Synergetic Dissolution of Branched Xylan and Lignin Opens the Way

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Synergetic Dissolution of Branched Xylan and Lignin Opens the Way for Enzymatic Hydrolysis of Poplar Cell Wall Xia Zhou, Dayong Ding, Tingting You, Xun Zhang, Keiji Takabe, and Feng Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00320 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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Journal of Agricultural and Food Chemistry

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Synergetic Dissolution of Branched Xylan and Lignin Opens the Way for Enzymatic

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Hydrolysis of Poplar Cell Wall

Xia Zhoua, Dayong Dinga, Tingting Youa, Xun Zhanga, Keiji Takabeb, and Feng Xua*

3 4

a

5

100083, China

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b

7

606-8502, Japan.

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*Corresponding author: [email protected]

9

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing

Laboratory of Tree Cell Biology, Graduate School of Agriculture, Kyoto University, Kyoto,

Tel/Fax: +86-10-62337993

10 11 12 13 14 15 16 17 18 1 Environment ACS Paragon Plus


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ABSTRACT

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As the main hemicellulose of poplar, the interaction of xylan with lignin was expected to have

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profound effect on biomass recalcitrance. In this paper, the dynamic changes of xylan and lignin

22

in poplar cell wall during a mild pretreatment using γ-valerolactone (GVL) was investigated

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using chemical and microscopic techniques. Synergetic dissolution of branched xylan and lignin

24

from the secondary wall of fibre cell was found to play a major role in openning the cell wall

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structure for enzymatic attack. In the case of the removal of xylan and lignin reaching a certain

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level, β-O-4′ cleavage of lignin which destroyed its interaction with hydrophobic cellulose face

27

was found to make great contribution to the enhanced enzymatic hydrolysis. The deep

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understanding of this process could lead to a new insight into the understanding of the plant cell

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wall architecture and provide basic information for biomass processing.

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Keywords: Xylan; Lignin; Topochemistry; γ-Valerolactone; Enzymatic hydrolysis.

31

INTRODUCTION

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Due to the deterioration of the global environment, there has recently been increasing interest

33

in searching for environment-friendly and renewable resource.1 Lignocellulosic biomass is a

34

particularly promising resource because it is low cost and renewable. With growing demand for

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renewable fuels, the production of bioethanol from the polysaccharides of lignocellulosic

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biomass is one of the most attractive routes.2 However, the chemical and structural complexity of

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biomass cell walls contributes to their recalcitrance, which resists chemical or microbial

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degradation.3-5 In order to deconstruct the compact cell wall structure, a series of thermochemical

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pretreatment strategies have been developed to overcome cell wall recalcitrance and make

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cellulose more accessible. However, most of them result in sugar degradation or significant

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structural changes in lignin at high severities.6-9 To make full use of biomass materials, an ideal

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pretreatment method should maximally recover all cell wall components and produce a cellulosic

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substrate that can be easily hydrolysed. Thus, the challenge now is to effectively and

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economically fractionate or extract cell wall components under mild conditions.

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GVL is a promising green solvent that is non-toxic and water-soluble, has a low melting point

46

and a high boiling point.10 Importantly, GVL can be produced from lignocellulose, and some

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excellent works have been done regarding the application of GVL in biomass processing. For

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biomass conversion, GVL was initially applied in the depolymerization of polysaccharides. An

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integrated degradation of hemicellulose and cellulose to furfural and levulinic acid was realized

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using GVL as solvent.11 Using solid acid catalysts in GVL, high yield of furfural was obtained

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for conversion of hemicellulose.12 In addition, a complete solubilization of biomass and effective

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sugar production directly from biomass were realized by adding trace amount of sulfuric acid in

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GVL/ H2O systerm.13 Different GVL-based solvent systems had been evaluated on their

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performance of in dissolving lignin fraction, among which GVL/ H2O was proved to be the most

55

efficient one.14 Given the partial degradation of sugars caused by high temperature (160-220 °C)

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and the ability of GVL to solubilize lignin, a mild biomass pretreatment (120 °C) using

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GVL/H2O has been developed.15 Combining with the following hydrolysis, this method achieved

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up to 99% total glucose and xylose yield. Unfortunately, current works provided mostly

59

qualitative results on polysaccharides conversion or focused on structural transformation of

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lignin. So far, there is a lack of knowledge about local distribution of hemicelluloses and lignin

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in biomass cell wall under GVL based system. It is well known that the topochemistry of both

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the hemicelluloses and lignin has profound effect on biomass recalcitrance.



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In this work, we investigated the subcellular distribution of hemicelluloses and lignin in poplar

64

during GVL based pretreatment (GVL/H2O with 0.1 M H2SO4). In particular, the dissolution of

65

unsubstituted xylan and branched xylan was monitored under confocal laser scanning

66

microscopy (CLSM) by immunofluorescence labelling, and the correlation with lignin removal

67

was uncovered. Combining with NMR analysis of extracted lignin and enzymatic hydrolysis, a

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schematic model was proposed to explain the deconstruction of poplar cell wall during GVL

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based pretreatment. It is anticipated to shed light on fundamental information as to the

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mechanism of GVL based pretreatment facilitating enzymatic hydrolysis.

