Comparison of the Structural Characteristics of Cellulolytic Enzyme

Nov 2, 2016 - Lignin structure has been considered to be an important factor that significantly influences the biorefinery processes. In this work, th...
0 downloads 3 Views 784KB Size
Subscriber access provided by University of Otago Library

Article

Comparison of the structural characteristics of cellulolytic enzyme lignin preparations isolated from wheat straw stem and leaf Bo Jiang, Tingyue Cao, Feng Gu, Wenjuan Wu, and Yongcan Jin ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01710 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 8, 2016

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 33

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

ACS Sustainable Chemistry & Engineering

1

Comparison of the structural characteristics of cellulolytic enzyme lignin preparations

2

isolated from wheat straw stem and leaf

3 4

Bo Jiang,† Tingyue Cao,† Feng Gu,‡ Wenjuan Wu,† Yongcan Jin*,†

5 6



7

Nanjing Forestry University, Nanjing 210037, China

8



9

Yancheng 224051, China

Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources,

School of Chemistry and Chemical Engineering, Yancheng Institute of Technology,

10 11

Mailing address: [email protected]; [email protected];

12

[email protected]; [email protected]; [email protected]

13 14

* Corresponding author

15

Dr. Yongcan Jin

16

Laboratory of Wood Chemistry

17

Department of Paper Science and Technology

18

Nanjing Forestry University

19

159 Longpan Rd., Nanjing 210037, China

20

E-mail address: [email protected]

21

Tel.: +86(25)8542 8163; Fax: +86(25)8542 8689

1

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

22 23

ABSTRACT Lignin structure has been considered as an important factor that significantly

24

influences the biorefinery processes. In this work, the effect of ball milling on the structural

25

components and extractable lignin in enzymatic residues was evaluated, and the structural

26

characteristics of the cellulolytic enzyme lignin preparations isolated from wheat straw

27

stem (SCEL) and leaf (LCEL) were comparatively investigated by a combination of

28

nitrobenzene oxidation (NBO), ozonation, infrared spectroscopy and 1H–13C heteronuclear

29

single quantum coherence nuclear magnetic resonance (2D HSQC NMR). The results

30

showed that 4 h ball-milled samples were good enough for structural analysis with high

31

lignin yield. Both of CELs are typical p-hydroxyphenyl-guaiacyl-syringyl lignin which

32

associated with p-coumarates and ferulates. However, the structure of lignin in wheat straw

33

stem is rather different from that in leaf. Compared to stem lignin, leaf lignin has lower

34

products yield of NBO and ozonation, lower erythro/threo ratio and higher condensation

35

degree. The analysis of 2D HSQC NMR indicated that the S/G ratio of SCEL was 0.8,

36

which is about twice as much as that of LCEL. The flavone tricin is incorporated into both

37

stem and leaf lignins. The content of tricin in LCEL is higher than that in SCEL.

38 39

KEYWORDS: Wheat straw, Stem, Leaf, Cellulolytic enzyme lignin (CEL), Structural

40

characteristics

2

ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33

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

41 42

ACS Sustainable Chemistry & Engineering

INTRODUCTION Lignin is one of the most abundant aromatic biopolymers and a major component of

43

plant cell walls. It is mainly composed of the monolignols p-coumaryl, coniferyl, and

44

sinapyl alcohols which give rise to the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S)

45

lignin units.1 The chemical utilization of lignin is supposed to be an important part of the

46

lignocellulosic biorefinery, but the complex morphological structure restricts its wide

47

application. Lignin is even associated with carbohydrates (in particular with hemicelluloses)

48

via covalent bonds to form a tight compact structure such as lignin-carbohydrate complex

49

(LCC).2 In herbaceous plants, hydroxycinnamic acids (p-coumaric and ferulic acids) are

50

attached to lignin and hemicelluloses via ester and ether bonds as bridges between them

51

forming carbohydrate-ether-hydroxycinnamate-ester-lignin complexes,3, 4 which result in

52

the structure of non-wood lignin being much more complex than wood lignin. Therefore, it

53

is nearly impossible to separate lignin from lignocellulose solely and keep their native state.

54

Alternatively, cellulolytic enzyme lignin (CEL) has commonly been used for the

55

structural analysis of cell wall lignin, which utilizes cellulolytic enzyme hydrolysis prior to

56

dioxane/water extraction of ball-milled wood meal to remove carbohydrates and achieve

57

lignin with high yield and purity.5, 6 For decades, a lot of work was devoted to

58

understanding the structural features of lignin from plant cell wall, for example, the

59

monomeric content of lignin polymer and some other structural features were analyzed with

60

different chemical degradation methods such as alkaline nitrobenzene oxidation,7

61

ozonation,8 thioacidolysis,9 and derivatization followed by reductive cleavage that uses

62

acetyl bromide for derivatization and zinc for reductive cleavage.10 These wet chemical

63

methods can be very precise for specific functional groups and structural moieties. 3

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

64

However, each chemical method gives limited information (mainly uncondensed lignin

65

units) and is not able to provide a general picture of the entire lignin structure.

66

In recent years, the analytical methods of nuclear magnetic resonance (NMR) for

67

lignin characterization have been significantly improved. NMR has the advantages of the

68

analysis of the whole lignin structure and direct detection of lignin moieties, including the

69

presence of aryl ether, condensed and uncondensed aromatic and aliphatic carbons.11, 12

70

Additionally, two-dimensional heteronuclear single quantum coherence (2D HSQC) NMR

71

has been developed to quantify the lignin structures and LCC linkages. The

72

semi-quantitative 2D NMR could be an ideal experiment for the estimation of specific

73

lignin structures, and provides information on the interunit linkages.13, 14 For example, del

74

Río et al.15 investigated milled wood lignin from wheat straw and found it is a G-S-H type

75

lignin associated with p-coumarates and ferulates. Rencoret et al.16 investigated cellulolytic

76

lignin in brewer’s spent grain and found the lignin presents a predominance of G units and

77

the main substructures present are β–O–4’ followed by β–5’, β−β’ and 5–5’ linkages. The

78

flavone tricin was present in these lignins, as also occurred in other grasses.17, 18

79

Wheat straw has been considered as one of the most important feedstocks for the

80

production of chemicals, materials and fuels via biorefinery technology. Lignin structure is

81

one of the key factors that influence the processes of biorefinery such as pretreatment and

82

enzymatic hydrolysis. Researches indicated that the structures of herbaceous lignin in leaf

83

are rather different from that in stem. For instance, Markovic et al.19 studied the structure of

84

acid detergent lignin (ADL) in alfalfa leaf and stem by the attenuated total reflectance

85

Fourier transform infrared (FTIR), and the results indicated the spectra of ADL from leaf

86

and stem are similar in frequency of absorption bands, but different in their intensities. Min 4

ACS Paragon Plus Environment

Page 4 of 33

Page 5 of 33

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

ACS Sustainable Chemistry & Engineering

87

et al.20 investigated the structure of lignin in corn stover and pointed out that stem lignin

88

had higher contents of p-coumaric acid, ferulic acid and β–O–4’ linkages but with lower

89

contents of β–5’, β–β’ linkages and lower ratio of p-hydroxyphenyl/guaiacyl (H/G). The

90

alkaline nitrobenzene oxidation (NBO) data showed stem lignin had higher products yield

91

and syringaldehyde/vanillin (S/V) ratio than leaf lignin. These findings suggest that

92

structural differences between stem and leaf in herbaceous lignin may result in different

93

biorefinery processes. As Jin et al.21 reported that, the enzymatic sugar recovery of sodium

94

carbonate-pretreated wheat leaf was higher than that of wheat stem, and the different

95

structure of lignin in stem and leaf might be one of the important influence factors.

