

remained from G units for hololignin. The intensity of the cinnamyl alcohol (Cα=Cβ) peak at

266

~130 ppm which overlaps with the side-chain olefinic CβH group of cinnamaldehyde is greatly

267

reduced in hololignin. In kraft pulping process, it has been showed that the cinnamyl

268

alcohol-type moieties were actually much more abundant in residual lignin than in the MWL.36

269

The depletion of cinnamyl aldehyde signal around 193 ppm is also further confirmed in the

270

DEPT NMR spectrum of hololignin. Hololignin

ppm MWL

OMe Cα=Cβ CβHO

g-5 g-6 g-2

s-2/6

CβH

CαH

Cγ HO

Cγ H2

ppm 271 272

Figure 4. DEPT-135 13C NMR spectra of poplar MWL and hololignin dissolved in DMSO-d6.

273

HSQC NMR analysis. Relative abundance of the lignin interunit linkage (e.g., β-O-4) and

274

monolignol compositions (e.g., S/G) were semi-quantitatively evaluated by using volume

275

integration of contours in HSQC spectra of MWL and hololignin.37, 38 Figure 5 represents the

276

aromatic and aliphatic regions of HSQC NMR spectra of MWL and hololignin isolated from 15



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

277

poplar. The cross peak assignments are also presented in Table S1 in supporting materials. In

278

aromatic region, both MWL and hololignin appeared mainly composed of guaiacyl (G) and

279

syringyl (S) along with considerable amounts of p-hydroxybenzoate (PB) units especially in

280

MWL. G unit shows correlations around 110.9/6.95, 115.1/6.75, and 118.8/6.78 ppm for C2/H2,

281

C5/H5, and C6/H6, respectively, whereas S unit shows major cross peaks centered at 103.9/6.65

282

ppm for C2,6/H2,6 correlations. α-Oxidized S units are observed in both MWL and hololignin

283

around 106.4/7.26 ppm for S’2/6 with significant increase of the oxidized S units signal intensity

284

in the hololignin. The oxidized G unit is only observed in hololignin with a cross peak centered

285

at 111.6/7.48 ppm for G’2. In aliphatic region of MWL, the C-H correlations in β-O-4, β-β, and

286

β-5 linkages are well recognized for α, β, and γ positions. On the other hand, the C-H correlation

287

signal in β-O-4 becomes significantly weak, while the β-β and β-5 linkages have disappeared in

288

the aliphatic region of hololignin spectra. The semi-quantitative analysis of S, G, and PB units is

289

conducted by integrating peaks of S2/6, G2, α-oxidized S2/6 and G2, PB2/6. For the lignin interunit

290

linkages, the α position of β-O-4, β-β, and β-5 is used. Table 2 presents the relative amount of

291

lignin subunits as well as their inter-linkages for MWL and hololignin. Results indicate that a

292

significant amount of S and G units are oxidized S and G in hololignin. The lower content of S

293

units and higher content of G units in hololignin result in a large decrease in S/G ratio, indicating

294

that S units appear to be more easily removed during the acid chlorite delignification process.

295

Meanwhile, all the distribution of lignin inter-linkages especially the predominance of β-O-4

296

linkages are significantly decreased after chlorite pulping in hololignin.

297 16


Page 16 of 26

Page 17 of 26

MWL

Bβ

Cβ

-OMe

δ13C/ ppm

298 Hololignin -OMe 60

Aγ + others Iγ

A’γ

Bγ X5

A’γ

Dioxane 70

Aα(G) Aα(S)

Aα

Cγ X3

80

X4 Aβ(S) Bα

Aβ(G)

Aβ(S) 90

Cα 4.5

4.0

3.0 δ1H/ppm 6.0

3.5

MWL

5.5

5.0

4.5

4.0

Hololignin

S2/6

3.5

3.0 δ1H/ppm δ13C/ppm

5.0

S2/6

S’2/6

S’2/6 G2

110

5.5

G2 G5+PB3/5

G’2

G5+PB3/5

G6

G6

120

6.0

130

Jβ

PB2/6

PB2/6 140

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


8.0

7.5

7.0

6.5

δ1H/ppm 8.0

7.5

7.0

6.5

δ1H/ppm

299

17



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

300

Figure 5. Aliphatic regions (top) and aromatic regions (bottom) of the HSQC NMR spectra of

301

MWL and hololignin. A: β-O-4, B: β-5, C: β-β, I: cinnamyl alcohol, S: syringyl, S’: oxidized

302

syringyl, G: guaiacyl, G’: oxidized guaiacyl, PB: p-hydroxybenzoate, and J: cinnamaldehyde.

