Self-sufficient bioethanol production system using a lignin-derived

Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori. 9. University, 4-101 Koyamacho-Minami, Tottori, Tottori 680-8552, ...
0 downloads 0 Views 909KB Size
Subscriber access provided by READING UNIV

Article

Self-sufficient bioethanol production system using a lignin-derived adsorbent of fermentation inhibitors Koichi Yoshioka, Masakazu Daidai, Yoshihiro Matsumoto, Rie Mizuno, Yoko Katsura, Tatsuya Hakogi, Hideshi Yanase, and Takashi Watanabe ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02915 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a 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

Self-sufficient bioethanol production system using a

2

lignin-derived adsorbent of fermentation inhibitors

3

Koichi Yoshiokaa, Masakazu Daidaib, Yoshihiro Matsumotoa, Rie Mizunoa, Yoko Katsurab,

4

Tatsuya Hakogic, Hideshi Yanasec and Takashi Watanabea*

5

a

6

University, Gokasho, Uji, Kyoto 611-0011, Japan

7

b

8

Osaka 332-0012, Japan

9

c

10

Laboratory of Biomass Conversion, Research Institute for Sustainable Humanosphere, Kyoto

Japan Chemical Engineering & Machinery Co. Ltd.4-6-23 Kashima, Yodogawaku, Osaka,

Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori

University, 4-101 Koyamacho-Minami, Tottori, Tottori 680-8552, Japan

11 12

*Corresponding author

13

Research Institute for Sustainable Humanosphere, Kyoto University

14

Gokasho, Uji, Kyoto 611-0011, Japan

15

TEL: +81-774-38-3640, FAX: +81-774-38-3681

16

E-mail: [email protected]

17 18

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

19

KEYWORDS: fermentation inhibitor; lignin; bioethanol; adsorbent; microwave-assisted

20

pretreatment; simultaneous saccharification and co-fermentation; Zymomonas mobilis;

21

Eucalyptus globulus

22

ABSTRACT: We have developed a new self-sufficient bioethanol producing system that

23

suppresses the inhibition of fermentation by thermally-processed residual lignin in a separate

24

hydrolysis and fermentation (SHF) and one-pot simultaneous saccharification and co-

25

fermentation (SSCF). The new fermentation process incorporates detoxification with the lignin-

26

derived adsorbent; thus, needs no purchased adsorbent, produces no waste adsorbent and relieves

27

waste water treatment load. Eucalyptus globulus wood was pretreated by microwave (MW)-

28

assisted hydrothermolysis in aqueous maleic acid and separated into soluble and insoluble

29

fractions. The insoluble fraction was hydrolyzed with cellulolytic enzymes, and the residual

30

lignin was separated. We found that thermal processing of the lignin under a normoxic

31

atmosphere efficiently adsorbed fermentation inhibitors without affecting monosaccharide

32

concentration by enzymatic saccharification. The processing was achieved at 250–350 °C, which

33

are much lower temperatures for wood charcoal production and resulted in higher yields of the

34

adsorbent. The residual lignin formed after SSCF was also converted to the selective adsorbent.

35

Using the lignin-derived adsorbent and genetically engineered Zymomonas mobilis, bioethanol

36

was produced at 54 g/L from the pretreated biomass mash by one-pot SSCF processes coupled

37

with prehydrolysis. The lignin-derived adsorbent is recyclable and potentially applicable to a

38

wide range of fermentation processes of lignocellulosics.

39

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

ACS Sustainable Chemistry & Engineering

40

INTRODUCTION: Excessive use of fossil resources causes global warming and depletes

41

accessible crude oil.1 Therefore, there is a growing demand to produce biofuels and chemicals

42

from renewable lignocellulosic biomass. Bioethanol production from lignocellulosic materials

43

relies on technologies that disintegrate cell wall structures to expose cellulose and

44

hemicelluloses, hydrolyze the cell wall polysaccharides to monosaccharides and ferment sugars

45

to bioethanol.2–9

46

To increase the economic feasibility of bioethanol production from lignocellulosics, a

47

decrease in the dosage of cellulolytic and hemicellulolytic enzymes, the use of monosaccharides

48

derived from hemicelluloses and a high rate of ethanol fermentation of sugars should be

49

accomplished, together with an optimized pretreatment for the overall process. The exposure of

50

cell wall polysaccharides for efficient enzymatic hydrolysis requires harsh pretreatment

51

conditions, but this process results in co-production of a higher amount of fermentation

52

inhibitors. Therefore, a new technology for suppression of fermentation inhibition contributes to

53

the overall processes of bioethanol production through intensive pretreatment, hydrolysis with a

54

smaller amount of enzyme and rapid fermentation.

55

In bioethanol production, pretreatments separating cell wall components result in the

56

production of fermentation inhibitors such as furfural, 5-hydroxymethylfurfural (5-HMF),

57

vanillin, vanillic acid, syringaldehyde, syringic acid, acetic acid and other organic compounds

58

from the cell wall polysaccharides and lignin (Figure 1).7–20 Molecular breeding of inhibitor-

59

resistant ethanologenic microorganisms and process engineering to remove fermentation

60

inhibitors have been studied for the production of bioethanol. Higher substrate concentration is

61

needed to produce bioethanol at a lower cost. However, this demand, like the harsh pretreatment

62

conditions, requires a concomitantly high level of technology to suppress the fermentation 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

63

inhibition. So far, a number of technologies have been employed for removing inhibitors. These

64

include adsorption of inhibitors on ion exchange resin and activated carbon, treatments with

65

calcium hydroxide, calcium carbonate, sodium hydroxide, ammonium hydroxide, dithionite,

66

hydrogen sulfite, membrane filtration, enzymatic and microbial detoxification, extraction with

67

organic solvents or supercritical fluid and evaporation before or during fermentation.7–20

68

However, the cost of bioethanol production is increased by additional processes, and the use of

69

purchased adsorbent for fermentation inhibition is impractical for the production of biofuels,

70

with a low cost that is competitive in the market.

71

To overcome problems caused by fermentation inhibitors, development of new adsorbent,

72

which is highly efficient, commercially available, environmentally friendly and easily

73

obtainable, is strongly required. Additionally, construction of highly efficient bioethanol process

74

is necessary for industrial use. In this paper, we found that thermally-processed residual lignin

75

produced after ethanol fermentation efficiently adsorbed fermentation inhibitors without

76

affecting monosaccharide concentration by enzymatic saccharification, and developed self-

77

sufficient bioethanol production system using the novel lignin-derived adsorbent for inhibitors.

78 79

EXPERIMENTAL SECTION:

80

Materials and general methods. All the reagents and activated carbon were of analytical

81

grade and purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and Nacalai

82

Tesque (Osaka, Japan). Ion exchange resin was obtained from Mitsubishi Chemical Corporation

83

(Tokyo, Japan). E. globulus wood chips were purchased from Oji paper Co., Ltd. (Tokyo, Japan).

