How pseudolignin is generated during dilute sulfuric acid pretreatment

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How pseudolignin is generated during dilute sulfuric acid pretreatment Guangcong Wan, Qingtong Zhang, Mingfu Li, Zhuan Jia, Chenyan Guo, Bin Luo, Shuangfei Wang, and Douyong Min J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02851 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 24, 2019

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Journal of Agricultural and Food Chemistry

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How pseudolignin is generated during dilute sulfuric acid pretreatment

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Guangcong Wan,a,b Qingtong Zhang,a,b Mingfu Li,a,b Zhuan Jia,a,b Chenyan Guo,a,b

3

Bin Luo,a,b Shuangfei Wang,a,b Douyong Min a,b*

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a. College of Light Industry and Food Engineering, Guangxi University, Nanning

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530004, PR China.

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b. Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control,

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Nanning 530004, PR China.

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Abstract

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Pseudolignin is generated from lignocellulose biomass during pretreatment with

10

dilute sulfuric acid, and it has a significant inhibitory effect on cellulase. However, the

11

mechanism of pseudolignin generation remains unclear. The following main points

12

have been addressed to help elucidate the pseudolignin generation pathway. Cellulose

13

and xylan were pretreated with sulfuric acid at different concentrations, and aliquots

14

were periodically collected, and the changes in the byproducts of the prehydrolysate

15

were quantified. Milled wood lignin (MWL) mixed with cellulose and xylan was

16

pretreated to evaluate the impact of lignin on pseudolignin generation. Furfural (FF), 5-

17

hydroxymethylfurfural (HMF), and MWL were pretreated as model compounds to

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investigate pseudolignin generation. The result indicated that the increasing acid

19

concentration significantly promoted the generation of pseudolignin. When the acid

20

concentration was increased from 0 wt% to 1.00 wt%, pseudolignin was increased from

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1.36 g to 4.05 g. In addition, lignin promoted the pseudolignin generation through the

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condensation between lignin and the generated intermediates.

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Key words: dilute acid pretreatment; pseudolignin; byproducts; milled wood lignin;

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mechanism

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1. Introduction

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With increasing problems associated with climate change and the increasing

27

emphasis on sustainable development, biomass, the most common material on earth,

28

has been attracting increasing attention (McKendry, 2002). Biomass-derived energy

29

has the advantages of renewability, as there are large reserve that can be converted into

30

a variety of biobased chemicals via biological or chemical pathways. Biomass energy

31

can reduce human overdependence on petrochemical products (Huber et al., 2006;

32

Ragauskas, 2006). The complex three-dimensional network formed by the intertwining

33

of cellulose, hemicellulose and lignin is a natural barrier to the degradation of

34

lignocellulosic fibers, and the direct enzymatic digestion of cellulose is limited, which

35

seriously hinders the industrial production of bioethanol (Zhao et al., 2012). As a result,

36

physical or chemical pretreatment is prerequisite for promoting enzymatic hydrolysis

37

for breaking down the dense structure formed by cellulose, hemicellulose and lignin,

38

reducing the degree of polymerization and crystallinity of cellulose, and increasing

39

cellulose accessibility, which subsequently increase the efficiency of the hydrolysis. In

40

the past decades, different physical and chemical pretreatment methods have been

41

developed to break down the natural barriers of biomass (Alvira et al., 2010; Kumar

42

and Wyman, 2009). For example, the maximum digestibility was increased from 15.4%

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to 61.4% after the acid pretreated bamboo was extracted with phosphoric acid, urea,

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and ethanol (Huang et al., 2019). With the optimal SO2-ethanol-water treatment, the

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enzyme digestibility of bamboo residue increased to 80.4% (Huang et al., 2018). Of the

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developed methods, dilute acid pretreatment is one of the most common and important

47

technologies as it can effectively dissolve hemicellulose, change its crystallinity,

48

increase pores, and increase the efficiency of enzymatic hydrolysis (Cao et al., 2012;

49

Chen et al., 2010; de Carvalho et al., 2017; Hsu et al., 2010). Dilute acid pretreatment

50

has no obvious effect on lignin extraction (Li et al., 2007). In contrast, it has been

