Accepted Manuscript

Short Communication

Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrys‐

talline cellulose and sugarcane bagasse pretreated by either ball milling or ionic

liquid [Emim][Ac]

Ricardo Sposina Sobral Teixeira, Ayla Sant’Ana da Silva, Han-Woo Kim,

Kazuhiko Ishikawa, Takashi Endo, Seung-Hwan Lee, Elba P.S. Bon

PII: S0960-8524(13)01442-9

DOI: http://dx.doi.org/10.1016/j.biortech.2013.09.019

Reference: BITE 12383

To appear in: Bioresource Technology

Received Date: 3 July 2013

Revised Date: 31 August 2013

Accepted Date: 3 September 2013

Please cite this article as: Teixeira, R.S.S., Silva, A.S.d., Kim, H-W., Ishikawa, K., Endo, T., Lee, S-H., Bon, E.P.S.,

Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline cellulose and sugarcane bagasse

pretreated by either ball milling or ionic liquid [Emim][Ac], Bioresource Technology (2013), doi: http://dx.doi.org/

10.1016/j.biortech.2013.09.019

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1

Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline 1

cellulose and sugarcane bagasse pretreated by either ball milling or ionic liquid 2

[Emim][Ac] 3

Ricardo Sposina Sobral Teixeiraa,1, Ayla Sant’Ana da Silvaa,1, Han-Woo Kimb, 4

Kazuhiko Ishikawab, Takashi Endob, Seung-Hwan Leeb,c*, Elba P. S. Bona* 5

aFederal University of Rio de Janeiro, Chemistry Institute, Av.Athos da Silveira Ramos, 149 - 6

Centro de Tecnologia, Bloco A, Cidade Universitária, CEP: 21941-909, Rio de Janeiro, RJ - 7

Brazil 8

bBiomass Refinery Research Center, National Institute of Advanced Industrial Science and 9

Technology (AIST), 3-11-32, Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan 10

cDepartment of Forest Biomaterials Engineering, College of Forest and Environmental 11

Sciences, Kangwon National University, Chuncheon 200-701, Korea 12

1Both authors contributed equally to the manuscript 13

14

*Corresponding authors: elba1996@iq.ufrj.br and lshyhk@kangwon.ac.kr 15

Contact information for Elba P. S. Bon: 16

Av. Athos da Silveira Ramos, 149 - Centro de Tecnologia, Bloco A - sala 539, Cidade 17

Universitária, Rio de Janeiro, RJ, Brazil, CEP: 21941-909. Tel: +55-21-2562-7358 18

Contact information for Seung-Hwan Lee: 19

Department of Forest Biomaterials Engineering, College of Forest and Environmental 20

Sciences, Kangwon National University, Chuncheon 200-701, Korea, Tel: +82-33-250-8323 21

22

Abstract 23

This study investigated the requirement of cellobiohydrolases (CBH) for 24

saccharification of microcrystalline cellulose and sugarcane bagasse pretreated either by 25

ball milling (BM) or by ionic liquid (IL) [Emim][Ac]. Hydrolysis was done using CBH-26

free blends of Pyrococcus horikoshii endoglucanase (EG) plus Pyrococcus furiosus β-27

2

glucosidase (EGPh/BGPf) or OptimashTM BG while Acremonium Cellulase was used as 28

control. IL-pretreated substrates were hydrolyzed more effectively by CBH-free 29

enzymes than were the BM-pretreated substrates. IL-treatment decreased the 30

crystallinity and increased the specific surface area (SSA), whereas BM-treatment 31

decreased the crystallinity without increasing the SSA. The hydrolysis of IL-treated 32

cellulose by EGPh/BGPf showed a saccharification rate of 3.92 g/L.h and a glucose 33

yield of 81% within 9 h. These results indicate the efficiency of CBH-free enzymes for 34

the hydrolysis of IL-treated substrates. 35

Keywords: cellobiohydrolase requirement; ball milling pretreatment; ionic liquid 36

pretreatment; cellulose crystallinity; sugarcane bagasse. 37

38

1. Introduction 39

The development of a biorefinery platform for the production of chemicals and 40

fuels, based on enzymatic processing of the main components of the plant cell wall (i.e. 41

cellulose, hemicellulose and lignin) is hindered by the lignocellulose materials’ 42

recalcitrance to enzymatic hydrolysis. Their recalcitrance depends on the cell wall 43

