Enzymatic Hydrolysis of Steam-Treated Sugarcane ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Enzymatic hydrolysis of steam-treated sugarcane bagasse: effect of enzyme loading and substrate total solids on its fractal kinetic modeling and rheological properties Douglas Henrique Fockink, Mateus Barbian Urio, Jorge Hernan Sanchez Toro, and Luiz Pereira Ramos Energy Fuels, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 22, 2017

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.

Energy & Fuels 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 38

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

Energy & Fuels

1

Enzymatic hydrolysis of steam-treated sugarcane bagasse: effect of enzyme

2

loading and substrate total solids on its fractal kinetic modeling and rheological

3

properties

4 5

Douglas H. Fockink1; Mateus B. Urio1; Jorge H. Sánchez2; Luiz P. Ramos1,3*

6 7

1

8

University of Paraná - UFPR, P. O. Box 1908 – Curitiba, PR – 81531-980 – Brazil

9

2

Research Center in Applied Chemistry (CEPESQ), Department of Chemistry, Federal

Pulp and Paper Research Group, Department of Chemical Engineering, Universidad

10

Pontificia Bolivariana – UPB, P.O. Box 56006 – Medellín – Colombia

11

3

INCT Energy & Environment (INCT E&A), Federal University of Paraná, Curitiba, Brazil

12 13

ABSTRACT

14

Rheological studies and fractal kinetic modeling were applied to investigate the enzymatic

15

hydrolysis of steam-exploded sugarcane bagasse (195°C, 7.5 min) using Cellic CTec3

16

cellulases (Novozymes). Initially, a central composite rotatable design (CCRD) was

17

performed to evaluate the effect of different enzyme loadings and substrate total solids (TS)

18

on both substrate apparent viscosity and kinetic parameters of enzymatic hydrolysis.

19

Hydrolysis at 20% TS for 12 and 96 h using 38.6 FPU g-1 glucan released 52 and 110 g L-1

20

glucose equivalents from the steam-exploded material, respectively, with cellobiose being

21

always below 1.7% of these readings. Fractal kinetic modeling provided a good fit of both

22

glucan and xylan conversions and the fractal kinetic parameters k and h had a strong

23

correlation with changes in both substrate TS and enzyme loading. At the center point of the

24

CCRD, Cellic CTec3 caused a decrease of one order of magnitude in the substrate apparent

1 ACS Paragon Plus Environment

Energy & Fuels

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

25

viscosity at every 6 h of hydrolysis. Cellic HTec3 had a boosting effect on the enzymatic

26

hydrolysis of cane bagasse glucans regardless of the low hemicellulose content of the steam-

27

treated material. Glucan hydrolysis was improved by 8% when 10% Cellic HTec3 was

28

added to a hydrolysis mixture containing Cellic CTec3 at 38.6 FPU g-1 glucan. With this, a

29

total production of 120 g L-1 glucose was achieved at 72 h using 20% TS.

30 31

Key-words: Sugarcane bagasse, enzymatic hydrolysis, apparent viscosity, fractal kinetics,

32

glucose yield.

33 34 35

1. INTRODUCTION

36 37

Renewable liquid fuels such as ethanol are recognized as sustainable alternatives to

38

overcome the negative environmental impact of fossil fuels in the transportation sector.

39

However, most of the fuel ethanol produced to date comes from sucrose or starch and these

40

primary feedstocks may not meet the sustainable criteria of modern biorefineries as well as

41

the rising demand for this biofuel worldwide. Ethanol can also be produced from

42

lignocellulosic materials but their highly ordered and tightly packed microfibrillar structure

43

is very hard to access. Therefore, a pretreatment method is needed to improve the

44

accessibility of both glucans (mostly cellulose) and hemicelluloses to enzymatic hydrolysis,

45

this without releasing substantial amounts of inhibitory compounds for the subsequent step

46

of fermentation. Besides, the lignin type, content and distribution also represent additional

47

barriers affecting the enzymatic hydrolysis of lignocellulosic materials.1 For these and other

48

reasons, pretreatment is still considered one of the most challenging steps for the optimal

49

performance of cellulosic ethanol production.2, 3 2 ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

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

Energy & Fuels

50

Different pretreatment strategies have been developed to date for a wide variety of

51

lignocellulosic materials.4 Among these, steam explosion is recognized as one of the most

52

efficient for hardwoods and agricultural residues and its selective fractionation is the core

53

technology for several cellulosic ethanol production facilities available worldwide.5-7 In this

54

process, lignocellulosic materials, with or without pre-impregnation with an exogenous

55

catalyst, are exposed to pressurized steam followed by a rapid decompression that results in

56

the explosive breakdown of the plant cell wall structure,8 promoting hemicellulose

57

depolymerization (mainly converted to water soluble oligomers) and structural changes in

58

lignin due to the use of relatively high pretreatment temperatures.9

59

Enzymatic hydrolysis of pretreated materials is also a key step towards the efficient

60

conversion of biomass into fuels and chemicals.2 However, technological improvements are

61

still needed to make the process economically viable.10 A potential route to reduce both

62

capital and production costs is to perform hydrolysis at high total solids (TS > 15%) with the

63

aims of lowering the use of water and increasing the sugar concentration in the liquid

64

stream. Economic analyses suggested that the concentration of fermentable sugars in

65

biomass hydrolysates must be high enough to produce ethanol concentrations above 4 wt.%.

66

This way, the energy consumption in the distillation step is reduced considerably and the

67

economics of the overall production process is greatly improved.11

68

Processing at high total solids (TS) is not an easy task since pretreated biomass

69

slurries are highly viscous and exhibit strong non-Newtonian flow properties.12-14 The low

70

availability of free water in these reaction systems introduces mechanical challenges that are

71

related to mixing and pumping,15 creating heat and mass transfer limitations that hinder the

72

optimal performance and uniform distribution of the enzymes, respectively.16 In addition,

73

soluble sugars rapidly accumulate in the reaction medium at high substrate TS, decreasing


Energy & Fuels

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

74

the water mobility and causing enzyme inhibition at the early stages of hydrolysis.14, 17, 18 As

75

a results, kinetic models for homogeneous reaction systems that are based on the Fick's law

76

of diffusion do not apply to the enzymatic hydrolysis of cellulose.19 By contrast, fractal

77

kinetics has been successfully used to fit such experimental data 20-22 but its application to

78

enzymatic hydrolysis with advanced cellulase systems such as Cellic® CTec3 (Novozymes)

79

at various substrate TS and enzyme loadings has not been reported as yet.

80

In the present study, sugarcane bagasse was pretreated by steam explosion at reaction

81

conditions that were pre-optimized by Pitarelo et al. 23 for optimal glucan recovery and

82

enzymatic hydrolysis. Then, a central composite rotatable design (CCRD) was employed to

83

evaluate the effect of different enzyme loadings and substrate TS on changes in both

84

substrate apparent viscosity and glucan conversion. The apparent viscosity was monitored

85

during the first 24 h of hydrolysis using different substrate TS and Cellic CTec3 enzyme

86

loadings. In addition, the fractal kinetic model was applied to fit the hydrolysis data and to

87

establish a correlation between the fractal kinetic parameters h and k and the susceptibility of

88

cane bagasse polysaccharides to enzymatic hydrolysis. The influence of Cellic HTec3

89

hemicellulases was also evaluated in order to assess their synergism with Cellic CTec3 in

90

the hydrolysis of steam-treated substrates.

