Dilute acid pretreatment of rapeseed straw for fermentable sugar generation

Eulogio Castro

, Manuel J. Daz, Cristbal Cara, Encarnacin Ruiz, Inmaculada Romero, Manuel Moya
Department of Chemical, Environmental and Materials Engineering, University of Jan, Campus Las Lagunillas, 23071 Jan, Spain
a r t i c l e i n f o
Article history:
Received 5 June 2010
Received in revised form 11 August 2010
Accepted 13 August 2010
Available online 22 August 2010
Keywords:
Agricultural residues
Dilute acid pretreatment
Enzymatic hydrolysis
Rapeseed straw
a b s t r a c t
The inuence of the main pretreatment variables on fermentable sugar generation from rapeseed straw is
studied using an experimental design approach. Low and high levels for pretreatment temperature
(140200 C), process time (020 min) and concentration of sulfuric acid (0.52% w/v) were selected
according to previous results. Glucose and xylose composition, as well as sugar degradation, were mon-
itored and adjusted to a quadratic model. Non-sugar components of the hydrolysates were also deter-
mined. Enzymatic hydrolysis yields were used for assessing pretreatment performance. Optimization
based on the mathematical model show that total conversion of cellulose from pretreated solids can
be achieved at pretreatment conditions of 200 C for 27 min and 0.40% free acid concentration. If optimi-
zation criteria were based on maximization of hemicellulosic sugars recovery in the hydrolysate along
with cellulose preservation in the pretreated solids, milder pretreatment conditions of 144 C, 6 min
and 2% free acid concentration should be used.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Rapeseed (Brassica napus) oil ranges third of oil consumption
worldwide. After oil extraction, rapeseed pomace can be used for
animal feed production due to its protein content. In the last years,
an increasing fraction of rapeseed oil has been used as raw material
for biodiesel production. According to FAO (2008), over 30 million
hectares of rapeseed were cultivated all over the world in 2007.
Agricultural residues from rapeseed cultivation are left behind
after seed harvesting. These renewable, costless materials must
be eliminated from the eld, which is usually done by burning.
Alternatively, the lignocellulosic nature of rapeseed straw can
be used for fuel ethanol production by a biochemical process
including pretreatment, enzymatic hydrolysis and fermentation.
There are relatively little reports focusing on the use of rapeseed
residues. Most of them deal with thermal applications like pyroly-
sis (Karaosmanoglu et al., 1999). Zabaniotou et al. (2008) reported
on the integrated utilization of rapeseed suitable to Greek condi-
tions for biodiesel production and parallel use of its solid residues
for energy and second generation biofuels production via fast pyro-
lysis. Reports dealing with rapeseed straw as raw material for eth-
anol production are also rare. The use of sulfuric acid-catalyzed
pretreatment with rapeseed straw at 180 C has been reported
(Lu et al., 2009). The same pretreatment was analyzed by Jeong
et al. (2010) for optimizing hemicellulose extraction form rapeseed
straw. Li et al. (2009) reported on rapeseed stover pretreatment
with phosphoric acidacetone for ethanol production by means
of simultaneous saccharication and fermentation. Biogas or etha-
nol production has also been reported (Petersson et al., 2007).
In a previous work (Daz et al., 2010) on the use of hydrothermal
pretreatment of rapeseed straw for fermentable sugars production,
it was concluded that 70% of the glucose in the raw material could
be obtained by liquid hot water pretreatment at 210220 C for
3050 min. In an attempt to improve previous results, this work
deals with the use of dilute sulfuric acid pretreatment on the same
rapeseed straw lot. This procedure has been applied to a variety of
agricultural residues (Cara et al., 2008) and it is claimed to effec-
tively hydrolyze hemicellulose and make the cellulose amenable
to enzymatic conversion (Lloyd and Wyman, 2005). The main oper-
ational variables, e.g. pretreatment temperature, residence time
and acid concentration are examined under an experimental de-
sign basis. The performance of the process is assessed by means
of the enzymatic hydrolysis yield and the sugar yield in the liquid
fractions issued from pretreatment.
2. Methods
2.1. Raw material
Rapeseed straw was locally collected after seed harvest. Then,
the raw material was air-dried at room temperature to equilibrium
moisture content of about 10%, milled using a laboratory hammer
mill (Retsch) to a particle size smaller than 1 cm, homogenised in a
single lot and stored until used.
The chemical composition of the raw material is (% w/w, dry ba-
sis): cellulose (as glucose), 36.6; hemicellulosic sugars, 24.1 (with
xylose as the main sugar accounting for 76% of hemicellulosic sug-
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.08.057

Corresponding author. Tel.: +34 953212163; fax: +34 953212143.