84

The wood chips were ground to 20 mesh using a Wiley mill, air-dried to approximately 10 %

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

85

moisture content and used for experiments. The wood contained 46.6 % cellulose, 30.2 %

86

hemicellulose and 22.2 % lignin.

87

The holocellulose and α-cellulose contents were determined according to the Wise chlorite

88

method 21 and TAPPI T203 om-93,22 respectively. Klason lignin was determined from the weight

89

ratio of solid residue after a two-step acid hydrolysis using 72 % and 1.9 % sulphuric acid

90

according to TAPPI T222 om-98 procedure.23

91

Preparation of the adsorbents from the residual lignin by enzymatic hydrolysis of

92

microwave-assisted pretreated pulp. The pretreatment of E. globulus for preparation of the

93

adsorbents was conducted using a tower-shaped 2.45 GHz microwave (MW) reactor equipped

94

with eight sets of 1.5 kW magnetrons and a 50 L reaction vessel (Japan Chemical Engineering &

95

Machinery Co. Ltd., Shiga, Japan).24 Wood powder of E. globulus (7.5 kg) was added to 42 kg of

96

tap water containing 0.5 kg of maleic acid. Microwave-assisted pretreatment of E. globulus wood

97

was performed at 170 °C for 30 min. After the treatment, the pulp residue was separated by

98

centrifugation and washed three times with tap water.

99

The pulp residue separated by microwave pretreatments was hydrolyzed with a commercial

100

cellulase preparation, Cellic CTec2 from Trichoderma reesei (Novozymes A/S, Bagsvaerd,

101

Denmark). Cellulase enzyme loading was 10 FPU/g of dry pulp. Enzymatic hydrolysis was

102

performed in 50 mM sodium succinate buffer (pH 4.5) at 50 °C for 72 h. After enzymatic

103

hydrolysis, the residual lignin was separated by filtration and washed with water. The residue

104

was added into hot water and stirred to remove buffer. After the solution was filtered and washed

105

with water, the purified residue was dried at 105 °C overnight.

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

106

For preparation of the adsorbent by thermal treatment under normoxic conditions, the dried

107

residual lignin was placed in a stainless steel tray, covered with an aluminium sheet with ten

108

small pinholes per 20 cm2, and heated in air at 250–350 °C for 1–4 h in an electric furnace

109

(FO810, Yamato Scientific Co. Ltd., Tokyo, Japan). The yield of the adsorbent treated at 350 °C

110

for 2 h was 39 % from the residual lignin. The yield of the adsorbent was calculated using the

111

following equation (1).

112 113

(1) Yield (wt%) = W1/W0 × 100, where W0 is the dry weight of the residual lignin and W1 is the dry weight of the adsorbent obtained by thermal treatment.

114

Recycled adsorbent was prepared by heating the treated adsorbent at 350 °C for 1 h in air

115

using the same apparatus. Yield of the recovered adsorbent was 69 %. The yields of the

116

adsorbent were calculated using the following equation (2).

117 118

(2) Yield (wt%) = W3/W2 × 100, where W2 is the dry weight of the treated adsorbent and W3 is the dry weight of the treated adsorbent obtained by thermal treatment.

119

For preparation of the lignin-derived adsorbents under anaerobic conditions, the dried

120

residue in a stainless steel dish was set in a gas-sealed electric furnace (Tokai Denki Co. Ltd.,

121

Osaka, Japan) under a nitrogen atmosphere. The thermal treatment was carried out at 250–350 ºC

122

for 2 h. The yield of each adsorbent was 52–82 % from the residual lignin. The yields of the

123

adsorbent were calculated using the equation (1).

124

Pretreatment of E. globulus wood using microwave irradiation for the fermentation

125

test and one-pot SSCF. Wood powder of E. globulus (7.5 kg) was added to 42.49 kg of tap

126

water containing 0.015 kg of maleic acid. Microwave-assisted pretreatment of E. globulus wood

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

127

was performed at 190 °C for 30 min. After the treatment, the pulp residue and the soluble

128

fraction were separated by centrifugation and washed three times with distilled water.

129

Analysis of fermentation inhibitors. Fermentation inhibitors were quantified using high

130

performance liquid chromatography (HPLC). Shimadzu Prominence system (Shimadzu Co., Ltd,

131

Kyoto, Japan) equipped with an LC-20AD pump, a CTO-20A column oven, and an SPD-M20A

132

photodiode array were used for HPLC analysis. Acetic acid, furfural and 5-HMF were analyzed

133

using an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad Laboratories, Inc., Hercules,

134

CA), with 8 mM aqueous sulfuric acid solutions as a mobile phase at a column temperature of

135

35 °C and detected at UV 210 nm. The elution was performed using a Unison UK-Phenyl

136

column (Imtakt, Inc., Kyoto, Japan), with 10 mM ammonium acetate buffer (pH 6.8) and

137

acetonitrile at 40 °C to quantify lignin-derived inhibitors vanillin, syringaldeyde, vanillic acid

138

and syringic acid with the detection at UV 280 nm.

139

HPLC analysis of monosaccharides. Neutral carbohydrates obtained by enzymatic

140

saccharification and adsorption experiments were determined by HPLC using a Prominence

141

HPLC post-labelling system (Shimadzu, Co., Ltd., Kyoto, Japan) equipped with a F-1080

142

fluorescence detector (HITACHI, Tokyo, Japan) and two tandemly connected Aminex HPX-87P

143

columns (300 mm × 7.8 mm: Bio-Rad Laboratories, Inc., Hercules, CA). For fluorescence

144

detection, samples were labelled at 150 °C using an aqueous solution of L-arginine (1 %) and

145

boric acid (3 %).25 The sample solution (5 µL) was injected and analyzed at 58 °C using water as

146

an eluent at a flow rate of 0.3 mL min−1. D-Glucoheptose was used as an internal standard.

147

Adsorptive removal of fermentation inhibitors and determination of monosaccharides in

148

MW-filtrate. The adsorbent (2.5 g) was added to 25 g of the soluble fraction obtained from the

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

149

microwave pretreatment of E. globulus at 190 °C. Each mixture was stirred at room temperature.

150

After 24 h, 1 mL of the solution was filtered through a Chromato-disc syringe filter with 0.45 µm

151

pore size (GL science Inc., USA) and subjected to the HPLC analysis of fermentation inhibitors

152

and monosaccharides. All adsorption tests were performed in triplicate.

153

Adsorption test and analysis of lignin-derived inhibitors. The adsorbent (2.5 g) was added

154

to 25 g of model aqueous solutions containing 20 mM of vanillin, syringaldehyde and p-

155

hydroxybenzaldehyde in each. Each mixture was stirred at room temperature. After 24 h, 1 mL

156

of the solution was filtered through the Chromato-disc syringe filter and subjected to the HPLC

157

analysis of lignin-derived inhibitors from the soluble fraction. All adsorption tests were

158

performed in triplicate.