chemical composition, the chemical bonding amongst their micro and macromolecular 44

components and their supra-molecular structures. Although there is a lack of consensus 45

in the literature regarding the importance of each biomass parameter to its recalcitrance, 46

the crystallinity of cellulose is recognized as having a significant influence on cellulosic 47

biomass enzymatic hydrolysis (Silva et al., 2010). Cellulose microfibrils contain 48

crystalline and amorphous regions. The crystalline regions consist of highly ordered 49

cellulose molecules derived from the organization of cellulose chains linked by 50

hydroxyl groups to form intra- and inter-molecular hydrogen bonds in different 51

arrangements, while the molecules are less ordered in the amorphous regions (Park et 52

al., 2010). The crystalline regions are more recalcitrant to enzymatic attack, while the 53

3

amorphous regions are readily hydrolyzed (Cao and Tan, 2005). Therefore, 54

pretreatments that alters the native structure and/or composition and/or crystallinity of 55

the plant cell wall are required to reduce biomass recalcitrance to enzymatic hydrolysis. 56

Ionic liquid (IL) and ball milling (BM) pretreatments significantly reduce the cellulose 57

crystallinity of lignocellulosic materials and alter the crystalline structure, as shown by 58

X-ray diffraction (Inoue et al., 2008; Silva et al., 2010; Silva et al., 2011). 59

The enzymatic hydrolysis of cellulose is carried out by cellobiohydrolases (CBHs) 60

that act efficiently in the crystalline regions of cellulose (Cao and Tan, 2005) and by 61

endoglucanases (EGs) that act preferentially in the amorphous regions (Al-Zuhair, 62

2008). Within this context, it is a reasonable hypothesis that materials with reduced 63

crystallinity require enzyme blends with lower CBH loads, decreasing the overall 64

production cost of biomass-derived sugar syrups. To investigate the CBH requirement 65

for the hydrolysis of materials with low crystallinity, we evaluated the performance of 66

enzyme cocktails lacking CBH activity for hydrolysis of IL- and BM-pretreated 67

microcrystalline cellulose and sugarcane bagasse. 68

2. Materials and Methods 69

2.1. Materials and enzyme activity assays 70

Microcrystalline cellulose (MCC) KC Flock W-100, without lignin and 71

hemicellulose, was purchased from Nippon Paper Chemicals Co., Japan. Sugarcane 72

bagasse, which was ground and fractionated through 0.250, 0.425 and 1.00 mm sieves, 73

was kindly provided by the Itarumã Sugar Mill (Goiás, Brazil). Three enzyme 74

preparations were used. The first preparation consisted of a blend of hyperthermophilic 75

endoglucanase (EGPh) and β-glucosidase (BGPf) from Pyrococcus horikoshii and P. 76

furiosus, respectively. Both enzymes were expressed in Escherichia coli and purified 77

according to Ando et al. (2002) and Kado et al. (2011). The 10 µM enzymes solution in 78

4

50 mM citrate buffer, pH 5.0 were blended, at a ratio of 5:1 EGPh:BGPf, and used in 79

the hydrolysis experiments. The second preparation was the commercial OptimashTM 80

BG (Genencor®, USA), which lacks CBH activity, diluted to 4% (v/v) in the same 81

aforementioned buffer. The third preparation was the commercial Acremonium 82

Cellulase (Meiji Seika Co., Japan), which contains high CBH levels besides a complex 83

set of biomass hydrolyzing enzyme activities, and was thus used as a control. 84

Endoglucanase activity was measured using carboxymethylcellulose (CMC) as a 85

substrate (Ghose, 1987), where the reaction mixtures containing 0.25 mL of the relevant 86

enzyme preparation and 0.25 mL of 2% CMC were incubated for 30 minutes at 85 °C 87

for the EGPh/BGPf blend and at 50 °C for the commercial enzymes. The filter paper 88

activity (FPase) was measured according to Ghose (1987). The 3,5-dinitrosalicylic acid 89

reagent was prepared according to Teixeira et al. (2012). 90

2.2. Ball milling and ionic liquid pretreatments and enzymatic saccharification 91

The BM- and IL-pretreatments were performed according to Silva et al. (2010) 92

and Silva et al. (2011), respectively. The determination of the structural carbohydrate 93

and lignin content of sugarcane bagasse was done according to Sluiter et al. (2011). 94