E-mail address: ecastro@ujaen.es (E. Castro).
Bioresource Technology 102 (2011) 12701276
Contents lists available at ScienceDirect
Bioresource Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
ars); acid-insoluble lignin (AIL), 15.6; acetyl groups, 3.65; and ash,
5.7 (Daz et al., 2010). The composition of raw material was deter-
mined according to the National Renewable Energy Laboratory
analytical methods for biomass, as described below.
2.2. Dilute acid pretreatment
Dilute acid pretreatment was performed in a laboratory scale
stirred Parr reactor. The reactor is built in Carpenter 20, an acid-
resistant alloy and has a total volume of 1 L, with an electric heater
and mechanic agitation. The temperature/speed controller is a
combination of furnace power control and motor speed control
with tachometer. 36 g of rapeseed straw (dry basis) and 600 mL
of the appropriate sulfuric acid solution were used for each pre-
treatment trial. Both sulfuric acid solution and raw material, ini-
tially at room temperature, were heated at 5 C/min. Agitation
was set at 350 rpm. Once the temperature of the reaction mixture
reached the target point, pretreatment time counting was initiated.
At the end of each run the reactor was removed from the heating
jacket and cooling water was charged through the serpentine coil.
The content of the reactor cooled down to 80 C in approximately
5 min. The reactor was kept sealed, and the slurry agitated until
the reactor was cooled to about 40 C. Then the wet material was
ltered for solid and liquid recovery.
The water-insoluble solids were washed thoroughly with water
until no colour in the resulting water was obtained, and analyzed
for hemicellulosic sugars, glucose and acid-insoluble lignin con-
tent, and used as substrate in enzymatic hydrolysis tests. Liquid
fraction issued from pretreatment (hydrolysate) was analyzed for
sugars, acetic acid and sugar-degradation products.
2.3. Experimental design
Rapeseed straw was pretreated at 13 different operational con-
ditions according to a BoxBehnken experimental design, includ-
ing one point and four replicates at the center of the domain
selected for each factor under study, as shown in Table 1 (17 runs).
Center values and intervals were chosen based on previous experi-
ence with agricultural residues to ensure a broad range of re-
sponses. Pretreatment experiments were performed in random
order. The recovery of cellulose and hemicelluloses in the pre-
treated solids, and the concentration of glucose and hemicellulosic
sugars in the liquid fractions issued from pretreatment were deter-
mined as responses. In addition, degradation of glucose and other
sugars as a consequence of pretreatment was also evaluated. The
experimental data were analyzed by the statistical software Design
Expert 8.0.2, Stat-Ease Inc., Minneapolis, USA.
2.4. Enzymatic hydrolysis tests
The washed water-insoluble residue of pretreated rapeseed
strawwas enzymatically hydrolysed by a cellulolytic complex (Cel-
luclast 1.5 L) kindly provided by Novozymes A/S (Denmark). Cellu-
lase enzyme loading was 15 Filter Paper Units (FPU)/g substrate.
Fungal -glucosidase (Novozym 188, Novozymes A/S) was used
to supplement the -glucosidase activity with an enzyme loading
of 15 International Unit (IU)/g substrate. Enzymatic hydrolysis
was performed in 0.05 M sodium citrate buffer (pH 4.8) at 50 C
on a rotary shaker (Certomat-R, B-Braun, Germany) at 150 rpm
for 72 h and at 5% (w/v) pretreated material concentration. Sam-
ples were taken every 24 h for glucose concentration determina-
tion. All enzymatic hydrolysis experiments were performed in
duplicate (standard deviations were in all cases <3%) and average
results are given.
2.5. Analytical methods
The composition of raw material was determined according to
the National Renewable Energy Laboratory analytical methods for
biomass (NREL, 19941998). Prior to other determinations, raw
material was extracted consecutively with water and with ethanol
(two-step extraction procedure). After the rst step, the sugar
composition of the water-extract was determined by high perfor-
mance liquid chromatography (HPLC) in a Varian Prostar liquid
chromatograph with refractive index detector. A Transgenomic
CHO-682 carbohydrate analysis column operating at 80 C with
ultrapure water as a mobile-phase (0.4 mL/min) was used. Free
and oligomeric sugar composition was determined before and after
a posthydrolysis process consisting in a treatment with sulfuric
acid (3% v/v) at 121 C and 30 min. The cellulose and hemicellulose
content of the extracted solid residue was determined based on
monomer content measured after a two-step acid hydrolysis pro-
cedure to fractionate the ber. A rst step with 72% (w/w) H
2
SO
4
at 30 C for 60 min was used. In a second step, the reaction mixture
was diluted to 4% (w/w) H
2
SO
4
and autoclaved at 121 C for 1 h.
This hydrolysis liquid was then analyzed for sugar content by HPLC
as described above. The remaining acid-insoluble residue is consid-