303

Table 2. Semi-quantitative information of lignin inter-linkages and subunits in MWL and

304

hololignin isolated from poplar. Lignin structures

MWL

Hololignin

Syringyl (S)

55.5

34.0

Oxidized syringyl (S’)

1.6

30.2

Guaiacyl (G)

44.5

66.0

Oxidized guaiacyl (G’)

0

29.1

S/G

1.25

0.52

14.7

9.7

β-ary ether (β-O-4)

57.6

19.4

Resinols (β-β)

7.9

0

Phenylcoumaran (β-5)

3.9

0

Sub-units

Hydroxycinnamates p-Hydroxybenzoate (PB) Inter-linkages

305 306

Note. Content (%) expressed as a traction of S + G + H.

Lignin 31P NMR analysis. Hydroxyl groups in lignin represent one of the most important

307 308

functionalities affecting physical and chemical properties of lignin. Quantitative 31P NMR 18


Page 18 of 26

Page 19 of 26

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


309

spectra of hololignin and MWL were used to determine the content of free hydroxyl groups

310

including guaiacyl, C5 substituted, p-hydroxyphenyl, aliphatic, and carboxylic hydroxyl groups.

311

The chemical structural differences between poplar MWL and hololignin as seen from 13C and

312

HSQC NMR are also reflected in the results from 31P NMR analysis of the phosphitylated lignin,

313

as summarized in Figure 6 and 7. There is a ~40% decrease in the content of aliphatic OH group

314

in hololignin after holocellulose pulping. The total amount of free phenolic OH groups in

315

hololignin is ~43% lower than that in MWL. This is quite different compared to kraft pulping in

316

which the residual lignins normally have a much greater quantity of phenolic groups than MWL

317

due to the cleavage of aryl ether linkages.39 The amount of guaiacyl and p-hydroxyphenyl

318

hydroxyl group is much lower in hololignin than MWL, while the amount of C5 substituted

319

phenolic OH is increased in holocellulose lignin, suggesting that holocellulose pulping results in

320

enriching condensed lignin fragments to some extent. Condensed structures were also found

321

quite resistant to a variety of bleaching agents and our results are consistent with these findings.15,

322

40, 41

323

of the hololignin as compared to poplar MWL. This could be due to the three-electron oxidation

324

of phenoxyl radical that is generated from phenol structure to a monomethyl ester of muconic

325

acid derivative.21 The nature of lower content of phenolic hydroxyl group and relatively higher

326

amount of carboxylic acid group in hololignin compared to MWL which could result in a

327

significantly decreased protein adsorption capacity in enzymatic hydrolysis.4, 42

In addition, 31P NMR analysis also shows a large increase in the carboxylic acid OH content

19



Hololignin

ppm MWL

Internal standard

Aliphatic

p-hydroxyl Guaiacyl C5 substituted Carboxylic

ppm

328 329

Figure 6. Quantitative 31P NMR spectra of poplar hololignin and MWL after phosphitylation of

330

the OH groups with TMDP. Internal standard: N-hydroxy-5-norbornene-2,3-dicarboximide. 8

MWL

Hololignin

7 OH content (mmol/g)

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

6 5 4 3 2 1 0

331 20


Page 20 of 26

Page 21 of 26

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

332


Figure 7. Hydroxyl group contents of poplar MWL and hololignin as determined by 31P NMR.

333 334

CONCLUSION

335

Lignin’s structural complexity requires comprehensive characterization to fully understand the

336

effect of delignification process on the structure of lignin. In this study, the fraction of poplar

337

lignin that is resistant to acid chlorite delignification, termed as hololignin, was isolated and

338

analyzed for its physicochemical structures for the very first time. Compared to poplar milled

339

wood lignin, hololignin has significantly lower molecular weight and much higher oxygenated

340

aromatic carbon content. In addition, an enrichment of condensed structures in hololignin is

341

observed which probably results in the resistance of residual lignin to acid chlorite. Hololignin is

342

also relatively enriched in guaiacyl units and has a lower S/G ratio, lower β-O-4 ether linkages,

343

lower aliphatic and phenolic hydroxyl group, and higher carboxylic acid group than the MWL.

344

ASSOCIATED CONTENT

345

Supporting Information

346

The supporting information is available free of charge on the ACS Publications website at DOI:

347

Assignments of the lignin 13C–1H correlation signals observed in the HSQC spectra of the

348

lignins. (PDF)

349

AUTHOR INFORMATION

350

Corresponding Author

351

*

352

Author Contributions

Art J. Ragauskas. Fax: 865-974-7076; Tel: 865-974-2042; E-mail: [email protected].

