Accepted Manuscript Cellulose conversion of corn pericarp without pretreatment Daehwan Kim, David Orrego, Eduardo A. Ximenes, Michael R. Ladisch PII: DOI: Reference:
S0960-8524(17)31470-0 http://dx.doi.org/10.1016/j.biortech.2017.08.156 BITE 18765
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 June 2017 23 August 2017 24 August 2017
Please cite this article as: Kim, D., Orrego, D., Ximenes, E.A., Ladisch, M.R., Cellulose conversion of corn pericarp without pretreatment, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.156
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Cellulose conversion of corn pericarp without pretreatment Daehwan Kima,b, David Orregoa,b, Eduardo A. Ximenesa,b, Michael R. Ladischa,b,c
a
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette,
IN 47907-2032, United States b
Department of Agricultural and Biological Engineering, Purdue University, West
Lafayette, IN 47907-2032, United States c
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN
47907-2032, United States
Corresponding author at: Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907-2032, United States. Tel.: +1 765 494 7022; fax: +1 765 494 7023. E-mail address:
[email protected] (M. Ladisch).
1
ABSTRACT
2
We report enzyme hydrolysis of cellulose in unpretreated pericarp at a cellulase
3
loading of 0.25 FPU/g pericarp solids using a phenol tolerant Aspergillus niger
4
pectinase preparation. The overall protein added was 5 mg/g and gave 98% cellulose
5
conversion in 72 hours. However, for double the amount of enzyme from
6
Trichoderma reesei, which is significantly less tolerant of phenols, conversion was
7
only 16%. The key to achieving high conversion without pretreatment is combining
8
phenol inhibition-resistant enzymes (such as from A. niger) with unground pericarp
9
from which release of phenols is minimal. Size reduction of the pericarp, which is
10
typically carried out in a corn-to-ethanol process, where corn is first ground to a fine
11
powder, causes release of highly inhibitory phenols that interfere with cellulase
12
enzyme activity. This work demonstrates hydrolysis without pretreatment of large
13
particulate pericarp is a viable pathway for directly producing cellulose ethanol in
14
corn ethanol plants.
15 16 17 18 19
Keywords: Lignocellulosic biomass; Corn pericarp; Enzyme; Inhibition; Enzymatic
20
hydrolysis
21 22
23 24
1. Introduction Pericarp is derived from the outer covering of corn kernels, and is ordinarily
25
ground together with the rest of the corn prior to cooking, hydrolysis, and
26
fermentation in commercial corn-to-ethanol facilities. Current practice in some of
27
these facilities involves recovering fermentation residuals after distillation, pretreating
28
them, and then carrying out cellulose hydrolysis and a second fermentation in order to
29
produce cellulose ethanol. Distillers’ grains that include pericarp as well as other
30
residual components has a composition of 12.6% cellulose, 14.9% xylan, 5.5%
31
arabinan, 5.9% starch and 37% crude protein (Kim et al., 2008a) while pericarp itself
32
has a higher fraction of structural polysaccharides: 18% cellulose, 35% hemicellulose,
33
and 20% starch (in addition to oil, protein, and lignin) (Gáspár et al., 2007). Hence,
34
residual by-products from corn ethanol plants have significant potential for producing
35
cellulose ethanol.
36
Pretreatment is usually required to reduce recalcitrance of cellulose to enzyme
37
hydrolysis. Pretreatment solubilizes hemicellulose and some lignin, disrupts cellulose
38
structure, and enhances accessibility of the cellulose to enzyme (Jönsson and Martin,
39
2016; Kim et al., 2015, 2013, 2011, 2009). Methods for corn fiber pretreatment
40
include alkali (Akin et al., 2008; Gáspár et al., 2007, 2005), heat extraction (Benkő et
41
al., 2007), and liquid hot water (Kim et al., 2008a, 2008b; Mosier et al., 2005).
42
This work shows enzymatic hydrolysis of cellulose in corn pericarp does not
43
require pretreatment. Comparison of three different enzyme preparations from
44
Trichoderma reesei and Aspergillus niger at high protein loadings (0.5 and 1.4 FPU
45
enzyme/g solids, which corresponds to 10 and 27 mg enzyme/g solids, respectively)
46
shows complete hydrolysis is possible for enzymes in an A. niger pectinase
47
preparation. The impact of phenolic inhibitors becomes pronounced at lower enzyme
48
levels of 1 - 5 mg protein/g solids, (equivalent to approximately 4.6 to 22 mg
49
protein/mg cellulose) particularly for enzymes from T. reesei, where cellulose
50
hydrolysis yields decrease. Grinding of pericarp to a smaller particle decreases
51
conversion significantly, rather than resulting in an expected increase. This is
52
attributed to release of phenolic compounds that inhibited the enzymes. This paper
53
presents the role of phenolic inhibitors on cellulose from pericarp hydrolysis and
54
applies these findings to achieve cellulose enzymatic hydrolysis without pretreatment.