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reported that the acid-insoluble lignin content of dilute acid-pretreated material is

52

higher than that in raw material. During acid pretreatment, carbohydrate including

53

cellulose and hemicellulose was hydrolyzed into glucose and xylose which was further

54

degraded into the corresponding compounds, e.g. FF and HMF. Under the severe

55

condition, the aromatic structure-enriched reaction intermediates were generated from

56

FF and HMF. Then, the intermediates reacted with FF and HMF to generate

57

pseudolignin (Kumar et al., 2013; Shinde et al., 2018). Scanning electron microscopy

58

was used study holocellulose pretreated with dilute acid under harsh conditions, and the

59

results showed that pseudolignin formed spherical droplets on the surface of the raw

60

material (He et al., 2018). FT-IR and NMR analyses showed that pseudolignin is rich

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in carbonyl, carboxyl, aromatic, methoxy, and aliphatic structures (Kumar et al., 2013;

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Ma et al., 2015; Sannigrahi et al., 2011). The nonproductive adsorption and blocking

63

of surface binding sites are the main effects of pseudolignin on enzymatic hydrolysis

64

(Hu et al., 2012). Despite a substantial amount of work having been conducted on

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pseudolignin, the pathway for pseudolignin production remains unclear. From the

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perspective of bioethanol and platform chemical production, it is important to

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understand the structure of pseudolignin and the mechanism by which pseudolignin is

68

produced because the formation of these compounds consumes hydrolysates such as

69

furfural and has a significant inhibitory effect on subsequent enzymatic treatments. The

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purpose of the current study is to quantify the amount of byproducts in the

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prehydrolysate and to explore the changes in the individual byproducts during the dilute

72

sulfuric acid pretreatment process to elucidate the structure of the byproducts and the

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mechanism of the formation of pseudolignin. More importantly, understanding the

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formation mechanism will facilitate preventing the formation of pseudolignin during

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dilute acid pretreatment and eventually enhance enzymatic hydrolysis of dilute acid-

76

pretreated biomass.

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2. Methods

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2.1 Raw materials

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Cellulose, xylan, furfural (FF), and 5-hydroxymethylfurfural (HMF) were purchased

80

from Aladdin Holdings Group (Shanghai, China). Sugarcane bagasse (SCB) was

81

provided by Gui Tang Group Co. Ltd. (Guigang, China), and it contained 43.45%

82

cellulose, 26.24% hemicellulose, and 22.15% lignin. The milled wood lignin (MWL)

83

was isolated from the sugarcane bagasse by Björkman’s method (Björkman, 1954), and

84

the MWL contained approximately 7.40% sugar.

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2.2. Biomass pretreatment

86

The details of the pretreatment conditions are presented in Table 1. The solid to

87

liquid ratio was 1:10, the temperature was 180ºC, the residence time was 45 min, and

88

the stirring speed was 600 rpm. When the temperature was increased to 180°C,

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uninterrupted sampling was employed every 5 min until the pretreatment was

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completed. Following pretreatment, the vessel was rapidly cooled to room temperature

91

with running water, and then the slurry was filtered into the pretreated sample and the

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prehydrolysate. The glucose and xylose in the prehydrolysate were quantified by ion

93

chromatography (IC). FF, HMF, formic acid (FA), acetic acid (HAc) and levulinic acid

94

(LA), defined as byproducts, were quantified by high-performance liquid

95

chromatography (HPLC). The pretreated sample was washed three times with

96

deionized water and then washed three times with ethanol. After vacuum drying, the

97

acid-insoluble lignin was quantified. Table 1．The details of the dilute acid pretreatment conditions

98

Reactant (g dry weight)†

Pretreatment conditions No.

Chemical

Temperatur

Residence time

Cellulose

Xylan

MWL

e

99 100

†

a

0 wt% H2SO4

180ºC

45 min

13.17

7.70

0

b

0.08 wt% H2SO4

180°C

45 min

13.17

7.70

0

c

1 wt% H2SO4

180°C

45 min

13.17

7.70

0

d

0 wt% H2SO4

180°C

45 min

13.17

7.70

6.45

e

0.08 wt% H2SO4

180°C

45 min

13.17

7.70

6.45

f

1 wt% H2SO4

180°C

45 min

13.17

7.70

6.45

The ratio of cellulose, xylan and MWL was designed according to the corresponding

components of SCB.