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EXPERIMENTAL SECTION

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Materials. A 5-year-old poplar tree (Populus×canadensis) was collected from Beijing

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Forestry University, China. The bark of the poplar tree was peeled off and the stem was used for

74

the experiment. Poplar samples were hand cut into small sticks (~1mm width and 1 cm length)

75

with a razor blade and air dried.

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GVL based pretreatment and lignin isolation. 4 g of poplar sticks were weighted and put

77

into a thick-walled glass reactor (Synthware, Beijing) with 40 mL GVL/H2O (80:20) and a trace

78

amount of H2SO4 (0.1 M) being added. The treatment was kept at 120 °C under oil bath with

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magnetic stirring for 20 min, 40 min, 60 min and 120 min respectively. After cooling down, the

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pretreated liquid and poplar sticks were separated by filtration. The poplar sticks were washed

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with deionized water till neutral. Then, a biphasic state of the filtrates was formed by adding

82

sodium chloride (NaCl). Finally, the water phase rich in carbohydrates and the GVL phase rich

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in lignin were separated. The lignin fraction was obtained by precipitation GVL phase by adding

84

a 5-fold volume of deionized water. The lignin fractions were denominated as L20, L40, L60 and


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L120 corresponding to the pretreatment time with 20 min, 40 min, 60 min and 120 min,

86

respectively. As a reference for comparison with GVL extracted lignin in the following NMR

87

analysis, the milled wood lignin of the untreated poplar (L0) was also isolated and purified

88

according to published literature methods.16,17

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Compositional analysis. Chemical composition analysis of the water-phase was conducted by

90

high performance anion exchange chromatography (HPAEC) (Dionex ISC 3000, USA). The

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column used was CarboPac PA20 (3×150 mm, Dionex).

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The composition of poplar sticks was analysed according to National Renewable Energy

93

Laboratory’s standard analytical procedure.18 Briefly, the milled sample was hydrolysed in 72%

94

H2SO4 for 1 h at 30 °C, followed by a complete hydrolysis in autoclave at 121 °C for 1 h. The

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acid insoluble lignin was determined by weighing the solid and the monosaccharides in the liquid

96

were detected with HPAEC as mentioned above.

97

Enzymatic digestions. Enzymatic hydrolysis was conducted in sodium acetate buffer (15mL,

98

pH 4.8). A substrate equivalent to 1 g glucan was added and commercial cellulase (Cellic®

99

CTec2 of Novozymes) was used at a concentration of 15 FPU/g glucan. Enzymatic hydrolysis

100

was conducted at 50 °C for 120 h. The released glucose was detected by a high performance

101

liquid chromatography (HPLC) system (Agilent, 1260). The Aminex HPX-87H organic acid

102

column (Bio-Rad) was used for analysis.

103

X-ray diffraction (XRD) analysis. For XRD analysis, untreated and pretreated sticks were

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milled in grinder and the powder of 40-60 mesh was used. XRD analysis was conducted on a

105

diffractometer Bruker D8 Advance (Bruker, Germany). All samples were scanned with the 2θ



106

ranging from 5° to 40°, with increments of 0.02° using the Cu Ka radiation (λ= 1.54 Å). The

107

crystallinity index (CrI) was calculated using Scherrer method19:

CrI =

108 109

‫ܫ‬002 − ‫ܫ‬஺ெ

‫ܫ‬଴଴ଶ

× 100%

where I002 is the intensity of the crystalline portion (2θ = 22.5°) and IAM is the intensity of amorphous portion (2θ = 18°).

110

Fixation and embedding. For fixation, small sticks (1cm×1mm×1mm) of untreated and

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pretreated poplar were put into 1% glutaraldehyde and 4% w/v paraformaldehyde in 50 mM

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phosphate buffer solution (PBS) (pH 7.2) at room temperature for 4 h. After fixation, the sticks

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were washed with deionized water three times 1h for each. Then the sticks were dehydrated by a

114

graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, 100%) 30min for each. The next step

115

was the infiltration of embedding resin (London Resin Co., UK) where the samples were put into

116

a series of mixture of LR White resin and ethanol (v/v = 1:3, 1:1, 3:1) 8h for each , and then in

117

100 % resin for 12 h. After infiltration, the sticks were transferred into gelatin capsules

118

containning pure LR White resin and the gelatin capsules were put into polymerizer at 60 °C for

119

24 h. For sectioning, the ultra-microtome (Leica EM UC7) was used and the thickness of

120

sections is 1µm. The sections were transferred on glass slides and dried at 70 °C for 5 min and

121

stored in the dark.

122

Immunofluorescence labelling and confocal laser scanning microscopy (CLSM)

123

detection. The sections were immersed in PBS containing 50 mM glycine for 15 min, and then

124

blocked with 3% v/v bovine serum albumin (BSA) in PBS for 30 min. After washing with PBS

125

three times 5 min each, sections were incubated with primary antibodies for at 4 °C for 2 days.