21



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

353

The manuscript was prepared through contributions of all authors. All authors have given

354

approval to the final version of the manuscript.

355

Notes

356

The authors declare that they have no competing financial interests.

357

ACKNOWLEDGMENTS

358

This research is funded by U.S. Department of Energy (DOE) Office of Science, Office of

359

Biological and Environmental Research under the Genomic Science Program (FWP ERKP752).

360

This manuscript has been authored by UT-Battelle, LLC, under Contract No.

361

DE-AC05-00OR22725 with the U.S. Department of Energy. The publisher, by accepting the

362

article for publication, acknowledges that the United States Government retains a non-exclusive,

363

paid-up, irrevocable, worldwide license to publish or reproduce the published form of this

364

manuscript, or allow others to do so, for United States Government purposes. The Department of

365

Energy will provide public access to these results of federally sponsored research in according

366

with the DOE Public Access Plan (https://www.energy.gov/downloads/doe-public-access-plan).

367 368

REFERENCES

369 370 371 372 373 374 375 376 377

1. Himmel, M. E.; Ding, S.-Y.; Johnson, D. K.; Adney, W. S.; Nimlos, M. R.; Brady, J. W.; Foust, T. D., Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production. Science 2007, 315, 804-807. DOI:10.1126/science.1137016 2. Foston, M.; Ragauskas, A. J., Biomass Characterization: Recent Progress in Understanding Biomass Recalcitrance. Ind. Biotechnol. 2012, 8, 191-208. DOI:10.1089/ind.2012.0015 3. DeMartini, J. D.; Pattathil, S.; Miller, J. S.; Li, H.; Hahn, M. G.; Wyman, C. E., Investigating plant cell wall components that affect biomass recalcitrance in poplar and switchgrass. Energy & Environmental Science 2013, 6, 898-909. DOI:10.1039/c3ee23801f 4. Guo, F.; Shi, W.; Sun, W.; Li, X.; Wang, F.; Zhao, J.; Qu, Y., Differences in the adsorption of 22


Page 22 of 26

Page 23 of 26

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

378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418


enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism. Biotechnol Biofuels 2014, 7, 38. DOI:10.1186/1754-6834-7-38 5. Zhang, Y.; Yu, G.; Li, B.; Mu, X.; Peng, H.; Wang, H., Hemicellulose isolation, characterization, and the production of xylo-oligosaccharides from the wastewater of a viscose fiber mill. Carbohydr. Polym. 2016, 141, 238-243. DOI:10.1016/j.carbpol.2016.01.022 6. Upton, B. M.; Kasko, A. M., Strategies for the Conversion of Lignin to High-Value Polymeric Materials: Review and Perspective. Chem. Rev. 2016, 116, 2275-2306. DOI:10.1021/acs.chemrev.5b00345 7. Li, C.; Zhao, X.; Wang, A.; Huber, G. W.; Zhang, T., Catalytic Transformation of Lignin for the Production of Chemicals and Fuels. Chem. Rev. 2015, 115, 11559-11624. DOI:10.1021/acs.chemrev.5b00155 8. He, W.; Fatehi, P., Preparation of sulfomethylated softwood kraft lignin as a dispersant for cement admixture. RSC Advances 2015, 5, 47031-47039. DOI:10.1039/C5RA04526F 9. Qian, Y.; Zhang, Q.; Qiu, X.; Zhu, S., CO2-responsive diethylaminoethyl-modified lignin nanoparticles and their application as surfactants for CO2/N2-switchable Pickering emulsions. Green Chemistry 2014, 16, 4963-4968. DOI:10.1039/C4GC01242A 10. Doherty, W. O. S.; Mousavioun, P.; Fellows, C. M., Value-adding to cellulosic ethanol: Lignin polymers. Industrial Crops and Products 2011, 33, 259-276. DOI:10.1016/j.indcrop.2010.10.022 11. Kai, D.; Tan, M. J.; Chee, P. L.; Chua, Y. K.; Yap, Y. L.; Loh, X. J., Towards lignin-based functional materials in a sustainable world. Green Chemistry 2016, 18, 1175-1200. DOI:10.1039/C5GC02616D 12. Mân Vu, T. H.; Pakkanen, H.; Alén, R., Delignification of bamboo (Bambusa procera acher): Part 1. Kraft pulping and the subsequent oxygen delignification to pulp with a low kappa number. Industrial Crops and Products 2004, 19, 49-57. DOI:10.1016/j.indcrop.2003.07.001 13. Banerjee, G.; Car, S.; Scott-Craig, J. S.; Hodge, D. B.; Walton, J. D., Alkaline peroxide pretreatment of corn stover: effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose. Biotechnol Biofuels 2011, 4, 16. DOI:10.1186/1754-6834-4-16 14. Yao, S.; Nie, S.; Zhu, H.; Wang, S.; Song, X.; Qin, C., Extraction of hemicellulose by hot water to reduce adsorbable organic halogen formation in chlorine dioxide bleaching of bagasse pulp. Ind. Crops Prod. 2017, 96, 178-185. DOI:10.1016/j.indcrop.2016.11.046 15. Yang, R.; Lucia, L.; Ragauskas, A. J.; Jameel, H., Oxygen Delignification Chemistry and Its Impact on Pulp Fibers. J. Wood Chem. Technol. 2003, 23, 13-29. DOI:10.1081/WCT-120018613 16. Mittal, A.; Katahira, R.; Donohoe, B. S.; Black, B. A.; Pattathil, S.; Stringer, J. M.; Beckham, G. T., Alkaline Peroxide Delignification of Corn Stover. ACS Sustainable Chemistry & Engineering 2017, 5, 6310-6321. DOI:10.1021/acssuschemeng.7b01424 17. Hubbell, C. A.; Ragauskas, A. J., Effect of acid-chlorite delignification on cellulose degree of polymerization. Bioresour. Technol. 2010, 101, 7410-7415. DOI:10.1016/j.biortech.2010.04.029 18. Collings, G. F.; Yokoyama, M. T.; Bergen, W. G., Lignin as Determined by Oxidation with Sodium Chlorite and a Comparison with Permanganate Lignin1. J. Dairy Sci. 1978, 61, 23