91 92

2. MATERIAL AND METHODS

93 94 95

2.1. Material Sugarcane bagasse was obtained from the São Martinho Mill (São Paulo, SP, Brazil)

96

with the logistical support of the Cane Technology Center - CTC (Piracicaba, SP, Brazil).

97

This sample was considered representative because it was collected directly from an

98

industrial site. Besides, little variance is observed in the compositional analysis of industrial 4 ACS Paragon Plus Environment

Page 4 of 38

Page 5 of 38

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

Energy & Fuels

99

samples of cane bagasse when suitable analytical methods are used for characterization.24, 25

100

The commercial enzymes used for hydrolysis (Cellic CTec3 and Cellic HTec3) were kindly

101

donated by Novozymes Latin America (Araucária, PR, Brazil).

102 103 104

2.2. Sugarcane bagasse pretreatment and chemical characterization Pretreatment was performed in a 10-L high pressure batch steam reactor at 195 °C

105

for 7.5 min using sugarcane bagasse with a final moisture content of 50 wt.%. Water-

106

washing was applied on the resulting pretreated material to remove water-soluble

107

hemicellulose and lignin components.23

108

The composition of both untreated and treated materials was characterized following

109

the National Renewable Energy Laboratory (NREL) standard procedures. The total

110

moisture, ash and total extractive contents were determined according to NREL/TP-510-

111

42621, 26 NREL/TP-510-42622 27 and NREL/TP-510-42619 28, respectively. Carbohydrate

112

and total lignin contents (acid-soluble and acid-insoluble lignin) were determined as

113

recommended by NREL/TP-510-42618.29 Substrate acid hydrolysates were analyzed by

114

high performance liquid chromatography (Shimadzu HPLC, LC-20AD series) using an

115

Agilent Hi-Plex H column (300 x 7.7 mm) with its corresponding guard column (50 x 7.7

116

mm) at 65 °C. Column elution was performed with 5 mmol L-1 H2SO4 at a flow rate of 0.6

117

mL min-1. Sample injection (20 µL) was carried out automatically using a Shimadzu SIL-

118

10AF autosampler. Quantitative analysis was carried out by external calibration using

119

differential refractometry (Shimadzu RID-10A) for detecting cellobiose, glucose, xylose,

120

arabinose and acetic acid, and UV spectrophotometry (Shimadzu SPD-M10AVP) at 280 nm

121

for detecting furfural and hydroxymethylfurfural. Each of these analytes was subsequently

122

converted to their original polysaccharide component by taking into account their


Energy & Fuels

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

123

corresponding anhydrous mass correction factors.

124 125 126

2.3 Enzymatic hydrolysis Enzymatic hydrolysis of steam-exploded cane bagasse was initially investigated

127

through a central composite rotatable design (CCRD) that involved four experiments at the

128

vertices and four experiments at the axial points of a 22 factorial design plus three replicates

129

at its center point for a total of 11 experiments. Enzyme loadings (Cellic CTec3) of 7.7 and

130

38.6 FPU g-1 glucan and substrate TS of 10 and 20% (w v-1) were adopted in the vertices of

131

this factorial design as shown in Table 1 (Design A). Statistical analysis was carried out with

132

the software Statistica 8.0 and the proposed CCRD was used to generate models that were

133

able to describe changes in the fractal kinetic parameters k and h for both glucan and xylan

134

conversions (see below for details).

135 136

Table 1

137 138

Total cellulase activity measured as filter paper units (FPU) was determined by the

139

I.U.P.A.C. method 30 with the adaptations proposed by Schwald et al. 31. All hydrolysis

140

experiments were carried out at 50 °C and 150 rpm in 125 mL Erlenmeyer flasks using 50

141

mmol L-1 sodium acetate buffer (pH 5.2). Aliquots were collected in reaction times of 0, 3,

142

6, 9, 12, 24, 48, 72 and 96 h, centrifuged at 10,000 g and analyzed in the same HPLC system

143

mentioned above. External calibration was used for the quantitative analysis of cellobiose,

144

glucose and xylose; however, cellobiose and glucose concentrations were accounted

145

together as glucose equivalents ([GlcEq] = [glucose] + 1.0526 [cellobiose]). Glucan

146

conversion was calculated by expressing GlcEq release in relation to the amount of glucans


Page 6 of 38

Page 7 of 38

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

Energy & Fuels

147

(mostly cellulose) that was present in the original steam-exploded material. However, small

148

volume variations in the substrate hydrolysates were not taken into account and, for this

149

reason, the resulting glucan conversions might have been a little overestimated.

150

Another simple 22 experimental design (two variables in two levels plus three

151

replicates at the center point) was carried out to evaluate the influence of Cellic HTec3

152

hemicelullases on hydrolysis efficiency (Table 1, Design B). These experiments were also

153

carried out in a rotary shaker incubator at 20% (w v-1) TS using 50 mmol L-1 sodium acetate

154

buffer (pH 5.2) at 50 °C and 150 rpm. Aliquots were collected in 12, 24, 48, 72 and 96 h,

155

centrifuged at 10,000 g and analyzed by HPLC. Quantitative analysis of cellobiose, glucose

156

and xylose was carried out by external calibration.

157 158 159 160

2.4 Fractal kinetics modeling The release of sugars (GlcEq and xylose) were fitted by fractal kinetics 20 using the pseudo first-order reaction equation originally proposed by Kopelman 32 (Equation 1),

161 162

P(t) = S0[1-exp(-k · t1-h)]

(1)

163 164

were P(t) is the product concentration (g L-1), S0 is the initial concentration of glucan or

165

xylan (represented as glucose or xylose in g L-1), k is the time dependent rate coefficient, h is

166

the fractal exponent, and t is the reaction time (h).

167 168 169 170

2.5 Apparent viscosity measurements Rheological measurements were performed in a stress/shear-rate controlled DHR-2 rheometer (TA instruments) using a Peltier concentric cylinder system. The geometry


Energy & Fuels

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

171

consisted of a four-bladed vane rotor, 42 mm in height and 28 mm in diameter, placed in a

172

30 mm diameter cup containing the sample, which resulted in a narrow gap. The vane

173

shaped rotor was used to reduce wall slip. The temperature was controlled and all

174

rheological experiments were carried out at the same temperature used for enzymatic

175

hydrolysis, i.e., 50 °C. In order to minimize evaporation, the cup was covered with a solvent

176

trap.

177

Enzymatic hydrolyses were carried out in shake-flasks under the selected conditions

178

of the CCRD (Table 1, Design A). The reaction was stopped after 3, 6, 9, 12 and 24 h of

179

hydrolysis and the suspension was transferred immediately to the rheometer for measuring

180

changes in its rheological properties. To obtain consistent rheological data and to eliminate

181

thixotropic effects after loading the sample into the measuring device, all suspensions were

182

pre-sheared to rupture any fiber bundles, followed by a relaxation time for the partial

183

recovery their fiber structure. Steady-state flow curves were obtained by a logarithmic shear

184

rate sweep of 1-1000 s-1, from which the apparent viscosity of the fiber suspension was

185

obtained.