55
2. Materials and methods
56
2.1. Materials
57
Corn kernels were provided by Purdue Farms (Fall, 2013 harvest, 3x7 gallon
58
buckets of Yellow #2 dent corn Dupont / Pioneer P1151AM1), dried at 45C for 24 h
59
to a 9.42% moisture content. Cracked corn was acquired from Salamonie Mills
60
(Warren, IN), and was referred to as “cracked pericarp derived intact,” and upon
61
grinding “ground pericarp.” Whole corn from Purdue Farms was hand-cut into quarter
62
and half sections in order to obtain defined particle sizes. Pericarp from these corn
63
samples are referred to as quarter-size and half-size derived pericarp in this work.
64
Spezyme CP (batch number: 3016295230, cellulase/hemicellulase from
65
Trichoderma reesei) and Multifect Pectinase (batch number: A216235001, pectinase,
66
cellulase, and hemicellulase from Aspergillus niger) were donated by Genencor,
67
Danisco Division (Palo Alto, CA). Promod 144GL (protease from papain) and Depol
68
692L (serial number: 11687615, cellulase/hemicellulase from T. reesei and A. niger)
69
were purchased from Biocatalysts (Wales, UK). Enzyme profiles and protein
70
concentrations in these preparations are summarized in Table 1.
71
Corn pericarp was collected after enzyme treatment of corn kernels, which
72
removed the starch and gave pericarp as a residue. Other substrates utilized for
73
enzyme activity measurement were Whatman filter paper No. 1 (Whatman
74
International Ltd, England, Cat. No. 1001125), carboxymethylcellulose (low viscosity,
75
Cat. No. C5678), oat spelt xylan (Cat. No. X0627), and para-nitrophenyl substrates
76
(para-nitropenyl β-D-glucopyranoside (p-NPG), Cat. No. N7006, and para-nitropenyl
77
β-D-xylopyranoside (p-NPX), Cat. No. N2123) from Sigma Aldrich (St. Louis, MO).
78
All other chemicals were from Fisher Scientific International, Inc. (Hampton, NH).
79
All reagents were of analytical quality.
80
2.2. Preparation of corn pericarp
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Cracked corn from Salamonie Mills yielded an average particle size of pericarp of
82
5.1 mm, while kernels from Purdue farms that had been manually cut into halves or
83
quarters, gave pericarp with a mean length of 13.7 mm and, 7.2 mm, respectively. 200
84
g of the kernels (cracked and manually cut in halves or quarters) was mixed with 800
85
mL citrate buffer (pH 5.0, (25% w/v), 0.2% (v/v) Spezyme CP (1.3 mg protein/g
86
solids corresponding to 0.84 FPU/g solids) and 0.15% (v/v) Promod 144GL (0.9 mg
87
protein/g solids), and incubated at 50C for 36 h at 100 rpm with an up-flow impeller
88
with a 3 inch diameter. Fractionation was conducted in triplicate.
89
The resulting slurry was filtered through a 35 mesh screen, which retained the
90
pericarp fraction. This pericarp fraction was then washed with 500 mL distilled water
91
and dried at room temperature overnight. The residual moisture of 6.6% was
92
measured by a Halogen moisture analyzer (Mettler Toledo HB43). Ground pericarp
93
was obtained from cracked pericarp ground to pass a 20-mesh screen using a hammer
94
mill. The pericarp, thus isolated from other corn kernel components, was used to
95
evaluate hydrolysis of cellulosic components in a corn kernel.
96
2.3. Particle size measurement
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The particle sizes used in this study ranged from 840 micron to 13.7 mm, and this
98
size range exceeded the capabilities of the Malvern Zetasizer instrument. Hence, sieve
99
analysis was carried out to determine the average particle size of pericarp. A sample
100
of 50 g of fractionated pericarp at 6.6% moisture was sieved over a set of stainless
101
steel screens ranging from 35 to 5/8 inches mesh, corresponding to opening sizes of
102
0.5 mm to 16 mm, respectively (USA standard testing sieve, ASTM E11 specification,
103
VWR, Philadelphia, PA). All tests were performed in triplicate in a sieve shaker
104
(Ro-Tap Model E, Test Sieve Shaker, W.S. Tyler Mentor, OH) at room temperature for
105
5 min. The samples were collected and weighed at 6.6% moisture to determine the
106
size distribution. The pericarp was stored at -4C until further use.