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101

The details of the pretreatment conditions for the model compounds are described in

102

Table 2. The compounds were pretreated in a 30 mL reactor, the solid-liquid ratio was

103

1:10, the temperature was 180ºC, the residence time was 45 min, and the stirring speed

104

was 600 rpm. Upon completion of the pretreatment, the reactor was quickly cooled to

105

room temperature with running water. The slurry was filtered into the pretreated solid

106

and the prehydrolysate. The FF, HMF, FA, HAc and LA in the prehydrolysate were

107

quantified by HPLC. The pretreated solid was washed three times with deionized water,

108

and then washed three times with ethanol. The acid-insoluble lignin content was

109

quantified after vacuum drying.

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Table 2．The details of the acid pretreatment conditions for the model compounds Pretreatment conditions No.

Reactant (g dry weight)

Temperatur Chemical

Time

FF

HMF

MWL

e

111

g

1 wt% H2SO4

180°C

45 min

1.00

0

0

h

1 wt% H2SO4

180°C

45 min

0

1.00

0

i

1 wt% H2SO4

180°C

45 min

1.00

1.00

0

j

1 wt% H2SO4

180°C

45 min

1.00

1.00

1.00

2.3 Compositional analysis

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The composition of the pretreated samples was analyzed according to the reported

113

method (Sluiter et al., 2008). The total hydrolysate was appropriately diluted with

114

deionized water and then filtered through a 0.22-μm filter. The FF, HMF, LA, and HAc

115

were quantified by HPLC using a Bio-Rad Aminex1 HPX-87H column (polystyrene6 / 32


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divinylbenzene sulfonic acid resin packing; 300 mm×7.8 mm) along with a microguard

117

cation cartridge (30 mm×4.6 mm; Bio-Rad Laboratories, Hercules, CA). The mobile

118

phase contained 5 mmol/L H2SO4. The flow rate was 0.6 mL/min, and the column

119

temperature was 50°C. Glucose and xylose were quantified by an IC system (Dionex

120

ICS-5000) with a Dionex CarboPacTM PA10 column using 0.2 mol/L NaOH as the

121

mobile phase. The flow rate was 0.6 mL/min, and the column temperature was 30 °C.

122

2.4 Solid-State 13C CP/MAS NMR and FT-IR analysis

123

The pseudolignin was air dried in a fume hood for 2 days and then ground into a

124

powder using a mortar and dried in a vacuum oven at 40°C for 48 h. For the solid-state

125

13C

126

kel-F caps and measured at a spinning speed of 10 KHz. The solid-state NMR spectra

127

were acquired on an Agilent 600 MHz spectrometer. The sample was also characterized

128

using FT-IR spectroscopy (Bruker, Germany) with a 400-4000 cm-1 scanning range and

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2 cm-1 resolution.

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2.5 Py-GC/MS analysis

CP/MAS NMR analysis, the sample was packed in 4-mm zirconia rotors fitted with

131

The Py-GC/MS analysis was performed on a CDS 5200 pyrolysis autosampler

132

(Oxford, PA, USA) connected to a 7890A-5975C Agilent gas chromatograph-mass

133

spectrometer. Approximately 2 mg of the sample was used for analysis. The carrier gas

134

was high-purity helium, and the heating rate was 20 °C/ms. The sample was heated

135

from 25 °C to 600 °C and then maintained at 600°C for 10 s. The carrier gas flow rate

136

was 50 mL/min. A DB-5 (Optima-5) wax column (30 m×0.25 mm, 0.25 μm, 5%

137

diphenyl and 95% polydimethylsiloxane) was used. The column temperature was

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initially 60 °C and was then increased to 270 °C at 10 °C/min in 6 min. The split ratio

139

was 1:20, and the chroma interface temperature was 270 °C.

140

2.6 Statistical analysis

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Each experiment was performed in triplicate. The average values with a confidence

142

interval (at level of significance≤0.05) were reported. Statistical analysis was

143

completed using MS Excel 2016.

