Changes of Chemical Composition and Hemicelluloses Structure in

Subscriber access provided by EKU Libraries

Agricultural and Environmental Chemistry

Changes of chemical composition and hemicelluloses structure in differently aged bamboo (Neosinocalamus affinis) culms Pan Pan Yue, Gen Que Fu, Ya Jie Hu, Jing Bian, Ming-Fei Li, Zheng-Jun Shi, and Feng Peng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03516 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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 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 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.

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 42

Journal of Agricultural and Food Chemistry

1

Changes of chemical composition and hemicelluloses structure in

2

differently aged bamboo (Neosinocalamus affinis) culms

3 4 5

Pan-Pan Yue, † Gen-Que Fu, † Ya-Jie Hu, † Jing Bian, † Ming-Fei Li, † Zheng-Jun Shi,

6

‡

Feng Peng†, *

8

†

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University,

9

No.35 Tsinghua East Road, Haidian District, Beijing, 100083, China

7

10 11

‡

12

Kunming, 650224, China

College of Material Science and Technology, Southwest Forestry University,

13 14

Corresponding Author:

15

Prof. Feng Peng

16

Beijing Forestry University

17

Beijing Key Laboratory of Lignocellulosic Chemistry

18

No.35 Tsinghua East Road, Haidian District, Beijing, 100083, China

19

Tel.: +86 10 62337250; Fax: +86 10 62337250.

20

*E-mail address: [email protected] (F. Peng).

21 22 1

ACS Paragon Plus Environment


23

Abstract

24

To study the differences in chemical composition analysis and spatial distribution of

25

young Neosinocalamus affinis bamboo, we used the method of standard of National

26

Renewable Energy Laboratory and confocal Raman microscopy, respectively. It was

27

found that the acid-soluble lignin and acid-insoluble lignin content showed an inverse

28

relationship with the increasing bamboo age. Raman analysis revealed that Raman

29

signal intensity of lignin both in the secondary cell wall (S) and the compound middle

30

lamella (CML) regions showed a similar increase trend with a growth of bamboo. In

31

addition, eight hemicellulosic fractions were obtained by successively treating

32

holocellulose of the 2, 4, 8, and 12 month-old Neosinocalamus affinis bamboo culms

33

with DMSO and alkaline solution. The ratio of arabinose to xylose of hemicelluloses

34

was increased with the growth of bamboo. FT-IR and NMR analysis revealed that

35

DMSO-soluble hemicelluloses of young bamboo culms are mainly composed of

36

highly substituted xylans and β-D-glucans.

37

Keywords: DMSO- and alkali-soluble hemicelluloses, Increasing age, Structural

38

characterization, HSQC, Confocal Raman microscopy

39 40 41 42

INTRODUCTION

43

Bamboo, a graminaceous, perennial and lignified plant, represents a potential

44

available sustainable energy feedstock.1 Properties and utilization of bamboo are 2


Page 2 of 42

Page 3 of 42


45

influenced by the chemical composition and structural changes during the growth of

46

bamboo culms.2 Bamboo age-related changes caused the chemical compositions

47

various in the cell wall. The chemical compositions of bamboo shoots changed every

48

few days after sprouting, and that the compositions of 20-day samples were basically

49

the same as those of the bamboo stem.3, 4 Therefore, in order to efficiently use the

50

compositions of bamboo, it is necessary to investigate the change of chemical

51

compositions at different immature bamboo growth stages. Meanwhile, an

52

observation of subcellular level in plant is necessary to understand the distribution and

53

clarify the changes of chemical compositions during the growth of bamboo. Currently,

54

confocal Raman microscopy (CRM) as a label-free and nondestructive analytical

55

technique has been applied for detecting chemical compositions of plant cell wall.5 As

56

for the chemical compositions, the content of lignin maintains unchanged or increases

57

slightly in bamboo culms older than one year, however, the holocellulose and

58

α-cellulose contents tend to reduce.6

59

It is well known that hemicelluloses make up about one-third of main chemical

60

compositions available in bamboo, which are interacted with cellulose and lignin.7

61

There are α-ether bonds and ester linkages between hemicelluloses and lignin and the

62

ester bonds between lignin and/or hemicelluloses and hydroxycinnamic acids, such as

63

ferulic acids and p-coumaric.8 Hemicelluloses play an essential part in the assembly,

64

maturation, and deconstruction of plant cell wall, especially in secondary cell walls of

65

woody plants and grass.9 On the other hand, because of the nontoxicity, bioactivity,

66

biodegradability and biocompatibility of hemicelluloses, it has a good applications in 3



67

some areas such as papermaking additive or flocculant, food, medicine, polymer

68

materials, and so on.10-13 Unlike cellulose, hemicelluloses are heteropolysaccharide

69

that are divided into four types of general classes of structurally different cell-wall

70

polysaccharides, that is xylans, xyloglucans, β-glucans with mixed linkages, and

71

mannans. The great structural varieties and diversities of hemicelluloses are one of the

72

greatest challenges for analysis of these cell-wall compounds. These types of

73

hemicelluloses are found in the cell walls of all terrestrial plants, except for the ones

74

that is in the form of β-(1→3, 1→4)-glucans, which are limited to Gramineae and

75

some other groups.14 Mannose can exist in the β-(1→4)-linked polysaccharides which

76

are widely distributed as the primary hemicelluloses in Charophytes.15 Xyloglucans is

77

the most abundant hemicelluloses in primary cell walls of spermatophytes except for

78

grasses. Shibuya and Misaki reported the structural features of a xyloglucan consists

79

of β-(1→4) glucan and arabinoxylan.16 However, xylans can be up to 50% in some

80

tissues of monocotyl plants (grasses and cereals), and they consist of

81

β-(1→4)-D-xylose linked with different kinds of branch units, mostly D-glucuronic

82

acid (and its 4-O-methyl derivative) and arabinose.17 There are similar basic chemical

83

structure of arabinoxylans presenting in various plant tissues, while they differ in the

84

terms of substitution of the xylan backbone.18 The primary differences are found in

85

the ratio of glucuronic acid to xylose (GlcA/Xyl) or arabinose to xylose (Ara/Xyl),

86

and they are commonly used to reflect the degree of branching or linearity of

87

hemicelluloses. There are O-acetyl groups located at some of the hydroxyl groups in

88

the natural xylan backbone, and acetyl groups can be retained during the pretreatment 4


Page 4 of 42

Page 5 of 42


89

of dimethyl sulfoxide (DMSO) solution. In the aspect of bamboo cell physiology, it is

90

necessary to investigate the nature of polysaccharides in bamboo cell walls. Kato et al.