159

Adsorption test and sugar analysis of xylooligosaccharides. The adsorbent (0.5 g) was

160

added to 5 g of the model aqueous solution containing 1 % of xylooligosaccharide (Wako Pure

161

Chemical Industries Ltd., Osaka, Japan). The solution was stirred at room temperature. After 24

162

h, total carbohydrates in the supernatant of mixture were determined by phenol-sulfuric acid

163

method.26 All adsorption tests were performed in triplicate.

164

Fermentation test using Z. mobilis. After preculturing the recombinant Z. mobilis6 for 48 h

165

in 10 ml of basal medium (RM) containing 10 g/L yeast extract, 2 g/L KH2PO4 and 20 g/L

166

glucose, cells were harvested and washed with the same volume of RM medium, except lacking

167

a carbon source. The washed cells were then inoculated into 100 ml of the following media (8:2

168

and 5:5) in a 200 ml bottle with a screw cap. The 8:2 medium was prepared by mixing eight parts

169

of the soluble fraction from MW-pretreated E. globulus at 190 °C for 30 min and a solution

170

obtained by treating the soluble fraction with various adsorbents, one part of the 10-fold

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

171

concentrate of RM medium without a carbon source and one part of 200 g/L of glucose. The 5:5

172

medium was prepared by mixing five parts of the soluble fraction, one part of the 10-fold

173

concentrate RM medium without a carbon source, one part of 200 g/L glucose and three parts of

174

the distilled water. The test media were adjusted to pH 5.5 and statically cultivated at 30 °C.

175

Structural analyses of adsorbents. The surface morphologies of adsorbents were analyzed

176

using scanning electron microscopy (SEM; JSM-6320, JEOL, Tokyo, Japan) at an acceleration

177

voltage of 5 kV. The surface area and pore size of adsorbents were analyzed using ASAP 2000

178

(Shimadzu, Co., Ltd., Kyoto, Japan) using nitrogen.

179

Enzymatic hydrolysis in the presence of adsorbents. Sodium succinate buffer (1 M),

180

distilled water and Cellic CTec2 were added to the soluble fraction (10 g) separated by MW

181

pretreatment from E. globulus at 190 °C for 30 min so that total amount of the solution was 20 g.

182

Cellulase enzyme loading was 5 mg protein per 20 g of the solution. After an adsorbent (1 g) was

183

added to the solution, enzymatic hydrolysis was performed in 50 mM sodium succinate buffer

184

(pH 4.5) at 50 °C on a rotary shaker (NTS-4000C, Rikakikai, Japan) at 140 rpm for 72 h. The

185

sugar concentration was based on the weight percentage of each sugar to the soluble fraction

186

using HPLC. All enzymatic hydrolysis experiments were performed in triplicate.

187

One-pot SSCF with prehydrolysis in a medium bottle. The pulp (3 g of dry weight),

188

soluble filtrate (13 ml) prepared by microwave-assisted reactions at 190 °C for 30 min, Cellic

189

CTec2 containing 15 mg protein and 0.15 g of 5 % polyethylene glycol 20,000 solution were

190

mixed in a bottle with a screw cap. Total volume of the fermentation broth was 20 mL. The

191

prehydrolysis was conducted at 50 °C on a shaker at 100 rpm for 24 h. After prehydrolysis, 2 g

192

of the lignin-derived adsorbent prepared by heating in air at 350 °C for 2 h (Entry 2 in Table 1)

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

193

was added to the bottle. After 24 h, the solution was cooled to 35 °C, and 2 ml of the 10-fold

194

concentrated RM medium without a carbon source and 2 ml of the seed culture were mixed to

195

start SSCF. The seed culture was prepared by cultivating the recombinant Z. mobilis6 with the

196

hydrolysates of MW-pretreated pulp. The concentration of glucose, xylose and ethanol was

197

monitored using HPLC as previously reported.6

198

One-pot SSCF with prehydrolysis in a jar fermenter. The pulp (90 g of dry weight) and

199

soluble filtrate (390 ml) prepared by microwave-assisted reactions at 190 °C for 30 min, Cellic

200

CTec2 containing 450 mg protein and 30g of the lignin-derived adsorbent prepared by heating in

201

air at 350 °C for 2 h (Entry 2 in Table 1) were mixed and adjusted to pH 5.0 with 5 N NaOH

202

solution in a 1 L jar fermenter. Total volume of the fermentation broth was 600 mL. The

203

prehydrolysis was conducted at 50 °C with gentle stirring at 100 rpm. After 48 h prehydrolysis,

204

the solution was cooled to 35 °C, and 60 ml of the 10-fold concentrated RM medium without a

205

carbon source and 60 ml of the seed culture were mixed to start SSCF. The seed culture was

206

prepared by cultivating the recombinant Z. mobilis

207

pulp. The concentration of glucose, xylose, cellobiose and ethanol was monitored using HPLC as

208

previously reported.6

6

with the hydrolysates of MW-pretreated

209 210

RESULTS and DISCUSSION:

211

Preparation and properties of lignin-derived adsorbents. E. globulus wood was

212

pretreated by microwaves in an aqueous solution containing 1 % maleic acid at 170 °C for 30

213

min. The pretreated biomass was separated into soluble and insoluble fractions. The insoluble

214

pulp fraction was hydrolyzed with cellulolytic enzymes, and the residual lignin was separated

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

215

from the enzymatic hydrolysates by centrifugation. The residual lignin was washed with distilled

216

water, dried and heated at 250 °C or 350 °C for 1–4 h under normoxic or nitrogen atmosphere in

217

an electric furnace (Table 1). The lignin-derived adsorbent was subjected to adsorption

218

experiments for inhibitors. In adsorption experiments for pretreated solutions, we used the filtrate

219

separated by microwave pretreatment in an aqueous solution containing 0.02 % maleic acid at

220

190 °C for 30 min. After mixing the soluble fraction with the lignin-derived adsorbent, the

221

solution was filtrated, and the concentration of inhibitors in the filtrate was determined by HPLC.