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Two monoclonal antibodies (LM10 and LM11) produced by PlantProbes were diluted 1:20 in

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PBS and applied in this study. Following washing with PBS, the sections were labelled with anti-

128

rat IgG-FITC (Invitrogen, USA; diluted 1:100 in PBS) for 2 h at 35 °C. Controls were incubated

129

without primary antibody. After washing with PBS, sections were air dried overnight. For CLSM

130

detection, the sections were mounted in Citifluor immersion oil containing an antifade reagent

131

and examined by fluorescence microscope (Leica TCS SP5) using a laser excitation at a

132

wavelength of 405 nm and 488 nm for lignin and xylan detection respectively. Spectral images

133

were analysed using Image-Pro Plus software. For all control samples where the primary

134

antibody was omitted, there was no fluorescence signal of the secondary antibody observed. NMR analysis. 2D heteronuclear single quantum coherence nuclear magnetic resonance (1H-

135 136

13

137

of the lyophilized lignin sample was completely dissolved with 0.5 mL of DMSO-d6. The peak

138

of DMSO at δC/δH 39.5/2.49 ppm was used as chemical shift reference. Assignment of lignin

139

cross-signals and the semi-quantitative analysis were conducted according to those reported in

140

the literature. 20-21

C HSQC NMR) detection was conducted on a Bruker Avance spectrometer (400 MHz). 40mg

141

Transmission electron microscopy (TEM). For TEM observation, ultrathin sections (90 nm)

142

were obtained by an ultramicrotome (Leica EMUC7). After sectioning, the ultrathin sections

143

were transferred on copper grids. In order to observe the lignin distribution in poplar cell wall,

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0.1% sodium citrate containing 1% w/v KMnO4 was used to stain the sections. Images were

145

obtained by JEM1220 (Japan).

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RESULT AND DISCUSSION



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Chemical composition analysis. Fig 1 shows the effect of GVL based pretreatment on the

148

chemical composition of poplar. Untreated poplar consisted of 68% carbohydrate and 20.3%

149

lignin. Most of the carbohydrate comprised of glucose, xylose and mannose, reflecting 48.5%

150

cellulose (glucose) and 19.4% hemicellulose (xylose and mannose). The results showed that

151

GVL pretreatment was efficient in lignin and hemicelluloses removal. About half of xylan was

152

removed within 20 min of pretreatment, after which the xylan showed lower dissolution rate. For

153

the sample pretreated with 120 min, 80% of xylan removal was achieved. The rest 20% of xylan

154

retained in cell wall may be tightly bound to cellulose surface which is embedded in microfibrils

155

and hardly to reach. The dissolved xylan was mostly recovered as monomers in the pretreatment

156

liquor with no sugar degradation products detected (Table S1). In contrast, the content of mannan

157

only had a slight decrease during the ﬁrst 20 min of pretreatment. Almost no further decrease

158

could be observed even the residence time was extended to 120 min. The difference between

159

xylan and mannan dissolution is likely attributable to that xylan is more associated with lignin

160

and glucomannan is more associated with cellulose which is embedded in the cell wall matrix.22

161

Moreover, GVL based pretreatment was proved to be an excellent system in dissolving lignin

162

with 60% lignin removal being achieved for the poplar pretreated by 120 min.

163 164


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Fig 1. Chemical composition of untreated and pretreated substrates with various residence times (expressed as the percentage based on the oven dry weight of poplar before pretreatment).

168

Enzymatic hydrolysis. To evaluate the enzymatic digestibility, enzymatic hydrolysis was

169

performed on the resulting solids. As shown in Fig 2, due to the compact structure of untreated

170

sample, only less than 10% glucose yield was obtained. Various degrees of enhancement in

171

enzymatic hydrolysis were observed for samples pretreated with different residence times. It was

172

noted that the improvement in enzymatic hydrolysis of poplar pretreated by 60min relative to

173

40min was mitigated compared with the increase between poplar pretreated by 40min and

174

20min. This might be attributed to the limited increase in lignin and xylan removal. Interestingly,

175

the glucose yield of poplar pretreated by 120 min achieved 81%, almost twice as high as the 48%

176

obtained in poplar pretreated by 60 min. The compositional analysis showed not that obvious

177

decrease in xylan and lignin content between these two samples. It is well known that two major

178

strategies to improve cellulose enzymatic hydrolysis include increasing the accessibility of

179

cellulase and decreasing the cellulose crystallinity. The variance of cellulose crystallinity was

180

investigated by XRD (Fig S1). The result showed that the cellulose crystallinity of samples 9 Environment ACS Paragon Plus


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pretreated by 60 min and 120 min were close. Therefore, the impressive improvement in

182

enzymatic hydrolysis could be attributed to the increased accessibility of cellulase. Given the

183

limited increase of lignin and xylan removal from 60 min to 120 min, the increased accessibility

184

of cellulase was most likely correlated with the disruption of crosslink in cell wall and the

185

structural alterations of cell wall components. In order to understand the process more deeply,

186

subcellular localization of xylan and lignin was performed, also the lignin structural alteration

187

was characterized to make a full explanation of the enhanced enzymatic hydrolysis.