96

In this paper, the CEL protocol was used to isolate lignin preparations from wheat

97

straw stem and leaf. The CEL preparations were characterized by destructive (alkaline

98

nitrobenzene oxidation and ozonation) and nondestructive methods (2D HSQC NMR and

99

FTIR spectroscopy) for understanding the difference of the structural characteristics

100

between lignin in wheat straw stem and leaf.

101

MATERIALS AND METHODS

102

Materials. Wheat straw (Triticum aestiuium) was collected from Yancheng, Jiangsu,

103

China in May, 2011. The materials were classified into stem and leaf (sheath included) by

104

hands, and then were ground using a Wiley mill. The particles passed through 20 mesh

105

(0.85 mm) sieve were collected. The straw meals were extracted with ethanol/benzene (1:2,

106

v/v) for 48 h to obtain extractive-free samples. No specific step was carried out to remove

107

protein. The extracted samples were air dried and subsequently vacuum dried.

108

Cellulase from Trichoderma reesei (NS 50013, 84 FPU/mL), β-glucosidase from

109

Aspergillus niger (NS 50010, 350 CBU/mL) and xylanase (NS 50014, 850 FXU/mL) were 5

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

110

generously provided by Novozymes (Novo Nordisk A/S, Demark). The chemicals used in

111

this study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and/or

112

Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

113

Isolation of CEL. The procedure for the isolation of CEL from wheat straw stem and

114

leaf is illustrated in Figure 1. The vacuum dried straw (2 g in each bowl) was milled in a

115

planetary ball mill (QM-3SP2, Nanjing Nanda Instrument Plant, China) at a fixed

116

frequency of 600 rpm. Two 100 mL zirconium dioxide bowls with 16 zirconium dioxide

117

balls (1 cm diameter) in each bowl were used in the milling. The milling time was 2–6 h to

118

obtain milled straw samples with different milling degrees. An interval of 5 min was set

119

between every 15 min of milling to prevent overheating. After ball milling, the straw

120

powder (MS and ML for stem and leaf, respectively) was carefully collected and dried

121

under vacuum.

122

The ball-milled sample (5 g) was suspended in 100 mL acetate buffer at pH 4.8, and an

123

enzyme cocktail mixed by NS 50013, NS 50014 and NS 50010 with a ratio of 1 FPU : 1.2

124

FXU : 1 CBU was added in a 250 mL Erlenmeyer flask and then incubated in a shaker

125

(DZH-2102, Jinghong, Shanghai, China) at 180 rpm and 50 °C. The charge of mixed

126

enzyme based on cellulase activity was 60 FPU/g-cellulose. After 72 h of enzymatic

127

hydrolysis, the mixture was centrifuged to remove the supernatant. The residue was washed

128

by centrifugation for 3 times using sodium acetate buffer and deionized water, respectively.

129

The washed enzymatic residues of ball-milled stem and leaf (EMS and EML) were

130

freeze-dried and then extracted twice (2 × 24 h) with 50 mL of 96% aqueous dioxane (v/v)

131

under nitrogen atmosphere. The supernatants were combined and the solvent was removed

132

by vacuum evaporation. The dried crude lignin samples were purified by 90% (w/w) acetic 6

ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33

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

ACS Sustainable Chemistry & Engineering

133

acid.22 The obtained CEL preparations from stem and leaf were named SCEL and LCEL,

134

respectively. No further purification was performed for the preservation of the structural

135

features of the lignin preparations.

136

Extractable lignin measurement. Extractable lignin23 was used to evaluate the

137

solubility of lignin in enzymatically hydrolyzed residues. Twenty milligram of sample was

138

suspended in 10 mL 96% (v/v) aqueous dioxane. The mixture was magnetically stirred for

139

48 h at room temperature. The extract was separated by centrifugation and 5 mL of the

140

supernatant was reduced with 1 mg of NaBH4 in 1 mL of 0.05 M NaOH for 24 h and then

141

was neutralized with 4 mL of glacial acetic acid. The same process was duplicated on 96%

142

(v/v) aqueous dioxane without sample suspension for the preparation of reference. The UV

143

absorbance at 280 nm was measured to calculate the amount of lignin using 13 L/(g·cm)

144

from sweetgum23 as the gram absorptivity.

145

Analytical methods. Lignin and sugar content of the samples were analyzed using the

146

NREL protocol.24 The Klason lignin (KL) content was taken as the ash free residue after

147

acid hydrolysis. The hydrolysate was collected for the determination of the acid-soluble

148

lignin (ASL) and the structural sugars. The ASL was measured by absorbance at 205 nm in

149

a UV-vis spectrometer (TU-1810, Beijing Puxi, China) and 110 L/(g·cm) as absorptivity

150

value was used which is an average of several reported values. The monomeric sugars were

151

quantitatively measured with a high performance liquid chromatography (HPLC, Agilent

152

1200 Series, Santa Clara, CA) equipped with the refractive index detector (RID). The

153

HPLC analysis was carried out using a Bio Rad Aminex HPX-87H 20n exclusion column

154

(300 × 7.8 mm, Bio-Rad Laboratories, Hercules, CA) with a Cation-H Refill Cartridge

155

guard column (30 × 4.6 mm, Bio-Rad Laboratories, Hercules, CA). The ash content was 7

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

156

determined by combustion at 575 °C. Alkaline nitrobenzene oxidation and ozonation were carried out according to the

157 158

procedure reported by Chen25 and Akiyama et al.,8 respectively. 2D NMR spectra of the CELs were recorded at 25 °C on an AVANCE III 600 MHz

159 160

instrument (Bruker, Switzerland) equipped with a cryogenically cooled 5 mm TCI

161

z-gradient triple-resonance probe. The lignin preparations (50 mg) were dissolved in 0.5

162

mL of deuterated dimethyl sulfoxide (DMSO-d6) according to the method previously

163

described.26, 27 The central solvent peak was used as the internal reference (δC/δH 39.5/2.50).