186 187

3. RESULTS AND DISCUSSION

188 189 190

3.1. Chemical characterization of sugarcane bagasse Both untreated and pretreated sugarcane bagasse were characterized for their

191

chemical composition (Table 2) and these values were used to calculate both pretreatment

192

and hydrolysis yields. Both glucan (measured as anhydroglucose) and lignin contents were

193

higher in the steam-treated materials as a result of the high susceptibility of hemicelluloses

194

to acid hydrolysis. The xylan content decreased from 16.7 wt.% in the native material to 3.4

195

wt.% after pretreatment. Also, acetyl groups and arabinose were not detectable in the 8 ACS Paragon Plus Environment

Page 8 of 38

Page 9 of 38

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

Energy & Fuels

196

pretreated cane bagasse acid hydrolysates.

197 198

Table 2

199 200

Similar carbohydrate and lignin contents were observed in other studies involving

201

steam explosion of sugarcane bagasse, even when more drastic pretreatment conditions were

202

applied. Ewanick and Bura 33 carried out pretreatment at 205 °C for 10 min to produce

203

substrates with glucan, hemicellulose and total lignin contents of 51.3, 3.6 and 33.9 wt.%,

204

respectively. Rocha et al. 34 pretreated sugarcane bagasse for 15 min at 190 °C and obtained

205

substrates with 58.0, 4.5 and 33.0 wt.% of glucan, hemicellulose and total lignin contents,

206

respectively. Amores et al. 35 found similar glucan (58.1 wt.%) and lignin (28.5 wt.%)

207

contents in pretreated samples that were steam-exploded at 200 °C for 5 min but their

208

hemicellulose content (14.0 wt.%) was much higher; however, the raw material used for

209

pretreatment had a high hemicellulose content as well (25.0 wt.%). Sugar losses after acid

210

hydrolysis have been attributed to pentose and hexose dehydration under acidic conditions to

211

form furfural and hydroxymethylfurfural (HMF), respectively. In cane bagasse acid

212

hydrolysates, HMF derives from hexoses such as glucose and galactose whereas furfural

213

comes from pentoses such as xylose and arabinose. On the basis of this clear uncertainty in

214

their origin, the quantification of furfural and HMF in acid hydrolysates was indicated in

215

Table 2 as unidentified pentoses and hexoses, respectively.36

216

The mass recovery yield after steam explosion was 86.2 wt.%, being 65.5 wt.% in

217

the water-insoluble fraction and 20.7 wt.% in the water-soluble fraction. The mass losses

218

observed during pretreatment (13.8 wt.%) were attributed to the volatilization of extractives

219

and byproducts (furfural, HMF and acetic acid) that are hard to recover from the high


Energy & Fuels

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

220

pressure steam reactor.37, 38 Also, furan compounds derived from sugar dehydration and

221

phenolic compounds derived from lignin may be partially involved in condensation

222

reactions that lead to the accumulation of acid-insoluble polymeric materials.39 Nevertheless,

223

a total glucan recovery of 98.9 wt.% was obtained in this study, with most of it being found

224

in the water-insoluble fraction (95.8 wt.%).

225 226

3.2. Enzymatic hydrolysis of steam-treated cane bagasse

227

Figure 1 shows the enzymatic hydrolysis profiles of steam-exploded materials that

228

were derived from the proposed experimental conditions of the CCRD. The largest glucan

229

conversions in 48 h of hydrolysis were obtained from experiments A7, A9 and A10 (Figure

230

1a), which corresponded to 57.5, 85.5 and 44.6 g L-1 GlcEq, respectively (Figure 1b).

231

Cellobiose was always a minor component in GlcEq measurements. However, cellobiose

232

accumulation increased with time in all hydrolysis runs, reaching the highest level when

233

high enzyme loadings were used at high levels of substrate TS. In average, cellobiose

234

accounted for 1.7 ± 0.9% of GlcEq for all hydrolysis data involved in the CCRD (additional

235

data are given in Table S1 and S2 of the Supporting Information) but, for experiments

236

carried out at the highest substrate TS and enzyme loadings, cellobiose exceeded the IC50

237

inhibitory levels proposed by Teugjas and Väljamäe 39 for Trichoderma reesei

238

cellobiohydrolases (2.6 mmol L-1 or 0.9 g L-1). Although not necessarily applicable to this

239

study, this information suggests that Cellic CTec3 may need some β-glucosidase

240

supplementation to avoid end-product inhibition, particularly at longer reaction times.

241 242

Figure 1

243


Page 10 of 38

Page 11 of 38

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

Energy & Fuels

244

A good correlation between substrate TS and both glucan conversion [Conversion

245

(%) = 0.098 TS2 – 5.157 TS + 115.440; R2 > 0.99] and glucose release [Glc (g L-1) = -0.044

246

TS2 + 3.234 TS + 15.570; R2 > 0.99] was observed in experiments A3, A4, A5 (center

247

point), A10 and A11, in which the same enzyme loading of 23.1 FPU g-1 glucan was applied

248

for hydrolysis. In general, the observed decrease in glucan conversion at high TS was

249

attributed to poor mass and heat transfers in viscous slurries, which is connected to the lack

250

of free water in the reaction environment.41 Likewise, the applied enzyme loading (EL)

251

correlated well with glucan conversion [Conversion (%) = - 0.028 EL2 + 2.951 EL + 7.154,

252

R2 > 0.99] and glucose release [Glc (g L-1) = - 0.026 EL2 + 2.657 EL + 6.431; R2 > 0.99] for

253

experiments carried out at 15% TS with different enzyme loadings of 1.4 (A8), 23.1 (A3, 4,

254

5) and 44.9 (A9) FPU g-1 glucan. The best fit of the experimental data was always obtained

255

through a second order polynomial regression, already revealing the non-linear correlation

256

that exists among the experimental data. Nevertheless, the observed differences in glucan

257

conversion (in %) and glucose release (in g L-1) were strongly related to the

258

enzyme/substrate ratio that was used for hydrolysis. Hydrolysis under different conditions

259

using almost the same enzyme/substrate ratio, as observed in experiments A9 (44.9 FPU g-1

260

glucan at 15% TS) and A10 (23.1 FPU g-1 glucan at 8% TS), resulted in similar patterns of

261

glucan conversion but completely different glucose releases. In addition, experiments at the

262

highest enzyme/substrate ratio (A7) resulted in both the fastest initial hydrolysis rates

263

(Figure 1) and the lowest apparent viscosity observed in this study (Table 4).