Enzymatic hydrolysis experiments were carried out in 35 mL reaction mixtures 95

containing 1% (dry weigh) BM- or IL-treated materials. In all cases, EG load 96

corresponded to 345 UI of CMCase/g biomass. This load was mandatory as it is found 97

in the control experiments with Acremonium Cellulase containing 15 FPU/g biomass, 98

which is frequently used in hydrolysis tests. The hydrolysis experiments were incubated 99

for 72 h at 85 °C for the EGPh/BGPf blend and stirred on a 12 Plus Carousel hotplate 100

(Radleys, United Kingdom), or at 45 °C for OptimashTM BG and Acremonium Cellulase 101

with stirring on a rotatory shaker. Mixtures were filtered and the solid residues, after 102

washing with 1L of water, were kept in wet state for further analysis. Monosaccharide 103

5

and cellobiose quantifications were done using an HPLC system according to Silva et 104

al. (2013). The cellulose and xylan conversion yields into glucose and xylose were 105

calculated based on the 0.90 and 0.88 conversion factors, respectively, while a 0.95 106

factor was used for conversion of glucose from cellobiose. The hydrolysis experiments 107

were done in duplicates and the data variations were lower than 2%. 108

2.3. Relative crystallinity and specific surface area analysis 109

The wide-angle X-ray diffraction (WAXD) pattern and specific surface area 110

(SSA) measurements were performed in duplicates for the untreated and pretreated 111

materials, as well as for the solid residue after enzymatic hydrolysis, according to Silva 112

et al. (2011). The samples were thoroughly washed with t-butyl alcohol for water 113

removal and freeze-dried aiming to preserve their surface patterns and morphology. 114

3. Results and discussion 115

3.1. Enzymatic hydrolysis of pretreated microcrystalline cellulose 116

The time courses for enzymatic hydrolysis of MCC pretreated by IL or BM, using 117

the three enzyme preparations, are shown in Figure 1. The combined effect of the IL-118

pretreatment and the action of the EGPh/BGPf blend at 85 °C produced the highest 119

initial hydrolysis rate of 3.92 g/L.h with a glucose yield of 81% within 9 h. The high 120

temperature might have accelerated the hydrolysis reaction by increasing the substrate 121

solubility (Kim and Ishikawa, 2010) besides the usual increase in reaction kinetics 122

associated with high temperatures. It is also important to consider that both thermophilic 123

enzymes possess particular catalytic features. The EGPh is able to attack crystalline 124

cellulose at some extent (Ando et al., 2002) and to release cellobiose after an initial 125

endo-type attack (Kim and Ishikawa, 2010). Moreover, BGPf is able to hydrolyze 126

cellooligosaccharides at high temperatures (Kado et al., 2011). Acremonium Cellulase, 127

used as control for a complete cellulase blend, reached equivalent hydrolysis yields, of 128

6

85%, within 24 h, while OptimashTM BG plateaued within 24 h at 26% hydrolysis yield. 129

The hydrolysis yields, specially at 24 h, for the BM-treated MCC were significantly 130

lower indicating a inferior efficiency to reduce MCC recalcitrance in comparison to IL. 131

Glucose yields for the Acremonium Cellulase, of 44%, were higher than that for the 132

EGPh/BGPf blend and OptimashTM BG of 27% and 15%, respectively, indicating the 133

need for CBH to achieve higher glucose yields. As expected, glucose yields for 134

untreated MCC were comparatively lower, reaching 68%, 13% and 2.8% for 135

Acremonium Cellulase, EGPh/BGPf blend and OptimashTM BG, respectively, after 72 h. 136

3.2. Evaluation of microcrystalline cellulose relative crystallinity 137

The diffraction patterns of the untreated and pretreated MCC and the hydrolysis 138

residues are shown in Supplementary Fig. 1. Three peaks at 2θ equal to 15.00° (1 139

10), 16.38° (110) and 22.52° (200) were observed for the untreated MCC, confirming 140

the presence of the cellulose I structure. An evident crystallinity decrease and similar 141