222

The concentration of furfural and 5-HMF in the original soluble fraction was 22.62 mM and 4.79

223

mM, respectively (Table 1). When the solution was treated with the lignin-derived adsorbents

224

prepared by thermal processing at 350 °C or 250 °C under normoxic oxygen atmosphere,

225

concentration of furfural and 5-HMF decreased to 0.83–1.40 mM and 0.58–0.90 mM,

226

respectively (Entry 1–4 in Table 1). The drastic decrease in the concentration of fermentation

227

inhibitors was also found for lignin-derived degradation compounds, such as vanillin and

228

syringaldehyde (Table 2). The concentration of vanillin decreased from 0.17 mM to a trace

229

amount and that of syringaldehyde decreased from 0.70 mM to 0.01 mM. Removal of acetic acid

230

was less effective, but the adsorbent decreased the concentration of the organic acid from 1.20 %

231

to 0.59–0.73 %. Thus, we found that thermal processing of residual lignin at 350 and 250 °C

232

under normoxic atmosphere gave the adsorbent a high adsorptivity (Entry 1–4 in Table 1). The

233

high adsorptivity of the lignin-derived adsorbent against the five fermentation inhibitors (Table

234

1) was comparable with that of activated carbon and anion exchange resin. The adsorbent

235

processed at 250 °C removed the inhibitors as effective as that of 350 °C but much less effective

236

to improve of fermentability. When the treatment was conducted with the lignin-derived

237

adsorbent prepared at the same temperature under nitrogen atmosphere, removal of these

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

238

inhibitors was much less effective as found in the very low levels of decrease in the

239

concentrations of 5-HMF, acetic acid and syringaldehyde (Entry 5 in Table 1).

240

Adsorptive removal of lignin-derived inhibitors was evaluated using a model solution

241

containing 20 mM vanillin, syringaldehyde and 4-hydroxybenzaldehyde (Table 2). As found in

242

adsorption treatments of the soluble fraction from E. globulus wood, the adsorbent made in air

243

was effective at removing inhibitors as well as activated carbon and ion exchange resin. The

244

lignin-derived adsorbent prepared under nitrogen atmosphere was ineffective at removing the

245

lignin monomers both in the pretreated biomass (Table 1) and in the model solution containing

246

inhibitors (Table 2).

247

The effect of inhibitor removal on bacterial growth, carbohydrate consumption and ethanol

248

production by the lignin-derived adsorbent was examined by fermentation experiments with Z.

249

mobilis (Figure 2 and Table 1). When the untreated soluble fraction was subjected to

250

fermentation experiments, at either ratio of the filtrate and RM medium (8:2 or 5:5), the bacterial

251

growth was very slow, and almost no ethanol production was observed. Dilution of the solution

252

to 2:8 ratio was necessary to produce ethanol without removal of inhibitory compounds.

253

However, treatment of the soluble fraction with the lignin-derived adsorbent drastically

254

improved the fermentability.

255

We found that the efficiency of inhibitor removal increased with increasing the weight

256

percentage of the adsorbent, and 10 % was the minimum concentration of the adsorbent under

257

the fermentation conditions employed. The concentration of inhibitors in the broth is another

258

factor affecting the fermentation rate. In this study, we used two different concentrations of the

259

filtrate by changing the ratio between the filtrate and RM medium. When the soluble fraction

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

260

was processed with 10 % lignin-derived adsorbent and fermented at a ratio of 8:2 of the filtrate

261

and RM medium, glucose was consumed, and ethanol production increased during 72 h (Figure

262

2C). When fermented at a ratio of 5:5, glucose was consumed within 24 h with concomitant

263

production of ethanol at the theoretical yield (Figure 2D). Thus, the new adsorbent derived from

264

the residual lignin was effective for detoxification of the pretreated biomass.

265

Recycling of the lignin-derived adsorbent increases the feasibility of practical applications

266

to the bioethanol production process. Therefore, after ethanol fermentation, the used adsorbent,

267

which was originally prepared at 350 °C for 2 h (Entry 2 in Table 1), was separated, processed at

268

350 °C for 1 h in an electric furnace and repeatedly used for detoxification experiments. As

269

shown in Figure 3, the recycled adsorbent slightly decreased the final ethanol concentration but

270

effective enough to produce ethanol at the ratio of the filtrate and RM medium (5:5). After

271

repeated use, the adsorbent containing inhibitors from the filtrate could be burned to recover heat

272

energy. This process is beneficial not only for energy recovery but also to decrease the

273

biochemical oxygen demand and chemical oxygen demand in the waste water, which is one of

274

the major cost factors in bioethanol production.

275

To apply adsorbents to bioethanol production, a high level of sugar recovery from

276

adsorbents is necessary. Therefore, loss of mono- and oligosaccharides by the adsorption process

277

was evaluated using HPLC. The lignin-derived adsorbent made at 350 °C for 2 h did not adsorb

278

monosaccharides. The low adsoptibity to carbohydrates is different from activated carbon and

279

ion exchange resin, which adsorbed around 20 % of monosaccharides in MW-filtrate and more

280

than 40 % of xylooligosaccharide in the model sugar solution (Entry 7 and 8 in Table 3). Thus,

281

ion exchange resin and activated carbon are not selective to the inhibitors and economically

282

feasible for biofuel production. When 1 % of xylooligosaccharides were treated with these 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

283

adsorbents, it was found that activated carbon and ion exchange resin strongly adsorbed

284

xylooligosaccharides. Adsorption of oligosaccharides on the lignin-derived adsorbent prepared at

285

350 °C in air (Entry 1–3 in Table 1) was much smaller than that on the activated carbon and ion

286

exchange resin, but around 20 % loss of the oligosaccharides was found. The problem of

287

adsorption of oligosaccharides can be avoided by prehydrolysis or simultaneous saccharification

288

in the presence of adsorbent if enzymatic hydrolysis was not inhibited by the presence of the

289

adsorbent. The latter process can be extended to a one-pot SSCF process if co-presence of the

290

adsorbent did not inhibit ethanol fermentation. Therefore, we evaluated the effect of co-presence

291

of the adsorbent on enzymatic saccharification and the SSCF process.

292

Enzymatic saccharification of pretreated biomass in the presence of lignin-derived

293

adsorbents. To evaluate the applicability of the lignin-derived adsorbents to the SSCF process,

294

we examined effects of co-presence of adsorbents on enzymatic saccharification of pretreated

295

biomass. Yields of monosaccharides after hydrolysis with Cellic Ctec2 at 50 °C for 72 h are

296

shown in Table 4. The sugar yields obtained in the presence of the lignin-derived adsorbents

297

prepared in air were close to that obtained in the absence of adsorbents. When the residual lignin

298

from the SSCF process was used for the adsorbent, higher sugar yields were obtained. These

299

results are highly contrasted to activated carbon, which decreased the sugar yield by around 20 %

300

(Table 4). Thus, the thermally-processed lignin adsorbents are applicable to in situ enzymatic

301

hydrolysis of pretreated biomass.

302

One-pot SSCF after prehydrolysis using lignin-derived adsorbent. The high

303

adsorptivity of fermentation inhibitors without interference from enzymatic saccharification

304

attracts our interest to apply the lignin-derived adsorbents to the one-pot SSCF process using Z.