164

The HSQC experiments used Bruker’s “hsqcetgpsp.2” adiabatic pulse program with

165

spectral widths from 0 to 16 ppm (9615 Hz) and from 0 to 165 ppm (24900 Hz) for the 1H-

166

and 13C-dimensions. The number of collected complex points was 2048 for the

167

1

168

increments were recorded in the 13C-dimension. The 1JCH used was 145 Hz. Processing

169

used typical matched Gaussian apodization in the 1H-dimension and squared cosine-bell

170

apodization in the 13C-dimension. Prior to Fourier transformation, the data matrices were

171

zero-filled to 1024 points in the 13C-dimension.

172

H-dimension with a recycle delay of 1.5 s. The number of transients was 64, and 256 time

FTIR spectra of SCEL and LCEL were recorded using a VERTEX 80V FTIR

173

spectrometer (Bruker, Germany). Around 2 mg of lignin samples was mixed with 400 mg

174

KBr, then determined after grinding and tabletting. The scan resolution was 4 cm–1 and the

175

scan area was 4,000–400 cm–1.

176

RESULTS AND DISCUSSION

177 178

Effect of ball milling time on structural components and extractable lignin in enzymatic residues. The chemical composition of wheat straw stem was rather different 8

ACS Paragon Plus Environment

Page 8 of 33

Page 9 of 33

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

ACS Sustainable Chemistry & Engineering

179

from that of leaf as shown in Table 1. After enzymatic hydrolysis, the glucan and xylan

180

content was very low in the hydrolyzed residues of both wheat straw stem and leaf.

181

However, the target products, SCEL and LCEL, still contained a certain amount of xylan

182

after 1,4-dioxane/water extraction. The removal of xylan was less than that of glucan, it

183

indicated that the lignin and hemicelluloses are present in cell walls not only as a simple

184

mixture but through chemical linkages.28 Ball milling leads to the structural modification of

185

total lignin, such as the increase of carbonyl content, the decrease of molar mass and

186

cleavage of aryl ether bonds. Lu and Ralph29 pointed out that ball milling destroys the

187

side-chain structure of lignin by the cleavage of β–O–4’ bonds and the increase of

188

α-carbonyl group in a certain degree. Ikeda et al.30 investigated the effect of ball milling on

189

lignin structure and found the drops of etherified β–O–4’ linkages and the increases of

190

phenolic β–O–4’ linkages occurred during the ball-milling process. However, the effect of

191

ball milling on total lignin was different from isolated lignin preparations,31 in particular,

192

the amount of β–O–4’ in the total lignin decreases progressively with ball milling, but it is

193

rather constant in MWL and CEL.6, 32 Furthermore, Capanema et al.32 compared MWL and

194

CEL preparations from three kinds of hardwood and pointed out that the yield of the lignin

195

preparations increases linearly with the milling time in the interval of 2.5–12.5 h and the

196

yields of CEL preparations are about twice as high as those of the corresponding MWLs. In

197

contrast, the S/G ratio does not change in the total lignin, but fluctuate in MWL and CEL

198

depending on the yield. Ball milling in this work helps improve substrate enzymatic

199

digestibility because more saccharides, especially hemicelluloses, were released.

200 201

Fujimoto et al.23 and Hu et al.6 described that if the extractable lignin yields from milled woods are the same, the structural changes of lignin caused by the ball milling are 9

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

202

similar regardless of the difference in milling conditions and apparatus. In this study,

203

extractable lignin was introduced as a general criterion to evaluate the milling degree and

204

the potential yield of isolated lignin. Figure 2 shows the extractable lignin yield of

205

enzymatically hydrolyzed stem and leaf. Extractable lignin yield of stem was significantly

206

improved when the ball milling time improved from 2 h to 4 h. The increase of extractable

207

lignin yield leveled off when the ball milling time was over 4 h. This result indicated that 4

208

h ball milling is good enough for isolating lignin by 96% 1,4-dioxane/water extraction and

209

the yield of extractable lignin in stem, on the basis of lignin in raw material, could reach

210

68.3%. On the basis of lignin in the enzymatic residue, more than 85% of the lignin in 4 h

211

ball-milled stem could be extracted after enzymatic hydrolysis. The extractable lignin of

212

leaf was much lower than that of stem. This was potentially caused by the structural

213

differences between leaf lignin and stem lignin, or by the more non-lignin components in

214

leaf.33

215

Fujimoto et al.23 studied the quantitative evaluation of milling effects on lignin

216

structure during the isolation process of milled wood lignin (MWL) by ozonation. The

217

results indicated that the proportion of β–O–4’ linkages showed declining trend as the

218

extension of ball milling time. The ozonation products yield and erythro to threo ratio (E/T)

219

decreased with the increase of extractable lignin yield. The structure of erythro form β–O–4’

220

was broken preferentially in the process of ball milling, but the degree would not exceed

221

25%.6 The degree for 4 h ball-milled wheat straw stem and leaf in this work was only 3.2%

222

and 4.7%, respectively. Therefore, CEL preparations obtained through 4 h ball milling time

223

in this work were representative for investigation of lignin interunit linkages.

224

Structural characteristics of lignin during CEL isolation. Alkaline nitrobenzene 10

ACS Paragon Plus Environment

Page 10 of 33

Page 11 of 33

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

ACS Sustainable Chemistry & Engineering

225

oxidation and ozonation were performed to investigate the effects of ball milling and

226

enzymatic hydrolysis on the structural characteristics of lignin in wheat straw stem and leaf.

227

The results are given in Table 2. The difference of NBO products yield between stem (2.23

228

mmol/g-lignin) and leaf (1.60 mmol/g-lignin) suggested the condensation degree of lignin

229

in leaf was higher than that in stem. Compared to lignin in raw materials and enzymatic

230

residues, the isolated CELs had higher NBO products yield. It implied that wheat straw

231

CEL featured with a lower condensation degree than original lignin. Due to the high

232

proportion of condensed guaiacyl units, only about 30% of them are converted to vanillin.

233

On the contrary, the conversion of syringyl units to syringaldehyde may be as high as 90%

234

due to the low proportion of condensation.25, 34 After 4 h ball milling, the decrease of NBO

235

products yield was 1.3% for stem, while it was 6.3% for leaf. The effects of ball milling

236

time in this work (2–6 h) on aromatic structure of lignin were not significant since no

237

obvious changes of S/V/H ratio were observed. However, while CEL well represents the

238

total biomass lignin in softwood6 and hardwood32, this is apparently not the case for

239

non-wood lignins, specifically for the wheat straw ones in this work, as indicated S/V/H

240

ratio data of CELs vs total lignin (Table 2). This is likely due to more heterogeneous

241

structure of non-wood lignins.