91

studied several immature plants.19-22 They obtained evidence that most of the glucose

92

residues in the hemicelluloses fraction, which were extracted from cell walls of

93

immature barley plants, were found to be derived from the β-(1→3,1→4)-glucan.21

94

They

95

O-(5-O-feruloyl-α-L-arabinofuranosyl)-(1→3)-O-β-D-xylopyranosyl-(1→4)-D-xylop

96

yranose from the Zea cell wall.22

also

obtained

97

As we all know, previous studies on Neosinocalamus affinis were more limited to

98

bamboo cultivation, mechanical and physical properties.23, 24 Nevertheless, there were

99

barely researches on the spatial distribution of main chemical composition, the

100

structural characterization of hemicelluloses, and chemical compositions from

101

different ages of Neosinocalamus affinis bamboo. According to the previous studies,

102

the chemical compositions and hemicelluloses structure of one-, two-, and

103

three-year-old bamboo had small differences, since the bamboo had grown well above

104

one years old.25 Therefore, in this study, the chemical and instrumental analysis were

105

used to investigate the spatial distribution, changes of chemical compositions, and

106

hemicelluloses structure of 2-month-, 4-month-, 8-month-, and 12-month-old

107

Neosinocalamus affinis bamboo culms. This study provides a theoretical basis for the

108

rational development, subsequent transformation, and utilization of Neosinocalamus

109

affinis bamboo. The results of this study could lay a theoretical foundation for the

110

efficient transformation and utilization of Neosinocalamus affinis bamboo, especially 5



111

for the comprehensive utilization of it.

112

MATERIAL AND METHODS

113

Materials

114

Neosinocalamus affinis bamboo culms (2, 4, 8, and 12-month-old, respectively)

115

were obtained from Sichuan Province, China. They were named as B2, B4, B8, and B12,

116

respectively. The samples were dried at room temperature and split into small pieces

117

(1-3 cm). Subsequently, all samples were grounded and screened with a micro plant

118

grinding machine to obtain the section of sized of 40-60 mesh. Bamboo powder was

119

extracted using a Soxhlet apparatus with a mixed solution of toluene and ethanol (2:1,

120

v/v) for 6 h.26 The dewaxed samples were further dried at 50 °C in an oven until a

121

constant weight and stored in plastic packaging. For Raman detection, the samples (B2,

122

B4, B8, and B12) were cut into small blocks (approximately 1 cm × 0.5 cm × 2 cm).

123

Without any further specimen pretreatment, the thickness 10 µm sections were

124

obtained by a sliding microtome, then rapidly covered with a coverslip on a clean

125

glass slide.

126 127

Preparation of hemicelluloses

128

The overall procedure for the extraction of hemicelluloses from the holocellulose

129

is listed in Figure 1. The samples were extracted successively with DMSO and

130

alkaline solution (3% aqueous NaOH).27 The wax-free bamboo culms flour was

131

delignified for 2 h at 78 °C with 6% sodium chlorite and regulated pH by 10% acetic

132

acid (pH 3.6-3.8). For the subsequent study, the residue was dried at 50 ºC in an oven. 6


Page 6 of 42

Page 7 of 42


133

The holocellulose was sequentially extracted with DMSO (1:25, g/mL) under stirring

134

for 7 h at 80 ºC. Then the filtrate (labeled as filtrate 1) was concentrated at reduced

135

pressure to a certain volume, precipitated in three times of volumes of ethanol under

136

stirring, and let it sit for a while. The centrifuge precipitate was separated from

137

solution (3500rpm, 15 min) and then freeze-dried. The dry precipitation was

138

hemicelluloses, and labelled as HD.

139

The residues (labeled as residues1) were treated with 3% aqueous NaOH (1:25,

140

g/ml) for 3 h at 60 °C. The filtrate (labeled as filtrate 2) was neutralized with 6 M HCl

141

(pH 5.5-6.0), dialyzed for one week, concentrated at reduced pressure. The precipitate

142

was collected by ethanol, freeze-dried. The dry powder was hemicelluloses, and

143

labelled as HA.

144 145

Analysis methods

146

The standard of National Renewable Energy Laboratory (NREL) was used for the

147

chemical composition of bamboo culms (B2, B4, B8, and B12) processing and

148

analysis.28 Sugars of bamboo and hemicelluloses were determined by high

149

performance anion exchange chromatography (HPAEC).29 Briefly, the 4~6 mg samples

150

were hydrolyzed at 105 ºC for 2.5 h with 6% sulphuric acid. After hydrolysis, the

151

hydrolysates were filtered, 50-fold diluted and then analyzed with chromatography.

152

The standard solutions of L-rhamnose, L-arabinose, D-mannose, D-glucose, D-xylose,

153

D-galactose, glucuronic acids and galacturonic acids were used for calibration. The

154

sugar analysis of hemicelluloses was performed in duplicate. 7



155

Confocal Raman microscopy (CRM) was used to conduct Raman detection of

156

lignin and carbohydrate distribution in different growth years of bamboo. The

157

procedure of Raman detection is based on the method used by Li et al.30 The spectra

158

were obtained by a LabRam Xplora confocal Raman microscope (Horiba Jobin Yvon,

159

Longjumeau, France) combined with a confocal microscope (Olympus BX51, Tokyo,

160

Japan) and a motorized x and y stage. To obtain a high spatial resolution,

161

measurements were conducted with an MPlan 100 × oil immersion microscope

162

objective from Olympus (NA=1.40) and a linear polarized laser (γ=532 nm) with a

163

diffraction-limited spot size of 0.61 γ/NA. The Raman light was detected with an

164

air-cooled back-illuminated spectroscopic charge coupled device (CCD) behind the

165

spectrograph. The image processing and spectral analysis applied Labspec 5 software.

166

All measurements in this study were performed in duplicate.

167

The molecular weights of hemicelluloses were analyzed by gel permeation

168

chromatography (GPC). The polysaccharides were dissolved in 5 mM sodium

169

phosphate buffer (pH 7.5) including 0.02 M NaCl, kept a concentration of 0.1%. Finally,

170

about 20 µL filtered solution was injected into the system.29 The experiments were

171

determined in duplicate.

172

The FT-IR spectrophotometer was used to obtain the FT-IR spectra of

173

hemicelluloses, there were spectral pure potassium bromide including 1% finely

174

ground hemicelluloses. Each spectrum was recorded at a 4 cm-1 resolution and in the

175

range of 4000 cm-1 to 650 cm-1. The 1H nuclear magnetic resonance (1H-NMR), 13C

176

nuclear magnetic resonance (13C-NMR), and heteronuclear single quantum coherence 8


Page 8 of 42

Page 9 of 42


177

(2D-HSQC) NMR spectra were determined on a Bruker 400 MHz spectrometer. The

178

hemicelluloses (15, 80, and 30 mg, for 1H, 13C, and HSQC NMR, respectively) were

179

dissolved in 0.55 mL DMSO-d6. The standard Bruker Topspin-NMR software was used

180

to process data. Based on the literature, the degree of acetylation (DSAC) was obtained

181

from the integral of signals of the acetyl group at 1.9~2.0 ppm and carbohydrate bands

182

at 3.0~5.5 ppm in 1H NMR spectra. 31 The following equation was used:

183

sum on integrals for acetyl groups at 1.9~2.0 ppm⁄3

DSAC = sum of integrals for carbohydrate signals at 3.0~5.5 ppm⁄6

(1)

184 185

RESULTS AND DISCUSSION

186

The chemical composition of bamboo

187

Table 1 lists the chemical components of 2-month (B2), 4-month (B4), 8-month (B8),

188

and 12-month-old (B12) bamboo, as well as the yields of hemicelluloses. The yields of

189

B2, B4, B8, and B12 treated with DMSO solution were 10.10%, 6.76%, 6.28%, and 7.36%

190

(relative to holocellulose, w/w), respectively. The maximum yield of hemicelluloses

191

(10.10% of holocellulose) was observed in H2D. It is well known that the main chemical

192

compositions of bamboo can vary depending on the different maturity. However, in the

193

previous work, the chemical compositions were no significant changes from 1-year to

194

3-year-old bamboo,25 suggested that one-year-old later bamboo would reach a stable

195

period of growth, the cell wall and the total amount of material of intercellular layer are

196

no longer changed apparently. As shown in Table 1, the ash (2.1-11.2%) and

197

extractives (1.8-11.1%) obviously decreased with age of bamboo culms increasing. In

198

contrast, the ash and extractives increased with age of bamboo culms from 2-year to

9



199

3-year-old,25 which revealed that 1~2 year-old bamboo would be applied properly to

200

raw materials of pulp and paper and other fiber products. In this study, ash (11.2%) and

201

extractives (11.1%) were relatively high in 2-month-old bamboo culms. The reason:

202

2-month-old bamboo is not fully developed and formed into bamboo pole, the amounts

203

of cellulose and hemicelluloses accumulate relatively small, and the lignification is

204

very weak.