144

Results and discussion

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3.1 Changes in the byproducts during dilute acid pretreatment

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Fig. 1 shows that the amount of byproducts (FF, HMF, LA, FA, and HAc)

147

generally decreased as the acid concentration increased, which implied that a higher

148

acid concentration promoted the formation of pseudolignin. Fig. 1a, b and c

149

demonstrate that the xylose amount decreased as the acid concentration increased

150

because it was degraded into byproducts. Comparing Fig. 1b and c, trace xylose was

151

detected, and the byproducts decreased as the acid concentration increased to 1.00

152

wt%. Therefore, it was proposed that byproducts originated from xylose were

153

degraded into intermediates and then converted to pseudolignin under harsh

154

pretreatment conditions (Kumar et al., 2013). As a result, xylose amount decreased

155

from 0.23 g to nearly 0 g, meanwhile FF amount decreased from 1.78 g to 0.42 g (Fig.

156

1c). It was reported that FF degraded from xylose was converted to the corresponding

157

intermediates during harsh acid pretreatment (Popoff and Theander, 1972; Kumar et

158

al., 2013; Shinde et al., 2018). In comparison, glucose amount increased as the acid

159

concentration increased (Fig. 1a, b and c). When the acid concentration was increased

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to 1.00 wt%, glucose amount decreased slightly as the treatment time increased

161

because it was degraded to HMF (Li et al., 2007; Thananatthanachon and Rauchfuss

162

et al., 2010). For example, HMF amount increased from 0.26 g to 0.49 g in 20 min

163

and then decreased to 0.20 g at 45 min when 1.00 wt% acid was employed. In

164

addition, the amount of LA degraded from HMF increased from 0 g to 0.65 g (Fig.

165

1c). It was also reported that HMF degraded from glucose was converted to the

166

corresponding intermediates under harsh acid pretreatment (Luijkx and Horst, 1994;

167

Shinde et al., 2018). Under the corresponding conditions, the byproducts (e.g., FF and

168

HMF) decreased with the addition of lignin (Fig. 1d, e and f). For instance, as 1.00

169

wt% of acid was employed, FF amount decreased from 1.78 g to 1.36 g, while HMF

170

amount decreased from 0.49 g to 0.27 g when lignin was applied which implied lignin

171

promoted the generation of pseudolignin.

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174

175

Fig. 1. The quantitative changes in FF, HMF, LA, FA, HAc, glucose, and xylose

176

during acid pretreatment with (a) 0 wt% H2SO4, (b) 0.08 wt% H2SO4, (c) 1.00 wt%

177

H2SO4, (d) 0 wt% H2SO4 with lignin addition, (e) 0.08 wt% H2SO4 with lignin

178

addition, (f) 1.00 wt% H2SO4 with lignin addition

179

3.2 Quantification of pseudolignin and byproducts

180

The change of solid after the pretreatment and byproducts in prehydrolyzate is

181

summarized in Table 3. Generally, little xylan was quantified in the pretreated solids

182

because hemicellulose (mainly composed by xylan) was hydrolyzed into water-soluble

183

xylose in an acidic aqueous solution, and further degraded to FF under the harsh

184

condition. When acid concentration was increased from 0 wt% to 1.00 wt%, FF amount

185

was increased from 1.16 g to 1.18 g, and then decreased to 0.42 g which was 10 / 32


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186

corresponding to the pseudolignin yield. Technically, a severe acid pretreatment

187

facilitated the hydrolysis of cellulose into glucose, followed with glucose degradation.

188

When acid concentration was increased from 0 wt% to 1.00 wt%, cellulose amount of

189

the solid was decreased from 10.82 g to 0.62 g, however glucose amount was only

190

increased from 1.27 g to 3.62 g. Meanwhile, a little increase of HMF degraded from

191

glucose was quantified. For example, HMF amount was increased from 1.27 g to 3.62

192

g. However, LA amount was increased from 0.19 g to 2.23 g, and then decreased to

193

0.65 g, and FA amount was increased from 0.83 g to 2.14 g, and then increased to 1.31

194

g. As a result, pseudolignin was correspondingly increased from 1.36 g to 4.05 g, which

195

indicated severer acid pretreatment promoted the generation of pseudolignin. As a

196

comparison, HMF was mainly degraded to FA and LA under mild acid pretreatment

197

(0.08%).