107
2.4. Analytical assays
108
Cellulase activities in Spezyme CP (Spezyme), Depol 692L (Depol), Multifect
109
Pectinase, and the carry-over liquid (filtered from fractionated corn kernels) were
110
determined with Whatman number 1 filter paper strips, carboxymethylcellulose
111
sodium salt (CMCase, endo-glucanase), oat spelt xylan (xylanase), and
112
para-nitrophenyl substrates (β-glucosidase and β-xylosidase) as substrates (Dien et al.,
113
2008). Specific enzyme activities were determined at 50C in 0.05 M sodium acetate
114
buffer (pH 4.8) according to described protocols. One unit of enzyme activity refers to
115
the amount of enzymes required to produce one micromole of substrate per min under
116
the specified conditions.
117
Amylase activity in each enzyme and for the carry-over liquid was measured
118
according to Campos and Felix (1995). A starch solution (0.5% w/v) in 0.5 M sodium
119
acetate buffer (pH 6.0) was used as a substrate in order to test for this enzymatic
120
activity at 50C. Released glucose was measured by HPLC. One unit of amylase was
121
defined as the release of one micromole glucose per min from soluble starch. The
122
protein concentration in each sample was measured using a Pierce BCA protein assay
123
kit (Thermo Scientific, IL). The remaining starch content in the pericarp was
124
determined using a starch assay kit (Megazyme, Wicklow, Ireland). Glucose formed
125
from the remaining starch, as well as residual glucose in the carry-over liquid, was not
126
counted in the total glucose concentration after the cellulose hydrolysis. Total phenols
127
in the carry-over liquid were analyzed by Folin-Ciocalteu colorimetry assay at 765 nm.
128
The goal here was to quantify the concentration of phenols in the different samples
129
tested rather than individually identify them, and then correlate that to inhibitory
130
effects. Our previous work (Kim et al., 2013, 2011; Ximenes et al., 2011, 2010) and
131
that of others (Oliva-Taravilla et al., 2015; Tejirian and Xu, 2011) had identified
132
individual phenols as a cause of major inhibitory effect on cellulases, hemicellulases
133
and/or pectinases. Gallic acid was used as the standard in order to obtain a calibration
134
curve against which concentrations of phenols were determined based on Singleton et
135
al (1999) and Waterhouse (2002). The control was the same buffer as used for
136
obtaining the standard curve except for that phenols were not added.
137
2.5. Compositional and HPLC analyses
138
Chemical composition of initial pericarp and extractive free solids were analyzed
139
by NREL (National Renewable Energy Laboratory) using standard analytical
140
protocols described by Sluiter et al. (2008, 2005a, 2005b). Sugars, acetate, and other
141
solubles in hydrolyzed samples were determined by HPLC as described by Kim et al.
142
(2013). Hydrolyzed samples were filtered through a 0.45 µm filter to remove solids.
143
The HPLC system was equipped with a Bio-Rad Aminex HPX-87H ion exchange
144
column (300 mm x 7.8 mm, Bio-Rad Laboratories Inc., Hercules, CA) connected to
145
liquid chromatography system. 5 mM sulfuric acid diluted in distilled water was used
146
as the mobile phase at 0.6 mL/min flow rate, and the column temperature was kept
147
constant at 60C controlled by an Eppendorf TC-50 (Eppendorf, Wetbury, NY) (Kim
148
et al., 2013, 2011).
149
2.6. Enzyme hydrolysis of corn pericarp
150
Pericarp was enzymatically hydrolyzed in the presence of liquid (pH 4.8) carried
151
over from the pericarp preparation step. The carry-over liquid was clear with a light
152
gray color. It contained residual enzyme activities, sugars, and inhibitors (phenols) at
153
low concentrations, and was used to dilute the stock solutions of each enzyme to
154
concentrations corresponding to protein loadings of 1-27 mg protein/g solids. This
155
liquid simulates what we believe would represent conditions encountered in a
156
dry-grind facility. In addition to cellobiose (0.1 g/L), glucose (2.5 g/L), acetate (1.1
157
g/L), and total phenols (0.3 mg/L), the carry over liquid also had low levels of
158
residual enzyme activities of FPU (0.1), endoglucanase (6.2 U/mL), and xylanase (2.1
159
OSX/mL), with a total protein concentration of 3.4 mg/mL. A total of 100 mL mixture
160
(carry-over liquid combined with each enzyme preparation) and 15 g dry pericarp (15%
161
w/v) were mixed into a 250 mL flask that was capped with a stopper. Enzyme
162
activities corresponding to a loading of 10 mg/g solids are summarized in Table 1.