ered as acid-insoluble lignin (AIL).
Table 1
Experimental design for dilute acid pretreatment of rapeseed straw. t
0
and A
0
are pretreatment time and temperature, while t and A stand for the same variables considering
instantaneous heat up and cooling that would result in the same severity factor (Ro) (see text for details).
Run t
0
(min0 T (C) A
0
(% w/v) Ro Factors for design
Coded Real Coded Real Coded Real A (% w/v) t (min)
1 0 10 1 140 +1 2 232.9 1.91 15.47
2 0 10 +1 200 1 0.5 14813.7 0.41 16.84
3 1 0 0 170 1 0.5 833.7 0.41 7.24
4 +1 20 +1 200 0 1.25 23847.6 1.16 27.11
5 +1 20 1 140 0 1.25 391.1 1.16 25.97
6 1 0 1 140 0 1.25 93. 6 1.16 6.21
7 0 10 0 170 0 1.25 1915.3 1.16 16.64
8 +1 20 0 170 +1 2 2995.8 1.91 26.03
9 0 10 +1 200 +1 2 14563.9 1.91 16.55
10 1 0 +1 200 0 1.25 5575.6 1.16 6.34
11 1 0 0 170 +1 2 693.9 1.91 6.03
12 0 10 0 170 0 1.25 1884.4 1.16 16.37
13 +1 20 0 170 1 0.5 3017.2 0.41 26.21
14 0 10 0 170 0 1.25 1919.1 1.16 16.67
15 0 10 0 170 0 1.25 1862.4 1.16 16.18
16 0 10 0 170 0 1.25 1878.2 1.16 16.32
17 0 10 1 140 1 0.5 233.0 0.41 15.48
E. Castro et al. / Bioresource Technology 102 (2011) 12701276 1271
After LHW-pretreatment, the composition of solid fraction was
determined as described for raw material except that no extraction
is used. The sugar content (glucose, xylose, arabinose, mannose
and galactose) of the liquid fraction after pretreatment (prehydro-
lyzate) was determined by HPLC using the system described above.
The inhibitor composition (acetic acid, formic acid, furfural and
HMF) was determined using the HPLC system with refractive index
detector mentioned above; a Bio-Rad HPX-87H column at 65 C
temperature was used. The mobile phase was 5 mM H
2
SO
4
, at a
ow rate of 0.5 mL/min. Glucose concentration from enzymatic
hydrolysis samples was measured by HPLC with the above de-
scribed Varian equipment. All analytical determinations were per-
formed in duplicate and average results are shown. Relative
standard deviations were in all cases below 5%.
3. Results and discussion
3.1. Effective pretreatment conditions
Factors in the experimental design were modied for taking ac-
count of two facts that can affect pretreatment performance, e.g.
the neutralizing capacity (NC) of biomass, and the effect of heating
and cooling periods.
Concerning the rst of these facts, suspensions of lignocellulosic
materials have been shown to partially neutralize acid solutions
due to the presence of basic cations in the lignocellulosic matrix.
When considering pretreatment by dilute acids, it is important to
take into account the concentration of acid that is actually used
to bond cleavage, and that which is just neutralized by biomass.
To determine the neutralizing capability of rapeseed straw, a mod-
ication of the procedures described by Esteghlalian et al. (1997)
and Lloyd and Wyman (2005) was applied. Briey, triplicate sam-
ples of rapeseed straw were calcinated at 525 C for 8 h and then
the resulting ash was dissolved in 0.5% (w/v) sulfuric acid solution.
The difference of pH of the sulfuric acid solutions before and after
adding the ash was used for calculating the neutralizing capacity of
the raw material. The same procedure was also followed using the
entire rapeseed straw, instead of ash. The neutralizing capacity re-
sulted to be 19.7 1.6 mg H
2
SO
4
/g dry rapeseed straw. These re-
sults compare with those reported by Esteghlalian et al. (1997)
for corn stover, poplar and switchgrass (43.7, 25.8 and 16.7 mg
H
2
SO
4
/g dry feedstock, respectively) and by Lloyd and Wyman
(2005) for corn stover (17.3 mg H
2
SO
4
/g dry feedstock).
From NC values, it is possible to determine the actual acid con-
centration that is available for pretreatment purposes, which re-
sulted to be 0.41, 1.16 and 1.91% (w/v) at initial concentration of
0.5, 1.25 and 2.0% (w/v) respectively. These actual concentrations,
also named free acid concentrations, have been considered for the
discussion of the experimental results, as hydronium ion concen-
tration directly affects the rates of hydrolysis of the carbohydrate
polymers (Springer and Harris, 1985). Other authors have also re-
ported on neutralization of sulfuric acid by the minerals in biomass
and the need of being taken into account for models to accurately
predict hydrolysis performance (Lloyd and Wyman, 2004).
On the other hand, pretreatment was performed on a Parr pres-
sure reactor at temperatures ranging from 140 C to 200 C. As de-
scribed in the precedent section, heat up was done at 5 C/min,
while cooled down to 80 C lasted for 5 min (approximately,
depending on the target temperature). A typical temperature pro-
le can be found elsewhere (Daz et al., 2010). To take account of
the effect of temperature, not only when maintained at the target
value, but also during heat up and cool down periods, the severity
factor Ro, adapted from Overend and Chornet (1987) by Abatzog-
lou et al. (1992), was applied following this equation:
Ro t exp
T 100
14:75

