Optimization of H

2
SO
4
-catalyzed hydrothermal pretreatment of rapeseed straw
for bioconversion to ethanol: Focusing on pretreatment at high solids content
Xuebin Lu
a,b
, Yimin Zhang
a
, Irini Angelidaki
b,
*
a
Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
b
Department of Environmental Engineering, Technical University of Denmark, Lyngby 2800, Denmark
a r t i c l e i n f o
Article history:
Received 30 September 2008
Received in revised form 7 January 2009
Accepted 12 January 2009
Available online 5 March 2009
Keywords:
Hydrothermal pretreatment
H
2
SO
4
Rapeseed straw
Bioethanol
a b s t r a c t
A central composite design of response surface method was used to optimize H
2
SO
4
-catalyzed hydrother-
mal pretreatment of rapeseed straw, in respect to acid concentration (0.52%), treatment time (520 min)
and solid content (1020%) at 180 C. Enzymatic hydrolysis and fermentation were also measured to
evaluate the optimal pretreatment conditions for maximizing ethanol production. The results showed
that acid concentration and treatment time were more signicant than solid content for optimization
of xylose release and cellulose recovery. Pretreatment with 1% sulfuric acid and 20% solid content for
10 min at 180 C was found to be the most optimal condition for pretreatment of rapeseed straw for eth-
anol production. After pretreatment at the optimal condition and enzymatic hydrolysis, 75.12% total
xylan and 63.17% total glucan were converted to xylose and glucose, respectively. Finally, 66.79% of the-
oretical ethanol yielded after fermentation.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
World faces the progressive depletion of its energy resources
mainly based on non-renewable fuels. At the same time, energy
consumption grows at rising rates which will result in signicant
fuel price increase, before the fuel supply eventually will run short
(Davis et al., 2005; Cazetta et al., 2007; Ohgren et al., 2006). Biofuels
are promising substitute for fossil fuels because they can be pro-
duced from biomass which is renewable. Biofuels also provide the
opportunity for non-oil-producing countries to be self-sufcient
in fuels. Biofuels include biodiesel, bioethanol, biogas, biohydrogen
and others. Intensive research on biomass conversion to bioenergy
is currently carried out; however, most of the studies focus on indi-
vidual conversion processes and specic end-products. To gain full
benet, it is important to investigate production and utilization cy-
cles in an integrated way and to consider all important aspects in-
volved: e.g. crop production, residues, supply chain, exibility of
end-products and environmental aspects (Angelidaki et al., 2007).
Bioethanol is an energy carrier that can be used as fuel in road
vehicles and can be produced from sugars. Its advantage over bio-
gas is that it is a liquid fuel that can readily be integrated into exist-
ing fuel supply systems and directly substitute fossil fuels in the
transportation sector (Karakashev et al., 2007). Bioethanol has tra-
ditionally been produced from starch or sugars containing feed-
stocks such as sugarcane and starch from corn or wheat (Bothast
and Schlicher, 2005; Gulati et al., 1996). The technology for bioeth-
anol production from these feedstocks is mature. It doesnt need
chemical/physical pretreatment of biomass before enzymatic
hydrolysis. Additionally, optimized commercial enzymes are avail-
able. However, due to the high prices of feedstocks that account for
almost 4075% of the total ethanol production and the competition
with food, alternative feedstocks are needed for ethanol production
such as wastes or agricultural residues (lignocellulosic materials)
which include straw, wood and waste.
Lignocellulose is the most abundant organic material on earth
and also is a promising raw material for bioenergy production
(Lu et al., 2007; Zhao et al., 2008). Lignocellulosic materials contain
cellulose and hemicellulose that are bound together by lignin. Cel-
lulose and hemicellulose are both polymers built up by long chains
of sugar monomers, which after pretreatment and hydrolysis can
be converted into sugars and produce bioenergy by microbial fer-
mentation. Rapeseed crop is an attractive feedstock for bioenergy
production. Its seeds can be used to produce biodiesel while the ra-
peseed straw is good for bioethanol and biohydrogen production
(Karakashev et al., 2007).
The so-called second generation technologies, based on waste
or residues as feedstocks for biofuels production, emerged to
accommodate the new feedstocks. While the production of bioeth-
anol from sugars and starch is more straightforward, bioethanol
production from lignocellulose creates additional technical chal-
lenges, such as a need for chemical/physical pretreatment to loos-
en up the lignocellulosic structure and facilitate enzymatic
hydrolysis (Ballesteros et al., 2004; Galbe and Zacchi, 2002). Bio-
mass pretreatment technology has been widely investigated in re-
cent years and various biological, chemical and physical
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.01.008
* Corresponding author. Tel.: +45 45251429; fax: +45 45932850.
E-mail address: ria@env.dtu.dk (I. Angelidaki).