163
Activities at other tested enzyme levels are proportionately higher or lower,
164
depending on enzyme loadings.
165
The enzymatic hydrolysis of pericarp was initially performed in flasks in a
166
shaking incubator at 50C and 290 rpm, with an enzyme loading of 27 mg protein/g
167
solids. The results showed 50 and 72% conversion at 12 and 24 h, respectively
168
compared to 98% using a dual impeller mixing. Therefore, all the following
169
enzymatic hydrolyses were carried out in triplicate at 50C and 290 rpm mixing with
170
a dual impeller overhead mixer (IKA Eurostar power control-visc, Wilmington, NC)
171
for 72 h in a water bath (VWR, Radnor, PA). The dual impeller had two elephant ear
172
impeller blades (2 inch, 5.08 cm) with a 45 angle and a ¼ in shaft bushing
173
(Cole-Parmer, Vernon Hills, IL). The appearance of pericarp after hydrolysis is
174
illustrated in Supplementary data. Hydrolysis of pericarp in 0.05 M citrate buffer (pH
175
4.8) and in carry-over liquid gave similar results indicating that differences in
176
hydrolysis of the pericarp were due to the pericarp rather than other components that
177
may have carried over from the corn kernel.
178
2.7 Statistical Analysis
179
Statistical analysis was performed with Minitab 16. The T-test was conducted,
180
with 95% significant difference, for enzymatic hydrolysis of different particle sizes of
181
pericarp.
182
3. Results and discussion
183
3.1. Compositional analysis of corn pericarp
184
Chemical composition of initial pericarp and extractive free solids were analyzed
185
by NREL (National Renewable Energy Laboratory) standard analytical protocols
186
described by Sluiter et al. (2008, 2005a, 2005b). Corn pericarp, washed with distilled
187
water after the fractionation step, was composed of glucan with 22.5% cellulose, and
188
with 3.2% remaining starch (Table 2). After water and ethanol extractions, the
189
pericarp was enriched in polysaccharide components with the composition of glucan,
190
xylan, and arabinan increased by 60% (Table 2).
191
Corn fiber includes 11-12% protein (free amino acids, globulins, and albumins),
192
3-4% crude fat, 2% acetyl groups, and other components such as waxes, free fatty
193
acids, sterols, sterol esters, sterol ferulates, tocopherols, and phyto sterols (Moreau et
194
al., 1996; Saha et al., 1998; Watson and Ramstad, 1987; Wu and Norton, 2001). Of
195
the total pericarp solids, water and ethanol steps extracted 14.1% and 25.6% of the
196
dry weight, respectively. We used the initial (non-extractive free) corn pericarp for the
197
study of conversion of glucan to sugars; extraction was only carried out to
198
characterize compositional changes in the pericarp.
199 200
3.2. Cellulose hydrolysis in corn pericarp with different enzyme preparations Hydrolysis of un-pretreated pericarp was initially carried out at high enzyme
201
loadings (10 and 27 mg protein/g solids, respectively) (Fig. 1A and B) in order to
202
facilitate comparison of different commercial enzyme preparations with activity
203
profiles given in Table 1 A and B for pericarp compositions in Table 2. A loading of
204
27 mg Multifect Pectinase protein/g solids (equivalent to 6.2 FPU/g cellulose)
205
incubated with un-pretreated fiber gave 100% conversion compared to Spezyme CP
206
(equivalent to 77.3 FPU/g cellulose, 120 mg protein/g cellulose) and Depol 692L (8.6
207
FPU/g cellulose equivalent to 170 mg protein/g cellulose) where conversions were 22
208
to 60% (Fig. 1B). The enzyme profile of Multifect Pectinase has been reported before
209
(Dien et al., 2008), and it has accessory enzymes including α-arabinofuranosidase,
210
α-galactosidase, feruloyl esterase, and p-coumaroyl esterease. These later two
211
enzymes were not present in enzyme preparations derived from T. reesei. The known
212
reduced sensitivity to inhibition of the enzymes derived from A. niger (Ximenes et al.,
213
2011, 2010), together with the larger proportion of endoglucanase (EG) and
214
β-glucosidase (β-G) relative to the other enzyme preparations coincide with the higher
215
conversions.