Z
t
0
exp
Tt 100
14:75

dt 1
The evaluation procedure started by calculating the integral
from curves T versus t, and then the value of time is derived from
the second term of Eq. (1). This value is the time assuming instan-
taneous heating-up and cooling down that would yield the same
severity factor as the real process. For example, run 4 was per-
formed by heating the reaction mixture (1.25% w/v acid concentra-
tion) up to 200 C, then hold for 20 min and cooling down
afterwards. The same effect in terms of severity factor would have
been reached if 200 C were maintained for 27.11 min, provided
instantaneous heating and cooling was possible.
Taking into account the two modications in process variables,
Table 1 summarizes the pretreatment conditions used for the
experimental design.
3.2. Mathematical model. Factors and responses
The study of the pretreatment performance by dilute sulfuric
acid was addressed by performing the experimental design in
which pretreatment temperature, process time, and free acid con-
centration, as detailed above, were retained as factors. The ratio li-
quid to solid or solid content in the pretreatment reactor was kept
constant at 6% w/v; this value was chosen after some preliminary
trials as the highest one causing no mixing problems inside the
reactor. Solid recovery, glucose and other sugars recoveries, includ-
ing the degradation taking place as a consequence of pretreatment,
Table 2
Coefcients of mathematical model Eq. (2)
a
.
Solid recovery G
s
G
l
X
s
X
l
Y Y
p
G
d
X
d
0
210.9 539.0 10.08 89.5 74.6 576.5 76.3 745.9 53.9
a
1
1.62 4.42 0.74 0.46 0.69 0.90 2.54 8.64 1.51
a
2
1.20 7.29 0.056 0.82 0.88 6.95 0.76 8.15 1.31
a
3
23.55 99.6 4.39 12.4 16.0 91.0 42.7 116.1 27.0
a
4
7.9210
3
28.410
3
2.7810
3
1.4210
3
3.2010
3
NS 19.610
3
46.210
3
NS
a
5
NS 0.359 0.106 60.610
3
0.148 NS NS 0.842 1.61
a
6
NS 0.760 NS 21.010
3
85.310
3
0.579 2.12 0.670 0.42
a
7
NS NS 6.2710
3
3.0810
3
NS 33.310
3
NS 29.810
3
NS
a
8
1.9810
3
20.610
3
NS 2.0810
3
2.3910
3
18.610
3
NS 22.310
3
6.4210
3
a
9
6.58 9.27 1.19 2.44 NS 5.93 11.2 NS 20.8
R
2
0.9918 0.9968 0.9737 0.9972 0.9745 0.9801 0.9789 0.9956 0.9711
NS: no statistical signicance.
a
G
s
, G
l
: percentage of glucose present in the raw material that remains after pretreatment in solids and hydrolysates; X
s
, X
l
: percentage of hemicellulosic sugars (sum of
xylose, galactose, mannose and arabinose) present in the raw material that remain after pretreatment in solids and hydrolysates; Y, Y
P
: Enzymatic hydrolysis yields referred
to either raw material or pretreated material (g glucose/100 g); G
d
, X
d
: percentage of glucose and other sugars present in the raw material that resulted degraded as a
consequence of pretreatment; Coefcients for G
l,
X
l
and X
s
were obtained after factor transformation, as proposed by the statistical software, as follows:

G
p
l
1;

X
p
l
1;