264

In general, the amount of glucose released at 10 and 15% TS in 48 h (A7 and A9,

265

respectively) was higher than those obtained from liquid hot water pretreated olive tree

266

prunings at 20% (w v-1) TS using 65.8 FPU g-1 glucan, which produced 52 g L-1 glucose in

267

72 h of enzymatic hydrolysis.16 On the other hand, Rosgaard et al. 42 obtained similar sugar


Energy & Fuels

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

268

concentrations (78 g L-1) from steam-treated barley straw at 15% (w w-1) TS using 12.9 FPU

269

g-1 glucan but only after 72 h of hydrolysis.

270

Experiments A1 and A8 had the lowest glucan conversion due the use of low enzyme

271

loadings at high TS. All other conditions achieved glucan conversions of approximately

272

70% in 48 h (Figure 1a). According to Arantes and Saddler 1, when 50-70% of the original

273

material has been hydrolyzed, a decrease in the reaction rate is observed due the

274

accumulation of adverse factors such as the higher recalcitrance of the remaining cellulosic

275

material, high levels of end-product inhibition, and enzyme losses due to their irreversible

276

and/or unproductive adsorption onto lignin-carbohydrate fragments, which is aggravated by

277

the build-up of lignin in the reaction mixture. By contrast, trends of decreasing viscosities at

278

longer reaction times are a result of the collapse of the cell wall structure, which also

279

involves a decrease in cellulose degree of polymerization and an increase in fiber porosity.42

280

Hydrolysis at 20% TS using 38.6 FPU g-1 glucan (A2) resulted in the highest GlcEq

281

release of 110 g L-1 after 96 h of hydrolysis. However, around 52 g L-1 GlcEq was already

282

achieved in 12 h in several experiments including A2 (Figure 1b). While 110 g L-1 is

283

promising for separate hydrolysis and fermentation, 52 g L-1 in 12 h seems to be very good

284

for fed-batch hydrolysis or simultaneous hydrolysis and fermentation.

285

Xylan hydrolysis profiles were similar to those of glucans, with higher conversions

286

being achieved in experiments A7, A9 and A10 (Figure 2a). The highest xylose

287

concentration of 6.6 g L-1 was obtained in A2 and this corresponded to an 85% conversion at

288

96 h of hydrolysis (Figure 2b). In general, xylans are more accessible to enzymatic

289

hydrolysis than glucans such as cellulose. However, when embedded in the structure of

290

pretreated cellulosic materials, xylans are not immediately available for hydrolysis but they

291

are gradually exposed to the concerted action of the enzymes as glucan hydrolysis goes on.


Page 12 of 38

Page 13 of 38

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

Energy & Fuels

292

Therefore, xylans are present in the chemical composition of partially hydrolysed cellulosic

293

materials even after most of the cellulose component had been hydrolysed to soluble sugars.

294 295

Figure 2

296 297

The fractal kinetic parameters k and h as well as the R2 values for the regression

298

models of Figures 1 and 2 are given in Table 3. The high R2 values for all hydrolysis

299

experiments reveal the ability of the fractal kinetic model to describe both glucan and xylan

300

conversions as a function of time; therefore, both k and h were analyzed in relation to the

301

process variables using second-order model equations and ANOVA to evaluate the

302

adequacy of the fitted models (Table S3 of the Supporting Information). The pure error,

303

calculated from the experimental error at the center point of the experimental design, ranged

304

from 0.1 to 2.8% according to the total sum of squares, indicating the good reproducibility

305

of the experimental data. Based on the F-test, the resulting models are predictive since the

306

calculated F-value for all responses are greater than the corresponding tabulated F-value

307

within the confidence level of 95%. Also, the high R2 values demonstrate that most of the

308

variance was explained by the fitted models.

309 310

Table 3

311 312

Equations 2 and 3 were generated to fit the fractal kinetic parameters of glucan

313

conversion (kGlc and hGlc) while Equations 4 and 5 were used to fit the fractal kinetic

314

parameters of xylan conversion (kXyl and hXyl). The coded models expressed in these

315

equations were subsequently used to generate the response surfaces of Figures 3 and 4,


Energy & Fuels

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

316

Page 14 of 38

where EL is the enzyme loading and TS is the substrate total solids.

317 318

kGlc = 0.098 + 0.047 EL - 0.003 EL2 - 0.027 TS + 0.031 TS2 - 0.044 EL x TS

(2)

319

hGlc = 0.287 - 0.040 EL + 0.012 EL2 + 0.059 TS + 0.054 TS2 - 0.060 EL x TS

(3)

320

kXyl = 0.172 + 0.083 EL - 0.004 EL2 - 0.029 TS + 0.020 TS2 - 0.052 EL x TS

(4)

321

hXyl = 0.468 - 0.030 EL + 0.012 EL2 + 0.044 TS + 0.036 TS2 - 0.053 EL x TS

(5)

322 323

Figure 3

324

Figure 4

325 326

As shown in Figures 3a and 4a, the rate coefficient k for both glucan and xylan

327

conversion was strongly affected by changes in the applied enzyme loading (see Figure S1

328

in Supporting Information). However, this effect was more pronounced in reactions that

329

were carried out at lower substrate TS. For instance, at 10% TS (A6 and A7 in Table 1), k

330

increased from 0.083 to 0.299 h-1 for glucan conversion and from 0.105 to 0.386 h-1 for

331

xylan conversion when the enzyme loading was increased from 7.7 to 38.6 FPU g-1 glucan

332

but, at 20% TS, increased enzyme loadings (A1 and A2) gave similar k values for glucan

333

conversion (0.102 and 0.108 h-1) and slightly higher k values for xylan conversion (0.134

334

and 0.194 h-1), respectively. This was so because, at high TS, the mass transport of products

335

and enzymes is much more restricted by the low availability of water in the fiber slurry. The

336

k value decreased from 0.299 to 0.108 h-1 for glucans and from 0.386 to 0.194 h-1 for xylans

337

when the substrate TS increased from 10 to 20% at 38.6 FPU g-1 glucan (A7 and A2,

338

respectively). Also, this effect was observed in smaller proportions at 23.1 FPU g-1 glucan

339

(A10 and A11). By contrast, the negative effect of TS on k is apparently overcome at low


Page 15 of 38

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

Energy & Fuels

340

enzyme loadings of 7.7 FPU g-1 glucan (A1 and A6) and this may be associated with higher

341

substrate availability and less enzyme traffic jamming at these hydrolysis conditions. These

342

observations justify the statistical significance of both the TS quadratic term and the binary

343

interaction between TS and enzyme loading, which was also observed in Pareto Charts for

344

the k parameter (see Figure S1 in Supporting Information).

345

Wang and Feng 21 reviewed the effects of enzyme loading and non-ionic surfactants

346

on the enzymatic hydrolysis of lignocellulosic materials using fractal analysis. Changes in

347

the fractal exponent h were associated with substrate accessibility, with good substrates for

348

hydrolysis tending to present low h values.43 However, there is still a lack of studies

349

involving the correlation between h and the initial substrate content at high TS using

350

different enzyme loadings. Response surfaces for the fractal exponent h are shown in

351

Figures 3b and 4b for glucan and xylan conversions, respectively.