305

mobilis (Figure 4). We designed the process starting from prehydrolysis and subsequent SSCF in 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

306

the presence of the lignin-derived adsorbents prepared by heating in air at 350 °C for 2 h (Entry 2

307

in Table 1). The prehydrolysis increased the initial concentration of sugars and resulted in an

308

acceleration of the ethanol production rate by SSCF. We pretreated E. globulus wood by

309

microwave-assisted hydrothermolysis and separated wood into soluble and insoluble fractions.

310

Preliminary experiments of one-pot SSCF of the pulp and the MW-filtrate obtained from the

311

MW-pretreated E. globulus with and without the lignin-derived adsorbent in a bottle were

312

investigated using Z. mobilis (Figure 5). Figure 5A indicates that one-pot SSCF of the pulp and

313

MW-filtrate without the lignin-derived adsorbent cannot produce ethanol. Additionally, amount

314

of glucose and xylose was hardly consumed. In contrast, ethanol was gradually produced in the

315

mixture with the lignin-derived adsorbent until 48 h, and the concentration of ethanol reached at

316

around 20g/L (Figure 5B). Glucose was used up for fermentation in Figure 5B. From these

317

results, it was found that one-pot SSCF of the pulp and MW-filtrate with the lignin-derived

318

adsorbent is available for scale-up process.

319

To a 1 L jar fermenter, the insoluble pulp fraction, soluble fraction and lignin-derived

320

adsorbent were added. The prehydrolysis was started by addition of Cellic Ctec2. The

321

concentration of pretreated substrate and adsorbent was 15 % and 5 %, respectively. After

322

incubation at 50 °C for 48 h, the enzymatic hydrolysates were cooled down to 35 °C. To the

323

same reactor, the nutrient broth and seed culture of Z. mobilis were added and fermented at 35 °C

324

for 72 h. The profile of fermentation by the one-pot SSCF process is shown in Figure 6. Ethanol

325

was produced at an ethanol concentration over 5 % for 70 h after SSCF was started.

326

Thus, ethanol was successfully produced from pretreated biomass mash by the one-pot

327

SSCF using lignin-derived adsorbents and Z. mobilis. The one-pot SSCF reduces the production

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

328

cost of bioethanol by eliminating the solid-liquid separation process and adsorption facilities

329

using a large scale column.

330

Structural analyses of lignocellulose-based adsorbents. Structures of the lignin-derived

331

adsorbents were analyzed to understand the differential adsorptivity caused by the thermal

332

treatment. Table 5 shows the surface area and pore volumes of the series of adsorbents analyzed

333

by the N2-BET method. Surface area of the original residual lignin was 0.39 m2/g. The surface

334

area of adsorbent 2, which was prepared at 350 ºC for 2 h in air was 525 m2/g, indicating that

335

surface area of the lignin increased over 1,000-fold. The surface area of adsorbent 4 prepared at

336

250 °C for 2 h in air was 26.9 m2/g, whereas the surface area of adsorbent 6 prepared under

337

nitrogen atmosphere at the same temperature and heating time was 0.15 m2/g. Thus, thermal

338

treatment in air significantly increased the surface area. Pore volumes of the lignin-derived

339

adsorbent between 10 and 1000 Å increased from 0.001 to 0.239 ml/g, respectively, over 200

340

times by the thermal treatment at 350 °C in air, and the values were comparable with those of

341

activated carbon.27–30 SEM images of adsorbents prepared in air and in nitrogen atmosphere

342

supported the increase in pore structure by the thermal treatment under a normoxic atmosphere

343

(Figure 7). Lignin adsorbent recycled by thermal treatment in air after ethanol fermentation kept

344

the similar indices in the surface morphology as those of the adsorbent initially prepared

345

(Supporting information S1). We assume that the oxygenated aromatic core structures with a

346

propane side chain remained in part after the dehydration process at low temperature in air,

347

giving higher affinity to inhibitors by the balance of hydrophilic and hydrophobic interactions,

348

including π-π stacking.31–33 So far, activated carbon has been prepared by thermal treatments of

349

lignin for the adsorption of metal ions and environmental pollutants.34 Chemical or physical

350

activation was applied to produce the activated carbon with high surface area and pore volume.

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

ACS Sustainable Chemistry & Engineering

351

For instance Cotoruelo et al. converted kraft lignin into activated carbon by carbonization at

352

600–1100 °C followed by physical activation with CO2 to remove environmental pollutants. The

353

adsorbent effectively adsorbed a synthetic dye, crystal violet.35 Compared with the previous

354

studies, the residual lignin can be converted to the adsorbent at much lower temperatures without

355

physical and chemical activation, giving big advantage in terms of cost, energy and material

356

balance. Yields of the adsorbent from the original lignin prepared at 350 °C for 1h, 2 h and 4 h

357

were 69 %, 41 % and 13 %, respectively. Under the higher temperatures over 800 °C for

358

charcoal production, it would be impossible to complete the material flow using the biomass

359

components due to very low recovery of the thermally processed adsorbent.

360 361

CONCLUSIONS: In this study, we have developed novel lignin-derived adsorbent, which can

362

strongly adsorb fermentation inhibitors with less adsorption of saccharides and interference of

363

enzymatic saccharification, by thermally treating of residual lignin from E. globulus. The high

364

selectivity of lignin-derived adsorbent to inhibitors shown by the lower level of monosaccharide

365

adsorption, minimum interference to enzymatic saccharification and high adsorptivity to furfural,

366

5-HMF and lignin monomers can be emphasized. In addition, one-pot SSCF process coupled

367

with the novel lignin-derived adsorbent using Z. mobilis was successfully constructed, and

368

ethanol was produced at 54 g/L. This new self-sufficient bioethanol system has a lot of

369

advantages, such as no waste material, no purchase adsorbents, simplification and cost reduction

370

of facilities, recyclable, energy recovery from soluble inhibitors, decrease load of waste water.

371

The lignin-derived adsorbent and one-pot SSCF can be potentially applied to various

372

fermentation processes of lignocellulosics, depending on the tolerance of microorganisms to

373

remaining fermentation inhibitors. 17

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

374 375

FIGURES:

376 377 378 379 380 381

Figure 1. Chemical structures of fermentation inhibitors.

382

0.6

10

0.4

5

0.2

0

389 390 391 392 393 394 395 396 397 398

0.0 0

24 48 Time (h)

25

1.0

20

0.8

15

0.6

10

0.4

5

0.2

0

72

24 48 Time (h)

72

(D)

25

2.0

20

1.6

15

1.2

10

0.8

5

0.4

0

0.0 0

0.0 0

(C)

Growth (OD660nm)

15

Sugars and ethanol (g/l)

388

0.8

Sugars and ethanol (g/l)

387

20

Growth (OD660nm)

386

1.0

Growth (OD660nm)

385

Sugars and ethanol (g/l)

384

(B)

(A) 25

24 48 Time (h)

:Glucose

25

1.0

20

0.8

15

0.6

10

0.4

5

0.2 0.0

0 0

72

:Xylose

:EtOH

399

18

ACS Paragon Plus Environment

24 48 Time (h)

72

:Growth(OD)

Growth (OD660nm)

383

Sugars and ethanol (g/l)

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 18 of 33

Page 19 of 33

400

Figure 2. Ethanol fermentation of the filtrate from the microwave-pretreated E. globulus using Z.