198

Table 3 demonstrates that lignin promoted the generation of pseudolignin. For

199

example, as the additional lignin was employed, FF and HMF were respectively

200

decreased from 3.62 g to 3.18 g and from 0.20 g to 0.15 g when 1.00 wt% acid was

201

applied, however pseudolignin was increased from 4.05 g to 4.59 g. This result implied

202

the potential reaction between FF, HMF, and lignin that enhanced the generation of

203

pseudolignin (Zhang et al., 2015; Zhang et al., 2016).

204

In addition, lignin retained more cellulose because the added lignin and the

205

generated pseudolignin precipitated on the cellulose surface, hindering its acid

206

hydrolysis. For example, when 1.00 wt% acid was employed, the retained cellulose

207

increased from 0.62 g to 1.52 g when lignin was applied.

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Table 3. Yields of cellulose and pseudolignin

208

Prehydrolyzate (g)

Pretreated solid (g)

No.

a

b

c

d

e

f

Glucose

Xylose

FF

HMF

LA

FA

AIM†

Cellulose

Pseudolignin‡

1.27

4.02

1.16

0.16

0.19

0.83

1.36

10.82

1.36

±0.02

±0.05

±0.09

±0.02

±0.03

±0.03

±0.03

±0.33

±0.05

3.39

2.10

1.18

0.20

2.23

2.14

2.03

8.10

2.03

±0.08

±0.03

±0.05

±0.01

±0.10

±0.07

±0.04

±0.21

±0.10

3.62

0.02

0.42

0.20

0.65

1.31

4.05

0.62

4.05

±0.10

±0.01

±0.02

±0.03

±0.04

±0.05

±0.12

±0.03

±0.16

1.26

4.02

1.10

0.14

0.05

0.73

7.87

11.11

1.42

±0.03

±-.09

±0.02

±0.01

±0.01

±0.03

±0.26

±0.31

±0.03

3.53

2.01

1.12

0.17

2.07

2.17

8.68

8.23

2.23

±0.10

±0.04

±0.03

±0.04

±0.06

±0.05

±0.14

±0.26

±0.11

3.18

0

0.37

0.15

0.59

1.31

11.04

1.52

4.59

±0.06

±0

±0.04

±0.02

±0.04

±0.06

±0.31

±0.02

±0.12

209

Note: AIM†, Acid-insoluble matter; Pseudolignin‡ was calculated as the discrepancy

210

between the AIM and the MWL.

211

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3.3 Quantification of pseudolignin generated from model compounds

213

The model compounds were pretreated with acid to investigate their degradation and

214

the generation of black polymer (BP) (Table 4). Technically, 0.24 g of BP (nominated

215

as BP-1) and 0.07 g of FA were generated when 1.00 g of FF was acid pretreated, and

216

this result was consistent with reported results (Cheng et al., 2018). In addition, 0.15 g

217

of BP (denoted BP-2), 0.54 g of LA and 0.14 g of FA were generated when 1.00 g of

218

HMF was acid pretreated. However, the production of BP (nominated as BP-3)

219

increased to 0.62 g when 1.00 g of FF and 1.00 g and HMF were simultaneously acid

220

pretreated. BP (nominated as BP-4) further increased to 0.83 g when 1.00 g of FF, 1.00

221

g of HMF and 1.00 g of MWL were simultaneously acid pretreated. The results showed

222

that the synergetic effect between the model compounds and MWL facilitated BP

223

generation. In addition to BP, detectable amounts of derivatives generated from the

224

model compounds were also quantified. Table 4. Yields of derivatives and BP after pretreatment

225 No

g

FF

0.57±0.06

HMF

N.D.

LA

FA

MWL

AIM

0.07±

0

0.24±0.03

N.D.