188 189

Fig 2. Enzymatic hydrolysis of pretreated solids with various pretreatment times (expressed as

190

the percentage based on the initial glucan content).

191

Subcellular dissolution of xylan. As the predominant component of poplar hemicelluloses,

192

xylan interacts with cellulose and lignin by forming physical and chemical bonds.23 Thus, the

193

detailed structure of xylan and its topochemistry are important for understanding plant cell wall

194

deconstruction during biomass conversion. The monoclonal antibodies (LM10 and LM11) were

195

used to detect the distribution of xylan by immunofluorescence. The LM10 antibody is directed

196

to unsubstituted xylan while LM11 antibody having greater affinity to highly substituted xylan.24


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Given that fibre cell is the major cell type and account for most of the mass of hardwood, this

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work focused on the distribution of xylan in fibre cell wall.

199

As seen in Fig 3, LM10 and LM11 showed different labelling in fibre walls of untreated

200

poplar. LM10 exhibited a strong and fairly uniform labelling pattern showing distribution across

201

the secondary cell wall, with only sparse labelling in the cell corner and middle lamella. In

202

contrast, LM11 revealed a heterogeneous labelling with higher labelling intensity in the S1 (outer

203

secondary wall) and S3 (inner secondary wall). The differences of hemicelluloses matrix in cell

204

walls might be correlated with the variance in lignin content and structure.22 As the pretreatment

205

progressed, there were varying degrees of decreased labelling for LM10 and LM11. To make a

206

fairer comparison between these two antibodies, a semi-quantitative analysis was conducted by

207

calculating average labelling intensity of whole cell wall in each fluorescence image (Fig 3).

208

LM11 showed decreased labelling as the pretreatment time prolonged. Although the decrease of

209

LM10 labelling was also be observed, the semi-quantitative analysis showed the overall constant

210

intensity except for the obvious decrease in the first 20min. The LM10 labelling intensity still

211

observed in poplar pretreated by 120 min may be explained by its tight association with

212

cellulose.25 The results of LM11 was corresponding to the HPAEC results (Table S1) which

213

showed constant increase in glucuronic acid content released in the pretreatment liquor despite of

214

the limited increase of released xylose after 20 min. It is well known that, the xylan in hardwood

215

is mainly substituted with glucuronic acid or 4-O-methyl-glucuronic acid residues.26 The

216

coincidence of the LM11 labelling and HPAEC results also confirmed the preferential binding of

217

high substituted xylan of LM11.

218

The labelling of LM10 and LM11 demonstrated that the dissolved xylan primarily originated

219

from secondary wall. With the extension of pretreatment time, the dissolution of branched xylan



220

predominated with the S2 (middle secondary wall) showing almost no labelling signal. A

221

detailed knowledge of lignification in hardwood described that the deposition of S lignin was

222

later than G lignin.27 In poplar, condensed lignin subunits were found localized in cell corners

223

while non-condensed lignin units present in secondary wall.28 Therefore, the branched xylan may

224

be associated with S lignin which mainly deposited in the S2. Non branched xylans have been

225

reported to preferably associate with the cellulose and branched xylan are preferably deposited

226

together with lignin.25, 29 To clarify the complex associations in plant cell wall, the interaction

227

between branched xylan (LM11) and lignin were further investigated.

228 229

Fig 3. Immunoflourescence images of poplar sticks pretreated with various residence times

230

(histogram right-hand showing average labelling intensity of whole cell wall corresponding to

231

left fluorescence image).

232

Interrelationship between xylan and lignin dissolution. The dissolution of xylan and lignin

233

during GVL based pretreatment is depicted in Fig 4. For both the xylan and lignin, the

234

solubilization could be divided into two stages, initial fast reaction followed by a slower reaction

235

rate. In addition, the overall tendency seems similar for the dissolution curve of lignin and xylan.

236

We speculate that the manner of dissolution for both lignin and xylan was likely related to their

237

structure and there interwoven linkages with other components in the compact cell wall. For


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xylan, the fast hydrolysing fraction accounted for 50%, with the slow hydrolysing fraction

239

accounting for 30% and 20% being remained in the cell wall when the pretreatment time is 120

240

min. During the first 20 min, xylan exhibited higher dissolution rate than lignin, suggesting the

241

efficient glycosidic bond hydrolysis under GVL/H2O system. As the pretreatment time

242

prolonged, the content of xylan decreased with the reduction becoming slower and showed

243

synchronism with lignin removal. The biphasic behaviour of xylan removal was also found in the

244

study regarding the role of lignin on xylan hydrolysis. To deeply understand the behaviors of

245

xylan and lignin and investigate the relationship between them during GVL based pretreatment,

246

colocalization of xylan and lignin was performed on the poplar cell wall under CLSM. To make

247

the pretreatment more complete, the thin sections (8um) of poplar were put into reaction vials,

248

and the reaction times were 20 min, 40 min and 60 min.