401

mobilis (A) and (B): Filtrate without detoxification was used; (C) and (D): Filtrate after

402

processing with lignin-derived adsorbent was used; (A) and (C): The filtrate was mixed with RM

403

medium at a ratio of 8:2; (B) and (D): The filtrate was mixed with RM medium at a ratio of 5:5.

404

Diamond and closed circle stand for glucose and xylose concentration. Closed triangle and

405

square show ethanol concentration and bacterial growth expressed by OD 660 nm. The lignin

406

adsorbent for (C) and (D) was prepared at 350 °C for 2 h under O2 atmosphere.

407

409 410 411 412 413 414 415 416

25

2.0

20

1.6

15

1.2

10

0.8

5

0.4

0

0.0 0

417

24

48

Growth (OD660nm)

408

Sugars and ethanol (g/l)

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

72

Time (h)

418 419

:Glucose

:Xylose

:EtOH

:Growth(OD)

420 421

Figure 3. Ethanol fermentation of the filtrate from the microwave-pretreated E. globulus using Z.

422

mobilis after the treatment with the recycled lignin adsorbent (Entry 9 in Table 1). Diamond and

423

closed circle stand for glucose and xylose concentration. Closed triangle and square show

424

ethanol concentration and bacterial growth expressed by OD 660 nm.

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

425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444

Figure 4. Bioethanol production system from lignocellulosic biomass by SSCF (A)

445

Conventional process. (B) One-pot SSCF using lignin-derived adsorbent.

446 447 448 449 450

20

ACS Paragon Plus Environment

Page 20 of 33

Page 21 of 33

451 452 453

457 458 459 460

80

6

60

5.5

40

5

20

4.5

0

461

24

48 72 Time (h)

96

100

6.5

80

6

60

5.5

40

5

20

4.5

0

4 0

Sugars and ethanol (g/l)

456

6.5

pH

455

(B)

(A) 100

4 0

120

pH

454 Sugars and ethanol (g/l)

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

24

48 72 Time (h)

96

120

462

:Glucose

463

:Xylose

:EtOH

:pH

464 465

Figure 5. One-pot SSCF of the pulp and MW-filtrate obtained by microwave-pretreatment of E.

466

globulus (A) without detoxification and (B) with detoxification using the lignin-derived

467

adsorbent prepared at 350 °C for 2 h under O2 atmosphere using Z. mobilis. Closed diamond,

468

circle and triangle stand for glucose, xylose ethanol concentration. Opened square shows pH in

469

the solution.

470 471 472 473 474 475 476

21

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering

477 478 479 480

120

Prehydrolysis

481

100

483

60

SSCF

Inoculate rec Zm. mobilis

482

EtOH

50

485 486 487 488 489 490

40

80

Glc

60

30

40

20

20

10

Ethanol and xylose (g/liter)

484 Released glucose (g/liter)

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 22 of 33

491 492 493 Xyl

494 495

0

0 0

24

48

72

96

120

144

168

192

Time (hr)

496 497 498

Figure 6. One-pot SSCF coupled with prehydrolysis of microwave-pretreated E. globulus using

499

Z. mobilis and lignin-derived adsorbent.

500 501 502

22

ACS Paragon Plus Environment

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

ACS Sustainable Chemistry & Engineering

503 504 505 506 507 508

(A)

(B)

(C)

(D)

509 510 511 512 513 514 515 516 517 518 519 520

Figure 7. SEM of adsorbents prepared by thermal treatment at 350 °C for 2 h under oxygen

521

atmosphere (A, B) and nitrogen atmosphere (C, D) with 1,000 (B, D) and 10,000 (A, C)

522

magnifications.

523 524 525 526 527 528

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

Page 24 of 33

529 530

TABLES:

531

Table 1. Concentrations of fermentation inhibitors in MW-filtrate after treatment with lignin-

532

derived adsorbents and fermentability by Z. mobilis.

Entry

Adsorbent

Furfural (mM)

5-HMF (mM)

Acetic acid (%)

Vanillin (mM)

Syringaldehyde (mM)

Fermentabilityb



Original soluble Fr.

22.62

4.79

1.20

0.17

0.70

2:8 24 h

1

350°C-4h-O2

1.40

0.65

0.59

n.d.

0.01

5:5 48 h

2

350°C-2h-O2

0.83

0.58

0.71

n.d.

0.01

5:5 24 h

3

350°C-1h-O2

1.29

0.65

0.60

n.d.

0.01

5:5 24 h

4

250°C-2h-O2

1.33

0.90

0.73

0.01

0.49

n.p.

5

350°C-2h-N2

18.52

4.96

1.19

0.05

0.64

n.p.

6

250°C-2h-N2

14.11

4.22

1.16

0.02

0.37

5:5 72 h

7

Activated carbon

1.12

0.50

0.46

n.d.

n.d.

5:5 72 h

8

Ion exchange resin

0.78

0.48

0.10

n.d.

0.01

8:2 24 h

9

350°C-2h-O2 recyclea

1.07

0.53

0.54

n.d.

0.01

5:5 24 h

533 534

a

535

b

536

n.d.: Not detected.

537

n.p.: No production of bioethanol at 8:2 and 5:5 ratios.

Residual lignin was recovered after adsorption treatment, thermally processed and used. Cultivation time and ratio between the filtrate and RM medium giving ethanol yield over 90 %.

538 539 540 541 542 543 544 24

ACS Paragon Plus Environment

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

545 546

Table 2. Adsorptive removal of three lignin degradation products with adsorbents.

547

1

350°C-4h-O2a

Vanillin (mM) i 0.12

2

350°C-2h-O2b

0.28

0.37

0.36

3

350°C-1h-O2

c

0.65

0.98

0.67

250°C-2h-O2

d

13.90

18.27

3.60

e

19.31 14.46 n.d. 0.07

19.11 15.63 n.d. 0.02

20.22 14.81 0.02 0.12

Entry

4 5 6 7 8

Adsorbent

350°C-2h-N2 250°C-2h-N2f Activated carbong Ion exchange resinh

Syringaldehyde (mM) i 0.09

4-hydroxybenzaldehyde (mM) i 0.21

548 549

a

Entry 1, bEntry 2, cEntry 3, dEntry 4, eEntry 5, fEntry 6, gEntry 7 and hEntry 8 in Table 1.

550

i

Concentration of each compound in the original solution was 20 mM.

551

n.d.: Not detected.

552 553 554 555 556 557 558 559 560 561 562 563 564

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

Page 26 of 33

565 566

Table 3. Analysis of monosaccharides in MW-filtrate and xylooligosaccharide in water after

567

treatment with adsorbents.