X
p
s 1.
1272 E. Castro et al. / Bioresource Technology 102 (2011) 12701276
and enzymatic hydrolysis yields referred to both feedstock and to
pretreated materials, were determined as model responses (Y). Re-
sults are shown in Tables 3 and 5.
The statistic interpretation of the results was formulated by
using the quadratic equation:
Y a
0
a
1
t a
2
T a
3
A a
4
t T a
5
t A a
6
T A
a
7
t
2
a
8
T
2
a
9
A
2
2
which allows the inuence of each factor on the responses as well
as interactions among factors to be determined, according to
parameters a
i
. Table 2 summarizes the model coefcients obtained
from ANOVA table for the different measured responses, together
with the statistic parameter R
2
. The model tted the experimental
data well (P < 0.05). In addition, F values were <0.0001, that is, there
is only a 0.01% chance that a Model F-Value this large could occur
due to noise. As an example, Fig. 1 shows a good agreement be-
tween predicted and experimental values for one of the responses
(solid recovery).
3.3. Effect of pretreatment on rapeseed straw
The dilute acid pretreatment of rapeseed straw resulted in a
wide variety of pretreated solids and hydrolysates in terms of
material recovery and composition. Solid recovery ranged from
29% to 66% depending on effective pretreatment conditions. As
can be seen in Fig. 2, free acid concentration exerted lower inu-
ence on material recovery than pretreatment temperature. For
example, increasing temperature from 140 to 200 C, at the lowest
free acid concentration and pretreatment time resulted in an in-
crease of material solubilisation by 28.8% (solid recovery varied
from 70.4% to 41.6%). By contrast, increasing effective acid concen-
tration from the lowest to the highest level, at 200 C and low pre-
treatment time represented only a 12.7% increase in material
solubilisation.
The composition of pretreated solids and hydrolysates in terms
of recovery of the main sugars (glucan and xylan) is summarized in
Table 3, where the percentages of glucose and other sugars present
in the raw material that are retained in the pretreated solids and
those which enter the liquid fractions are shown.
Concerning pretreated solids, it can be seen a wide range of
variation in terms of cellulose retained in the solids after pretreat-
ment. The cellulose virtually disappeared from solids of experi-
ments 4 and 9, which showed the highest severity factors, while
Table 5
Glucose enzymatic hydrolysis yields referred to raw material (Y, g glucose/100 g
glucose of raw material) and pretreated materials (Y
p
, g glucose/100 g glucose in
pretreated material) and degradation of glucose (G
d
) and other sugars (X
d
) (%).
Run number Y Y
p
G
d
X
d
1 21.20 22.25 0.00 1.11
2 53.70 98.21 42.91 57.29
3 56.82 62.96 5.18 15.87
4 nd nd 98.36 100.00
5 35.52 42.30 9.77 5.99
6 30.99 36.61 10.79 12.64
7 43.32 62.14 17.32 43.81
8 29.17 53.41 45.38 75.88
9 nd nd 91.65 99.35
10 25.86 71.99 41.42 90.46
11 29.51 39.03 13.30 38.97
12 41.66 56.96 14.34 51.34
13 61.54 76.25 12.12 6.43
14 44.15 60.86 15.22 46.37
15 46.82 68.33 20.47 46.09
16 46.61 64.75 15.31 41.04
17 28.73 35.32 13.32 0.00
nd: not determined.
Actual
P
r
e
d
i
c
t
e
d
Solid recovery, %
20
30
40
50
60
70
20 30 40 50 60 70
Fig. 1. Predicted versus experimental values for solid recovery after pretreatment.
Table 3
Severity factor (Ro) and recovery (%) of glucose (G) and hemicellulosic sugars (X) in
pretreated solids (subscript s) and hydrolysates (subscript l) from rapeseed straw
pretreatment experiments.
Run number Ro G
s
% G
l
% X
s
% X
l
%
1 232.9 95.28 6.71 16.64 82.25
2 14813.7 54.68 8.41 10.43 32.28
3 833.7 90.25 4.57 25.73 58.40
4 23847.6 0.00 1.64 0.00 0.00
5 391.1 83.97 6.26 19.64 74.37
6 93. 6 84.64 4.57 38.98 48.38
7 1915.3 69.71 12.97 0.00 56.19
8 2995.8 54.62 0.00 0.00 24.12
9 14563.9 0.00 8.35 0.65 0.00
10 5575.6 35.92 22.66 0.00 9.54
11 693.9 75.60 11.10 0.00 61.03
12 1884.4 73.14 12.52 0.00 48.66