205

Carbohydrates and lignin are the main chemical constituents in bamboo culms.

206

Based on the current knowledge, the degree of lignification and the compositions of

207

hemicelluloses in long and short parenchyma cells of bamboo are different. According

208

to the chemical compositions analysis (Table 1), the glucose (32.2-43.8%) was

209

increased gradually with the increasing bamboo age roughly (the glucose represents

210

cellulose). It should be noted that all nutrients can be absorbed rapidly for metabolic

211

processes, and a young bamboo stem may not contain starch during the growing

212

phase.2 The relative proportions of the arabinose and galactose decreased from

213

2-month- to 8-month-old of bamboo culms. More importantly, the content of total

214

lignin (12.8-25.4%) exhibited a drastically increasing trend with the growth of

215

bamboo age. The lignin enhances the mechanical strength of bamboo and ensures a

216

good capacity of bearing. The extent of lignification in culms from 2 to 14 years was

217

investigated by Itoh,32 the content of lignin remains unchanged or slightly increased,

218

showing no further lignification. Besides, as the age of bamboo culms increases, the

219

content of acid-insoluble lignin (4.5-21.7%) increased, while the content of

220

acid-soluble lignin (8.3-3.7%) showed a decreased trend. The reason for this 10


Page 10 of 42

Page 11 of 42


221

phenomenon, inverse relationship of acid-soluble lignin and acid-insoluble lignin

222

content, is as follows: the acid-soluble lignin content was calculated using the

223

measured absorbance which was measured at 240 nm by an ultraviolet/visible

224

spectrophotometer. The impurities such as protein and furfural have a certain

225

influence on the absorbance of bamboo acid-soluble lignin at 240 nm. In addition,

226

with the growth of bamboo age, the protein content decreased. Therefore, the value of

227

acid-soluble lignin decreased. 4

228

Confocal Raman microscopy analysis

229

The distribution of lignin and carbohydrates in cell wall of 2, 4, 8, and

230

12-month-old bamboo was determined by CRM in in-situ monitor. Raman spectra of

231

bamboo cell wall contain the vibrational modes primarily from three main

232

compositions: cellulose, hemicellulose (mostly xylan), and lignin. In the Raman

233

images, intensity scale locates at the right. As shown in Figure 2, the different

234

concentrations of carbohydrates and lignin in the morphologically distinct cell wall

235

were reflected by the varied intensities.

236

It was found that a heterogeneous distribution of the compositions existed in the

237

bamboo of different age within morphologically distinct regions since the varied

238

intensities reflected different concentrations. Clearly, a high intensity of carbohydrates

239

and lignin were observed in older bamboo. As a result of the multi-compositions

240

nature of bamboo, its vibrational spectrum is fairly complex with broad overlapping

241

bands.33 It is well known that hemicelluloses and cellulose microfibrils are linked by

242

hydrogen bonds and van der Waals forces, making cellulose and hemicelluloses 11



243

possess similar chemical bonds.34 Therefore, the peak at 2889 cm-1 is attributed to

244

C-H2 and C-H stretching of carbohydrates.34 Raman images of the carbohydrates and

245

lignin in the bamboo cell walls were integrated from 2800 to 2920 cm-1

246

(carbohydrates) and from 1541 to 1687 cm-1 (lignin), respectively. Lignification of

247

cells in the bamboo begins at the outside of the culm and proceeds inwardly. Besides,

248

lignification of the cell wall started from the cell corner middle lamella (CCML) and

249

compound middle lamella (CML; middle lamella adjacent primary walls), and

250

progressively reduced from the outer layer to the inner layer of the wall. The chemical

251

images of the CML revealed an increasing intensity with a growth of bamboo and

252

showed higher lignin concentration than the S2 layer. Lignin plays a decisive role in

253

its physiological growth and mechanical stability. Additionally, Raman analysis

254

revealed that the intensity of carbohydrates had a slight increase in the S regions with

255

the growth of bamboo.

256

Figure 3 showed the corresponding average Raman spectra of the secondary cell

257

wall (S) and CML regions of bamboo cell walls. By comparing the spectra from B2,

258

B4, B8 and B12 samples, the lignin intensity in the S and CML (Figure 3a and 3b)

259

increased with the growth of bamboo age from 2 to 12-month-old. In addition, the

260

lignin intensity both in the CCML and S regions exhibited a similar increase trend

261

with a growth of bamboo. Similarly, Raman signal intensity of carbohydrates

262

exhibited a similar increasing trend in the regions of S and CCML, respectively

263

(Figure 3c and 3d). Significantly, combining with the result of composition analysis,

264

it was found that the contents of carbohydrates and lignin increased with a growth of 12


Page 12 of 42

Page 13 of 42


265

young bamboo.

266 267

Sugar composition of hemicelluloses

268

In bamboo cell wall, hemicelluloses have a wide variation in chemical structure and

269

content with the growth of bamboo age. As shown in Table 2, DMSO-soluble and

270

alkali-soluble hemicelluloses both mainly consist of arabinose (6.5-25.5%), xylose

271

(56.1-89.6%), and glucuronic acid (1.4-14.6%). Among the eight hemicelluloses,

272

xylose is the main sugar compositions. In addition, small amounts of glucose (except

273

for 2-month-old bamboo) and galactose were also detected.

274

The sugar components of H2D and H2A were quite different from that of

275

hemicelluloses from 4, 8, and 12 month-old bamboo culms. The xylose content of H2D

276

and H2A was lower than those of other hemicelluloses. In addition, the content of

277

glucose in H2D is relatively high, which probably originated from β-glucan.35 Kozlova

278

et al. studied that the mixed linkage glucan arranged in cell walls of growing maize

279

roots and reported that a model was devised in which the mixed-linkage glucan works

280

as a gel-like filler of the space between the separating domain of cellulose microfibrils

281

and glucuronoarabinoxylan.36 It is also possible that the non-crystalline cellulose has a

282

degradation at high temperature under the treatment of DMSO, the obtained glucose

283

is partly dissolved in the system,37, 38 which need to be further investigated.