249 250

Fig 4. Dissolution of xylan and lignin during GVL based pretreatment.

251

The localization of branched xylan was performed by immunolabeling while the distribution

252

of lignin was realized by its autofluorescence (Fig 5). The CLSM results showed that the

253

dissolved lignin mainly stemmed from secondary wall of fibre with the cell corner and middle



254

lamella still showed high lignin fluorescence intensity for the sections pretreated by 120 min. For

255

the sections pretreated by 20 min, the intensity of branched xylan showed significant decrease in

256

the secondary wall. Interestingly, the retention of LM11 antibody signal near the cell lumen and

257

middle lamellae was observed. This pattern was also detected in maize cell walls after dilute acid

258

pretreatment.30 Yet, the explanation for this special picture hadn’t been clarified. Extending the

259

pretreatment time to 60 min, the cell wall detachment and separation was observed due to the

260

robust removal of cell wall components with weak labelling being observed for xylan and bright

261

signal for lignin existing merely in the cell corner and middle lamella which also showed

262

decreased intensity. The colocalization demonstrated the concomitant removal of the slow

263

hydrolysing fraction of xylan with further lignin dissolution, suggesting the association between

264

branched xylan and lignin. For comparison, colocalization of LM10 and lignin was conducted

265

(Fig S2). LM10 showed decrease in labelling intensity for the first 20min, after which almost no

266

obvious changes being observed. This is also corresponding to the histogram showed in Fig 3. In

267

contrast, the lignin showed significant decrease during the process, excluding the possibility of

268

concomitant dissolution between unsustituted xylan and lignin.


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269 270

Fig 5. Colocalization of branched xylan and lignin on poplar cross sections pretreated with

271

various residence times.

272

Taken together, these results revealed that the concomitant dissolution of branched xylan and

273

lignin from S2 of fibre cell wall promoted enzymatic hydrolysis of the pretreated poplar.

274

However, given the limited increase of lignin and xylan removal from 60 min to 120 min, the

275

noticeable rise of glucose yield in poplar pretreated by 120 min couldn’t be simply explained by

276

the dissolution of cell wall components. There must be other correlated fundamental reasons.

277

Besides the subcellular behaviour of xylan and lignin dissolution, lignin’s structural alteration is

278

another vital feature to be evaluated for biomass conversion.

279

Chemical structure of GVL extracted lignin. To elucidate the effects of GVL based

280

pretreatment on lignin structure, 2D-NMR was performed on the lignin fractions obtained by



281

precipitation from pretreated liquor with adding water. Fig. 6 shows aromatic and aliphatic

282

regions of 2D

283

according to the literature.20 The relative abundance of those functionalities and also the S/G

284

ratios showed difference for samples with various residence times.

13

C-1H HSQC spectra for the lignin samples and the cross peaks were assigned

285 286

Fig 6. 2D-HSQC-NMR spectra of extracted lignins. (A) Side chain (δC/δH 45–90/2.5–6.0 ppm)

287

regions; (B) Aromatic (δC/δH 95–140/6.0–8.0 ppm) regions.

288

In the side chain region, the spectra showed primary signals of β-O-4′ ether units with minor

289

amounts of β-β′, β-5′. Extracted lignin from the GVL based pretreatment contained most of the

290

native lignin functionalities typically found in native poplar. However, the relative abundance of

291

the lignin substructures showed difference for the lignin fractions obtained under various

292

residence times. The quantitative results from NMR analysis showed that the decreased order of

293

β-O-4′ linkages was L20 > L40 > L60 > L120, indicating the increasing degrees of β-O-4′ cleavage


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during this process (Table 1). Initially, due to the short residence time, the cleavage of β-O-4′

295

linkages was limited and the resulting lignin preserved nearly native lignin structure. As the

296

pretreatment time prolonged, the cleavage of β-O-4′ linkages became more serious, which

297

coincided with the previous reports.8-9 Nevertheless, unlike common acid pretreatment under

298

aqueous environment where serious cleavage of β-O-4′ linkages and concurrent formation of

299

condensed structures occurred, the GVL based pretreatment here still preserved high β-O-4′

300

contents. This can be attributed to the excellent lignin solubility in GVL/H2O system, which was

301

demonstrated exceptional properties for dissolving various types of lignin even at low

302

temperatures.14 During the process, a declining trend was also observed for the signal intensities

303

of β-5′ linkages with only trace amount β-5′ linkages being detected for L120. Due to the fact that

304

S unit can’t form C-C bond at the 5-C position, the decrease of β-5′ linkages can be largely

305

attributed to the less abundance of G units in extracted lignin with the extension of the

306

pretreatment time.