13 3017.2 80.71 7.17 11.32 82.25
14 1919.1 72.54 12.24 0.00 53.63
15 1862.4 68.52 11.01 0.00 53.91
16 1878.2 71.99 12.70 0.00 58.96
17 233.0 81.34 5.34 67.90 34.70
Table 4
Inhibitor composition (g/100 g raw material) of hydrolysates.
Run number Acetic acid Formic acid Furfural HMF
1 4.90 0.21 0.50 0.00
2 4.98 1.03 5.32 0.83
3 1.50 1.03 0.17 2.52
4 3.45 0.35 2.20 0.00
5 4.28 1.07 0.33 0.00
6 1.75 0.35 0.00 0.00
7 5.67 0.00 4.70 0.43
8 3.48 1.58 7.05 0.60
9 3.63 7.22 4.20 0.10
10 5.08 1.70 8.45 2.23
11 5.35 0.00 3.42 0.17
12 6.12 0.00 5.57 0.45
13 3.92 0.00 1.47 0.18
14 5.80 0.00 5.32 0.45
15 5.57 0.00 4.30 0.43
16 5.60 0.00 4.55 0.42
17 0.37 0.28 0.00 0.00
E. Castro et al. / Bioresource Technology 102 (2011) 12701276 1273
remained almost unaltered at run 1. Considering xylan, most of the
pretreatment experiments resulted in the total loss of this compo-
nent in the pretreated solids. By contrast, most of it entered the li-
quid fraction issued from pretreatment. The inuence of
pretreatment variables in glucan recovery of the pretreated solids
is depicted in Fig. 3. It can be deduced that glucan remained in
the solid at the high level of acid concentration if temperature
was kept at the low assayed level (140 C). Temperature was also
a determining factor for xylan dissolution, and only at low levels
of both temperature and acid remained almost unaltered in the so-
lid fraction.
Glucose and xylose were also found in hydrolysates as a conse-
quence of glucan and xylan dissolution due to lignocellulosic struc-
ture breakdown at pretreatment conditions. Fig. 4 shows, using
contour plots, how temperature and acid concentration conditions
determined the hydrolysate composition in terms of hemicellulo-
sic sugars. In agreement with the above discussion, xylose was par-
ticularly found in the hydrolysates obtained at high acid
concentrations and low temperatures and xylose recoveries as high
as 82% were found under selected conditions. Concerning glucose,
most of the hydrolysates showed an average glucose recovery
about 810%, except two extreme experiments (0.0% and 22.6%
glucose).
The hydrolysates contained also non-sugars compounds, as
shown in Table 4. Acetic acid is a product of hemicellulose acetyl
group cleavage. Degradation of glucose and xylose due to severe
pretreatment conditions resulted in furfural and HMF generation.
Formic acid is also a degradation product of furfural. All these com-
pounds have been described as potential inhibitors of yeast fer-
mentation at varying operational conditions and, at the same
time represent a monomeric sugar loss (Lau et al., 2009). In addi-
tion, a synergistic inhibitory effect has been reported (Daz et al.,
2009). Acetic acid and furfural were the inhibitors commonly
found at higher concentrations in this work. The highest acetic acid
and HMF concentrations (6.12 and 2.52 g/100 g raw material),
compare with the highest reported values when rapeseed straw
was pretreated by liquid hot water (5.95 and 2.58 g/100 g, respec-
tively) which were obtained at 210 C and 50 min pretreatment
conditions (Daz et al., 2010). Formic acid and furfural concentra-
tions were however higher in hydrolysates from dilute acid
pretreatment than those obtained from liquid hot water pretreat-
ment. Similar results to those reported here were obtained by
Shuai et al. (2010) on dilute acid pretreated spruce (180 C, 5% acid,
30 min). Lu et al. (2009), in a work on sulfuric acid pretreated rape-
seed straw, also reported on furfural, HMF and acetic acid, with
highest results of 1.21 (furfural + HMF) and 4.40 g/100 g raw mate-
rial (acetic acid), although the pretreatment temperature was not
studied and remained constant in all experiments (180 C).
3.4. Enzymatic hydrolysis yields
The enzymatic hydrolysis yields referred to both raw material
and pretreated solids after 72 h enzymatic action are shown in Ta-
0.4
0.8
1.2
1.6
2.0 140
150
160
170
180
190
200
0
20
40
60
80
100