216
Since EG and β-G activities in Multifect Pectinase, Spezyme, and Depol were
217
measured with respect to carboxymethylcellulose (CMC) and para-nitrophenyl-β-D-
218
glucopyranoside (p-NPG) in buffer, respectively, the activities reported in Table 1
219
were unaffected by inhibition. Although Spezyme showed higher filter paper activity
220
in the absence of phenols (Table 1), its hydrolysis of un-pretreated pericarp, in the
221
presence of phenols, was markedly lower. At 10 mg protein/g solids, yields after 72
222
hours were distinctly lower for Spezyme (30% conversion) and Depol (18%
223
conversion) compared to Multifect Pectinase (close to 100% conversion) (Fig. 1A).
224
Corn fiber releases phenolics in the form of p-coumaric acid and ferulic acid
225
(Akin et al., 2008; Carpita, 1996; Hartley and Ford, 1989). Akin et al. (2008) reported
226
that corn fiber had ester-linked p-coumaric and ferulic acids (1.56 and 14.84 mg/g
227
pericarp, respectively), with 80% of these phenolic compounds released after
228
pretreatment with 4 M sodium hydroxide at 170C. Release of inhibitors was also
229
noted for liquid hot water pretreated corn fiber i.e., pericarp. (Dien et al., 2008). Even
230
without pretreatment, and using enzyme hydrolysis, phenols are still released.
231
Inhibition is a key determinant of enzyme activity in the hydrolysis of pericarp.
232
3.3. Effect of cellulase loadings on hydrolysis in corn pericarp
233
Considering previous results, Multifect Pectinase was selected for further
234
experiments to test lower enzyme dosage (1 or 5 mg protein/g solids corresponding to
235
0.24 FPU or 1.1 FPU/g cellulose) (Fig. 2). When this activity load, which is
236
equivalent to 5 mg protein/g solids (22 mg/g cellulose), was used at the same
237
experimental conditions 98% glucose conversion occurred in 72 h. The lowest
238
enzyme dose at 1 mg protein/g solids (Fig. 2) gave 40% yield after 72 h, which was
239
higher than the results obtained with loadings of 10 mg protein/g solids for Spezyme
240
(16% conversion) and Depol (32% conversion) (Fig. 1A). There was no difference
241
between enzyme to buffer or in carry-over liquid both for protein loadings of 1 and 27
242
mg protein/g solids, respectively.
243
We also evaluated hydrolysis of the hemicellulosic fraction, however, xylan
244
hydrolysis was negligible despite the high amount of hemicellulase (mainly xylanase
245
and β-xylosidase) in Multifect Pectinase. The hemicellulase activity, without a
246
pretreatment step to increase enzyme accessibility to substrate, is insufficient to
247
effectively hydrolyze hemicellulose. Pericarp xylan has complex inter-linkages with
248
arabinan and ferulic acid and these may play a role in protecting the xylan backbone
249
from enzymatic hydrolysis (Dien et al., 2008).
250
3.4. Effect of particle size of pericarp on enzymatic hydrolysis
251
The difference in conversion between cracked, half, and quarter sized pericarp in
252
Fig. 3 was statistically insignificant, based on a T-test with 95% significant difference.
253
While a decrease in size from half (13.7 mm) to quarter (7.2 mm) to cracked (5.1 mm)
254
led to slightly lower rates of cellulose conversion at 27 mg Multifect Pectinase
255
protein/g solids loadings, cellulose hydrolysis was still 95% after 24 h for all three
256
cases (Fig. 3). In comparison, ground pericarp (0.84 mm) was 87% less, even when
257
the hydrolysis time was extended to 72 h. Deactivation due to shear has been noted in
258
the literature for amylase (van der Veen et al., 2004). However, when comparing
259
conversion of the pericarp with agitation by impeller to the conversion obtained in the
260
incubator shaker with agitation at 290 rpm, we observed significant improvement in
261
hydrolysis, indicating that loss in activity due to high stirring speed was not a major
262
factor. We observed an increased release of phenols with decreased pericarp particle
263
size contributed to the lower cellulose conversions. Ground pericarp released 22 times
264
more total phenols (0.44 mg/L) than pericarp half size (0.02 mg/L) and 1.7 than
265 266
cracked pericarp (Fig. 4). The inhibitory effect of phenols is not just a matter of their concentration but also
267
reflects chemical compositions as shown in our earlier work. Our previous work has
268
shown that strong enzyme inhibition occurred at low phenol concentration levels (for
269
instance, 80% loss of β-glucosidase activity with 1 mg tannins from wood/mg enzyme
270
protein, equivalent to 5 mg/mL in solution) (Ximenes et al., 2011; Kim et al., 2011;
271
2013). The inhibition effect became less pronounced as enzyme loadings increased.