284

Based on previous studies, it is known that bamboo hemicelluloses are composed

285

of a backbone of D-xylopyranosyl units with branches of arabinose and glucuronic

286

acid or 4-O-methyl-D-glucuronic acid. 39, 40 Therefore, the ratio of arabinose to xylose 13



287

(Ara/Xyl) or glucuronic acid to xylose (GlcA/Xyl) are commonly represent the degree

288

of branching or linearity of hemicelluloses, thus reflecting the binding capacity of

289

hemicelluloses and other compositions. The proportion of GlcA/Xyl (0.02-0.06%) and

290

Ara/Xyl (0.07-0.26%) from DMSO-soluble hemicellulosic fractions are slightly lower

291

than the proportion of GlcA/Xyl (0.06-0.21%) and Ara/Xyl (0.09-0.45%) from

292

alkali-soluble hemicellulosic fractions. These results suggested that alkali-soluble

293

hemicellulosic fractions mainly composed of highly substituted xylan, while

294

DMSO-soluble hemicelluloses primarily consisted of slightly high substituted xylan,

295

which was in good agreement with other studies from Valent and Albersheim.41

296

Additionally, the ratio of Ara/Xyl decreased with the growth of bamboo age, which

297

indicated the hemicelluloses of younger bamboo culms contain relatively high

298

substituted structures. The data above showed that the hemicelluloses in bamboo cell

299

wall mainly consists of glucuronoarabinoxylans.

300 301

Molecular weight analysis

302

The Mw and Mn of eight hemicelluloses are listed in Table3. During the extracting

303

process of hemicelluloses, different reagents and temperature were chosen, therefore

304

the molecular weight of hemicelluloses exhibited a certain difference.42, 43 As for the

305

two types of hemicelluloses (except for H4D and H4A) isolated from the same age of

306

bamboo, the average molecular weight of DMSO-soluble hemicelluloses was lower

307

than that of alkali-soluble hemicelluloses, which suggested that alkali played a crucial

308

part in releasing the high-molecular-weight hemicelluloses. A similar result was 14


Page 14 of 42

Page 15 of 42


309

observed in the research of Bian et al.38 Besides, the seven hemicellulosic fractions

310

(except for H12D) showed a relatively lower polydispersity index (1.30-1.53), which

311

indicated that hemicelluloses obtained by DMSO and alkaline solution had a

312

relatively narrow molecular mass distribution. Furthermore, the polydispersity of H12D

313

fraction showed a slightly higher polydispersity index with Mw/Mn values of 5.93,

314

suggested a broad distribution of molecular sizes for the H12D obtained from 12

315

month-old bamboo.

316 317

FT-IR spectra analysis

318

The FT-IR spectra of DMSO-soluble and alkali-soluble hemicelluloses are shown in

319

Figure 4 and 5, respectively. The predominate band around 1738 cm-1 is

320

corresponded to the C=O stretching of acetyl groups in the region of the carbonyl

321

stretching vibration. The peak at 1738 cm-1 is observed in spectra of DMSO-soluble

322

hemicelluloses while it disappeared in the spectra of alkali-soluble hemicelluloses,

323

which suggested that alkali-soluble hemicellulosic fractions were completely

324

saponified the acetyl groups and methyl esters in this study.44 The absorption peak at

325

around 1245 cm-1 is assigned to the C-O linkage in xylan.45 Evidently, this peak in the

326

spectra of DMSO-soluble hemicelluloses is stronger than that of alkali-soluble

327

hemicelluloses. The signals at 1633 and 1437 cm-1 are related to the symmetric

328

stretching of -COOH salt in 4-O-methyl-glucuronic acid carboxylate.46 Additionally,

329

the peak at 1633 cm-1 is presumably due to the H-O-H angle vibration, suggesting the

330

hemicelluloses have a strong affinity for water. 27, 37 The absorption at about 3427, 2980, 15



331

and 2826 cm-1 are assigned to the stretching of -OH and C-H in the polysaccharide,

332

respectively. The occurrence of a slightly intense signal at 1521 cm-1 in the spectrum of

333

H2D (spectrum a) is originated from aromatic skeletal vibrations in bound lignin,

334

suggesting the H2D was slightly contaminated by lignin bonds.47 Although it is difficult

335

to determine the exact band of polysaccharides distribution in the region of 1200-800

336

cm-1, each particular polysaccharide has a specific band maximum.48 The peaks at

337

1046 and 1170 cm-1 are corresponded to the C-OH bending mode and C-O stretching

338

in C-O-C glycosidic linkages, respectively.29, 49 The signal at 892 cm-1 is originated

339

from the C-1 group frequency, which is characteristic of β-glycosidic linkages in the

340

sugar units.31

341 342

NMR spectra analysis

343

NMR is considered to be a powerful tool to obtain a deeper insight into the

344

molecular structures of hemicellulosic fractions. The signals of NMR spectra are

345

assigned based on the previous references.50,

346

(DSAC) can be calculated by 1H NMR spectra, the DSAC was acquired by the signals

347

integration assigned to those of all carbohydrates and acetyl groups.52 The acetyl

348

substitution of hemicelluloses are shown in Table 2. It should be noted that the DSAC

349

in the DMSO-soluble hemicelluloses showed an increasing trend with the growth of

350

bamboo age. 1H NMR spectra of the H2D and H2A fractions are shown in Figure 6.

351

The peaks of β-anomeric and α-anomeric protons were observed in the range of

352

4.34-4.93 ppm and 5.21-5.35 ppm, respectively. The peaks at 3.14-4.35 ppm are

51

The degree of acetyl substitution

16


Page 16 of 42

Page 17 of 42


353

originated from β-D-xylose residues. The intensive signal at 2.49 ppm is assigned to

354

DMSO-d6 solvent.53 In addition, the signals at 1.97, 1.93, and 1.86 ppm in H2D are

355

ascribed to acetyl groups of DMSO-soluble hemicellulosic fractions.54

356

The

13

C NMR spectra of H2D and H2A are demonstrated in Figure 7. The peaks at

357

171.43 and 21.30 ppm were detected in H2D, which is because of the existed O-acetyl

358

groups in the H2D, but the signals of O-acetyl groups did not appear in H2A.18 This

359

result is consistent with the results of FT-IR and 1H NMR. Besides, the peaks at

360

101.72, 86.38, 75.95, 71.60, 68.67, and 60.65 ppm in 13C NMR of H2D are assigned to

361

C-1, C-3, C-5, C-2, C-4, and C-6 of Glcp residue linked by (1→3) linkage,

362

respectively.32 The signals at 103.18, 73.35, 74.17, 80.72, 75.95, and 60.65 ppm are

363

originated from C-1, C-2, C-3, C-4, C-5 and C-6 of 4-O-linked Glcp, respectively.32

364

The 2D-HSQC NMR spectra of H2D, H4D, H8D, H12D, and H2A are illustrated in

365

Figure 8, and Table 4 listed the chemical shift assignments of H2D. In the HSQC

366

NMR spectra, the anomeric and the O-acetylated xylose region are 1H 4.4-5.5

367

ppm/13C 90-110 ppm and 1H 4.5-5.5 ppm/13C 70-80 ppm, respectively. The

368

chemical shifts of 102.21/4.23, 73.36/3.14, 74.17/3.34, 75.32/3.52, 63.63/3.87

369

(H-5eq), and 63.63/3.14 (H-5ax) ppm are corresponded to C-1/H-1, C-2/H-2, C-3/H-3,

370

C-4/H-4, and C-5/H-5 of the (1→4)-β-D-xylan backbone, respectively.29 The signals

371

at 107.48/5.40, 86.38/3.87, 80.73/3.78, 78.11/3.52, 62.27/3.34 (H-5ax), and

372

62.27/3.52 (H-5eq) ppm are assigned to C-1/H-1, C-4/H-4, C-2/H-2, C-3/H-3,

373

C-5/H-5 (H-5ax) and C-5/H-5 (H-5eq) of α-L-arabinofuranosyl residues at O-3.35, 54