BP†

0.24±0.02 0.02

h

i

j

0.02±

0.54±

0.14±

0.01

0.03

0.01

0.04±

0.50±

0.18±

0

0.15±0.02

0.03±0.01

0.15±0.03

0

0.62±0.04

0.43±0.04

0.31±0.01

0.62±0.01 0.01

0.04

0.03

0.05±

0.46±

0.12±

1.00

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1.83±0.03

0.83±0.03


0.02

0.01

0.01

226

Note: † The quantity of the BP was calculated from the discrepancy between the AIM

227

and the MWL.

228

3.4 Characterization of pseudolignin

229

3.4.1 FT-IR characterization

230

The FT-IR spectra of the model compounds and BPs are shown in Fig. 2. The

231

assignments of the main peaks are shown in Table 5. The broad bands at 1512, 1604,

232

1462 and 1422 cm−1, attributable to aromatic C=C bonds, indicated that the

233

pseudolignin involved aromatic structures (Chen et al., 1994; Hu and Ragauskas, 2012).

234

The strong band at 1697 cm−1 was assigned to conjugated C=O motifs (carbonyl and/or

235

carboxylic). The bands at 1020 cm-1 and 1163 cm−1 were assigned to C–O stretching,

236

e.g., furans, alcohols, ethers, or carboxylic acids. The strong and broad peak at 3238

237

cm−1 was assigned to hydroxyl O-H stretching (Kumar et al., 2013; Rodrigues et al.,

238

2002; Sumerskii et al., 2010). Compared to FF and HMF, the decreasing broad band of

239

pseudolignin at 1697 cm−1 indicated that the carbonyl group (C=O) is the reactive site

240

for BP generation.

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241 242

Fig. 2. FT-IR spectra of the model compounds and BP

243

Table 5 The assignments of the main FT-IR bands Number

Wavenumber (cm-1)

Assignment

1

788, 760

-CH2 aliphatic C–H rocking

2

823

-CH3 hydroxymethyl group

3

1020, 1163

C–O stretching in alcohols, FF, HMF, or carboxylic acids

4

1360

Aliphatic C–H rocking

5

1604, 1512, 1462,

C=C stretching in benzene or furan

1422 6

1697

C=O stretching in carboxylic acids, conjugated aldehydes or ketones

7

2923

Aliphatic C–H stretching

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8

244 245

3238

O–H stretching in alcohols, phenols or carboxylic acids

3.4.2 Solid-state CP/MAS 13C NMR characterization The solid-state

13C

NMR spectrum is shown in Fig. 3, and the main peak

246

assignments are presented in Table 6. As for cellulose, the strong signals at δC 106.8,

247

72.5, 74.8, 87.6, 79.4 and 63.4 ppm are the characteristic peaks of glucose, which are

248

respectively assigned to C-1, C-2, C-3, C-4, C-5 and C-6 (Wada et al., 2001). As for

249

hemicellulose, the strong signals at δC 101.6, 76.3, 73.6, 72.6 and 62.9 ppm are the

250

characteristic peaks of xylose, which are respectively assigned to C-1, C-2, C-3, C-4

251

and C-5 (Peng et al., 2010; Shen et al., 2009). As for MWL, the intensified signals at

252

δC 110-160 ppm are the characteristic peaks of lignin, which are assigned to the

253

unsaturated carbons (aromatic carbons) of MWL. Generally, the different structures

254

were observed from black polymers compared to the starting substrates (e.g. glucose,

255

xylose and MWL) indicating the generation reaction was occurred during the severer

256

acid pretreatment. Meanwhile, the generated black polymers possessed the similar

257

structures. For example, the intensified signals at δC 160-210 ppm assigned to carbonyl,

258

δC 150-160 ppm assigned to unsaturated carbon, and δC 10-55 ppm assigned to aliphatic

259

carbon were identified from the black polymers. Therefore, it was proposed that during

260

the severer acid pretreatment, cellulose and hemicellulose hydrolyzed to glucose and

261

xylose which then were respectively degraded to 5-HMF and FF, and eventually

262

generated the black polymers. As for BP-1, BP-2, and BP-3, the signal at δC 28 ppm

263

was assigned to CH3 moiety of diketone, and the signal at δC 12 ppm was assigned to

264

CH3 of aliphatic group. Therefore, the condensation of the derivatives converted from 16 / 32


Page 16 of 32

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265

the ring-opening of FF and HMF was proposed as the main generation pathway of black

266

polymer which was consistent to the reported result (Cheng et al., 2018). As a

267

comparison, BP-4 possessed the strong signal at δC 56 ppm which was assigned to

268

methoxyl group. Thus, it was proposed that methoxyl group of pseudolignin is mainly

269

derived from lignin. Conclusively, FF and HMF underwent ring-opening reaction

270

during the acid pretreatment, and the reactive carbonyl groups and unsaturated groups

271

promoted the condensation between FF, HMF and MWL to generate pseudolignin.