352

Regarding the fractal exponent h, substrate TS was the most significant effect for

353

xylan conversion and the only significant effect for glucose conversion (see Figure S1 in

354

Supporting Information). High substrate TS resulted in high fractal exponents in both cases

355

and this can be observed by comparing experiments A1 and A6, in which 7.7 FPU g-1 glucan

356

were applied at 20 and 10% TS (h values of 0.536 and 0.295, respectively), and A10 and

357

A11, in which 23.1 FPU g-1 glucan were applied at 8 and 22% TS (h values of 0.305 and

358

0.475, respectively). In addition to end-product inhibition, the accumulation of lignin in the

359

reaction mixture is higher at high TS and this may have increased the non-productive and/or

360

irreversible binding of enzymes by hydrophobic interactions. By contrast, this was not

361

observed at high enzyme loadings (A2 and A7) because the extent of enzyme adsorption on

362

lignin and lignin-carbohydrate fragments was probably not a critical barrier to achieve

363

relatively good hydrolysis performances.


Energy & Fuels

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

364

Using substrate TS not higher than 5%, some studies have shown that the fractal

365

exponent increases with an increase in enzyme loading and this has been attributed to the

366

crowding of cellulases on the cellulose surface, as well as to the formation of aggregates due

367

to protein-to-protein interactions.21, 44 This effect was also observed in experiments A6 and

368

A7 at 10% TS but the use of higher fiber slurries of 15% TS (A8, A3, 4, 5 and A9) and 20%

369

TS (A1 and A2) resulted in the opposite trend.

370

Finally, all experiments showed higher k and h values for xylan conversion when

371

compared to glucan conversion. Such high k values are due the high hemicellulase content

372

of Cellic CTec3, which is able to hydrolyse most of the hemicellulose components of the

373

steam-exploded cane bagasse. By contrast, high h values are probably expressing the low

374

accessibility of residual hemicelluloses that are embedded in and more closely bound to the

375

lignocellulosic matrix, being accessible only after glucans are gradually removed by the

376

concerted action of the cellulolytic enzymes.

377 378

3.3 Viscosity changes during enzymatic hydrolysis

379

Rheological measurements for all CCRD conditions showed that hydrolysis caused a

380

gradual decrease in substrate apparent viscosity and this was primarily associated to changes

381

in substrate TS and fiber morphology. Also, the substrate apparent viscosity decreased with

382

increasing shear rate at all times used for hydrolysis, indicating the non-Newtonian “shear-

383

thinning” behavior that is shown in Figure 5. Previous works with pretreated slurries have

384

reported similar pseudoplastic behavior.12, 14 The shear-thinning nature of the material was

385

explained by Ebeling et al. 45, who reported that the orientation of cotton cellulose

386

microcrystals is dependent on shear rate. Above a certain shear rate value, changes in

387

apparent viscosity are less pronounced because cellulose microcrystals align horizontally


Page 16 of 38

Page 17 of 38

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

Energy & Fuels

388

along the shear direction and the resistance to the flow becomes approximately constant.

389

However, this orientation phenomenon is completely reversible. On the other hand, Du et al.

390

46

391

lignocellulose, which are broken by an increase in the shear rate. Thus, the density of the

392

entanglements is reduced and the apparent viscosity decreases.

attributed this pseudoplasticity to the formation of highly entangled structures of

393 394

Figure 5

395 396

The apparent viscosity of fiber slurries also undergoes dynamic and dramatic

397

changes with time due to the progressive action of cellulolytic enzymes. Figure 5 shows that

398

the apparent viscosity is reduced just about one order of magnitude at every 6 h of

399

hydrolysis. This change is due to a loss in lignocellulose structure by the combined effects

400

of cell wall deconstruction and a gradual decrease in the average cellulose and hemicellulose

401

chain lengths.42 Various chemical bonds within the fiber suspension are hydrolyzed while

402

several components are solubilized into the liquid phase, thus reducing the contact between

403

neighboring particles.14, 47

404

Variations in viscosity at shear rates higher than 100 s-1 are probably due to a gradual

405

change from laminar to turbulent flow during the tests. For this reason, changes in apparent

406

viscosity were compared at 10 s-1 and these data are presented in Table 4 as function of

407

hydrolysis time. As expected, the initial apparent viscosities were proportional to the

408

substrate TS, providing values at 15, 20 and 22% TS that were approximately two, four and

409

five times higher than that of 10% TS slurries, respectively. For slurries of 15% TS and

410

higher, high viscosities can be due to the lower availability of free-water and greater

411

hydrogen bonding among cellulose chains. Also, as the substrate TS increases, the average


Energy & Fuels

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

412

distance between particles decreases, leading to an increase in the particle-to-particle

413

interactions.42 This phenomenon results in more friction and resistance to flow and therefore

414

to higher apparent viscosities. Furthermore, as a result of the pretreatment process, fiber

415

porosity is increased by partial hemicellulose and lignin removal, improving both the

416

accessibility of hydrolases to fiber polysaccharides and the fiber swelling by water

417

absorption, which also reduces the free-water content of the slurry.41

418 419

Table 4

420 421

Different enzyme/substrate ratios led to a considerable reduction in substrate

422

viscosity within the first 3 h of hydrolysis but the most significant changes were observed in

423

experiments where the highest enzyme loadings were used. Table 4 shows that reductions of

424

81%, 97%, and 89% were achieved for runs A2, A7 and A9, respectively. In general, higher

425

substrate viscosities were observed at lower glucan conversions but these values dropped

426

rapidly due to a combination of a gradual reduction in substrate TS and cellulose

427

fragmentation into smaller particles.

428 429

3.4 Effect of Cellic HTec3 on hydrolysis efficiency

430

According to Figures 1 and 2, glucans and xylans followed similar hydrolysis

431

profiles and the latter were present in partially hydrolysed substrates up until 96 h of

432

hydrolysis. For this reason, Cellic HTec3 hemicellulases (10% in relation to Cellic CTec3)

433

were added to the hydrolysis mixture to investigate their boosting effect on glucan

434

conversion by facilitating the removal of xylan oligomers from the surface of cellulose

435

aggregates. These experiments were carried out at 20% TS as part of a typical 22 factorial


Page 18 of 38

Page 19 of 38

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

Energy & Fuels

436

design (see conditions at Table 1, Design B). The results reported in Figure 6a were

437

statistically different for different levels of Cellic CTec3 (p < 0.05 in the ANOVA) and this

438

motivated the use of the Tukey’s test to differentiate the individual reaction responses. Until

439

48 h of hydrolysis, there were no differences (p > 0.05) in both response variables when

440

hemicellulases were added to the reaction medium (see experiments B1 and B6; B2 and B7

441

in Figure 6). However, at 72 h, significant differences (p < 0.05) were observed at the

442

highest levels of cellulase activity (see experiments B2 and B7). As a result, the use of Cellic

443

HTec3 at the highest level (10%) caused an increase of 8% in glucan conversion, which

444

corresponds to a GlcEq release of 10.5 g L-1 after 72 or 96 h of hydrolysis. It is also

445

noteworthy that, in 96 h, the combination of Cellic CTec3 and Cellic HTec3 (B3, B4 and

446

B5) produced the same glucan conversion and GlcEq release that was produced by Cellic

447

CTec3 alone at the highest level of the experimental design (B7).