374

The cross-peaks at 99.92/5.06, 70.56/3.52, 73.36/3.78, 86.38/3.14, 60.65/3.34 ppm are 17


13

C/1H


to

the

C-1/H-1,

C-2/H-2,

C-3/H-3,

C-4/H-4,

Page 18 of 42

375

assigned

-OCH3

of

the

376

4-O-methylglucuronic acid units at position O-2, respectively. The signals at

377

4.66/73.36 and 4.93/74.17 ppm (data not shown in H2A) are corresponded to H-2/C-2

378

and H-3/C-3 due to the acetylation at position 2 and 3 of 1,4-linked β-Xylp residues,

379

respectively.52 Additionally, the signal of (1→4)-D-Xylp-2-O-(4-OMe-D-GlcpA)

380

units is detected at 99.92/4.66 (C-1/H-1) ppm (data not shown in H2A).55More

381

significantly, the signals at 103.18/4.23, 80.73, 75.96 ppm, and 60.65 arises from

382

β-glucan,56, 33 these signals are not shown in H2A HSQC spectrum, indicating no such

383

polysaccharides exist in alkaline-soluble hemicelluloses, while DMSO-soluble

384

hemicelluloses partly consisted of β-glucans. By comparing with our previous study,

385

DMSO-soluble hemicelluloses structure of maturation bamboo were mainly

386

composed of O-acetyl arabino-4-O-methylglucurono-(1→4)-β-D-xylan, there were no

387

glucans.25 However, in this study, the DMSO-soluble hemicelluloses in the young

388

bamboo consisted of highly substituted xylans and glucans.

389

It is well known that β-(1→3)(1→4)-D-glucan in the young bamboo stem is

390

distributed in the short parenchyma cell wall and the intercellular layer of long

391

parenchyma, respectively. The β-glucans in young bamboo have a functional role as a

392

cell wall component, this polysaccharide is bound to cellulose microfibrils by a bond,

393

and this model in the cell wall contribute to enhancing the mechanical strength of

394

young bamboo culm. The structural characteristics of mixed-linkage glucans,

395

investigated by Fincher, shows that mixed-linkage glucans form a gel-like matrix in the

396

wall, sometimes constituted junction zones with cellulose.57 Additionally, the peaks 18


Page 19 of 42


397

intensity of α-L-arabinofuranosyl in DMSO-soluble hemicelluloses are weaker than

398

that of H2A (Figure 8), because the proportion of the arabinose in the DMSO-soluble

399

hemicelluloses is significantly lower than that of H2A (Table 2). Taken together,

400

DMSO-soluble

401

O-acetyl-α-L-arabino-4-O-methylglucurono-(1→4)-α-D-xylan

402

β-(1→3)(1→4)-glucans, while H2A are mainly composed of highly substituted

403

arabino-4-O-methylglucurono-(1→4)-β-D-xylan.

hemicelluloses

of

bamboo

primarily

composed and

of some

404 405

Notes

406

The authors declare no competing financial interest.

407

Acknowledgements

408

This work was supported by Fundamental Research Funds for the Central

409

Universities (JC2015-03), Natural Science Foundation of China (31470417), Author of

410

National Excellent Doctoral Dissertations of China (201458), and the National

411

Program for Support of Top-notch Young Professionals.

412

References

413

[1] Scurlock, J. M. O.; Dayton, D. C.; Hames, B. Bamboo: an overlooked biomass

414 415 416

resource?. Biomass Bioenerg. 2000, 19, 229-244. [2] Liese, W.; Weiner, G. Ageing of bamboo culms. A review. Wood Sci. Technol. 1996, 30, 77-89.

417

[3] Shibamoto, T.; Shoji, R.; Kubota, S. Studies on some properties of stem and shoot

418

of bamboo (Phyllostachys edulis RIV.)II. Bull. Tokyo Uni. For. 1954, 47, 203-207. 19



419

Page 20 of 42

[4] Fujii, Y.; Azuma, J.; Marchessault, R. H.; Morin, F. G.; Aibara, S.; Okamura, K.

420

Chemical

composition

change

421

Holzforschung 1993, 47, 109-115.

of

bamboo

accompanying

its

growth.

422

[5] Chu, L. Q.; Masyuko, R.; Sweedler, J. V.; Bohn, P. W. Base-induced

423

delignification of miscanthus x giganteus studied by three-dimensional confocal

424

raman imaging. Bioresour. Technol. 2010, 101, 4919-4925.

425

[6] Chen, Y.; Qin, W.; Li, X.; Gong, J. The chemical composition of ten bamboo

426

species. In Recent Research on Bamboo, Rao, A.N., Dhanarajan, G., Sastry, C.B.

427

(Eds.), Workshop Hangzhou, China, 1985; pp. 110-113.

428 429 430

[7] Hoch, G. Cell wall hemicelluloses as mobile carbon stores in non-reproductive plant tissues. Funct. Ecol. 2007, 21, 823-834. [8] Spencer, R. R.; Akin, D. E. Rumen microbial-degradation of potassium

431

hydroxide-treated

coastal

bermudagrass

leaf

432

electron-microscopy. J. Anim. Sci., 1980, 51, 1189-1196.

blades

examined

by

433

[9] Wang, X. Q.; Ren, H. Q.; Zhang, B.; Fei, B. H.; Burgert, I. Cell wall structure and

434

formation of maturing fibres of moso bamboo (Phyllostachys pubescens) increase

435

buckling resistance. J. R. Soc. Interface 2012, 9, 988-996.

436

[10] Landim, A. S.; Filho, G. R.; Sousa, R. M. F.; Ribeiro, E. A. M.; Souza, F. R. B.;

437

Vieira, J. G.; Assungao, R. M. N.; Cerqueira, D. A. Application of cationic

438

hemicelluloses produced from corn husk as polyelectrolytes in sewage treatment.

439

Polimeros-Ciencia E Tecnologia 2013, 23, 468-472.

440

[11] Yi, G.; Zhang, Y. One-pot selective conversion of hemicellulose (xylan) to xylitol 20


Page 21 of 42


441

under mild conditions. ChemSusChem 2012, 5, 1383-1387.

442

[12] Bian, J.; Peng, F.; Peng, X. P.; Peng, P.; Xu, F.; Sun, R. C. Structural features and

443

antioxidant activity of xylooligosaccharides enzymatically produced from

444

sugarcane bagasse. Bioresour. Technol. 2013, 127, 236-241.

445

[13] Guan, Y.; Zhang, B.; Tan, X.; Qi, X. M.; Bian, J.; Peng, F.; Sun, R. C.

446

Organic-inorganic composite films based on modified hemicelluloses with clay

447

nanoplatelets. ACS Sustainable Chem. Eng. 2014, 2, 1811-1818.

448 449 450 451

[14] Scheller, H. V.; Ulvskov, P. Hemicelluloses. Ann. Rev. Plant Biol. 2010, 61, 263-289. [15] Popper, Z. A. Evolution and diversity of green plant cell walls. Curr. Opin. Plant Biol. 2008, 11, 286-292.

