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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: [email protected] and [email protected] 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
1. Ago, M., Endo, T., Hirotsu, T., 2004. Crystalline transformation of native cellulose 242
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
12
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
16. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 282
2011. Determination of structural carbohydrates and lignin in biomass. Laboratory 283
Analytical Procedure (LAP). NREL/TP-510-42628. 284
17. Teixeira, R.S.S., Silva, A.S., Ferreira-Leitão, V.S., Bon, E.P.S., 2012. Amino acids 285
interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid 286
assay mislead carbohydrase activity measurements. Carbohydr. Res. 363, 33-37. 287
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
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