272
This is consistent with our current work where enzyme loadings of 1, 5, and 10 mg
273
Multifect Pectinase protein/g ground solids gave 33%, 57%, and 74% conversion
274
yields, respectively (Fig. 5A, B, and C).
275
These data are opposite of the observations of Kim et al. (2015) for hydrolysis of
276
liquid hot water pretreated mixed hardwood, where conversion increased by 50%
277
when the average particle size of substrate decreased by 33% from 3 mm to 2 mm.
278
Differences in the biomass material help to explain this. Lignin in pericarp is less than
279
5% (Table 2), while hardwood after pretreatment is more than 30% (Kim et al., 2015;
280
Ko et al., 2015a). Lignin adsorbs cellulases and decreases conversion (Kim et al.,
281
2015; Ko et al., 2015a, 2015b). Also, in these referred studies with hardwood the
282
pretreated materials were exhaustively washed to remove inhibitors, such as phenols,
283
and a lignin blocking agent such as bovine serum albumin, BSA, was used to
284
minimize enzyme adsorption on lignin. This effectively minimized enzyme adsorption
285
on lignin exposed by pretreatment and directed the cellulolytic enzymes to the
286
cellulose substrate whose exposure increased with decreased particle size (Kim et al.,
287 288
2015; Ko et al., 2015a). In the case of pericarp, lignin content is only 5%, so its effect is minimal.
289
However, grinding to a smaller particle size released more soluble inhibitors (mainly
290
phenols), which were not washed away from the pericarp after it was ground. The
291
increase in phenol concentration in the liquid and decreased conversions indicate an
292
inhibitory effect by the released phenols. Kim et al. (2013, 2011) showed hydrolysis
293
of lignin-free cellulose (Solka Floc) in buffer gave 70% conversion at 1 mg protein/g
294
glucan enzyme loading, while Solka Floc at the same conditions in liquid containing
295
soluble phenol inhibitors gave a conversion of less than 40%. Hence as lignin content
296
increases, cellulase activity decreases due to adsorption on lignin.
297
4. Conclusions
298
Aspergillus niger cellulolytic enzymes in a pectinase preparation effectively
299
hydrolyzed pericarp, with a 5 mg protein/g pericarp resulting in 98% yield in 72 h, but
300
only if the pericarp size was at 5.1 mm. When pericarp was ground to a smaller size
301
of 0.84 mm, conversion decreased. This was found to coincide with the release of
302
phenols by the ground material. Confirming previous reports, phenols were identified
303
as the major potential enzyme inhibitors, whose effect could be minimized by using
304
enzymes from A. niger and a larger particle size of pericarp where phenol release was
305
minimal.
306
Acknowledgements
307
This research was supported by Indiana Corn Marketing Council grant 209346,
308
Hatch fund 199225 and 0205217, the Department of Agricultural and Biological
309
Engineering, and Purdue University Agricultural Research Programs. We thank
310
Genencor for providing Spezyme and Multifect Pectinase enzymes. We also thank
311
Ximing Zhang, Iman Beheshti, and Raymond RedCorn for internal review, and Carla
312
Carie for excellent assistance in preparing this manuscript.
313 314
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10. Kim, Y., Kreke, T., Ko, J.K., Ladisch, M.R., 2015. Hydrolysis-determining
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substrate characteristics in liquid hot water pretreated hardwood. Biotechnol.
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11. Kim, Y., Kreke, T., Hendrickson, R., Parenti, J., Ladisch, M.R., 2013.
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Fractionation of cellulase and fermentation inhibitors from steam pretreated mixed
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hardwood. Bioresour. Technol. 135, 30-38.
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2011.
Soluble
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inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.
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13. Kim, Y., Hendrickson, R., Mosier, N.S., Ladisch M.R., 2009. Liquid hot water pretreatment of cellulosic biomass. Methods Mol. Biol. 581, 93-102.
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14. Kim, Y., Mosier, N., Ladisch, M.R., 2008a. Process simulation of modified dry
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grind ethanol plant with recycle of pretreated and enzymatically hydrolyzed
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distillers’ grains. Bioresour. Technol. 99, 5177-5192.
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AFEX pretreated distillers’ grains at high-solids loadings. Bioresour. Technol. 99,
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5206-5215.
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pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis
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of cellulose. Biotechnol. Bioeng. 122, 252-262.
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lignins of liquid hot water pretreated hardwoods. Biotechnol. Bioeng. 112,
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18. Moreau, R.A., Powell, M.J., Hicks, K.B., 1996. Extraction and quantitative analysis of oil from commercial corn fiber. J. Agric. Food Chem. 44, 2149-2154.