448 449

Figure 6

450 451

Regardless of the hydrolysis time, all xylan conversions and xylose concentrations

452

described in Figure 6b were statistically different in the ANOVA (p < 0.05) for different

453

levels of Cellic CTec3. Likewise, at the highest levels of enzyme loading (B2 and B7),

454

Cellic HTec3 resulted in significant differences (p < 0.05) in all reaction times except 72 h,

455

providing a 9.4% higher xylan conversion and an average increase of 0.7 g L-1 in xylose

456

concentration. However, at the lowest levels of Cellic CTec3 (B1 and B6), supplementation

457

with Cellic HTec3 activity was only statistically meaningful after 96 h of hydrolysis, in

458

which an increase of 8.7% was observed in xylan conversion.

459

Cellic HTec3 had a boosting effect on the enzymatic hydrolysis of cane bagasse


Energy & Fuels

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

460

glucans regardless of the low hemicellulose content of the steam-treated material. This

461

unexpected performance is justified by the relatively high recalcitrance of the hemicellulose

462

fraction that withstood pretreatment and fractionation. Being more intimately associated

463

with cane bagasse glucans and/or lignin, these residual hemicellulose fractions are more

464

difficult to access and their hydrolysis progresses slowly as substrate glucans are gradually

465

removed.

466 467

4. CONCLUSION

468 469

Successful ethanol fermentation is only achievable if high sugar concentrations are

470

obtained upon enzymatic hydrolysis. Cellic CTec3 was able to release more than 100 g L-1

471

GlcEq from steam-treated cane bagasse in 72 h at 20% TS using 38.6 FPU g-1 glucan.

472

Biomass slurries exhibited poor rheological properties above 15% TS, however, a

473

remarkable decrease in substrate apparent viscosity was observed after 3 h of hydrolysis.

474

Fractal kinetics provided a good fit of both glucan and xylan hydrolysis data. The rate

475

coefficient k and the fractal exponent h were strongly affected by changes in the applied

476

enzyme loading and these trends were useful to predict substrate accessibility and the ideal

477

enzyme loading for optimal hydrolysis performance at high substrate TS. Finally, Cellic

478

HTec3 improved the total glucan conversion by 8% but only when a relatively high enzyme

479

loading was used for hydrolysis.

480 481

Supporting Information

482

Table S1. Release of glucose equivalents from steam-treated sugarcane bagasse as a function

483

of hydrolysis time under the conditions given in the CCRD experimental design.


Page 20 of 38

Page 21 of 38

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

Energy & Fuels

484

Table S2. Release of cellobiose from steam-treated sugarcane bagasse as a function of

485

hydrolysis time under the conditions given in the experimental design of the CCRD.

486

Table S3. Analysis of variance (ANOVA) of the fractal kinetic parameters that were derived

487

from the central composite rotatable design (CCRD, Design A).

488

Figure S1. Pareto chart describing the primary and secondary effects of different total solids

489

and enzyme loadings in: (a) parameter k (h-1) and (b) parameter h for glucan conversion; and

490

(c) parameter k (h-1) and (d) parameter h for xylan conversion.

491

Figure S2. Correlation between the observed and predicted values for the fractal parameters

492

(a) k (h-1) and (b) h for glucan conversion and (c) k (h-1) and (d) h for xylan conversion.

493

Figure S3. Apparent viscosity at 10 s-1 of steam-treated sugarcane bagasse during enzymatic

494

hydrolysis with Cellic CTec3 under the conditions given in Table 1 (CCRD).

495 496

Corresponding author

497

*E-mail: [email protected]. Telephone: +55 4133613175.

498

Note: the authors declare no competing financial interest.

499 500

Acknowledgements

501

The authors are grateful to CNPq (grants 551404/2010-8 and 311554/2011-3) and to

502

the INCT in Energy and Environment for providing financial support to carry out this study,

503

as well as to Novozymes Latin America (Araucária, PR, Brazil) for donating the enzyme

504

preparations used for hydrolysis. Also, the authors wish to thank CAPES for providing

505

scholarships to M.B.U. and D.H.F. and CYTED (Red Provalor, grant 312RT0456) for

506

funding the student exchange between UFPR and UPB.

507 508

References 21 ACS Paragon Plus Environment

Energy & Fuels

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

509

(1)

Arantes, V.; Saddler, J. N. Cellulose accessibility limits the effectiveness of

510

minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic

511

substrates. Biotechnol. Biofuels 2011, 4:3.

512

(2)

513

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

514

production. Science 2007, 315, 804-807.

515

(3)

516

economic estimates for the production cost of lignocellulosic bio-ethanol. Renewable

517

Sustainable Energy Rev. 2013, 26, 307-321.

518

(4)

519

Bogel-Łukasik, R.; Andreaus, J.; Ramos, L. P. Current Pretreatment Technologies for the

520

Development of Cellulosic Ethanol and Biorefineries. ChemSusChem 2015, 8, 3366-3390.

521

(5)

522

an efficient bioethanol production process based on enzymatic hydrolysis: a review.

523

Bioresour. Technol. 2010, 101, 4851-4861.

524

(6)

525

combined with an intermediate separation of fiber cells-optimization of fermentation of corn

526

straw hydrolysates. Bioresour. Technol. 2012, 121, 100-104.

527

(7)

528

method coupling steam explosion and mechanical carding fractionation. Bioresour. Technol.

529

2013, 139, 59-65.

530

(8)

531

biochemical pathway: a review. Energy Convers. Manage. 2011, 52, 858-875.

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

Chovau, S.; Degrauwe, D.; Van der Bruggen, B. Critical analysis of techno-

Silveira, M. H. L.; Morais, A. R. C.; da Costa Lopes, A. M.; Olekszyszen, D. N.;

Alvira, P.; Tomás-Pejó, E.; Ballesteros, M.; Negro, M. Pretreatment technologies for

Zhang, Y.; Fu, X.; Chen, H. Pretreatment based on two-step steam explosion

Wang, N.; Chen, H.-Z. Manufacture of dissolving pulps from cornstalk by novel

Balat, M. Production of bioethanol from lignocellulosic materials via the


Page 22 of 38

Page 23 of 38

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

Energy & Fuels

532

(9)

Lee, J. M.; Shi, J.; Venditti, R. A.; Jameel, H. Autohydrolysis pretreatment of

533

Coastal Bermuda grass for increased enzyme hydrolysis. Bioresour. Technol. 2009, 100,

534

6434-6441.

535

(10)

536

lignocellulosic biomass through biorefining. Prog. Energy Combust. Sci. 2012, 38, 583-598.

537

(11)

538

Ethanol from Softwood: Comparison of SSF and SHF and Identification of Bottlenecks.

539

Biotechnol. Prog. 2003, 19, 1109-1117.

540

(12)

541

biorefinery applications. J. Rheol. 2009, 53, 877-892.

542

(13)

543

concentration and yield stress of biomass slurries during enzymatic hydrolysis at high‐

544

solids loadings. Biotechnol.Bioeng. 2009, 104, 290-300.