272 273

Fig. 3. Solid-state CP/MAS 13C NMR spectra of the samples

274

Table 6. Assignments of the main peaks of the samples Chemical shift (ppm)

Assignment

220–196

C=O in aldehydes or ketones

178–168

C=O in carboxylic acids or esters

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155–142

Aromatic or furan C–O

142–125

Aromatic or furan C–C

125–102

Aromatic or furan C–H

110-160

Aromatic C-H in MWL

106.8

C-1 in cellulose

101.6

C-1 in hemicellulose C-2, C-3, C-4 and C-5 in cellulose and C-2, C-3,

72-89 C-4 in hemicellulose

275 276 277

64.9

C-5 in hemicellulose

63.4

C-6 in cellulose

60–55

Methoxy related to aromatic rings

50–10

Aliphatic carbons

3.4.3 Py-GC/MS analysis The pyrolytic products of the samples are shown in Fig. 4. The assignments of the main derivatives are listed in Table 7.

278

279

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280

281 282 283

Fig. 4. Typical ion chromatograms of BP

19 / 32



284

Table 7. Assignments of the peaks of the main pyrolytic degradation products

Number

Retention time (min)

Compound

1

1.69

Acetone

2

1.82

Methyl vinyl ketone

3

1.91

2-Methylfuran

4

2.48

Furan, 2,5-dimethyl

5

3.63

Furan, 2-ethyl-5-methyl

6

3.87

Furan, 2,3,5-trimethyl

7

4.30

Furfural

8

6.91

2-Furancarboxaldehyde, 5-methyl

9

7.37

Phenol

10

8.15

Phenol, 2-methoxy

11

8.20

Phenol, 3-methyl

12

8.78

Furan, 2-(2-furanylmethyl)-5-methyl

13

8.82

Phenol, 4-ethyl

14

8.88

Creosol

15

9.10

Benzofuran, 4,7-dimethyl

16

9.15

Benzofuran, 2,3-dihydro

17

9.51

1H-inden-1-one, 2,3-dihydro

18

9.58

2-Methoxy-4-vinylphenol

19

9.74

Phenol, 2,6-dimethoxy

20 / 32


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20

10.19

3,5-Dimethoxy-4-hydroxytoluene

285

All of the solid samples were achieved from the corresponding starting materials

286

which were pretreated by 1.00 wt% acid at 180 °C for 40 min. Compared to FF, the

287

pyrolytic products of BP-1 were dominated by furan derivatives such as FF, 2-

288

methylfuran and 2,5-dimethylfuran. The generation of BP was proposed to occur via

289

an aldol condensation between FF and xylose, an acetal reaction between FF, and an

290

aldol condensation/acetal reaction between FF and its derivatives (Li et al., 2015; Mittal

291

et al., 2017). Therefore, the pathway of BP-1 formation was proposed in Scheme 1

292

(Zeitsch, 2000). The carbonyl group of FF was attacked by hydrogen ions to form a

293

carbocation, which further attacked C5 of another FF to eventually generate the furan

294

dimer (FD) containing a hydroxyl group. Then, FD reacted with FF to form a polymer

295

containing a hydroxyl group (FPHG). In addition, FD reacted with FF to form another

296

polymer containing carbonyl groups and double C=C bonds (FPCD) (Ilgen et al., 2009;

297

Cheng, 2018). A little amount of 2, 3-dihydro, 1H-inden-1-one (No. 16 in Table 7) was

298

identified in the pyrolytic degradation products, indicating the generation of 3, 8-

299

dihydroxy-2-methylchromone (DMC) during acid pretreatment (Popoff and Theander,

300

1972; Shinde et al., 2018).