452

[16] Shibuya, N.; Misaki, A. Structure of hemicellulose isolated from rice endosperm

453

cell wall: mode of linkages and sequences in xyloglucan, β-glucan and

454

arabinoxylan. Agric. Biol. Chem. 1978, 42, 2267-2274.

455 456 457 458

[17] Lzydorczyk, M. S.; Biliaderis, C. G. Cereal arabinoxylans: advances in structure and physicochemical properties. Carbohydr. Polym. 1995, 28, 33-48. [18] Chaikumpollert, S.; Methacanon, P.; Suchiva, K. Structural elucidation of hemicelluloses from Vetiver grass. Carbohydr. Polym. 2004, 57, 191-196.

459

[19] Kato, Y.; Iki, K.; Matsuda, K. Cell-wall polysaccharides of immature barley

460

plants. I. isolation and characterization of a β-d-glucan. Agric. Biol. Chem. 1981,

461

45, 2737-2744.

462

[20] Kato, Y.; Iki, K.; Matsuda, K. Cell-wall polysaccharides of immature barley 21



Page 22 of 42

463

plants. II. characterization of a xyloglucan. Agric. Biol. Chem. 1981, 45,

464

2745-2753.

465 466 467

[21] Kato, Y., Ito, S., Iki, K., Matsuda, K., Xyloglucan and β-D-glucan in cell walls of rice seedlings. Plant Cell Physiol. 1982, 23, 351-364. [22]

Kato,

Y.;

Nevin,

D.

J.

Isolation

and

identification

of

468

O-(5-O-feruloyl-α-L-arabinofuranosyl)-(1→3)-O-β-D-xylopyranosyl-(1→4)-D-x

469

ylopyranose as a component of Zea shoot cell walls. Carbohydr. Res. 1985, 137,

470

139-150.

471 472 473 474

[23] Ray A. K.; Das, S. K.; Mondal, S.; Ramachandrarao, P. Microstructural characterization of bamboo. J. Mater. Sci. 2004, 39 , 1055-1060. [24] Wang, Y.; Wang, G.; Tian, G.; Liu, Z.; Xiao, Q. F.; Zhou, X.; Han, X.; Gao, X. Structures of bamboo fiber for textiles. Text. Res. J. 2010, 80, 334-343.

475

[25] Zhang, B.; Guan, Y.; Bian, J.; Peng, F.; Ren, J. L.; Yao, C. L.; Sun, R. C.

476

Structure of hemicelluloses upon maturation of bamboo (Neosinocalamus affinis)

477

culms, Cell Chem. Technol. 2016, 50, 189-198.

478

[26] Sun, R. C.; Tomkinson, J. Characterization of hemicelluloses isolated with

479

tetraacetylethylenediamine activated peroxide from ultrasound irradiated and

480

alkali pre-treated wheat straw, Eur. Polym. J. 2003, 39, 751-759.

481

[27] Peng, P.; Peng, F.; Bian, J.; Xu, F.; Sun, R. C.; Kennedy, J. F. Isolation and

482

structural characterization

483

Phyllostachys incarnata Wen, Carbohydr. Polym. 2011, 86, 883-890.

484

of hemicelluloses from the bamboo species

[28] Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, 22


Page 23 of 42


485

D. Determination of structural carbohydrates and lignin in biomass;

486

NREL/TP-510-42618; National Renewable Energy Laboratory: Golden, CO,

487

2008.

488

[29] Peng, F.; Ren, J. L.; Xu, F.; Bian, J.; Peng, P.; Sun, R. C. Fractionation of

489

alkali-solubilized hemicelluloses from delignified Populus gansuensis: structure

490

and properties. J. Agric. Food Chem. 2010, 58, 5743-5750.

491

[30] Li, H. Y.; Sun, S. N.; Wang, C. Z.; Sun, R. C. Structural and dynamic changes of

492

lignin in Eucalyptus cell walls during successive alkaline ethanol treatments. Ind.

493

Crop. Prod. 2015, 74, 200-208.

494

[31] Peng, F.; Bian, J.; Peng, P.; Xiao, H.; Ren, J. L.; Xu, F.; Sun, R. C. Separation

495

and characterization of acetyl and non-acetyl hemicelluloses of Arundo donax by

496

ammonium sulfate precipitation. J. Agric. Food Chem. 2012, 60 (16), 4039-4047.

497 498 499 500

[32] Itoh, T. Lignification of bamboo (Phyllostachys heterocycla Mitf.) during its growth. Holzforschung 1990, 44, 191-200. [33] Gierlinger, N.; Schwanninger, M. Chemical imaging of poplar wood cell walls by confocal Raman microscopy. Plant Physiol. 2006, 140, 1246-1254.

501

[34] Agarwal, U. P.; Ralph S. A. FT-Raman spectroscopy of wood: identifying

502

contributions of lignin and carbohydrate polymers in the spectrum of black spruce

503

(Picea mariana). Appl. Spectrosc. 1997, 51, 1648-1655.

504

[35] Cui, W.; Wood, P. J.; Blackwell, B.; Nikiforuk, J. Physicochemical properties and

505

structural characterization by two-dimensional NMR spectroscopy of wheat

506

β-D-glucan-comparison with other cereal β-D-glucans. Carbohydr. Polym. 2000, 23



507

Page 24 of 42

41, 249-258.

508

[36] Kozlova, L. V.; Ageeva, M. V.; Ibragimova, N. N.; Gorshkova, T. A.

509

Arrangement of mixed-linkage glucan and glucuronoarabinoxylan in the cell walls

510

of growing maize roots. Ann. Bot. 2014, 114, 1135-1145.

511

[37] Ebringerová, A.; Hromádková, Z.; Heinze, T. Polysaccharides I, Structure,

512

Characterisation and Use. In Hemicellulose, Heinze, T. (Ed.), Springer, Berlin,

513

Heidelberg, 2005; pp. 1-67.

514

[38] Bian, J.; Peng, F.; Xu, F.; Sun, R. C.; Kennedy, J. F. Fractional isolation and

515

structural characterization of hemicelluloses from Caragana korshinskii.

516

Carbohydr. Polym. 2010, 80, 753-760.

517

[39] Kacurakova, M.; Belton, P. S.; Wilson, R. H.; Hirsch, J.; Ebringerova, A.

518

Hydration

properties

of

xylan-type

structures:

519

xylooligosaccharides. J. Sci. Food Agric. 1998, 77, 38-44.

an

FTIR

study

of

520

[40] Wen, J. L.; Xiao, L. P.; Sun, Y. C.; Sun, S. N.; Xu, F.; Sun, R. C.; Zhang, X. L.

521

Comparative study of alkali-soluble hemicelluloses isolated from bamboo

522

(Bambusa rigida). Carbohydr. Res. 2011, 346, 111-120.

523 524

[41] Valent, B. S.; Albersheim, P. The structure of plant cell walls. Plant Physiol. 1973, 51, 327-370.

525

[42] Persson, T.; Ren, J. L.; Joelsson, E.; Jönsson, A. S. Fractionation of wheat and

526

barley straw to access high-molecular-mass hemicelluloses prior to ethanol

527

production. Bioresour. Technol. 2009, 100, 3906-3913.

528

[43] Tunc, M. S.; Heiningen, A. R. P. V. Characterization and molecular weight 24


Page 25 of 42


529

distribution of carbohydrates isolated from the autohydrolysis extract of mixed

530

southern hardwoods. Carbohydr. Polym. 2011, 83, 8-13.