G
s
,

%


Free acid, %
Temperature, C
Fig. 3. Glucan recovery in pretreated solids as a function of free acid concentration
and pretreatment temperature according to the model (process time, 15 min).
0.4 0.8 1.2 1.6 2.0
140
150
160
170
180
190
200
Xl, %
Free acid, %
T
e
m
p
e
r
a
t
u
r
e
,

C
0
20
40
60
60
70
80
20
40
0
60
60
70
80
Fig. 4. Contour plots for hemicellulosic sugars recovery in hydrolysates as a
function of free acid concentration and pretreatment temperature according to the
model (process time, 6 min).
0.4
0.8
1.2
1.6
2.0
140
150
160
170
180
190
200
20
30
40
50
60
70
80


S
o
l
i
d

r
e
c
o
v
e
r
y
,

%


C: Free acid
Temperature, C
Fig. 2. Response surface for material recovery as a function of free acid concen-
tration and pretreatment temperature according to the model (process time,
15 min).
1274 E. Castro et al. / Bioresource Technology 102 (2011) 12701276
ble 5 and the inuence of pretreatment temperature and effective
acid concentration are depicted in Fig. 5a and b, respectively. Pre-
treated solids obtained from experiments 4 and 9 were not submit-
ted to enzymatic hydrolysis, since no cellulose was left as a result
of conditions of pretreatment. For the rest of solids, enzymatic
hydrolysis yields referred to raw material varied in a wide range
2162%, depending on pretreatment conditions.
From Fig. 5a, it can be deduced that the highest values of this
parameter are obtained from relatively high pretreatment temper-
atures (in the assayed range, pretreatment temperatures below the
medium point, 170 C, resulted in comparative lower enzymatic
hydrolysis yields). High temperatures of dilute acid hydrolysis
have been shown to result in increased enzymatic digestibility,
and that has been potentially attributed to the glass transition of
lignin (Jensen et al., 2010) which improves enzyme access to cellu-
lose pore. However, this fact can be counteracted by lignin recon-
densation reactions that occur at pretreatment conditions (Selig
et al., 2007).
Regarding the inuence of acid concentration on enzymatic
hydrolysis yields, it is enough using that necessary for the neutral-
izing capacity of biomass to be reached.
Enzymatic hydrolysis yields referred to pretreated material (g
glucose released/100 g glucose in pretreated material) show also
a wide range of variation (2298%). It is worth noting that, accord-
ing to Fig. 5b, relatively high results can be obtained from a wide
interval of free acid concentration pretreatment conditions, and
especially at high temperatures.
Comparing enzymatic hydrolysis yields with those previously
reported on the same feedstock, the best experimental result after
72 h enzyme action (61.54 g glucose/100 g raw material, equiva-
lent to 36.9 g/L) reported here compares well with that by Lu
et al. (2009), who reported 28.0 g glucose/L after 24 h. Enzymatic
digestibility as high as 95.4% after 72 h were reported by Jeong
et al. (2010) using pretreated rapeseed straw at optimized condi-
tions and favourable enzymatic dosage (60 FPU/g of glucan versus
15 FPU/g substrate in the present work) and substrate concentra-
tion (1%). If results are compared to other pretreatment methods
on the same raw material, it is found that enzymatic hydrolysis
yields based on raw material as high as 67.6 g/100 g were reported
after liquid hot water pretreatment at 210 C for 50 min, using the
same enzyme complex, while the present pretreatment conditions
were 170 C for 20 min and 0.5% w/v acid sulfuric concentration.
When results of glucose released by enzymes are referred to glu-
cose content of pretreated solids, values of enzymatic hydrolysis
yields are higher than those previously described. Even an experi-
mental result as high as 98.2 g glucose/100 g of glucose in the pre-
treated material was attained.
As a consequence of pretreatment, some sugars resulted de-
graded, and hence were neither available for enzymatic release
nor present in the hydrolysates. For example, Lau et al. (2009) re-
ported that about 13% of xylan was lost through chemical degrada-
tion of dilute acid pretreated corn stover. By contrast,
hydrothermal pretreatment of lignocellulosic materials is known
to result in a cellulose-enriched solid, which remains almost unal-
tered (Garrote et al., 2008).
The proportion of degraded sugars is also summarized in
Table 5, calculated as the difference between 100 and the sum of
the amount of each type of sugar in both the pretreated solid
and the liquid fraction. In general, sugar degradation occurred as
a function of pretreatment severity, and enzymatic hydrolysis
yields increased as sugar degradation did.
3.5. Model optimization
The mathematical model that was developed from the experi-
mental results is able to predict the operational conditions that
should be used in pretreatment to optimize model responses. Table
6 summarizes these conditions for several optimization criteria.
For example, optimization can be directed to the highest preserva-
tion or recovery of sugars, both in the solid (cellulose) and in
hydrolysates (mainly xylose). So if hemicellulosic sugars are to
140
150
160
170
180
190
200
0.4
0.8
1.2
1.6
2.0
0
10
20
30
40
50
60
70