21 / 32



Scheme 1. The proposed generation pathway of BP-1

301

302

Compared to HMF, the pyrolytic degradation products of BP-2 mainly included

303

furan derivatives and ketones. Therefore, the generation pathway of BP-2 was proposed

304

in Scheme 2 (Patil and Lund, 2011). C5 of HMF was attacked by hydrogen ions to

305

generate 2, 5-dioxo-6-hydroxyhexanal (DHH) (Horvat et al., 1985). Then, DHH reacted

306

with HMF through an aldol condensation to form humins (Patil et al., 2012; Hoang et

307

al., 2013; Pin et al., 2014). In addition, several aromatic products such as benzofuran 2,

308

3-dihydro (No. 15 in Table 7) were identified in the pyrolytic degradation products,

309

confirming the generation of aromatic intermediates (Luijkx and Horst, 1994; Shinde

310

et al., 2018).

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311 312

The pyrolytic degradation products of BP-3 mainly included furan derivatives,

313

ketones, and aromatic compounds, indicating that the synergetic effect between FF and

314

HMF promoted the generation of pseudolignin. Although HMF hardly polymerized

315

with FF by an aldol condensation, DHH generated by the degradation of HMF reacted

316

with FF/HMF to form humins, and then the addition of FF to humins further produced

317

BP-3 (Scheme 3). Additionally, the phenolic aldehyde condensation reaction of FF,

318

HMF and humins with the intermediates further increased the generation of BP-3

319

(Scheme 3).

23 / 32



320


321

The pyrolytic degradation products of BP-4 included more complicated derivatives,

322

such as various aromatic compounds and furan derivatives. The results showed that the

323

addition of lignin significantly contributed to the formation of BP-4. During acid

324

pretreatment, the aryl ether bonds (β-O-4) in lignin were cleaved, and then the

325

fragments underwent acid-catalyzed recondensation (Shuai et al., 2010). Due to the

326

cleavage of β-O-4, a massive amount of molecular fragments containing phenolic

327

hydroxyl groups was produced, and they reacted with FF, HMF and their degradation

328

products, including those containing carbonyl groups, to form BP-4. Consequently, the

329

generation pathway of BP-4 was proposed in Scheme 4 (Bu et al., 2011).

330

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331 332

Under harsh acid pretreatment conditions, the hemicellulose and cellulose in

333

biomass material were hydrolyzed into xylose and glucose, which were then degraded

334

into

335

polycondensation/aggregation of FF. Humins as the degradation product of HMF

336

formed humins through the polymerization. Technically, FPCD, FPHG and humins

337

formed the backbone of pseudolignin. FF and HMF were linked to the backbone of the

338

polymer via aldol and acetal reactions. Simultaneously, DMC, BTO and lignin

339

fragments were also linked to the backbone through phenolic aldehyde condensation

340

reactions. The generation pathway of pseudolignin is thus confidently proposed in

341

Scheme 5. Under the harsh condition, cellulose and hemicellulose in bagasse underwent

342

acid-catalyzed hydrolyzed to afford glucose and xylose. Then, monosaccharides were

343

degraded into HMF and FF, which were further converted into the key intermediates,

344

including humins, FPHG and FPCD. The intermediates were used to form the backbone

FF

and

HMF.

FPCD

and

FPHG

were

25 / 32


formed

through

the


345

of pseudolignin, then HMF, FF, lignin fragments and their derivatives were linked to it

346

through phenolic aldehyde condensations, aldehyde addition and acetal reaction.

347 348

Scheme 5. The proposed generation pathway of pseudolignin Acknowledgments

349

This research was financially supported by the Natural Science Foundation of

350

China (31400514), Postdoctoral Science Foundation of China (2015M570419),

351

Guangxi Natural Science Fund (2018JJA130224), and Guangxi Key Laboratory of

352

Clean Pulp & Papermaking and Pollution Control Fund (ZR201805-7). 26 / 32


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353

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461

Graphic Abstract (TOC)

462 463

How pseudolignin is generated during dilute sulfuric acid pretreatment

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How pseudolignin is generated during dilute sulfuric acid pretreatment

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