531

[44] Sun, R. C.; Sun, X. F.; Ma, X. H. Effect of ultrasound on the structural and

532

physiochemical properties of organosolv soluble hemicelluloses from wheat straw.

533

Ultrason. Sonochem. 2002, 9, 95-101.

534

[45] Sun, S. L.; Wen, J. L.; Ma, M. G.; Sun, R. C. Successive alkali extraction and

535

structural characterization of hemicelluloses from sweet sorghum stem. Carbohydr.

536

Polym. 2013, 92, 2224-2231.

537

[46] Chatjigakis, A. K.; Pappas, C.; Proxenia, N.; Kalantzi, O.; Rodis, P.; Polissiou, M.

538

FT-IR spectroscopic determination of the degree of esterification of cell wall

539

pectins from stored peaches and correlation to textural changes. Carbohydr. Polym.

540

1998, 37, 395-408.

541

[47] Vena, P. F.; Brienzo, M.; Garcíaaparicio, M. D. P.; Görgens, J. F.; Rypstra, T.

542

Hemicelluloses extraction from giant bamboo (Bambusa balcooa Roxburgh) prior

543

to kraft or soda-AQ pulping and its effect on pulp physical properties,

544

Holzforschung 2013, 67, 863-870)

545

[48] Robert, P.; Marquis, M.; Barron, C.; Guillon, F.; Saulnier, L. FT-IR investigation

546

of cell wall polysaccharides from cereal grains. Arabinoxylan infrared assignment.

547

J. Agric. Food Chem. 2005, 53, 7014-7018.

548 549 550

[49] Kacurakova, M., Ebringerova, A., Hirsch, J., Hromadkova, Z. Infrared study of arabinoxylans. J. Sci. Food Agric. 2010, 66, 423-427. [50] Sun, X. F.; Cang, R.; Fowler, P.; Baird, M. S. Extraction and characterization of 25



551

original lignin and hemicelluloses from wheat straw. J. Agric. Food Chem. 2005,

552

53, 860-870.

553

[51] Li, M. F.; Fan, Y. M.; Xu, F.; Sun, R. C. Structure and thermal stability of

554

polysaccharide fractions extracted from the ultrasonic irradiated and cold alkali

555

pretreated bamboo. J. Appl. Polym. Sci. 2011, 121, 176-185.

556

[52] Teleman, A.; Lundqvist, J.; Tjerneld, F.; Stalbrand, H.; Dahlman, O.

557

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen

558

employing 1H and 13C NMR spectroscopy. Carbohydr. Res. 2000, 329, 807-815.

559

[53] Xu, F.; Sun, R. C.; Zhai, M. Z.; Sun, J. X.; Jiang, J. X.; Zhao, G. J. Comparative

560

study of three lignin fractions isolated from mild ball-milled Tamarix

561

austromogoliac and Caragana sepium. J. Appl. Polym. Sci. 2008, 108, 1158-1168.

562

[54] Xu, F.; Sun, R. C.; Zhai, M. Z.; Sun, J. X.; She, D.; Geng, Z. C.; Lu, Q.

563

Fractional separation of hemicelluloses and lignin in high yield and purity from

564

mild ball-milled Periploca sepium. Sep. Sci. Technol. 2008, 43, 3351-3375.

565

[55] Sun, S. N.; Cao, X. F.; Li, H. Y.; Xu, F.; Sun, R. C. Structural characterization of

566

residual hemicelluloses from hydrothermal pretreated Eucalyptus fiber. Int. J. Biol.

567

Macromol. 2014, 69, 158-164.

568

[56] Irakli, M.; Biliaderis, C. G.; Izydorczyk, M. S.; Papadoyannis, I. N. Isolation

569

structural features and rheological properties of water-extractable β-glucans from

570

different Greek barley cultivars. J. Sci. Food Agric. 2004, 84, 1170-1178.

571 572

[57] Fincher, G. B. Exploring the evolution of (1,3;1,4)-β-d-glucans in plant cell walls: comparative genomics can help! Curr. Opin. Plant Biol. 2009, 12, 140-147. 26


Page 26 of 42

Page 27 of 42


573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588

Figure Captions and tables

589 590 591

Figure 1. Scheme for isolation of hemicelluloses from bamboo (Neosinocalamus

592

affinis).

593 594

Figure 2. Raman images of the lignin (L) and carbohydrates (C) distribution in the 27



Page 28 of 42

595

bamboo cell walls (2, 4, 8, and 12-month-old ) were integrated from 1541 to 1687

596

cm-1 (lignin) and from 2800 to 2970 cm-1 (carbohydrate), respectively. Intensity scale

597

locates at the right. Bright white/yellow regions suggest a high concentration of lignin

598

and carbohydrates; dark blue/black locations indicate very low concentration.

599 600

Figure 3. Zoom into average Raman spectra extracted from the S and the CML

601

regions of bamboo cell walls, 1525-1700 and 2760-3050 cm-1, respctively.

602 603

Figure 4. FT-IR spectra of DMSO-soluble hemicellulosic fractions H2D (spectrum a),

604

H4D (spectrum b), H8D (spectrum c), and H12D (spectrum d).

605 606

Figure 5. FT-IR spectra of alkali-soluble hemicellulosic fractions H2A (spectrum a),

607

H4A (spectrum b), H8A (spectrum c), and H12A (spectrum d).

608 609

Figure 6. 1H NMR spectra of hemicellulosic fractions H2A and H2D.

610 611

Figure 7. 13C NMR spectra of hemicellulosic fractions H2A and H2D.

612 613

Figure 8. HSQC NMR spectra of hemicellulosic fractions H2D and H2A. Designations

614

are

615

(1→4)-D-Xylp-2-O-(4-OMe-D-GlcpA) units; G, β-D-Glcp.

as

follows:

X,

Xylp

unit;

A,

Araf

unit;

616 28


U,

GlcpA

unit;

XU,

Page 29 of 42


617

Table 1. Main chemical components of bamboo culms from Neosinocalamus affinis.

618 619

Table 2. Composition and degree of acetylation of hemicelluloses from bamboo

620

(Neosinocalamus affinis).

621 622

Table 3. Weight-average (Mw) and number-average (Mn) molecular weights (g/mol)

623

and polydispersity (Mw/Mn) of hemicelluloses from bamboo (Neosinocalamus affinis).

624 625

Table 4. 1H and 13C chemical shift (ppm) assignements for H2D.

626 627 628 629 630 631

29



632 633 634

Figure 1. Scheme for isolation of hemicelluloses from bamboo (Neosinocalamus

635

affinis).

636 637 638 639 640

30


Page 30 of 42

Page 31 of 42


641 642 643

Figure 2. Raman images of the lignin (L) and carbohydrates (C) distribution in the

644

bamboo cell walls (2, 4, 8, and 12-month-old ) were integrated from 1541 to 1687

645

cm-1 (lignin) and from 2800 to 2970 cm-1 (carbohydrate), respectively. Intensity scale

646

locates at the right. Bright white/yellow regions suggest a high concentration of lignin

647

and carbohydrates; dark blue/black locations indicate very low concentration.

648 649 650 31



651

652 653 654

Figure 3. Zoom into average Raman spectra extracted from the S and the CML

655

regions of bamboo cell walls, 1525-1700 and 2760-3050 cm-1, respctively. Different

656

colors represent different ages of bamboo.