XRD profiles were observed for BM- and IL-pretreated MCC. However, the hydrolysis 142

yields for the IL-treated MCC was rather higher, suggesting differences in the structure 143

for both materials. Indeed, the XRD analysis of the BM-treated MCC hydrolysis 144

residues displayed a weaker peak that was split into two peaks at 2θ equal to 20.1° (110) 145

and 21.53° (200), indicating the presence of residual crystallinity and a cellulose II 146

structure that may hinder effective enzymatic attack by CBH-free enzymes and could 147

explain their poor performance. The transformation of cellulose I to cellulose II has 148

been reported for BM-treated cotton-derived cellulose in the presence of different 149

amounts of water (Ago et al., 2004). Moreover, BM-pretreatment showed no significant 150

effect on the MCC SSA while IL-treated MCC increased 280-fold, contributing to its 151

fast hydrolysis. The lack of sufficient residue from the IL-treated MCC hydrolysis 152

prevented XRD analysis. 153

7

3.3. Enzymatic hydrolysis of sugarcane bagasse 154

BM- and IL-treated sugarcane bagasse was composed of 41.9% and 50.9% 155

cellulose, 25.0% and 22.5% xylan and 22.7% and 15.7% lignin, respectively. Figures 2 156

and 3 show the glucose and xylose hydrolysis yields for BM- and IL-treated bagasse. 157

Both pretreatments reduced the recalcitrance of bagasse and significantly increased the 158

hydrolysis yields. For Acremonium Cellulase, the glucose yields for BM- and IL-treated 159

bagasse were 91% and 99% respectively, within 24 h. The superior performance of 160

Acremonium Cellulase, in comparison to the CBH-free enzyme preparations, indicated 161

the presence of remaining crystalline structures that hinder an effective hydrolysis. 162

Similar hydrolytic profiles were observed between the BM- and IL-treated 163

bagasse for OptimashTM BG, reaching 42% and 47% glucose yield within 24 h, 164

respectively. However, a significant accumulation of cellobiose was detected during the 165

hydrolysis of IL-treated bagasse by OptimashTM BG, indicating a β-glucosidase load 166

deficiency. Indeed, the CMCase:BG activity ratio of 67:1 in OptimashTM BG is 167

excessively high, due to its low β-glucosidase level, compared to the 3:1 and 5:1 ratios 168

of Acremonium Cellulase and the EGPh/BGPf blend, respectively. The conversion of 169

the accumulated cellobiose to glucose would result in a significant increase in the 170

glucose yield from 67% to 87%, within 72 h, confirming the IL treatment efficiency and 171

a lower dependency on CBH activity. The glucose yields were consistently two-fold 172

higher for OptimashTM BG than for the EGPh/BGPf blend. This result could be 173

explained by the lack of xylanase activity in the EGPh/BGPf blend, that could remove 174

the hindrance caused by hemicellulose. Indeed, high xylose yields of 51% and 61% for 175

the hydrolysis of BM- and IL-treated bagasse, respectively, were observed upon the use 176

of OptimashTM BG. Thus, the presence of hemicellulases in OptimashTM BG enhances 177

the overall bagasse hydrolysis by digesting xylan and increasing cellulose accessibility. 178

8

Although EGPh has been reported to be inactive on xyloglucans (Ando et al., 2002), a 179

low release of xylose was observed. The use of Acremonium Cellulase resulted in low 180

xylose concentrations despite its high xylanase activity. This result could be related to 181

xylobiose accumulation in the hydrolysates, which confirmed that this enzyme has a 182

low β-xylosidase activity as reported by Inoue et al. (2008). OptimashTM BG also 183

performed better than EGPh/BGPf blend in terms of the theoretical conversion of 184

accumulated cellobiose during IL-treated bagasse hydrolysis, though only to a small 185

extent, because [Emim][Ac] removes part of the hemicellulose linked to lignin, 186

increasing the cellulose/hemicellulose ratio and exposing the cellulose. Those results are 187

in agreement with Barr et al. (2012), that described a negligible effect of CBH on the 188

hydrolysis of poplar and switchgrass pretreated with [Emim][Ac]. 189

3.4. Evaluation of sugarcane bagasse relative crystallinity 190

The EGPh/BGPf blend had better hydrolytic performance on IL-treated bagasse 191

than on BM-treated bagasse, achieving 73% and 33% glucose yields after 72 h, 192

respectively. Apart from the removal of some of the hemicellulose and lignin by IL, this 193

result can be explained by the change of cellulose crystallinity after pretreatment and 194

hydrolysis with EGPh/BGPf, as indicated by the diffraction patterns (Supplementary 195

Fig. 2). Cellulose I diffraction patterns were identified for untreated bagasse, with two 196

peaks at 2θ equal to 16.25° (110) and 22.52° (200). After BM- or IL-pretreatments, the 197

diffraction patterns changed, indicating that the cellulose crystallinity decreased. 198