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

419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459

1156-1160. DOI:10.3168/jds.S0022-0302(78)83700-X 19. Tarvo, V.; Lehtimaa, T.; Kuitunen, S.; Alopaeus, V.; Vuorinen, T.; Aittamaa, J., A Model for Chlorine Dioxide Delignification of Chemical Pulp. J. Wood Chem. Technol. 2010, 30, 230-268. DOI:10.1080/02773810903461476 20. Sarkanen, K. V., Chemistry of delignification in pulp bleaching. Pure Appl. Chem. 1962, 5, 219-31. DOI:10.1351/pac196205010219 21. Kolar, J. J.; Lindgren, B. O.; Pettersson, B., Chemical reactions in chlorine dioxide stages of pulp bleaching. Wood Science and Technology 1983, 17, 117-128. DOI:10.1007/BF00369129 22. Kumar, R.; Mago, G.; Balan, V.; Wyman, C. E., Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies. Bioresour. Technol. 2009, 100, 3948-3962. DOI:10.1016/j.biortech.2009.01.075 23. Ahlgren, P. A.; Goring, D. A. I., Removal of wood components during chlorite delignification of black spruce. Can. J. Chem. 1971, 49, 1272-5. DOI:10.1139/v71-207 24. Bauer, S.; Sorek, H.; Mitchell, V. D.; Ibáñez, A. B.; Wemmer, D. E., Characterization of Miscanthus giganteus Lignin Isolated by Ethanol Organosolv Process under Reflux Condition. J. Agric. Food. Chem. 2012, 60, 8203-8212. DOI:10.1021/jf302409d 25. Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D., Determination of structural carbohydrates and lignin in biomass. In Laboratory Analytical Procedure, National Renewable Energy Laboratory: 1617 Cole Boulevard, Golden, Colorado, 2008. 26. Guerra, A.; Mendonça, R.; Ferraz, A.; Lu, F.; Ralph, J., Structural Characterization of Lignin during Pinus taeda Wood Treatment with Ceriporiopsis subvermispora. Applied and Environmental Microbiology 2004, 70, 4073-4078. DOI:10.1128/AEM.70.7.4073-4078.2004 27. Foston, M. B.; Hubbell, C. A.; Ragauskas, A. J., Cellulose isolation methodology for NMR analysis of cellulose ultrastructure. Materials 2011, 4, 1985-2002. DOI:10.3390/ma4111985 28. Prinsen, P.; Rencoret, J.; Gutiérrez, A.; Liitiä, T.; Tamminen, T.; Colodette, J. L.; Berbis, M. Á.; Jiménez-Barbero, J.; Martínez, Á. T.; del Río, J. C., Modification of the Lignin Structure during Alkaline Delignification of Eucalyptus Wood by Kraft, Soda-AQ, and Soda-O2 Cooking. Industrial & Engineering Chemistry Research 2013, 52, 15702-15712. DOI:10.1021/ie401364d 29. Yoo, C. G.; Kim, H.; Lu, F.; Azarpira, A.; Pan, X.; Oh, K. K.; Kim, J. S.; Ralph, J.; Kim, T. H., Understanding the Physicochemical Characteristics and the Improved Enzymatic Saccharification of Corn Stover Pretreated with Aqueous and Gaseous Ammonia. BioEnergy Research 2016, 9, 67-76. DOI:10.1007/s12155-015-9662-6 30. Foston, M.; Samuel, R.; He, J.; Ragauskas, A. J., A review of whole cell wall NMR by the direct-dissolution of biomass. Green Chemistry 2016, 18, 608-621. DOI:10.1039/C5GC02828K 31. Samuel, R.; Pu, Y.; Foston, M.; Ragauskas, A. J., Solid-state NMR characterization of switchgrass cellulose after dilute acid pretreatment. Biofuels 2010, 1, 85-90. DOI:10.4155/bfs.09.17 32. Capanema, E. A.; Balakshin, M. Y.; Kadla, J. F., A Comprehensive Approach for Quantitative Lignin Characterization by NMR Spectroscopy. J. Agric. Food. Chem. 2004, 52, 1850-1860. DOI:10.1021/jf035282b 24