307 308 309

Table 1. Quantification of the lignins by quantitative 2D-HSQC method: results expressed per 100 Aryl units

Sample

β-O-4'

β-β'

β-5'

L0

58.2

7.5

3.2

L20

47.1

7.16

2.9



Page 18 of 26

L40

32.3

7.04

2.5

L60

30.5

5.8

0.68

L120

18

5.3

0.17

310 311

As can be seen from Fig 6, S/G ratio of extracted lignin was also altered during this process.

312

The aromatic region of NMR spectra showed correlation signals for lignin S and G units along

313

with p-hydroxyphenyl benzoate (PB) substructure, suggesting a typical G/S type hardwood

314

lignin. The spectra of these four lignin fractions suggested that S units dominated in the GVL

315

extracted lignin. The extracted lignin showed a slight increase of S/G ratio at earlier stages of the

316

GVL pretreatment (L40 > L20 by 3%), with the increase was augmented with the pretreatment

317

time prolonged (L60 > L20 by 61% and L120 > L20 by 54%). The results demonstrated that GVL is

318

more effective in dissolving S units in poplar. This may simply be because that the S units could

319

be dissolved more easily through the cleavage of β-O-4′ linkages. Moreover, an increase in

320

condensed structure in extracted lignin was observed with elongated pretreatment time.

321

Unexpectedly, the condensed structure was exclusively found in S units while almost no

322

condensed structure being observed in dissolved G units. As reported, the condensation reaction

323

was supposed to mainly occur on G unints.31 A rational explanation is that the lignin S units were

324

more prone to cleavage/acidic degradation during this process, thereby being preferably

325

dissolved and extracted by GVL even repolymerization of lignin fragments occurred

326

simultaneously. In contrast, for G units, due to its more condensed structure and the less

327

abundance of β-O-4′ linkages, the dissolution became more difficult especially with long


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328

residence time. To further understand the lignin behaviour at the ultrastructure level, the TEM

329

detection coupled with potassium permanganate (KMnO4) staining was performed (Fig 7). TEM

330

images showed the denser intensities in cell corner where the G units predominantly deposite. As

331

the pretreatment progressing, the contrast between cell corner and secondary wall became more

332

obvious with the former being darker and the latter showing light dyeing, indicating the retention

333

of lignin in cell corner. The common observation of coalesce and migration of lignin in form of

334

droplets on the cell wall surface in dilute acid pretreatment didn’t appear in our present work,

335

suggesting that the timely lignin dissolution by GVL could prevent the formation of lignin

336

globules which has negative impact on the accessibility of cellulose.

337 338

Fig 7. TEM images of poplar cell wall with various pretreatment times.

339

Integrating with the NMR results, a more comprehensive view of the lignin alteration has been

340

provided. GVL based pretreatment was effective in dissolving the S type lignin in secondary

341

wall, while most of the G type lignin in cell corner and middle lamella remaining steady in their

342

place. Given the above, the most obvious change of lignin between samples pretreated with

343

60min and 120 min lied in the β-O-4′ linkages. The obvious reduction in β-O-4′ linkages in L120



344

indicated the cleavage of this bond in lignin. Previous study reported that cellulose accessibility

345

was enhanced mainly by exposing the hydrophobic cellulose face.4 In this work, we speculate

346

that the lignin degradation caused by β-O-4′ cleavage can increase the accessible hydrophobic

347

cellulose face, thus resulting in improvement of the enzymatic hydrolysis. In addition, Raman

348

microprobe study suggested the parallel orientation of lignin aromatic rings relative to the

349

microfibrils.32 Follow-up studies demonstrated the β-O-4′ linked lignin adsorption onto the (200)

350

surface of cellulose and this interaction was weakened by structural alterations of lignin.33-34

351

Hence, the abrupt enhancement in glucose yield of poplar pretreated by 120 min could be

352

explained by the β-O-4′ cleavage which destroyed the interaction of lignin with the hydrophobic

353

cellulose face. However, for the utilization of lignin, the improved preservation of β-O-4′ favours

354

selective downstream depolymerisation of lignin to low Mw chemicals and is preferable for the

355

effective lignin valorisation.35 Therefore, during the biomass pretreatment, there should be a

356

balance between lignin removal and structural alteration.

357

This study provides evidence that the synergetic dissolution of branched xylan and lignin

358

plays an important role in opening the way for enzymatic hydrolysis. And what’s interesting is

359

that a prominent relationship between cleavage of β-O-4′ linkages of lignin and enhanced

360

enzymatic hydrolysis was discovered for the sample where most of the hemicelluloses was

361

removed. Thus, for enzymatic hydrolysis of cell wall with a small quantity of hemicelluloses or

362

high lignin content, the chemical transformation of lignin structure played an important role not

363

inferior to lignin dissolution.

364 365

SUPPORTING INFORMATION


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Page 21 of 26


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Fig S1. XRD analysis of pretreated samples with various residence time: (a) 0 min, (b) 20 min,

367

(c) 40 min, (d) 60 min, (e) 120 min.