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Welch, G., Dien, B.S., Aden, A., Ladisch, M.R., 2005. Industrial scale-up of
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pH-controlled liquid hot water pretreatment of corn fiber for fuel ethanol
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fiber current status and technical prospects. Appl. Biochem. Biotechnol. 70,
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D., 2008. Determination of structural carbohydrates and lignin in biomass.
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2008. National Renewable Energy Laboratory, Golden, Colorado.
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403 404
Figure captions
405
Fig. 1. Enzymatic hydrolysis of un-pretreated pericarp (15 g dry solids/100 mL) in the
406
presence of three different enzymes at (A) 10 and (B) 27 mg enzyme protein/g solids.
407
Experimental conditions: Spezyme CP, Depol 692L, and Multifect Pectinase were
408
incubated with pericarp slurry in carry-over liquid in an agitated vessel submerged in
409
a water bath to maintain temperature at 50°C, pH 4.8, for 72 h. Agitation provided by
410
a dual head impeller at 290 rpm.
411
Fig. 2. Enzymatic hydrolysis of un-pretreated pericarp (15 g dry solids/ 100 mL) with
412
or without carry-over liquid at different enzyme loadings (equivalent to 1, 5, 10, and
413
27 mg Multifect Pectinase protein/g total pericarp solids). Hydrolysis tests were
414
carried out at 50°C, pH 4.8, for 72 h with a dual head impeller; mixing at 290 rpm.
415
Fig. 3. Comparison of cellulose conversion of different sizes of un-pretreated pericarp
416
(15 g dry solids/100 mL) for enzyme hydrolysis. Enzymatic hydrolysis was performed
417
in the presence of carry-over liquid combined with Multifect Pectinase (27 mg
418
protein/g solids) at 50°C, pH 4.8, for 72 h with a dual impeller head mixing of 290
419
rpm. Control: no enzyme treatment with cracked pericarp. Half particle size pericarp:
420
13.7 mm; Quarter particle size pericarp: 7.2 mm; Cracked particle size pericarp: 5.1
421
mm; Ground particle size pericarp: 0.84 mm.
422
Fig. 4. Total phenols released as a function of particle size of corn pericarp (ground,
423
cracked, quarter, and half) after hydrolysis. Enzymatic hydrolysis (15 g dry solids/100
424
mL) was performed in the presence of carry-over liquid combined with Multifect
425
Pectinase (27 mg enzyme protein/g solids) at 50°C, pH 4.8, for 72 h with a dual head
426
impeller mixing of 290 rpm. Half particle size pericarp: 13.7 mm; Quarter particle
427
size pericarp: 7.2 mm; Cracked particle size pericarp: 5.1 mm; Ground particle size
428
pericarp: 0.84 mm.
429
Fig. 5. Enzymatic hydrolysis of cracked pericarp and ground pericarp (15 g dry
430
solids/100 mL) with different Multifect Pectinase loadings at (A) 1, (B) 5, and (C) 10
431
mg protein/g total solids. All runs were performed at 50°C, pH 4.8, for 72 h with a
432
dual impeller head mixing of 290 rpm. Cracked particle size pericarp: 5.1 mm;
433
Ground particle size pericarp: 0.84 mm.
434
435
Fig. 1. A. 10 mg protein/g solids
100 80 60 40
436 437 438 439 440 441 442 443 444 445 446 447 448
Cellulose conversion to glucose (%)
20 0
24
100
48
72
B. 27 mg protein/g solids
80 60
Fig. 2.
40 20 0 24
48 Hydrolysis time (h)
Spezyme CP
Depol 692L
72
Multifect Pectinase
27 mg protein
27 mg protein in buffer 5 mg protein
80
30
10 mg protein
60 20
1 mg protein in buffer 1 mg protein
40
10
20 0
0 0
24
48
72
Hydrolysis time (h)
449 450 451 452 453 454 455 456 457 458 459 460 461 462
27 mg protein in carry-over liquid
27 mg protein in buffer
10 mg protein in carry-over liquid
5 mg protein in carry-over liquid
1 mg protein in carry-over liquid
1 mg protein in buffer
Glucose concentration (g/L)
Cellulose conversion to glucose (%)
40
100
Fig. 3. 40
5.1 mm
100 80
30
0.84 mm 7.2 mm 13.7 mm
60
20
40 10 20 Control (no enzyme) 0
0 0
Control 464 465 466 467
12
24
13.7 mm
36 Time (h) 7.2 mm
48
5.1 mm
60
0.84 mm
72
Glucose concentration (g/L)
Cellulose conversion to glucose (%)
463
468
Fig. 4.