Page 24 of 26

Page 25 of 26

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

460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488


33. Capanema, E. A.; Balakshin, M. Y.; Kadla, J. F., Quantitative Characterization of a Hardwood Milled Wood Lignin by Nuclear Magnetic Resonance Spectroscopy. J. Agric. Food. Chem. 2005, 53, 9639-9649. DOI:10.1021/jf0515330 34. Xia, Z.; Akim, L. G.; Argyropoulos, D. S., Quantitative 13C NMR Analysis of Lignins with Internal Standards. J. Agric. Food. Chem. 2001, 49, 3573-3578. DOI: 10.1021/jf010333v 35. Pu, Y.; Chen, F.; Ziebell, A.; Davison, B. H.; Ragauskas, A. J., NMR Characterization of C3H and HCT Down-Regulated Alfalfa Lignin. BioEnergy Research 2009, 2, 198. DOI: 10.1007/s12155-009-9056-8 36. Pinto, P. C.; Evtuguin, D. V.; Pascoal Neto, C.; Silvestre, A. J. D.; Amado, F. M. L., Behavior of Eucalyptus globulus lignin during kraft pulping. II. Analysis by NMR, ESI/MS, and GPC. J. Wood Chem. Technol. 2002, 22, 109-125. DOI:10.1081/WCT-120013356 37. Kim, H.; Ralph, J., Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Organic & Biomolecular Chemistry 2010, 8, 576-591. DOI:10.1039/B916070A 38. del Río, J. C.; Rencoret, J.; Prinsen, P.; Martínez, Á. T.; Ralph, J.; Gutiérrez, A., Structural Characterization of Wheat Straw Lignin as Revealed by Analytical Pyrolysis, 2D-NMR, and Reductive Cleavage Methods. J. Agric. Food. Chem. 2012, 60, 5922-5935. DOI:10.1021/jf301002n 39. Froass, P. M.; Ragauskas, A. J.; Jiang, J.-e., Nuclear Magnetic Resonance Studies. 4. Analysis of Residual Lignin after Kraft Pulping. Industrial & Engineering Chemistry Research 1998, 37, 3388-3394. DOI:10.1021/IE970812C 40. Jääskeläinen, A. S.; Sun, Y.; Argyropoulos, D. S.; Tamminen, T.; Hortling, B., The effect of isolation method on the chemical structure of residual lignin. Wood Science and Technology 2003, 37, 91-102. DOI:10.1007/s00226-003-0163-y 41. Lai, Y.-Z.; Funaoka, M.; Chen, H.-T., Oxygen bleaching of kraft pulp. 1. Role of condensed units. Holzforschung 1994, 48, 355-9. DOI:10.1515/hfsg.1994.48.4.355 42. Pan, X., Role of functional groups in lignin inhibition of enzymatic hydrolysis of cellulose to glucose. J Biobased Mater Bio. 2008, 2. DOI:10.1166/jbmb.2008.005

489 490 491 492 493 494 25



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

495

Table of Contents

496

497 498 499 500 501 502

SYNOPSIS The nature of hololignin that remains associated with holocellulose after repeated acid chlorite treatments was characterized by various analytical techniques.

503

26


Page 26 of 26

The Nature of Hololignin - ACS Sustainable Chemistry & Engineering

Recommend Documents