However, the XRD profiles of the residual BM-treated material after hydrolysis by 199

EGPh/BGPf and OptimashTM BG, indicated an increase in crystallinity, suggesting that 200

this pretreatment did not completely eliminate the crystallinity, that was exposed in the 201

hydrolysis residue. The structure remaining after hydrolysis of BM-treated bagasse was 202

more crystalline than the residual material from IL-treated bagasse, demonstrating that 203

9

BM treatment is less efficient at deconstructing the crystalline structure in 204

lignocellulose materials, corroborating the hydrolysis results. 205

3.5. Specific surface area of the pretreated substrates 206

The native and BM-treated cellulose and bagasse SSAs were 1.2 and 0.6 m2/g and 207

0.8 and 1.3 m2/g, respectively, indicating that BM has no effect on the SSA. In contrast, 208

the IL-pretreatment increased the SSA of the bagasse and MCC from 0.8 to 135.2 m2/g 209

and 1.2 to 336.5 m2/g, respectively. Although BM did not affect the bagasse SSA, the 210

pretreatment was very effective for the hydrolysis using Acremonium Cellulase. 211

3.6. Discussion on the CBH requirement and IL use 212

Lower CBH requirements would facilitate one-step conversion of cellulose to 213

ethanol, as engineered microorganisms would be able to perform both cellulose 214

degradation and fermentation through the sole co-expression of EG and β-glucosidase. 215

This would be an interesting approach, as increasing the specific activity and secretion 216

of CBH by Saccharomyces cerevisiae is still a challenge (Haan et al., 2013). If IL-217

tolerant EG/BG can be efficiently expressed in fermenting microorganisms, a bio-218

consolidated process could be envisaged. Moreover, hyperthermophilic enzymes, such 219

as the ones that were hereby studied, represent promising biocatalysts for industrial 220

processes. Thermophilic IL-tolerant cellulase cocktails can be used for the one-step 221

biomass pretreatment and hydrolysis, as several thermophilic enzymes can tolerate low 222

amounts of IL (Park et al., 2012), while mesophilic enzymes usually become inactive in 223

the presence of trace amounts of IL. Nevertheless the application of the IL for biomass 224

pretreatment aiming the production of low added value products is still not viable for 225

large-scale operations, advances regarding the decrease of the IL:biomass ratio is 226

promising (Silva et al., 2013). At present, we have shown that enzyme blends with high 227

EG and β-glucosidase contents can be used, in spite of their hyperthermophilic sources, 228

10

if the IL is completely removed after pretreatment 229

Conclusion 230

Tailor-made enzyme blends for biomass hydrolysis can be investigated in terms of 231

different pretreatment options. Although IL and BM are known to decrease cellulose 232

crystallinity, remaining crystalline structures were observed after hydrolysis of the BM 233

samples. These structures hindered efficient hydrolysis by the enzyme blends that 234

lacked CBH activity. In contrast, IL pretreatment was highly effective at reducing the 235

biomass recalcitrance. Lower CBH loads were required to effectively hydrolyze the IL-236

treated cellulose and IL-treated bagasse. 237

Acknowledgements 238

This work was financed by FINEP, JICA and JST. Silva A.S. and Teixeira R.S.S. are 239

grateful to CNPq/Petrobras and to BIOMM S/A for research scholarships, respectively. 240

References 241

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from cellulose I to cellulose II polymorph by a ball-milling method with a specific 243

amount of water. Cellulose 11, 163-167. 244

2. Al-Zuhair, S., 2008. The effect of crystallinity of cellulose on the rate of reducing 245

sugars production by heterogeneous enzymatic hydrolysis. Bioresour. Technol. 99, 246

4078–4085. 247

3. Ando, S., Ishida, H., Kosugi, Y., Ishikawa, K., 2002. Hyperthermostable 248

endoglucanase from Pyrococcus horikoshii. Appl. Environ.68, 430-433. 249

4. Barr, C.J., Mertens, J.A., Schall, C.A., 2012. Critical cellulase and hemicellulase 250

activities for hydrolysis of ionic liquid pretreated biomass. Bioresour. Technol. 104, 251