Total phenols at 765 nm (mg/L)
0.5 0.84 mm
0.4
5.1 mm
0.3
7.2 mm 0.2
0.1 y = -0.0318x + 0.4529 R² = 0.9846
13.7 mm
3
15
0 0 469 470
6 9 12 Corn pericarp particle size (mm)
18
471
Fig. 5. A. 1 mg protein/g solids
B. 5 mg protein/g solids
26
Cellulose conversion to glucose (%)
40
100 80
30
Ground cracked
60 20
Cracked 40
10
Ground cracked
20 0
0 0
472
12
24
36
48
60
72 40
100 80
30
60 20 40 10
20 0 473
0 0
Cracked
12
24
36
48
60
72
27
40
100 80
30
60
20 40 10
20 0 474 475 476
0 0
12
24
36
48
60
72 Hydrolysis time (h)
477 478 479 480
28
481
Table 1. (A) Commercial enzyme profiles of Spezyme CP, Depol 692L, and Multifect
482
Pectinase, and (B) normalized enzyme activities (unit/g pericarp solid) in Spezyme CP,
483
Depol 692, and Multifect Pectinase at 10 mg protein/g solids enzyme loadings.
484
(A) Amy (U/ml)
FPU
EG (U/ml)
β-G (CBU/ml)
Xyl (OSX/ml)
β-X (U/ml)
Protein (mg/ml)
Origin
5.4
52.8
163
104.5
2622
7.3
82
T. reesei
1
Spezy me
485 486 487 488 489 490 491 492
493 494 495 496 497
2
Depol
26.1
5.9
44
11
1510
18.2
116
T. reesei +A. niger
3
MP
1.5
4.2
577
176
947
35
82
A. niger
1
Spezyme: Spezyme CP. Depol: Depol 692L. 3 MP: Multifect Pectinase. Amy: amylase; EG: endo-glucanase activity with respect to CMC; β-G: β-glucosidase activity with respect to p-NPG; Xyl: xylanase activity with respect to oat spelt xylan; β-X: β-xylosidase activity with respect to p-NPX. 2
(B) Amy
FPU
EG
β-G
Xyl
β-X
Origin
Spezyme CP
0.04
0.43
1.33
0.85
21.32
0.06
T. reesei
Depol 692L
0.15
0.03
0.25
0.06
8.69
0.1
T. reesei +A. niger
Multifect Pectinase
0.01
0.03
4.69
1.43
7.7
0.28
A. niger
Amy: amylase; EG: endo-glucanase; β-G: β-glucosidase; Xyl: xylanase; β-X: β-xylosidase. Normalized enzyme activity is calculated by the enzyme unit (unit/mg protein) times the enzyme loading concentration dividing by the total pericarp solids. enzyme activity mg protein
ormalized enzyme activity (unit g solid 29
enzyme dose mg protein g solid
498 499 500 501
Table 2. Composition (%) of initial corn pericarp and extractive free of initial pericarp.
502
Corn pericarp was collected after enzymatic fractionation of corn kernels at solid
503
concentration of 25% (w/v) in the presence of 0.2% (v/v) Spezyme CP and 0.15%
504
(v/v) Promod 144GL (protease). Compositional analysis was done in triplicate. Composition (% dry weight) Component Glucan
1
Initial
Extractive free
25.7 ± 0.61
42.6 ± 0.72
Cellulose (22.5 ± 0.11) Starch (3.2 ± 0.12) Xylan/galactan
505 506 507 508 509 510
15.5 ± 0.04
25.8 ± 0.61
Arabinan
8.2 ± 0.3
13.7 ± 0.30
Acetyl
1.8 ± 0.03
3.0 ± 0.02
Acid Insoluble Lignin
4.5 ± 0.15
7.1 ± 0.65
Acid Soluble Lignin
0.2 ± 0.01
0.3 ± 0.00
Ash
0.1 ± 0.02
0.1 ± 0.8
Extractives (water + ethanol)
39.7 ± 0.12
-
Mass Closure
95.7 ± 0.16
92.6 ± 0.44
1
20.0% (dry basis) corn pericarp was recovered after enzymatic fractionation of corn kernels.
30
511 512
HIGHLIGHTS:
513
Cellulose hydrolysis of corn pericarp is feasible without pretreatment.
514
A. niger cellulases in pectinase enzyme fraction tolerate phenol inhibitors.
515
Ground pericarp (0.84 mm) releases more phenols than cracked pericarp (5.1 mm).
516
Phenols from ground pericarp strongly inhibit enzymatic hydrolysis of pericarp.
517
31