242

The ozonation products yield and E/T ratio of lignin decreased with the ball milling

243

hours. For example, in 4 h ball-milled stem, the decrease of ozonation yield and E/T ratio

244

were 7.1% and 3.2%, respectively. However, the ozonation products yield of LCEL (0.46

245

mmol/g-lignin) was higher than that of raw material (0.33 mmol/g-lignin). Compared with

246

stem, leaf had lower ozonation yield. It might be caused by all these yields were based on

247

the sum of Klason lignin and acid-soluble lignin, while the Klason procedure cannot 11

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

248

discriminate between true lignin and lignin-like materials in leaf.35 Salamanca et al.33 also

249

pointed out that the Klason lignin may include both lignin and other non-hydrolyzable

250

products. In untreated leaf, the Klason lignin residue originated from components highly

251

resistant to degradation by H2SO4, and the Klason lignin residue greatly overestimated the

252

real lignin content of leaf. Some lignin-like materials in leaf contribute to the amount of

253

Klason lignin residue,36, 2 and these materials were easily removed during lignin isolation

254

process,36 as a result, the extracted leaf lignin showed both higher NBO and ozonation

255

products yield than lignin in original leaf and its enzymatic hydrolysis residue. The E/T

256

ratio of leaf lignin was lower than that of stem lignin, it is in good agreement with the result

257

of NBO analysis which showed leaf lignin had low S units content. The ratio of syringyl to

258

guaiacyl stereo-chemically governed the proportion of erythro and threo forms of β–O–4’

259

structures during lignin formation.8

260

1

H–13C HSQC NMR analysis. 1H–13C NMR is a powerful tool to probe structures of

261

lignin and its derivatives. The signals relate to the structural units and various linkages

262

between units of lignin in 2D NMR spectra can be assigned according to the published

263

literatures.12, 15-17, 37 The NMR spectra of CEL preparations from wheat straw stem and leaf

264

are illustrated in Figure 3, and the detailed assignments of main peaks of SCEL and LCEL

265

in NMR spectra (δC/δH 150–50/8.0–2.5) are listed in Table S1. Figure 4 depicts the major

266

lignin substructures shown in Figure 3. A semi-quantitative analysis based on HSQC

267

signals was performed using Bruker’s Topspin 2.1 processing software and the integral

268

method was according to the method described by del Río et al.15

269 270

As shown in Figure 3, signals from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units were observed clearly in the isolated SCEL and LCEL. The prominent signals 12

ACS Paragon Plus Environment

Page 12 of 33

Page 13 of 33

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

ACS Sustainable Chemistry & Engineering

271

corresponding to p-coumarate (PCA) and ferulate (FA) structures were observed which

272

were typically identified in gramineous plant lignin.12, 16, 17, 27, 37 The NMR spectra indicated

273

that β–O–4’ was the main interunit linkages of lignin, followed by phenylcoumarans and

274

other minor linkages, such as resinols, spirodienones, dibenzodioxocins and α, β-diaryl

275

ethers. The signals of the β–5’ structure in wheat straw is much more intensive than that of

276

β–β’ structure. It indicates that the condensation degree of G units is higher than that of S

277

units, which could be used to explain the reason of S/V increment in isolated lignin

278

samples.38 The intensive signals derived from tricin (T) were detected which acted as

279

antioxidants, antimicrobial and antiviral agents in vascular plants.18 Tricin is considered to

280

be fully compatible with lignification reactions and is an authentic lignin monomer.17 Tricin

281

linked to lignin units via 4’–O–β–ether bonds had been reported.17, 39

282

Polysaccharide signals (for X), mainly originated from hemicellulose, were found in

283

the spectra, including xylan correlations in the range δC/δH 65–80/2.5–4.5, which partially

284

overlapped with some lignin signals. As shown in Figure 3, the polysaccharide cross-peak

285

signals of X2 (δC/δH 72.9/3.14), X3 (δC/δH 74.1/3.32), X4 (δC/δH 75.6/3.63), X5 (δC/δH

286

63.2/3.26 and 3.95) were assigned to β-D-xylopyranoside.40 These polysaccharide signals

287

evidenced that lignin was mainly linked with xylan via covalent bonds which caused

288

difficulties in effective separation of components in a technical scale. LCC is primarily

289

composed of γ-ester, benzyl ether and phenyl glycoside and is one of the main factors

290

causing recalcitrance of biorefining.41 In the process of lignin purification by 90% (w/w)

291

acetic acid, LCC substructures especially benzyl ether and phenyl glycoside structures were

292

most removed which reduced the signals overlap of lignin and carbohydrate in the 2D

293

NMR spectra of lignins. 13

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

294

The relative amounts of the main lignin interunit linkages and the molar abundances of

295

the different lignin units (H, G and S), p-coumarates, ferulates, and the molar S/G ratios of

296

the lignins in wheat straw, estimated from volume integration of contours in the HSQC

297

spectra, are given in Table 3. The percentage of these structural units was calculated by

298

referring these structural unit signals to the total number of aromatic rings (H+G+S). The

299

data indicated that the main substructures present are β–O–4’ alkyl aryl ether both in SCEL

300

(64%) and LCEL (56%), while other linkages referring as the condensed structures (β–β’

301

resinols, β–5’ phenylcoumarans and β–1’ spirodienones) were present in minor amounts. In

302

particular, the contents of β–β’, β–1’ interunit linkages were similar, but the content of β–5’

303

substructures in LCEL was higher than that in SCEL, that was caused by the higher

304

condensation degree of leaf lignin, which may correlate with S/G ratio of stem and leaf

305

lignins. Furthermore, tricin and its derivatives were believed to protect plants from

306

pathogens,42 the high amounts of tricin in wheat straw are remarkable (12-17%) which may

307

induce the isolation and purification of tricin for potential application such as food and

308

medicine fields even though the physiological function of tricin in plants remains poorly

309

understood.

310

Comparatively, in aromatic/unsaturated region of the HSQC spectra, the signal of G

311

units is obviously more intensive than H and S units both in SCEL and LCEL. The result of

312

LCEL from semi-quantitative 2D NMR analysis is consistent with the results of NBO.

313

However, the data of SCEL from NMR are not consistent with the NBO results because the

314

condensation degree of G units is higher than that of S units. Additionally, the content of G

315

units in LCEL (71%) was higher than that in SCEL (54%) which had more S units content,

316

it induced a significant difference on S/G ratio between SCEL (0.8) and LCEL (0.4). The 14

ACS Paragon Plus Environment

Page 14 of 33

Page 15 of 33

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

ACS Sustainable Chemistry & Engineering

317

NBO results showed the leaf lignin exhibits a higher degree of condensation (Table 2). The

318

more branched and condensed G units such as 5–5’, 4–O–5’ units not only acted as a

319

surface barrier, but restricted the swelling of lignocellulose and reduced the accessible

320

surface area available to the enzyme.43 However, the linear lignin contained more S units

321

that could adsorb on the cellulose surface more tightly which blocked the accessibility of

322

cellulose dramatically.44 Comprehensively, the effects of lignin on lignocellulosic

323

saccharification may depend on S/G ratio as reports pointed out that lignin with high S/G

324

ratio is negative on biomass enzymatic digestibility in Miscanthus.45, 46 Therefore, the lower

325

S/G ratio in leaf lignin may explain why the enzymatic sugar recovery of sodium

326

carbonate-pretreated21 and green liquor-pretreated47 wheat leaf was higher than that of

327

wheat stem. Comparing the ratio of S/G/H in 2D HSQC NMR spectra with the ratio of