480-485. 252

5. Cao, Y., Tan, H., 2005. Study on crystal structures of enzyme-hydrolyzed cellulosic 253

11

materials by X-ray diffraction. Enzyme Microb. Technol., 36, 314-317. 254

6. Ghose T., 1987. Measurement of cellulase activities. Pure Appl. Chem., 59, 257-268. 255

7. Haan, R., Kroukamp, H., Van Zyl, J., Van Zyl, W., 2013. Cellobiohydrolase secretion 256

by yeast: Current state and prospects for improvement. Process Biochem. 48, 1-12. 257

8. Inoue, H., Yano S., Endo T., Sakaki T., Sawayama S., 2008. Combining hot-258

compressed water and ball milling pretreatments to improve the efficiency of the 259

enzymatic hydrolysis of Eucalyptus. Biotechnol. Biofuels. 1, 1-9. 260

9. Kado,Y., Inoue, T., Ishikawaa, K., 2011. Structure of hyperthermophilic β-261

glucosidase from Pyrococcus furiosus. Acta Crystallogr. 67, 1473-1479. 262

10. Kim, H-W., Ishikawa, K., 2010. Complete saccharification of cellulose at high 263

temperature using Endocellulase and β-Glucosidase from Pyrococcus sp. J. Microbiol. 264

Biotechnol. 20, 889–892. 265

11. Park, S., Baker, J.O., Himmel, M.E., Parilla, P.A., Johnson, D.K., 2010. Cellulose 266

crystallinity index: measurement techniques and their impact on interpreting cellulase 267

performance. Biotechnol. Biofuels. 3, 1-10. 268

12. Park, J.I., Steen, E.J., Burd, H., Evans, S.S., Redding-Johnson, A.M., et al. 2012. A 269

thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic 270

biofuels. Plos One. 7, e37010. 271

13. Silva, A.S., Inoue, H., Endo, T., Yano, S., Bon, E.P.S., 2010. Milling pretreatment of 272

sugarcane bagasse and straw for enzymatic hydrolysis and ethanol fermentation. 273

Bioresour. Technol. 101, 7402-7409. 274

14. Silva, A.S., Lee, S-H., Endo, T., Bon, E.P.S., 2011. Major improvement in the rate 275

and yield of enzymatic saccharification of sugarcane bagasse via pretreatment with the 276

ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]). Bioresour. Technol. 277

102, 10505-10509. 278

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15. Silva, A.S., Teixeira, R.S.S., H., Endo, T., Bon, E.P.S., Lee, S-H., 2013. Continuous 279

pretreatment of sugarcane bagasse at high loading in an ionic liquid using a twin-screw 280

extruder. Green Chem. 15, 1991-2001. 281

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Figure captions 288

Figure 1. Time course for the enzymatic hydrolysis of microcrystalline cellulose after 289

ball milling (dashed lines - open symbols) and ionic liquid [Emim][Ac] (solid lines - 290

closed symbols) treatments using the EGPh/BGPf blend at 85 °C ( , ), 291

OptimashTM BG ( , ) and Acremonium Cellulase ( , ) at 45 °C. 292

Figure 2. Time course for the enzymatic hydrolysis of sugarcane bagasse into glucose 293

after ball milling (A) or ionic liquid [Emim][Ac] pretreatment (B). EGPh/BGPf blend at 294

85 °C ( ). OptimashTM BG ( ). Acremonium Cellulase ( ). OptimashTM BG, 295

adding up to the glucose concentration the theoretical conversion of cellobiose to 296

glucose ( ), at 45 °C. The hydrolysis of untreated bagasse using Acremonium 297

Cellulase ( ),OptimashTM BG ( ) and EGPh/BGPf ( ) blend is presented. 298

Figure 3. Time course for the enzymatic hydrolysis of sugarcane bagasse into xylose 299

upon pretreatment by ball milling (dashed lines - open symbols) or ionic liquid 300

[Emim][Ac] (solid lines - closed symbols) using the EGPh/BGPf blend at 85 °C 301

( , ), OptimashTM BG ( , ) and Acremonium Cellulase ( , ) at 45 302

°C. 303

Figure 1

Figure 2

Figure 3

Graphical Abstract

1

Highlights 1

CBH was not required to efficiently hydrolyze amorphous IL-treated 2

cellulose. 3

CBH activity was necessary to hydrolyze BM-treated bagasse. 4

Low CBH loads can hydrolyze IL-treated sugarcane bagasse. 5

A crystalline residue was obtained after hydrolysis of the BM-treated 6

substrates. 7

IL greatly increased the specific surface area of both substrates, while BM 8

did not. 9

10