545

(14)

546

slurries at high solids concentrations - effects of saccharification and particle size.

547


548

(15)

549

loadings - A review. Biomass Bioenergy 2013, 56, 526-544.

550

(16)

551

solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive

552

tree biomass. Process Biochem. 2007, 42, 1003-1009.

553

(17)

554

polymers to constrain water with the potential to inhibit cellulose saccharification.

555

Biotechnol. Biofuels 2014, 7, 159.

Zhu, J.; Zhuang, X. Conceptual net energy output for biofuel production from

Wingren, A.; Galbe, M.; Zacchi, G. Techno‐Economic Evaluation of Producing

Knutsen, J. S.; Liberatore, M. W. Rheology of high-solids biomass slurries for

Roche, C. M.; Dibble, C. J.; Knutsen, J. S.; Stickel, J. J.; Liberatore, M. W. Particle

Viamajala, S.; McMillan, J. D.; Schell, D. J.; Elander, R. T. Rheology of corn stover

Modenbach, A. A.; Nokes, S. E. Enzymatic hydrolysis of biomass at high-solids

Cara, C.; Moya, M.; Ballesteros, I.; Negro, M. J.; González, A.; Ruiz, E. Influence of

Selig, M. J.; Thygesen, L. G.; Felby, C. Correlating the ability of lignocellulosic


Energy & Fuels

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

556

(18)

Hsieh, C. C.; Cannella, D.; Jørgensen, H.; Felby, C.; Thygesen, L. G. Cellulase

557

inhibition by high concentrations of monosaccharides. J. Agric. Food Chem. 2014, 62, 3800-

558

3805.

559

(19)

560

enzymatic catalysis: Contributions from the fractal and jamming (overcrowding) effects.

561

Appl. Catal. A 2007, 317, 70-81.

562

(20)

563

hydrolysis can be described in terms of fractal-like kinetics. Biotechnol. Bioeng. 2003, 84,

564

254-257.

565

(21)

566

cellulose under different conditions. Bioresour. Technol. 2010, 101, 7995-8000.

567

(22)

568

saccharification of acid pretreated corn stover: empirical and fractal kinetic modelling.

569


570

(23)

571

Ethanol production from sugarcane bagasse using phosphoric acid-catalyzed steam

572

explosion. J. Braz. Chem. Soc. 2016, 27, 1889-1898.

573

(24)

574

whole sugarcane lignocellulosic biomass. Biotechnol. Biofuels 2015, 8:44.

575

(25)

576

C. Influence of mixed sugarcane bagasse samples evaluated by elemental and physical–

577

chemical composition. Ind. Crops Prod. 2015, 64, 52-58.

578

(26)

579

Templeton, D.; Wolfe, J. Determination of total solids in biomass and total dissolved solids

Xu, F.; Ding, H. A new kinetic model for heterogeneous (or spatially confined)

Väljamäe, P.; Kipper, K.; Pettersson, G.; Johansson, G. Synergistic cellulose

Wang, Z.; Feng, H. Fractal kinetic analysis of the enzymatic saccharification of

Wojtusik, M.; Zurita, M.; Villar, J. C.; Ladero, M.; Garcia-Ochoa, F. Enzymatic

Pitarelo, A. P.; da Fonseca, C. S.; Chiarello, L. M.; Gírio, F. M.; Ramos, L. P.

Pereira, S. C.; Maehara, L.; Machado, C. M. M.; Farinas, C. S. 2G ethanol from the

Moraes Rocha, G. J.; Nascimento, V. M.; Goncalves, A. R.; Silva, V. F. N.; Martín,

Sluiter, A.; Hames, B.; Hyman, D.; Payne, C.; Ruiz, R.; Scarlata, C.; Sluiter, J.;


Page 24 of 38

Page 25 of 38

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

Energy & Fuels

580

in liquid process samples; Technical Report NREL/TP-510-42621. National Renewable

581

Energy Laboratory, Golden, 2008.

582

(27)

583

Determination of ash in biomass; Technical Report NREL/TP-510-42622. National

584

Renewable Energy Laboratory, Golden, 2008.

585

(28)

586

extractives in biomass; Technical Report NREL/TP-510-42619. National Renewable Energy

587

Laboratory, Golden, 2008.

588

(29)

589

Determination of structural carbohydrates and lignin in biomass; Technical Report

590

NREL/TP-510-42618. National Renewable Energy Laboratory, Golden, 2012.

591

(30)

Ghose, T. Measurement of cellulase activities. Pure Appl. Chem. 1987, 59, 257-268.

592

(31)

Schwald, W.; Chan, M.; Breuil, C.; Saddler, J. Comparison of HPLC and

593

colorimetric methods for measuring cellulolytic activity. Appl. Microbiol. Biotechnol. 1988,

594

28, 398-403.

595

(32)

Kopelman, R. Fractal Reaction Kinetics. Science 1988, 241, 1620-1626.

596

(33)

Ewanick, S.; Bura, R. The effect of biomass moisture content on bioethanol yields

597

from steam pretreated switchgrass and sugarcane bagasse. Bioresour. Technol. 2011, 102,

598

2651-2658.

599

(34)

600

explosion and diluted sulfuric acid pretreatments: Comparative study aiming the sugarcane

601

bagasse saccharification. Ind. Crops Prod. 2015, 74, 810-816.

Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.

Sluiter, A.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D. Determination of

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

Rocha, G.; Gonçalves, A.; Nakanishi, S.; Nascimento, V.; Silva, V. Pilot scale steam


Energy & Fuels

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

602

(35)

Amores, I.; Ballesteros, I.; Manzanares, P.; Sáez, F.; Michelena, G.; Ballesteros, M.

603

Ethanol production from sugarcane bagasse pretreated by steam explosion. Eletronic J.

604

Energy Eviron. 2013, 1, 25-36.

605

(36)

606

biomass for biorefineries: comparative composition of carbohydrate and non-carbohydrate

607

components of bagasse and straw. Carbohydr. Polym. 2014, 114, 95-101.

608

(37)

609

M. H. L. Enzymatic hydrolysis of steam-exploded sugarcane bagasse using high total solids

610

and low enzyme loadings. Bioresour. Technol. 2015, 175, 195-202.

611

(38)

612

analysis of volatile organic compounds of sugarcane (Cachaça) and fruit spirits. Food Anal.

613

Methods 2013, 6, 978-988.

614

(39)

615

chloride catalyzed dehydration and degradation of glucose. Energy Fuels 2015, 29, 2387-

616

2393.

617

(40)

618

cellulose substrates. Bioresour. Technol. 2013, 6, 104.

619

(41)

620

enzymatic saccharification of pretreated corn stover slurries. Energy Fuels 2009, 23, 492-

621

497.

622

(42)

623

substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw. Appl.

624

Biochem. Biotechnol. 2007, 143, 27-40.