328

S/V/H in nitrobenzene oxidation, apparent differences were observed and that was because

329

the p-coumarates and ferulates in wheat straw contributed to the H and V units under

330

alkaline nitrobenzene oxidation condition, respectively.48 Alkaline nitrobenzene oxidation

331

can only detect the non-condensed guaiacyl, syringyl and p-hydroxyphenyl units, and it

332

measures the S/V of releasable syringaldehyde (minor syringic acid) and vanillin (minor

333

vanillic acid) monomers from oxidative cleavage of the side chain.20 NMR does profile the

334

“entire” lignin (the isolated lignin) in principle including the condensed and the

335

non-condensed parts of lignin. Although some contours of aromatic parts of composing

336

units (G and H units) were overlapped with the contours of aromatic parts of p-coumarate

337

and ferulate esters, the value of S/G ratio was still calculated by using the reported

338

assignments of the aromatic contour of each composing unit.26 Even though including the

339

ferulates in G units and p-coumarates in H units when correlating 2D NMR data with the 15

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

340

NBO ones, the discrepancies were still considerably obvious. Santos et al.49 pointed out that

341

wood lignin has a good linear relation between S/G and S/V ratio, and the S/G ratio of

342

lignin can be predicted by multiplying the S/V value by a constant (0.806). However, an

343

earlier reported value of 0.59 may be more reasonable.50 In this work, the value of constant

344

was 0.62 and 0.50 for SCEL and LCEL, respectively, which was reasonable in

345

consideration of structural differences between non-wood and wood lignin. The different

346

forms of ferulic acid and lignin-like materials36 in stem and leaf lignins may cause the

347

different content of V and S units or the difference was simply derived from the enrichment

348

of certain types of subunits in the free phenolic groups.

349

FTIR spectroscopy. The FTIR spectra of LCEL and SCEL in wheat straw are shown

350

in Figure S1. The assignments of main absorption bands51-54 are listed in Table S2. The

351

spectra showed some common features but also vibrations that were specific to each lignin.

352

The stretching vibration of S-unit at 1330 cm–1 in SCEL was apparently stronger than that

353

in LCEL which means stem lignin has more S units content than that in leaf lignin, which is

354

consistent with the data of NBO products and the analysis of 2D NMR. In contrast, the

355

bending vibration of C–H and C–O at 1085 cm–1 in LCEL is showed clearly but is almost

356

undetectable in SCEL. These different stretching vibration positions between SCEL and

357

LCEL may be attributed to the difference of steric hindrance, electron cloud density and

358

field effect among functional groups. For example, Li et al.55 pointed out that S unit is

359

characterized with more methoxy groups on benzene ring which generates steric hindrance

360

effect. The oxygen atoms in phenolic hydroxyl and methoxy groups have shown strong

361

electronegativity. The unshared p electron pairs can form p-π conjugated system with the π

362

electron cloud of benzene ring. 16

ACS Paragon Plus Environment

Page 16 of 33

Page 17 of 33

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

363 364

ACS Sustainable Chemistry & Engineering

CONCLUSIONS The structure of lignin in wheat straw stem is rather different from that in leaf. Wet

365

chemistry results showed that leaf lignin has lower products yield of nitrobenzene oxidation

366

and ozonation, lower erythro/threo ratio and higher condensation degree than stem lignin.

367

The 2D HSQC NMR spectra and ozonation results showed that the stem lignin contains

368

more β–O–4’ linkages which are mainly composed by erythro form. Besides, leaf lignin has

369

less syringyl (S) content and more guaiacyl (G) content while the similar hydroxyphenyl (H)

370

content, which means the lower ratio of S/G in leaf lignin. The flavone tricin is

371

incorporated into both stem and leaf lignins. The content of tricin in leaf lignin is higher

372

than that in stem lignin. The analysis of FTIR spectra also showed that the functional

373

groups are structurally different between wheat straw stem and leaf lignins. The difference

374

of structural characteristics between stem and leaf lignins in herbaceous plants may be the

375

main influence factors on saccharification of lignocellulosic biomass which results in

376

different biorefinery processes.

377

17

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

FUNDING SOURCES This work was supported by the National Key Technology Research and Development

380

Program of China (grant number 2015BAD15B09), the National Natural Science

381

Foundation of China (grant numbers 31370571, 31400514), China Postdoctoral Science

382

Foundation (Grant No. 2016M591853), and the Priority Academic Program Development

383

of Jiangsu Higher Education Institutions, China.

384

18

ACS Paragon Plus Environment

Page 18 of 33

Page 19 of 33

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

ACS Sustainable Chemistry & Engineering

385

Supporting Information. The FTIR spectra of LCEL and SCEL; Assignment of the

386

1

387

the assignment of absorption peaks in CELs are supplied as Supporting Information.

H−13C correlation peaks in the 2D HSQC spectra of SCEL and LCEL; The positions and

388

19

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

389

REFERENCES

390

(1) Boerjan, W.; Ralph, J.; Baucher, M. Lignin biosynthesis. Annu. Rev. Plant Biol. 2003,

391 392 393 394 395 396

54, 519–546. (2) Buranov, A.U.; Mazza, G. Lignin in straw of herbaceous crops. Ind. Crop. Prod. 2008, 28, 237–259. (3) Baucher, M.; Monties, B.; Montagu, M.V.; Boerjan, W. Biosynthesis and genetic engineering of lignin. Crit. Rev. Plant Sci. 1998, 17, 125–197. (4) Sun, R.; Tomkinson, J. Comparative study of lignins isolated by alkali and

397

ultrasound-assisted alkali extractions from wheat straw. Ultrason. Sonochem. 2002, 9,

398

85–93.

399

(5) Chang, H.M.; Cowling, E.B.; Brown. W. Comparative studies on celluloytic enzyme

400

lignin and milled wood lignin of sweetgum and spruce. Holzforschung 1975, 29, 153–

401

159.

402

(6) Hu, Z.J.; Yeh, T.F.; Chang, H.M.; Matsumoto, Y.; Kadla, J.F. Elucidation of the

403

structure of cellulolytic enzyme lignin. Holzforschung 2006, 60, 389–397.

404 405

(7) Dzhumanova, Z.K.; Dalimova, G.N. Nitrobenzene oxidation of lignins from several plants of the family Gramineae. Chem. Nat. Compd. 2011, 47, 419–421.

406

(8) Akiyama, T.; Sugimoto, T.; Matsumoto, Y.; Meshitsuka, G. Erythro/threo ratio of beta–

407

O–4 structures as an important structural characteristic of lignin. I: Improvement of

408

ozonation method for the quantitative analysis of lignin side-chain structure. J. Wood

409

Sci. 2002, 48, 210–215.

410

(9) van de Pas, D.J.; Nanayakkara, B.; Suckling, I.D.; Torr, K.M. Comparison of

411

hydrogenolysis with thioacidolysis for lignin structural analysis. Holzforschung 2014,

412

68, 151–155.