Entry

Adsorbent

Monosaccharides (%)



MW-filtrate (No treatment)

1.60

100

1

350°C-4h-O2

a

1.47

77

2

350°C-2h-O2b

1.51

83

3

350°C-1h-O2c

1.53

82

4

250°C-2h-O2d

1.54

101

5

350°C-2h-N2e

1.54

100

6

250°C-2h-N2

f

1.60

102

7

Activated carbong

1.33

5

1.28

59

h

8

Ion exchange resin

9

350ºC-2h-O2 recyclei

568 569

a

b

c

570

n. d.: Not determined

d

Xylooligosaccharide (%)

1.56 e

f

n.d. g

Entry 1, Entry 2, Entry 3, Entry 4, Entry 5, Entry 6, Entry 7, Entry 8 and iEntry 9 given in Table 1.

571 572 573 574 575 576 577 578 579 580 581

26

ACS Paragon Plus Environment

h

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

ACS Sustainable Chemistry & Engineering

582

Table 4. Concentration of total monosaccharides after saccharification of MW-filtrate and MW-

583

filtrate under the adsorbent.

584

Entry

Adsorbent

Concentration of total monosaccharides (%)



MW-filtrate (No treatment)

1.95

2

350°C-2h-O2a

1.89

5

350°C-2h-N2b

1.85

7

Activated carbonc

1.65

10

SSCF-350°C-2h-O2

2.04

a

b

c

Entry 2, Entry 5 and Entry 7 in Table 1.

585 586 587 588

Table 5. Surface area and pore size of adsorbents. Temperature (°C)

Time (h)

Atmos -phere

Surface area (m2/g)

Pore volume 10-1000 Å (mL/g)

Pore volume < 10 Å (mL/g)

Total pore volume (mL/g)

Average pore diameter (Å)

Residual lignin







0.39

0.001/0.001



0.001

128

2

350°C-2h-O2a

350

2

Air

525

0.117/0.074

0.205

0.239

18

4

250°C-2h-O2

b

250

2

Air

26.9

0.015/0.002

0.006

0.017

25

6

250°C-2h-N2c

250

2

N2

0.15

n.d.f

n.d.f

n.d.f

126

7

Activated carbond







387g









350°C-2h-O2 recycle e

350

1

Air

565

0.131/0.092

0.219

0.261

18

Entry

Adsorbent



9

589

a

b

c

d

e

Entry 2, Entry 4, Entry 5, Entry 7 and Entry 9 given in Table 1.

590

f

Unmeasurable due to production of high amount of tar.

591

g

Value from the reference 31.

592 593 594 595

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

596

ASSOCIATED CONTENT:

597

Supporting Information. SEM images of the recycled adsorbents prepared by thermal treatment

598

of at 350 °C for 1 h under oxygen atmosphere (Figure S1).

599 600

AUTHOR INFORMATION:

601

Corresponding Author

602

E-mail: [email protected]; Tel: +81-774-38-3644; Fax: +81-774-38-3681

603 604

Present Addresses

605

†K. Y: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University,

606

Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.

607 608

Notes

609

The authors declare no competing financial interest.

610 611

ACKNOWLEDGMENT:

612

A part of this work was supported by New Energy and Industrial Technology Development

613

Organisation (NEDO) and Collaborative Research Program of RISH, Kyoto University. The

614

authors acknowledge Dr. Sensho Honma for preparing the adsorbent under N2 atmosphere and

615

Toyota motors for collaboration in the NEDO bioethanol project. The authors extend their

616

gratitude to Mr. Masakazu Kaneko, Mr. Masashi Tomita, Mr. Yosuke Kurosaki, Mrs. Yukari 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

617

Takahashi and Ms. Ai Yamada from RISH, Kyoto University for technical and scientific

618

discussion and support.

619 620

ABBREVIATIONS:

621

SHF, separate hydrolysis and fermentation; SSCF, simultaneous saccharification and co-

622

fermentation; 5-HMF, 5-hydroxymethylfurfural; HPLC, high performance liquid

623

chromatography;

624 625

REFERENCES:

626

(1) Masnadi, M. S.; Brandt, A. R. Climate impacts of oil extraction increase significantly with

627

oilfield age. Nat. Climate Change, 2017, 7, 551–556.

628

(2) Jin, M.; Gunawan, C.; Uppugundla, N.; Balanab, V.; Dale, B. E. A novel integrated

629

biological process for cellulosic ethanol production featuring high ethanol productivity, enzyme

630

recycling and yeast cells reuse. Energy Environ. Sci. 2012, 5, 7168–7175.

631

(3) Conde-Mejía, C.; Jiménez-Gutiérrez, A.; El-Halwagi, M. M. Assessment of Combinations

632

between Pretreatment and Conversion Configurations for Bioethanol Production. ACS

633

Sustainable Chem. Eng. 2013, 1 (8), 956−965.

634

(4) Verma, P.; Watanabe, T.; Honda, Y.; Watanabe, T. Microwave-assisted pretreatment of

635

woody biomass with ammonium molybdate activated by H2O2. Bioresour. Technol. 2011, 102

636

(4), 3941–3945.

637

(5) Liu, R. J.; Takada. R.; Karita, S.; Watanabe, T.; Honda, Y.; Watanabe, T. Microwave-

638

assisted pretreatment of recalcitrant softwood in aqueous glycerol. Bioresour. Technol. 2010,

639

101 (23), 9355–9360.

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

640

(6) Yanase, H.; Miyawaki, H.; Sakurai, M.; Kawakami, A.; Matsumoto, M.; Haga, K. Kojima,

641

M; Okamoto, K. Ethanol production from wood hydrolysate using genetically engineered

642

Zymomonas mobilis. Appl. Microbiol. Biotechnol. 2012, 94 (6), 1667–1678.

643

(7) Palmqvist, E.; Hahn-Hägerdal, B. Fermentation of lignocellulosic hydrolysates. I: inhibition

644

and detoxification. Bioresour. Technol. 2000, 74 (1), 17–24.

645

(8) Palmqvist, E.; Hahn-Hägerdal, B. Fermentation of lignocellulosic hydrolysates. II: inhibitors

646

and mechanisms of inhibition. Bioresour. Technol. 2000, 74 (1), 25–33.

647

(9) Jönsson, L. J.; Alriksson, B.; Nilvebrant, N. O. Bioconversion of lignocellulose: inhibitors

648

and detoxification. Biotechnol. Biofuels 2013, 6 (1), 16–25.

649

(10) Sainio, T.; Turku, I.; Heinonen, J. Adsorptive removal of fermentation inhibitors from

650

concentrated acid hydrolyzates of lignocellulosic biomass. Bioresour. Technol. 2011, 102 (10),

651

6048–6057.