Szczerbowski, D.; Pitarelo, A. P.; Zandoná Filho, A.; Ramos, L. P. Sugarcane

Ramos, L. P.; da Silva, L.; Ballem, A. C.; Pitarelo, A. P.; Chiarello, L. M.; Silveira,

Capobiango, M.; Oliveira, E. S.; Cardeal, Z. L. Evaluation of methods used for the

Zhang, X.; Hewetson, B. B.; Mosier, N. S. Kinetics of maleic acid and aluminum

Teugjas, H.; Väljamäe, P. Product inhibition of cellulases studied with 14 C-labeled

Dasari, R. K.; Dunaway, K.; Berson, R. E. A scraped surface bioreactor for

Rosgaard, L.; Andric, P.; Dam-Johansen, K.; Pedersen, S.; Meyer, A. S. Effects of


Page 26 of 38

Page 27 of 38

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

Energy & Fuels

625

(43)

Aguiar, R. S.; Silveira, M. H. L.; Pitarelo, A. P.; Corazza, M. L.; Ramos, L. P.

626

Kinetics of enzyme-catalyzed hydrolysis of steam-exploded sugarcane bagasse. Bioresour.

627

Technol. 2013, 147, 416-423.

628

(44)

629

surfactants to eliminate lignin inhibition in enzymatic saccharification of cellulose.

630


631

(45)

632

Shear-induced orientation phenomena in suspensions of cellulose microcrystals, revealed by

633

small angle X-ray scattering. Langmuir 1999, 15, 6123-6126.

634

(46)

635

liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a

636

horizontal rotating bioreactor. Bioprocess Biosyst. Eng. 2014, 37, 173-181.

637

(47)

638

and rheological properties in cellulosic slurries. Appl. Biochem. Biotechnol. 2007, 137, 289-

639

299.

Wang, Z.; Xu, J.-H.; Feng, H.; Qi, H. Fractal kinetic analysis of polymers/nonionic

Ebeling, T.; Paillet, M.; Borsali, R.; Diat, O.; Dufresne, A.; Cavaille, J.; Chanzy, H.

Du, J.; Zhang, F.; Li, Y.; Zhang, H.; Liang, J.; Zheng, H.; Huang, H. Enzymatic

Dasari, R. K.; Berson, R. E. The effect of particle size on hydrolysis reaction rates

640


Energy & Fuels

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 28 of 38

641

Table 1. Matrices of the experimental designs that were carried out to investigate the

642

enzymatic hydrolysis of steam-treated sugarcane bagasse (coded values are given in brackets

643

and experiments 3,4 and 5 are the center points of the experimental designs).

644

Design A

Design B

CTec3 loading

Total solids

Run

645

CTec3 loading

HTec3

(FPU g-1 glucan)

loading (%)*

Run (FPU g-1 glucan)

(% w v-1)

A1

7.7 (-1)

20 (+1)

B1

7.7 (-1)

10 (+1)

A2

38.6 (+1)

20 (+1)

B2

38.6 (+1)

10 (+1)

A3, 4, 5

23.1 (0)

15 (0)

B3,4,5

23.1 (0)

5 (0)

A6

7.7 (-1)

10 (-1)

B6

7.7 (-1)

0 (-1)

A7

38.6 (+1)

10 (-1)

B7

38.6 (+1)

0 (-1)

A8

1.4 (-1.41)

15 (0)

-

-

-

A9

44.9 (+1.41)

15 (0)

-

-

-

A10

23.1 (0)

8 (-1.41)

-

-

-

A11

23.1 (0)

22 (+1.41)

-

-

-

* Values expressed in relation to the mass of Cellic CTec3.


Page 29 of 38

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

Energy & Fuels

646

Table 2. Chemical composition of untreated and pretreated sugarcane bagasse.

647

Component (wt.%)

Untreated

Pretreated

Anhydroglucose1

39.5 ± 0.7

54.5 ± 0.9

Anhydroxylose2

16.7 ± 0.4

3.4 ± 0.2

Anhydroarabinose2

1.5 ± 0.03

bdl8

Acetyl groups2

4.0 ± 0.1

bdl

Unidentified anhydrohexoses3

0.69 ± 0.01

0.89 ± 0.19

Unidentified anhydropentoses4

2.99 ± 0.23

0.45 ± 0.02

Acid soluble lignin

5.5 ± 0.7

3.7 ± 0.2

Acid insoluble lignin

20.6 ± 0.7

30.2 ± 0.7

Ashes

5.1 ± 0.2

5.2 ± 0.5

Extractives in water

3.5 ± 0.1

nd8

Extractives in ethanol

1.7 ± 0.1

nd

Total

101.8

98.3

Total glucan content5

40.2

55.4

Total hemicellulose contet6

25.2

3.8

Total lignin content7

26.1

33.8

648

1

Present as β(1-4)-D-glucans (cellulose);

649

2

Present as heteroxylan components (hemicellulose);

650

3

Dehydration by-product from hexoses measured as hydroxymethylfurfural (HMF);

651

4

Dehydration by-product from pentoses measured as furfural;

652

5

Summation of anhydroglucose and unidentified hexoses that were detected a HMF;

653

6

Summation of anhydroxylose, anhydroarabinose, acetyl groups and unidentified pentoses

654

that were detected as furfural;

655

7

Summation of acid soluble and acid insoluble lignin;

656

8

bdl, below the detection limit of the method; nd, not determined. 29 ACS Paragon Plus Environment

Energy & Fuels

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 30 of 38

657

Table 3. Fractal kinetic parameters of the enzymatic hydrolysis data that was obtained from

658

steam-treated sugarcane bagasse under the conditions given in the experimental design of

659

the CCRD.

660

Run

Glucan conversion

Xylan conversion

k (h-1)

h

R2

k (h-1)

h

R2

A1

0.102

0.536

0.995

0.134

0.648

0.968

A2

0.108

0.330

0.999

0.194

0.496

0.991

A3, 4, 5 0.095 ± 0.012 0.272 ± 0.040

0.998

0.166 ± 0.019 0.471 ± 0.028

0.995

A6

0.083

0.295

0.999

0.105

0.443

1.000

A7

0.299

0.387

0.999

0.386

0.525

0.965

A8

0.014

0.352

0.997

0.036

0.555

0.992

A9

0.150

0.246

0.998

0.276

0.445

0.993

A10

0.178

0.305

0.999

0.238

0.499

0.993

A11

0.126

0.475

0.995

0.175

0.591

0.981

661 662


Page 31 of 38

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

Energy & Fuels

663

Table 4. Apparent viscosity at 10 s-1 of steam-treated sugarcane bagasse during enzymatic

664

hydrolysis with Cellic CTec3 under the conditions given in Table 1 (CCRD)*.

665

Apparent viscosity (Pa s) after enzymatic hydrolysis for: Run

666

0h

3h

6h

9h

12 h

24 h

A1

90.8

39.3

30.5

21.8

17.6

9.7

A2

97.0

18.1

9.4

4.0

1.8

0.4

A3, 4, 5

53.5 ± 8.1

15.7 ± 1.4

7.4 ± 0.8

2.6 ± 0.5

1.0 ± 0.1

0.4 ± 0.1

A6

26.0

16.8

7.9

2.5

1.1

0.3

A7

20.0

0.7

0.3

0.1

Enzymatic Hydrolysis of Steam-Treated Sugarcane ... - ACS Publications

Recommend Documents