413

(10) Partovi, T.; Abdol, H.M.; Seyyed, A. M. Investigation of structure of milled wood and

414

dioxane lignins of Populus nigra and Cupressus sempervirens using the DFRC method.

415

Chem. Pap. 2012, 66, 800–804.

416

(11) Capanema, E.A.; Balakshin M.Y.; Kadla J.F. A comprehensive approach for

417

quantitative lignin characterization by NMR spectroscopy. J. Agric. Food Chem. 2004,

418

52, 1850–1860. 20

ACS Paragon Plus Environment

Page 20 of 33

Page 21 of 33

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

ACS Sustainable Chemistry & Engineering

(12) Wen, J.L.; Sun, S.L.; Xue, B.L.; Sun, R.C. Recent advances in characterization of

420

lignin polymer by solution-state nuclear magnetic resonance (NMR) methodology.

421

Materials 2013, 6, 359–391.

422

(13) Balakshin, M.Y.; Capanema, E.A.; Gracz, H.; Chang, H.M.; Jameel, H. Quantification

423

of lignin–carbohydrate linkages with high-resolution NMR spectroscopy. Planta 2011,

424

233, 1097–1110.

425 426 427

(14) Crestini, C.; Melone, F.; Sette M.; Saladino, R. Milled wood lignin: A linear oligomer. Biomacromolecules 2011, 12, 3928–3935. (15) del Río, J.C.; Rencoret, J.; Prinsen, P.; Martínez, Á.T.; Ralph, J.; Gutiérrez, A.

428

Structural characterization of wheat straw lignin as revealed by analytical pyrolysis,

429

2D-NMR, and reductive cleavage methods. J. Agric. Food Chem. 2012a, 60, 5922–

430

5935.

431

(16) Rencoret, J.; Prinsen, P.; Gutieŕrez, A.; Martıńez, A.T.; del Río, J.C. Isolation and

432

structural characterization of the milled wood lignin, dioxane lignin, and cellulolytic

433

lignin preparations from brewer’s spent grain. J. Agric. Food Chem. 2015, 63,

434

603−613.

435

(17) Wu, L.; Lu, F.C.; Regner, M.; Zhu, Y.M.; Rencoret, J.; Ralph, S.A.; Zakai, U.I.;

436

Morreel, K.; Boerjan, W.; Ralph, J. Tricin, a flavonoid monomer in monocot

437

lignification. Plant Physiol. 2015, 167, 1284–1295.

438 439

(18) Zhou, J.M.; Ibrahim, R.K. Tricin—a potential multifunctional nutraceutical. Phytochem. Rev. 2010, 9, 413–424.

440

(19) Markovic, J.P.; Štrbanovic, R.T.; Terzic, D.V.; Djokic, D.J.; Simic, A.S.; Vrvic, M.M.;

441

Živkovic, S.P. Changes in lignin structure with maturation of alfalfa leaf and stem in

442

relation to ruminants nutrition. Afr. J. Agric. Res. 2012, 7, 257–264.

443

(20) Min, D.Y.; Jameel, H.; Chang, H.M.; Lucia, L.; Wang, Z.G.; Jin, Y.C. The structural

444

changes of lignin and lignin-carbohydrate complexes in corn stover induced by mild

445

sodium hydroxide treatment. RSC Adv. 2014, 4, 10845–10850.

446

(21) Jin, Y.C.; Huang, T.; Geng, W.H.; Yang, L.F. Comparison of sodium carbonate

447

pretreatment for enzymatic hydrolysis of wheat straw stem and leaf to produce

21

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

448 449 450

fermentable sugars. Bioresour. Technol. 2013, 137, 294–301. (22) Björkman, A. Studies on finely divided wood. Part I. Extraction of lignin with neutral solvents. Svensk Papperstidn. 1956, 59, 477–485.

451

(23) Fujimoto, A.; Matsumoto, Y.; Chang, H.M.; Meshitsuka, G. Quantitative evaluation of

452

milling effects on lignin structure during the isolation process of milled wood lignin. J.

453

Wood Sci. 2005, 51, 89–91.

454

(24) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D.

455

Determination of structural carbohydrates and lignin in biomass. Laboratory analytical

456

procedure, NREL, Report No. TP–510–42618, 2008.

457 458 459 460

(25) Chen, C.L. Nitrobenzene and cupric oxide oxidations. In Methods in Lignin Chemistry; Lin, S., Dence, C., Eds.; Springer-Verlag: Berlin, 1992; pp 301–321. (26) Kim, H.; Ralph, J.; Akiyama, T. Solution-state 2D NMR of ball milled plant cell-wall gels in DMSO-d6. Bioenergy Res. 2008, 1, 56−66.

461

(27) Rencoret, J.; Marques, G.; Gutiérrez, A.; Nieto, L.; Jiménez-Barbero, J.; Martínez, A.T.;

462

del Río, J.C. Isolation and structural characterization of the milled wood lignin from

463

Paulownia fortune wood. Ind. Crops Prod. 2009, 30, 137−143.

464

(28) Overend, R.P.; Johnson, K.G. Lignin carbohydrate complexes from

465

poplarwood-isolation and enzymatic degradation. ACS Symp. Ser. 1991, 460, 270–287.

466

(29) Lu, F.; Ralph, J. Lignin. In Cereal straw as a resource for sustainable biomaterials and

467

biofuels: Chemistry, Extractives, Lignins, Hemicelluloses and Cellulose; Sun, R.C. Ed.;

468

Elsevier Press: Amsterdam, 2010; pp 169–200.

469

(30) Ikeda, T.; Holtman, K.; John, F.K.; Chang, H.M.; Jameel, H. Studies on the effect of

470

ball milling on lignin structure using a modified DFRC method. J. Agric. Food Chem.

471

2001, 50, 129–135.

472

(31) Balakshin, M.Y.; Capanema, E.A.; Chang, H.M. Recent advances in the isolation and

473

analysis of lignins and lignin–carbohydrate complexes. In: Characterization of

474

Lignocellulosics; Hu, T. Ed.; Blackwell: Oxford, UK, 2008; pp 148–170.

475

(32) Capanema, E.A.; Balakshin, M.; Katahira, R.; Chang, H.M.; Jameel, H. How well do

476

MWL and CEL preparations represent the whole hardwood lignin? J. Wood Chem.

477

Techn. 2015, 35, 17–26. 22

ACS Paragon Plus Environment

Page 22 of 33

Page 23 of 33

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

478

ACS Sustainable Chemistry & Engineering

(33) Salamanca, E.F.; Kaneko, N.; Katagiri, S.; Nagayama, Y. Nutrient dynamics and

479

lignocellulose degradation in decomposing Quercus serrata leaf litter. Ecolog. Res.

480

1998, 13, 199–210.

481

(34) Sarkanen, K.V.; Hergert, H.L. Classification and distribution. In Lignins: Occurrence,

482

Formation, Structure and Reactions; Sarkanen, K.L., Ludwig, C.H., Eds.;

483

Wiley-Interscience: New York, 1971; pp 43–94.