657 658 659 660 661 662 663 664 32


Page 32 of 42

Page 33 of 42


665 666 667

Figure 4. FT-IR spectra of DMSO-soluble hemicellulosic fractions H2D (spectrum a),

668

H4D (spectrum b), H8D (spectrum c), and H12D (spectrum d). Different colors represent

669

different hemicelluloses.

670 671 672 673 674 675 676 677 678 679 680 33



681

682 683 684

Figure 5. FT-IR spectra of alkali-soluble hemicellulosic fractions H2A (spectrum a),

685

H4A (spectrum b), H8A (spectrum c), and H12A (spectrum d). Different colors represent

686

different hemicelluloses.

687 688 689 690 691

34


Page 34 of 42

Page 35 of 42


692 693 694

Figure 6. 1H NMR spectra of hemicellulosic fractions H2A and H2D.

695 696 697 698 699 700 701 702 703 704 705 706 35



707 708 709

Figure 7. 13C NMR spectra of hemicellulosic fractions H2A and H2D.

710 711 712 713 714 715 716 717 718 719 720 721 36


Page 36 of 42

Page 37 of 42


722 723 724

Figure 8. HSQC NMR spectra of hemicellulosic fractions H2D and H2A. Designations

725

are

726

(1→4)-D-Xylp-2-O-(4-OMe-D-GlcpA) units; G, β-D-Glcp. Different color represent

727

different structure units.

as

follows:

X,

Xylp

unit;

A,

Araf

unit;

728 729 730 731 732 37


U,

GlcpA

unit;

XU,


Page 38 of 42

733 734

Table 1

735

Main chemical components of bamboo culms from Neosinocalamus affinis.

Samples

B2

B4

B8

B12

Yielda (%)

10.10

6.76

6.28

7.36

Ash (%)

11.20±0.23

6.37±0.11

5.65±0.05

2.05±0.03

Extractives (%)

11.10±0.50

6.36±0.22

4.66±0.31

1.83±0.10

Rha

1.46±0.01

0.56±0.05

0.64±0.10

0.70±0.03

Ara

3.83±0.03

1.46±0.21

1.08±0.05

1.32±0.09

Gal

2.77±0.02

0.52±0.02

0.43±0.01

0.45±0.06

Glc

32.17±0.24

36.13±0.20

36.21±0.02

43.82±0.01

Xyl

11.56±0.11

23.22±0.03

21.15±0.05

19.57±0.30

Man

0.21±0.01

0.11±0.00

0.11±0.03

NDc

GlcA

0.12±0.31

0.22±0.20

0.14±0.09

0.58±0.01

GalA

0.30±0.04

0.08±0.05

0.11±0.00

0.10±0.03

ASL

8.29±0.02

4.63±0.13

4.25±0.26

3.67±0.15

AIL

4.46±0.01

8.57±0.14

13.94±0.36

21.73±0.20

Total lignin

12.75

13.20

18.19

25.40

Carbohydrates b (%)

Lignind (%)

736

a

the yields of DMSO-soluble hemicelluloses, relative to holocellulose.

737

b

Rha, rhamnose; Ara, arabinose; Gal, galactose; Glc, glucose; Man, mannose; Xyl, xylose; GlcA, glucuronic acid;

738

GalA, galacturonic acid.

739

c

ND, Not detectable.

38


Page 39 of 42


740

d

741

Table 2

742

Composition and degree of acetylation of hemicelluloses from bamboo (Neosinocalamus affinis).

ASL, acid soluble lignin; AIL, acid insoluble lignin.

Molar compositiona

Fractions

Molar ratiob

DSACc

(relative %, mol/mol)

Rha

Ara

Gal

Glc

Xyl

GlcA

GalA

GlcA/Xyl

Ara/Xyl

H2D

1.46±0.03

12.12± 0.03

4.98±0.01

21.25±0.04

56.27±0.01

3.61±0.05

0.30±0.01

0.06

0.26

0.105

H4D

0.55±0.01

5.98±0.02

0.51±0.01

1.96±0.05

89.57±0.05

1.36±0.02

0.08±0.02

0.02

0.07

0.112

H8D

0.64±0.22

6.48±0.02

0.92±0.05

2.46±0.04

87.34±0.01

2.04±0.01

0.11±0.01

0.02

0.07

0.166

H12D

0.30±0.11

7.27±0.05

0.73±0.02

1.23±0.13

87.30±0.03

2.97±0.02

0.20±0.01

0.03

0.08

0.387

H2A

0.99±0.01

25.48±0.03

6.42±0.14

4.03±0.06

56.12±0.22

6.75±0.01

0.20±0.02

0.12

0.45

-

H4A

0.78±0.21

11.56±2.11

1.34±0.12

0.73±0.60

80.53±0.35

4.80±0.11

0.25±0.01

0.06

0.14

-

H8A

0.72±0.11

9.43±0.31

1.45±0.30

1.10±0.15

72.66±0.51

14.57±0.35

0.07±0.02

0.21

0.13

-

H12A

0.55±0.02

7.82±0.11

1.50±0.20

0.51±0.36

83.81±0.21

5.52±0.02

0.28±0.04

0.07

0.09

-

743

a

Mannose was not detected.

744

b

Ara/Xyl, molar ratio of arabinose to xylose; GlcA/Xyl, molar ratio of glucuronic acid to xylose.

745

c

DSAC, the degree of acetylation of hemicelluloses.

746 747 748 749 750 39



Page 40 of 42

751 752

Table 3

753

Weight-average (Mw) and number-average (Mn) molecular weights (g/mol) and polydispersity (Mw/Mn) of

754

hemicelluloses from bamboo (Neosinocalamus affinis).

Samples

H2D

H4D

H8D

H12D

H2A

H4A

H8A

H12A

Mw

53250

67290

28440

45040

83080

50360

489503

51360

Mn

40750

50990

21840

7590

54460

34940

38390

35220

Mw/Mn

1.31

1.32

1.30

5.93

1.53

1.44

1.27

1.46

755 756 757 758 759 760 761 762 763 764 765 766 767 768 40


Page 41 of 42


769 770

Table 4

771

1

H and 13C chemical shift (ppm) assignements for H2D.

Chem shift (ppm) H/C Sugar residue 1

2

3

4

5axa

5eqb

4.23

3.14

3.34

3.52

3.14

3.87

102.21

73.35

74.17

75.32

63.63

63.0

3.52

3.78

3.14

3.34

70.56

73.86

86.38

60.65

5.40

3.78

3.52

3.87

3.34

3.52

107.48

80.71

78.38

86.68

62.39

62.39

6

OCH3

→4)-β-Xylp(1→

α-GlcAp-(1→2

α-Araf-(1→3

4.66

→4)-β-Xylp(1→,2-O-Ac 73.35

4.93

→4)-β-Xylp(1→,3-O-Ac 74.17

4.23 (1→4)-β-D-Glcp 103.18

(1→3)-β-D-Glcp

73.36

74.17

80.72

4.45

41


75.95

60.65


101.72

71.60

86.38

68.67

Page 42 of 42

75.95

60.65

772

TOC/Abstract graphic

773

774 775 776 777

Synopsis

778

Unveiling the chemical composition and hemicelluloses structure of differently aged

779

bamboo culms will promote the development for sustainable process.

780 781

42


Changes of Chemical Composition and Hemicelluloses Structure in

Recommend Documents