368

Fig S2. Colocalization of unsubstituted xylan and lignin on poplar cross sections pretreated with

369

various residence times.

370

Table S1 HPAEC analysis of water phase (% (w/w) of raw material)

371

ACKNOWLEDGMENTS

372

The authors gratefully acknowledge the financial support from National Key R&D Program of

373

China (2017YFD0600204).

374

REFERENCES

375

(1) Tuck, C. O.; Perez, E.; Horvath, I. T.; Sheldon, R. A.; Poliakoff, M. Valorization of biomass:

376

deriving more value from waste. Science 2012, 337 (6095), 695-699.

377

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

378

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

379

Science 2007, 315 (5813), 804-807.

380

(3) DeMartini, J. D.; Pattathil, S.; Miller, J. S.; Li, H.; Hahn, M. G.; Wyman, C. E. Investigating

381

plant cell wall components that affect biomass recalcitrance in poplar and switchgrass. Energy

382

Environ. Sci. 2013, 6 (3), 898-909.

383

(4) Ding, S. Y.; Liu, Y. S.; Zeng, Y.; Himmel, M. E.; Baker, J. O.; Bayer, E. A. How does plant

384

cell wall nanoscale architecture correlate with enzymatic digestibility? Science 2012, 338 (6110),

385

1055-1060.



386

(5) Zhang, T.; Zheng, Y.; Cosgrove, D. J. Spatial organization of cellulose microfibrils and

387

matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force

388

microscopy. Plant J. 2016, 85 (2), 179-192.

389

(6) Langan, P.; Petridis, L.; O'Neill, H. M.; Pingali, S. V.; Foston, M.; Nishiyama, Y.; Schulz,

390

R.; Lindner, B.; Hanson, B. L.; Harton, S.; Heller, W. T.; Urban, V.; Evans, B. R.; Gnanakaran,

391

S.; Ragauskas, A. J.; Smith, J. C.; Davison, B. H. Common processes drive the thermochemical

392

pretreatment of lignocellulosic biomass. Green Chem. 2014, 16 (1), 63-68.

393

(7) da Costa Sousa, L.; Jin, M.; Chundawat, S. P. S.; Bokade, V.; Tang, X.; Azarpira, A.; Lu, F.;

394

Avci, U.; Humpula, J.; Uppugundla, N.; Gunawan, C.; Pattathil, S.; Cheh, A. M.; Kothari, N.;

395

Kumar, R.; Ralph, J.; Hahn, M. G.; Wyman, C. E.; Singh, S.; Simmons, B. A.; Dale, B. E.;

396

Balan, V. Next-generation ammonia pretreatment enhances cellulosic biofuel production. Energy

397

Environ. Sci. 2016, 9 (4), 1215-1223.

398

(8) Pu, Y.; Hu, F.; Huang, F.; Davison, B. H.; Ragauskas, A. J. Assessing the molecular structure

399

basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnol.

400

Biofuels 2013, 6 (1), 15-48.

401

(9) Cao, S.; Pu, Y.; Studer, M.; Wyman, C.; Ragauskas, A. J. Chemical transformations of

402

Populus trichocarpa during dilute acid pretreatment. RSC Adv. 2012, 2 (29), 10925-10936.

403

(10) Tang, X.; Zeng, X.; Li, Z.; Hu, L.; Sun, Y.; Liu, S.; Lei, T.; Lin, L. Production of γ-

404

valerolactone from lignocellulosic biomass for sustainable fuels and chemicals supply.

405

Renewable Sustainable Energy Rev. 2014, 40, 608-620.


Page 22 of 26

Page 23 of 26


406

(11) Alonso, D. M.; Wettstein, S. G.; Mellmer, M. A.; Gurbuz, E. I.; Dumesic, J. A. Integrated

407

conversion of hemicellulose and cellulose from lignocellulosic biomass. Energy Environ. Sci.

408

2013, 6 (1), 76-80.

409

(12) Gürbüz, E. I.; Gallo, J. M. R.; Alonso, D. M.; Wettstein, S. G.; Lim, W. Y.; Dumesic, J. A.

410

Conversion of hemicellulose into furfural using solid acid catalysts in γ-valerolactone. Angew.

411

Chem. Int. Edit. 2013, 52 (4), 1270-1274.

412

(13) Luterbacher, J. S.; Rand, J. M.; Alonso, D. M.; Han, J.; Youngquist, J. T.; Maravelias, C. T.;

413

Pfleger, B. F.; Dumesic, J. A. Nonenzymatic sugar production from biomass using biomass-

414

derived gamma-valerolactone. Science 2014, 343 (6168), 277-280.

415

(14) Xue, Z.; Zhao, X.; Sun, R.-c.; Mu, T. Biomass-Derived γ-Valerolactone-Based Solvent

416

Systems for highly efficient dissolution of various lignins: dissolution behavior and mechanism

417

study. ACS Sustainable Chem. Eng. 2016, 4 (7), 3864-3870.