484

(35) Johansson, M.B.; Kogel, I., Zech, W. Changes in the lignin fraction of spruce and pine

485

needle litter during decomposition as studied by some chemical methods. Soil Biol.

486

Biochem. 1986, 18, 611–619.

487

(36) Jin, Z.F.; Akiyama, T.; Chung, B.Y.; Matsumoto, Y.; Iiyama, K.; Watanabe, S. Changes

488

in lignin content of leaf litters during mulching. Phytochemistry 2003, 64, 1023–1031.

489 490

(37) Kim, H.; Ralph, J. Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Org. Biomol. Chem. 2010, 8, 576−591.

491

(38) Gu, F.; Wu, W.J.; Wang, Z.G.; Toyoma, Y.; Jin, Y.C. Effect of complete dissolution in

492

LiCl/DMSO on the isolation and characteristics of lignin from wheat straw internode.

493

Ind. Crop. Prod. 2015, 74, 703–711.

494

(39) Chang, C.L.; Wang, G.J.; Zhang, L.J.; Tsai, W.J.; Chen, R.Y.; Wu, Y.C.; Kuo, Y.H.

495

Cardiovascular protective flavolignans and flavonoids from Calamus quiquesetinervius.

496

Phytochemistry 2010, 71, 271−279.

497

(40) del Río, J.C.; Prinsen, J.; Rencoret, J.; Nieto, L.; Jiménez-Barbero, J.; Ralph, J.;

498

Martínez, A.T.; Gutiérrez, A. Structural characterization of the lignin in the cortex and

499

pith of elephant grass (Pennisetum purpureum) Stems. J. Agric. Food Chem. 2012, 60,

500

3619–3634.

501 502 503 504 505 506

(41) Koshijima, T.; Watanabe, T. Association between lignin and carbohydrates in wood and other plant tissues. Forestry 2004, 77, 175–176. (42) Park, H.S.; Lim, J.H.; Kim, H.J.; Choi, H.J.; Lee, I.S. Antioxidant flavone glycosides from the leaves of Sasa borealis. Arch. Pharmacal Res. 2007, 30, 161–166. (43) Li, X.; Ximenes, E.; Kim, Y.; Slininger, M.; Meilan, R.; Ladisch, M.; Chapple, C. Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid

23

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

507 508

hot water pretreatment. Biotechnol. Biofuels 2010, 3, 27–34. (44) Min, D.Y.; Yang, C.M.; Shi, R. The elucidation of the lignin structure effect on the

509

cellulase-mediated saccharification by genetic engineering poplars (Populus nigra L.

510

and Populus maximowiczii A.). Biomass Bioenergy 2013, 58, 52–57.

511

(45) Li, M.; Si, S.; Hao, B.; Zha, Y.; Wan, C.; Hong, S.; Kang, Y.; Jia, J.; Zhang, J.; Li, M.;

512

Zhao, C.; Tu, Y.; Zhou, S.; Peng, L. Mild alkali-pretreatment effectively extracts

513

guaiacyl-rich lignin for high lignocellulose digestibility coupled with largely

514

diminishing yeast fermentation inhibitors in Miscanthus. Bioresour. Technol. 2014, 169,

515

447–454.

516

(46) Xu, N.; Zhang, W.; Ren, S.; Liu, F.; Zhao, C.; Liao, H.; Xu, Z.; Huang, J.; Li, Q.; Tu, Y.

517

Hemicelluloses negatively affect lignocellulose crystallinity for high biomass

518

digestibility under NaOH and H2SO4 pretreatments in Miscanthus. Biotechnol. Biofuels

519

2012, 5, 58.

520

(47) Jiang, B.; Wang, W.X.; Gu, F.; Cao, T.Y.; Jin, Y.C. Comparison of the substrate

521

enzymatic digestibility and lignin structure of wheat straw stems and leaves pretreated

522

by green liquor. Bioresour. Technol. 2016, 199, 181–187.

523

(48) Nakagame, S.; Chandra, R.P.; Kadla, J.F.; Saddler, J.N. Enhancing the enzymatic

524

hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the

525

associated lignin. Biotechnol. Bioeng. 2011, 108, 538–548.

526 527

(49) Santos, R.B.; Capanema, E.A.; Balakshin, M.Y.; Chang, H.M.; Jameel, H. Lignin structural variation in hardwood species. J. Agric. Food Chem. 2012, 60, 4923–4930.

528

(50) Capanema, E.A.; Balakshin, M.Y.; Katahira, R.; Chang, H.M.; Jameel, H. Structural

529

variations in hardwood lignins. Proc. 14th Intern. Symp. Wood Fibre Pulping Chem.,

530

CD-ROM. Durban, South Africa, 2007.

531

(51) Karmanov, A.P.; Derkacheva, O.Y. Application of Fourier transform infrared

532

spectroscopy for the study of lignins of herbaceous plants. Russ. J. Bioorg. Chem. 2013,

533

39, 677–685.

534

(52) Watkins, D.; Nuruddin, M.; Mahesh H.; Alfred T.N.; Jeelani, S. Extraction and 24

ACS Paragon Plus Environment

Page 24 of 33

Page 25 of 33

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

ACS Sustainable Chemistry & Engineering

535

characterization of lignin from different biomass resources. J. Mater. Res. Technol.

536

2015, 4, 26–32.

537 538 539

(53) Mansouri, N.E.E.; Yuan, Q.; Huang, F. Characterization of alkaline lignins for use in phenol-formaldehyde and epoxy resins. Bioresources 2011, 3, 2647–2662. (54) Joffres, B.; Lorentz, C.; Vidalie, M.; Laurenti, D.; Quoineaud, A.A.; Charon, N.;

540

Daudin, A.; Quignard, A.; Geantet, C. Catalytic hydroconversion of a wheat straw soda

541

lignin: Characterization of the products and the lignin residue. Appl. Catal. B 2014,

542

145, 167–176.

543

(55) Li, H.Q.; Qu, Y.S.; Xu, J. Microwave-assisted conversion of lignin. In Production of

544

Biofuels and Chemicals with Microwave; Fang, Z., Smith, R.L., Qi, X.H., Eds.;

545

Springer: Berlin, 2015; pp 61–82.

546

25

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

547

Comparison of the structural characteristics of cellulolytic enzyme lignin preparations

548

isolated from wheat straw stem and leaf

549 550

Bo Jiang, Tingyue Cao, Feng Gu, Wenjuan Wu, Yongcan Jin*

551 552

TOC graphic

553 554

555

Synopsis: The different structures of stem and leaf lignins in herbaceous plants influence

556

the saccharification efficiency and lead to different biorefinery processes.

557 558

26

ACS Paragon Plus Environment

Page 26 of 33

Page 27 of 33

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

559

ACS Sustainable Chemistry & Engineering

Figures and Tables

560

561 562

Figure 1. Isolation procedure of cellulolytic enzyme lignin from wheat straw stem and leaf.