652

(11) Carter, B.; Gilcrease, P. C.; Menkhaus, T. J. Removal and recovery of furfural, 5-

653

hydroxymethylfurfural, and acetic acid from aqueous solutions using a soluble polyelectrolyte.

654

Biotech. Bioeng. 2011, 108 (9), 2046–2052.

655

(12) Zhang, K.; Agrawal, M.; Harper, J.; Chen, R.; Koros, W. J. Removal of the Fermentation

656

Inhibitor, Furfural, Using Activated Carbon in Cellulosic-Ethanol Production. Ind. Eng. Chem.

657

Res. 2011, 50 (24), 14055–14060.

658

(13) Ranjan, R.; Thust, S.; Gounaris, C. E.; Woo, M.; Floudas, C. A.; von Keitz, M.; Valentas,

659

K. J.; Wei, J.; Tsapatsis, M. Adsorption of fermentation inhibitors from lignocellulosic biomass

660

hydrolyzates for improved ethanol yield and value-added product recovery. Micropor. Mesopor.

661

Mater. 2009, 122 (1), 143–148.

662

(14) Nilvebrant, N.-O.; Reimann, A.; Larsson, S.; Jönsson, L. J. Detoxification of lignocellulose

663

hydrolysates with ion-exchange resins. Appl. Biochem. Biotechnol. 2001, 91 (1), 35–49.

664

(15) Pienkos, P. T.; Zhang, M. Role of pretreatment and conditioning processes on toxicity of

665

lignocellulosic biomass hydrolysates. Cellulose 2009, 16 (4), 743–762.

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

666

(16) Parawira, W.; Tekere, M. Biotechnological strategies to overcome inhibitors in

667

lignocellulose hydrolysates for ethanol production: review. Crit. Rev. Biotechnol. 2011, 31 (1),

668

20–31.

669

(17) Cantarella, M.; Cantarella, L.; Gallifuoco, A.; Spera, A.; Alfani, F. Comparison of different

670

detoxification methods for steam-exploded poplar wood as a substrate for the bioproduction of

671

ethanol in SHF and SSF. Proc. Biochem. 2004, 39 (11), 1533–1542.

672

(18) Xiros, C.; Olsson, L. Comparison of strategies to overcome the inhibitory effects in high-

673

gravity fermentation of lignocellulosic hydrolysates. Biomass Bioenerg. 2014, 65, 79–90.

674

(19) Alriksson, B.; Cavka, A.; Jonsson, L. J. Improving the fermentability of enzymatic

675

hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents.

676

Bioresour. Technol. 2011, 102 (2), 1254–1263.

677

(20) Cavka, A.; Alriksson, B.; Ahnlund, M.; Jönsson, L. J. Effect of sulfur oxyanions on

678

lignocellulose-derived fermentation inhibitors. Biotechnol. Bioeng. 2011, 108 (11), 2592–2599.

679

(21) Wise, L. E.; Murphy, M.; D’Addieco, A. A. Chlorite holocellulose, its fractionation and

680

bearing on summative wood analysis and on studies on the hemicelluloses. Pap. Trade J. 1946,

681

122 (2), 35–43.

682

(22) T203 om-93. Alpha-, beta- and gamma-cellulose in pulp. In TAPPI Standard Method;

683

TAPPI: Atlanta, GA, 1993.

684

(23) T222 om-98. Acid-insoluble lignin in wood and pulp. In TAPPI Standard Method; TAPPI:

685

Atlanta, GA, 1998–1999.

686

(24) Hasegawa, N.; Mitani, T.; Shinohara, N.; Daidai, M.; Katsura, Y.; Sego, H.; Watanabe, T.

687

Pilot-plant scale 12 kW microwave irradiation reactor for woody biomass pretreatment. IEICE

688

Trans. Electronics 2014, E97-C (1), 986–993.

689

(25) H. Mikami and Y. Ishida, Bunseki Kagaku, 1983, 32, E207–E210.

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

690

(26) Dubois, M.; Gilles, K.; Hamilton, J.K.; Rebers, P.A.; Smith, F. A Colorimetric method for

691

the determination of sugars. Nature 1951, 168, 167.

692

(27) Biniak, S.; Szymański, G. S.; Siedlewski, J.; Światkowski, A. The characterization of

693

activated carbons with oxygen and nitrogen surface groups. Carbon 1997, 35 (12), 1799–1810.

694

(28) Pastor-Villegas, J.; Pastor-Valle, J.F.; Rodríguez, J. M. M. García, M. M. Study of

695

commercial wood charcoals for the preparation of carbon adsorbents. J. Anal. Appl. Pyrolysis

696

2006, 76 (1–2), 103–108.

697

(29) Biniak, S.; Pakuła, M.; Szymański, G. S. Światkowski, A. effect of activated carbon surface

698

oxygen-and/or nitrogen-containing groups on adsorption of Copper (II) ions from aqueous

699

solution. Langmuir 1999, 15 (18), 6117–6122.

700

(30) Pulido-Novicio, L.; Hata, T.; Kurimoto, Y.; Doi, S.; Ishihara, S.; Imamura, Y. Adsorption

701

capacities and related characteristics of wood charcoals carbonized using a one-step or two-step

702

process. J. Wood Sci. 2001, 47 (1), 48–57.

703

(31) Goncalves, M.; Molina-Sabio, M.; Rodriguez-Reinoso. F. Modification of activated carbon

704

hydrophobicity by pyrolysis of propene. J. Anal. Appl. Pyrolysis 2010, 89 (1), 17–21.

705

(32) Ahnert, F.; Arafat, H. A.; Pinto, N. G. A study of the influence of hydrophobicity of

706

activated carbon on the adsorption equilibrium of aromatics in non-aqueous media. Adsorption

707

2003, 9 (4), 311–313.

708

(33) Müller, E. A.; Gubbins, K. E. Molecular simulation study of hydrophilic and hydrophobic

709

behavior of activated carbon surfaces. Carbon 1998, 36 (10), 1433–1438.

710

(34) Carrott, P. J. M.; Carrott, M. M. L. R. Lignin – from natural adsorbent to activated carbon:

711

A review. Bioresour. Technol. 2007, 98 (12), 2301–2312.

712

(35) Cotoruelo, L. M.; Marqués, M. D.; Díaz, F. J.; Rodríguez-Mirasol, J.; Rodríguez, J. J.;

713

Cordero, T. Lignin-based activated carbons as adsorbents for crystal violet removal from

714

aqueous solutions. Environ. Prog. Sustain. Energy 2012, 31 (3), 386–396.

32

ACS Paragon Plus Environment

Page 32 of 33

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

715 716

For Table of Contents Use Only:

717

Synopsis: A new process suppressing the fermentation inhibition using the residual lignin from

718

the bioethanol production system has been developed.

719 720

Content graphic:

721 722 723 724 725 726

33

ACS Paragon Plus Environment