Y
,

%


Temperature, C Free acid, %
0.4
0.8
1.2
1.6
2.0 140
150
160
170
180
190
200
0
20
40
60
80
100


Y
p
,

%


Free acid, %
Temperature, C
(a)
(b)
Fig. 5. Enzymatic hydrolysis yields as a function of free acid concentration and
pretreatment temperature after 72 h enzymatic action. (a) Referred to raw material
(pretreatment time, 6 min). (b) Referred to pretreated material (pretreatment time,
27 min).
Table 6
Optimized pretreatment conditions according to the mathematical model for
different optimization criteria.
Optimization
criteria
Pretreatement variables Optimal value
Time Temperature Free
acid
Maximize X
l
6.0 144.62 2.0 X
l
= 92.3%, G
s
= 99.4%
Maximize X
l
and
G
s
6.0 142.84 2.0 X
l
= 92.2%, G
s
= 100%
Maximize Y 27.0 180.47 0.40 Y = 64.93%
Maximize Y
p
27.0 200.0 0.40 Y
p
= 100%
E. Castro et al. / Bioresource Technology 102 (2011) 12701276 1275
be recovered in hydrolysates, along with cellulose in the pretreated
solids, it is best to perform pretreatment at about 143 C for 6 min
and 2% free acid concentration. This result is in close agreement
with that obtained by Jeong et al. (2010), which reported optimum
conditions of 152.6 C, 21 min and 1.76% acid concentration.
Maximization of enzymatic hydrolysis yields is possible under a
variety of pretreatment conditions. If yields are referred to raw
material, the total conversion of cellulose into glucose is predicted
to be reached if pretreatment is performed at 200 C for 27 min and
0.4% free acid concentration. Considering the whole raw material, a
maximum of 65% of the present glucose can be obtained, under the
enzymatic hydrolysis conditions assayed, when rapeseed is pre-
treated at 180 C, 27 min 0.4% free acid concentration.
4. Conclusions
This work conrms that rapeseed straw can be considered a
suitable feedstock for sugar generation as a rst step toward fuel
ethanol production. Pretreatment with dilute sulfuric acid allows
lower temperature to be used compared to hydrothermal pretreat-
ment. Total conversion of the cellulose retained in the pretreated
materials into glucose can be attained by operation at 200 C for
27 min and 0.4% free acid concentration. By contrast, hydrolysates
will be more difcult to ferment into ethanol due to higher inhib-
itor concentration. Further research for taking full advantage of
sugars in hydrolysates, including detoxication procedures, would
improve ethanol production from rapeseed straw.
Acknowledgements
This work was partially nanced by Agencia Espaola de Coop-
eracin Internacional para el Desarrollo (AECID) under Projects ref.
D/016096/08 and D/023784/09.
References
Abatzoglou, N., Chornet, E., Belkacemi, K., Overend, R.P., 1992. Phenomenological
kinetics of complex systems. The development of a generalized severity
parameter and its application to lignocellulosics fractionation. Chem. Eng. Sci.
47, 11091122.
Cara, C., Ruiz, E., Oliva, J.M., Sez, F., Castro, E., 2008. Conversion of olive tree
biomass into fermentable sugars by dilute acid pretreatment and enzymatic
saccharication. Bioresour. Technol. 99, 18691876.
Daz, M.J., Ruiz, E., Romero, I., Cara, C., Moya, M., Castro, E., 2009. Inhibition of Pichia
stipitis fermentation of hydrolysates from olive tree cuttings. World J. Microbiol.
Biotechnol. 25, 891899.
Daz, M.J., Cara, C., Ruiz, E., Romero, I., Moya, M., Castro, E., 2010. Hydrothermal
pretreatment of rapeseed straw. Bioresour. Technol. 101, 24282435.
Esteghlalian, A., Hashimoto, A.G., Fenske, J.J., Penner, M.H., 1997. Modeling and
optimization of the dilute-sulfuric-acid pretreatment of corn stover, poplar and
switchgrass. Bioresour. Technol. 59, 129136.
FAO, 2008. http://faostat.fao.org/faostat (last accessed 11-21-2008).
Garrote, G., Cruz, J.M., Domnguez, H., Paraj, J.C., 2008. Non-isothermal
autohydrolysis of barley husks: product distribution and antioxidant activity
of ethyl acetate soluble fractions. J. Food Eng. 84, 544552.
Jensen, J.R., Morinelly, J.E., Gossen, K.R., Brodeur-Campbell, M.J., Shonnard, D.R.,
2010. Effects of dilute acid pretreatment conditions on enzymatic hydrolysis
monomer and oligomer sugar yields for aspen, balsam, and switchgrass.
Bioresour. Technol. 101, 23172325.
Jeong, T.S., Um, B.H., Kim, J.S., Oh, K.K., 2010. Optimizing dilute-acid pretreatment of
rapeseed straw for extraction of hemicellulose. Appl. Biochem. Biotechnol. 161,
2233.
Karaosmanoglu, F., Tetik, E., Gll, E., 1999. Biofuel production using slow pyrolysis
of the straw and stalk of the rapeseed plant. Fuel Process. Technol. 59, 112.
Lau, M.W., Gunawan, C., Dale, B.E., 2009. The impacts of pretreatment on the
fermentability of pretreated lignocellulosic biomass: a comparative evaluation
between ammonia ber expansion and dilute acid pretreatment. Biotechnol.
Biofuels 2 (30), 111.
Li, H., Kim, N.J., Jiang, M., Kang, J.W., Chang, H.N., 2009. Simultaneous
saccharication and fermentation of lignocellulosic residues pretreated with
phosphoric acidacetone for bioethanol production. Bioresour. Technol. 100,
32453251.
Lloyd, T.A., Wyman, C.E., 2004. Predicted effects of mineral neutralization and
bisulfate formation on hydrogen ion concentration for dilute sulfuric acid
pretreatment. Appl. Biochem. Biotechnol. 113116, 10131022.
Lloyd, T.A., Wyman, C.E., 2005. Combined sugar yields for dilute sulfuric acid
pretreatment of corn stover followed by enzymatic hydrolysis of the remaining
solids. Bioresour. Technol. 96, 19671977.
Lu, X., Zhang, Y., Angelidaki, I., 2009. Optimization of H
2
SO
4
-catalyzed hydrothermal
pretreatment of rapeseed straw for bioconversion to ethanol: focusing on
pretreatment at high solids content. Bioresour. Technol. 100, 30483053.
National Renewable Energy Laboratory (NREL). Chemical Analysis and Testing
Laboratory Analytical Procedures: LAP-002 (1996), LAP-010 (1994) and LAP
017(1998). NREL, Golden, CO, USA. http://www.eere.energy.gov/biomass/
analyticalprocedures.html.
Overend, R.P., Chornet, E., 1987. Fractionation of lignocellulosics by steam-aqueous
pretreatments. Philos. Trans. R. Soc. London A 321, 523536.
Petersson, A., Thomsen, M.H., Hauggaard-Nielsen, H., Thomsen, A.B., 2007. Potential
bioethanol and biogas production using lignocellulosic biomass from winter
rye, oilseed rape and faba bean. Biomass Bioenergy 31, 812819.
Selig, M.J., Viamajala, S., Decker, S.R., Tucker, M.P., Himmel, M.E., Vinzant, T.B., 2007.
Deposition of lignin droplets produced during dilute acid pretreatment of maize
stems retards enzymatic hydrolysis of cellulose. Biotechnol. Progr. 23, 1333
1339.
Springer, E.L., Harris, J.F., 1985. Procedures for determining the neutralizing capacity
of wood during hydrolysis with mineral acid solutions. Ind. Eng. Chem. Prod.
Res. Dev. 24, 485489.
Shuai, L., Yang, Q., Zhu, J.Y., Lu, F.C., Weimer, P.J., Ralph, J., Pan, X.J., 2010.
Comparative study of SPORL and dilute-acid pretreatments of spruce for
cellulosic ethanol production. Bioresour. Technol. 101, 31063114.
Zabaniotou, A., Ioannidou, O., Skoulou, V., 2008. Rapeseed residues utilization for
energy and 2nd generation biofuels. Fuel 87, 14921502.
1276 E. Castro et al. / Bioresource Technology 102 (2011) 12701276