418

(15) Shuai, L.; Questell-Santiago, Y. M.; Luterbacher, J. S. A mild biomass pretreatment using γ-

419

valerolactone for concentrated sugar production. Green Chem. 2016, 18 (4), 937-943.

420

(16) Björkman, A. Isolation of lignin from finely divided wood with neutral solvents. Nature

421

1954, 174, 1057−1058.

422

(17) You, T. T., Mao, J. Z., Yuan, T. Q., Wen, J. L., Xu, F. Structural elucidation of the lignins

423

from stems and foliage of Arundo donax Linn. J. Agric. Food Chem. 2013, 61 (22), 5361-5370.

424

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

425

Determination of structural carbohydrates and lignin in biomass. Laboratory analytical

426

procedure 2008, 1617, 1-16.



427

(19) Kurakake, M.; Ooshima, H.; Kato, J.; Harano, Y. Pretreatment of bagasse by nonionic

428

surfactant for the enzymatic hydrolysis. Bioresource Technol. 1994, 49 (3), 247-251.

429

(20) Wen, J. L.; Yuan, T. Q.; Sun, S. L.; Xu, F.; Sun, R.-C. Understanding the chemical

430

transformations of lignin during ionic liquid pretreatment. Green Chem. 2014, 16 (1), 181-190.

431

(21) Wen, J. L.; Sun, S. L.; Xue, B. L.; Sun, R. C. Quantitative structures and thermal properties

432

of birch lignins after ionic liquid pretreatment. J. Agric. Food Chem. 2013, 61 (3), 635-645.

433

(22) Salmén, L.; Olsson, A.-M. Interaction between hemicelluloses, lignin and cellulose:

434

structure-property relationships. J. Pulp Pap. Sci. 1998, 24 (3), 99-103.

435

(23) Reis, D.; Vian, B. Helicoidal pattern in secondary cell walls and possible role of xylans in

436

their construction. CR Biol. 2004, 327 (9), 785-790.

437

(24) Kim, J. S.; Sandquist, D.; Sundberg, B.; Geoffrey, D. Spatial and temporal variability of

438

xylan distribution in differentiating secondary xylem of hybrid aspen. Planta 2012, 235(6),

439

1315-1330.

440

(25) Dammström, S.; Salmén, L.; Gatenholm, P. On the interactions between cellulose and

441

xylan, a biomimetic simulation of the hardwood cell wall. BioResources 2008, 4 (1), 3-14.

442

(26) Ebringerova, A.; Heinze, T. Xylan and xylan derivatives– biopolymers with valuable

443

properties, 1. Naturally occurring xylans structures, isolation procedures and properties.

444

Macromol. rapid comm. 2000, 21 (9), 542-556.

445

(27) Donaldson, L. A. Lignification and lignin topochemistry—an ultrastructural view.

446

Phytochemistry 2001, 57 (6), 859-873.


Page 24 of 26

Page 25 of 26


447

(28) Grünwald, C.; Ruel, K.; Kim, Y. S.; Schmitt, U. On the cytochemistry of cell wall formation

448

in poplar trees. Plant Biology 2002, 4 (01), 13-21.

449

(29) Ruel, K.; Chevalier-billosta, V.; Guillemin, F.; BERRIO-SIERRA, J.; Joseleau, J.-P. The

450

wood cell wall at the ultrastructural scale-formation and topochemical organization. Maderas.

451

Ciencia y Tecnología 2006, 8 (2), 107-116.

452

(30) Brunecky, R.; Vinzant, T. B.; Porter, S. E.; Donohoe, B. S.; Johnson, D. K.; Himmel, M. E.

453

Redistribution of xylan in maize cell walls during dilute acid pretreatment. Biotechnol. Bioeng.

454

2009, 102 (6), 1537-1543.

455

(31) Gierer, J. Chemistry of delignification. Wood Sci. Technol. 1985, 19 (4), 289-312.

456

(32) Atalla, R. H.; Agarwal, U. P. Raman microbe evidence for lignin orientation in the cell

457

walls of native woody tissue. Science 1985, 227, 636-639.

458

(33) Besombes, S.; Mazeau, K. The cellulose/lignin assembly assessed by molecular modeling.

459

Part 1: adsorption of a threo guaiacyl β-O-4 dimer onto a Iβ cellulose whisker. Plant Physiol.

460

Biochem. 2005, 43 (3), 299-308.

461

(34) Besombes, S.; Mazeau, K. The cellulose/lignin assembly assessed by molecular modeling.

462

Part 2: seeking for evidence of organization of lignin molecules at the interface with cellulose.

463

Plant Physiol. Biochem. 2005, 43 (3), 277-286.

464

(35) Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.;

465

Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M.; Langan, P.; Naskar, A. K.; Saddler, J. N.;

466

Tschaplinski, T. J.; Tuskan, G. A.; Wyman, C. E. Lignin valorization: improving lignin

467

processing in the biorefinery. Science 2014, 344 (6185), 1246843-1246852.



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Synergetic Dissolution of Branched Xylan and Lignin Opens the Way

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