563

27

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

564 565

Figure 2. The effects of ball milling hours on the yield of extractable lignin on the basis of

566

lignin in raw materials.

567

28

ACS Paragon Plus Environment

Page 28 of 33

Page 29 of 33

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

ACS Sustainable Chemistry & Engineering

568 569

Figure 3. The signals of 2D HSQC NMR spectra in side chain (δC/δH 50−90/2.5−6.0) and

570

aromatic regions (δC/δH 90−150/6.0−8.0) of SCEL and LCEL.

571

29

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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

OMe R

HO

O

O O γ

HO

β O 4'

α

γ

OMe

HO

OMe

O α'

5'

γ β

HO

β O 4'

α

α

β' γ'

β

O

OMe

α O

OMe O

O

OMe

O

O A

OMe

A’

OMe

B

C

OMe

O

H

γ OH

α β

HO γ

O 1' α'

O

γ

β

α

OAr

OMe

OMe

OMe

O

O

O

OH

O F

I

OH

J

R

OH

α

FA

O

PCA

OMe

OH

α

α

α

8 9 HO 7 O

OMe O

β

α

OH

OMe

O

O

γ β

α

α

γ' β'

O

O γ

β

MeO

OMe O

MeO

OMe O

6

O

5 10

4

3' O 2' 4' 1' 2 6' 5' OMe 3

OH O

572

G

S

S’

H

T

573

Figure 4. Main structures present in the lignins of wheat straw: (A) β–O–4’ alkyl-aryl

574

ethers; (A′) β–O–4’ alkyl-aryl ethers with acylated γ-OH; (B) phenylcoumarans; (C)

575

resinols; (F) spirodienones; (I) cinnamyl alcohol end-groups; (J) cinnamyl aldehyde

576

end-groups; (FA) ferulates; (PCA) p-coumarates; (G) guaiacyl units; (S) syringyl units; (H)

577

p-hydroxyphenyl units; (T) tricin units connected with lignin polymer through β–O–4’

578

linkage.

579

30

ACS Paragon Plus Environment

Page 30 of 33

Page 31 of 33

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

ACS Sustainable Chemistry & Engineering

580

Table 1. Mass balance of original, ball-milled, enzyme hydrolyzed straw samples and CEL

581

preparations (%). Data are the mean of two measurements. Samplesa

Carbohydrate

Lignin

Ash

SRb

2.1 ± 0.1

7.6 ± 0.3

100

22.3 ± 0.3

0.8 ± 0.1

3.0 ± 0.0

42.4

0.7 ± 0.0

19.6 ± 0.0

0.5 ± 0.1

3.0 ± 0.0

33.2

2.9 ± 0.0

0.6 ± 0.0

18.9 ± 0.0

0.4 ± 0.0

2.8 ± 0.1

30.7

0.2 ± 0.0

1.1 ± 0.0

0.2 ± 0.0

14.8 ± 0.2

0.2 ± 0.0

0.0 ± 0.0

16.6

Leaf

35.1 ± 0.1

22.7 ± 0.1

4.8 ± 0.3

14.2 ± 0.5

2.4 ± 0.0

11.3 ± 0.5

100

EML-2h

2.6 ± 0.1

3.6 ± 0.0

0.8 ± 0.0

11.8 ± 0.2

0.7 ± 0.0

6.7 ± 0.0

30.7

EML-4h

0.9 ± 0.1

1.6 ± 0.0

0.4 ± 0.0

10.7 ± 0.1

0.5 ± 0.0

6.5 ± 0.0

25.9

EML-6h

0.7 ± 0.1

1.6 ± 0.1

0.3 ± 0.0

10.1 ± 0.0

0.5 ± 0.1

6.2 ± 0.1

21.3

LCEL

0.1 ± 0.0

0.6 ± 0.0

0.1 ± 0.0

6.9 ± 0.1

0.1 ± 0.0

0.1 ± 0.0

7.6

Glucan

Xylan

Arabinan

KL

ASL

Stem

40.7 ± 0.6

22.4 ± 1.0

2.8 ± 0.1

21.7 ± 0.3

EMS-2h

5.7 ± 0.0

5.6 ± 0.1

0.9 ± 0.0

EMS-4h

1.9 ± 0.1

3.4 ± 0.1

EMS-6h

1.7 ± 0.2

SCEL

582

a

583

respectively; EMS: enzymatic residue of the ball-milled stem (2–6 h); EML: enzymatic residue of the

584

ball-milled leaf (2–6 h); SCEL: CEL isolated from stem; LCEL: CEL isolated from leaf.

585

b

The content of benzene-ethanol extractives from wheat straw stem and leaf was 4.6% and 6.5%,

SR: solid recovery on the basis of starting material.

586

31

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

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 32 of 33

587

Table 2. The yields and ratios of nitrobenzene oxidation (NBO) and ozonation products of

588

lignin in wheat straw stem and leaf. Data are the mean of two measurements. Samplesa

Nitrobenzene oxidation

Ozonation

Yield (mmol/g-lignin)

S/V/H

Stem

2.23 ± 0.05

MS

b

Yield (mmol/g-lignin)

E/T

41/47/12

0.84 ± 0.07

1.58 ± 0.08

2.20 ± 0.02

40/48/12

0.78 ± 0.03

1.53 ± 0.02

EMS

84.5 ± 0.7

2.33 ± 0.08

45/45/10

0.83 ± 0.03

1.53 ± 0.02

SCEL

63.0 ± 0.9

2.67 ± 0.10

51/40/9

0.77 ± 0.09

1.51 ± 0.00

Leaf

1.60 ± 0.03

29/59/12

0.33 ± 0.04

1.29 ± 0.03

ML

1.50 ± 0.03

29/58/13

0.33 ± 0.02

1.23 ± 0.10

1.42 ± 0.06

30/58/12

0.36 ± 0.03

1.23 ± 0.10

EML 589

Lignin yield (%)

67.5 ± 0.5

LCEL 42.2 ± 0.7 2.18 ± 0.06 41/51/8 0.46 ± 0.12 1.18 ± 0.03 a MS: 4 h ball-milled stem; ML: 4 h ball-milled leaf; EMS: enzymatic residue of MS; EML: enzymatic

590

residue of ML; SCEL: CEL isolated from stem; LCEL: CEL isolated from leaf.

591

b

592

p-hydroxybenzoic acid.

S = syringaldehyde + syringic acid; V = vanillin + vanillic acid; H = p-hydroxybenzaldehyde +

593

32

ACS Paragon Plus Environment

Page 33 of 33

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

ACS Sustainable Chemistry & Engineering

594

Table 3. Structural characteristics (lignin interunit linkages, aromatic units and S/G ratio,

595

p-coumarate/ferulate, tricin) from integration of C–H correlation peaks in the HSQC

596

spectra of the SCEL and LCEL. SCEL

LCEL

β–O–4’ substructures (A/A’)

64

56

β–5’ phenylcoumaran substructures (B)

2

5

β–β’ resinol substructures (C)