Bioresource Technology 100 (2009) 30483053
Contents lists available at ScienceDirect
Bioresource Technology
j our nal homepage: www. el sevi er. com/ l ocat e/ bi or t ech
pretreatment approaches have been proven to increase the suscep-
tibility of cellulose to enzymatic attack. Among the different exist-
ing pretreatment methods, hydrothermal pretreatment such as
liquid hot water pretreatment has been reported to have the po-
tential to improve cellulose digestibility, sugar extraction and pen-
tose recovery in pretreatment, with the advantage of producing
hydrolysates that result in little or no inhibition of sugar fermenta-
tion (Cara et al., 2007a,b; Zeng et al., 2007). Liquid hot water pre-
treatment is proved to be an efcient pretreatment method to
remove the hemicellulose from raw material. However, this kind
of pretreatment is carried out at lower solid loading (510%, w/
w) which requires a lot of energy for heating the water (Laser
et al., 2002; Negro et al., 2003). Therefore, technologies, requiring
low water addition to the straw, should be developed. Recently,
Wyman et al. (2005) evaluated different pretreatment technologies
on corn stover and reported on the efciency of methods based on
the use of water, acids, lime or ammonia. It is stated that an acid or
a base is necessary to effectively disrupt biomass structure by
removing hemicellulose or lignin at a reasonable cost. During the
pretreatment process, some non-sugar compounds are also re-
leased because of the degradation of sugars. These compounds in-
clude furfural, hydroxymethylfurfural (HMF) and acetic acid which
are known to inhibit fermentation process at a certain concentra-
tion level (Liu, 2006; Lu et al., 2008). In order to optimize the over-
all process, attention must be paid to several partial objectives, e.g.
cellulose recovery in the solid residue, hemicellulosic-derived sug-
ars recovery and prevention of inhibitors release in the ltrate
(hydrolysate) both in the pretreatment step, hydrolysis yield in
the enzymatic step, and ethanol yield in fermentation process.
In this study, H
2
SO
4
-catalyzed hydrothermal pretreatment was
carried out to pretreat rapeseed straw. A central composite design
of response surface method was used to evaluate the effect of acid
concentration, treatment time and solid content on the hydrother-
mal pretreatment. Evaluation criteria for optimization of the pre-
treatment conditions were based on high xylose yield and low
inhibitors (VFA, furfural and HMF) content in the hydrolysate and
on high cellulose recovery from the solid residues obtained after
ltration of whole pretreated materials. The bioethanol yield and
productivity from the solid phase were also evaluated by enzy-
matic hydrolysis and fermentation to evaluate the optimal pre-
treatment conditions.
2. Methods
2.1. Rapeseed straw and cellulase
Raw rapeseed straw was obtained from a local farm and was cut
to about 1 cm in length.
The commercial cellulase enzyme used in this study was cellu-
clast 1.5 L. b-Glucosidase Novozym 188 was also used in this study
for the hydrolysis of rapeseed straw. Both enzymes were kindly
provided by Novozymes. The yeast of Saccharomyces cerevisiae
was from a local supermarket.
2.2. H
2
SO
4
-catalyzed hydrothermal pretreatment
Pretreatment was performed in a laboratory scale stainless steel
cylindrical reactor with a total volume of 500 mL. A salt bath with a
liquid mixture of NaNO
2
and KNO
3
in a proportion 1:1 was used to
give the wanted temperature. The reactor was heated at a rate be-
tween 15 and 20 C min
1
. When the desired temperature inside
the reactor was reached, the treatment time was started to be
counted. After the target treatment time was reached, the reactor
was taken out from the salt bath and sunk into a water bath to cool
it down to 30 C for about 5 min.
The wet material was ltrated by vacuum pump, obtaining a so-
lid phase and a liquid phase. The solid phase was washed thor-
oughly with water, weighed and analyzed for carbohydrate
content. Liquid phase was analyzed for sugar, acetic acid, furfural
and HMF content.
2.3. Enzymatic hydrolysis and fermentation
After pretreatment, enzymatic hydrolysis was carried out at a
solid loading of 10% DM. All experiments were done in duplicates.
Hydrolysis was performed at 52 C and pH 5.0 with an enzyme
loading of 0.11 g celluclast 1.5 L/g cellulose and 0.05 g b-glycosi-
dase/g cellulose for 24 h. After cooling to room temperature, 10 g
yeast/L liquid was added. The fermentation was carried out in
100 mL Pyrex asks. These asks were equipped with yeast locks
lled with glycerol for the release of produced CO
2
from the asks,
and then incubated at 37 C for a period of time. The concentra-
tions of sugars and ethanol were determined by high performance
liquid chromatography (HPLC).
2.4. Analysis of carbohydrates in solid phase
The raw material composition was determined according to the
National Renewable Energy Laboratory (NREL, Golden, CO) analyti-
cal methods for biomass (NREL, 19941998). To quantify the sugar
polymers inthe rawmaterial andthe solidphase after pretreatment,
a two-step acid hydrolysis was performed. The rst hydrolysis step
was performed at 30 C for 60 min with 1.5 mL of H
2
SO
4
(72%) for
0.15 g DM. Then 42 mL water was added and the second step was
performed at 121 C for 60 min. The hydrolysate was ltrated and
the dried lter cake subtracted for ash content is called Klason lig-
nin. The ltrate was then analyzed for sugar content by HPLC.
2.5. HPLC analysis
The released sugar monomers in the hydrolysate as well as con-
centrations of ethanol were determined by HPLC (Agilent) using a
column (BioRad Aminex HPX-87H, 300 7.8 mm) at 64 C and
4 mM H
2
SO
4
as eluent at a ow rate of 0.6 mL min
1
. A refractive
detector was used for sugars, acetic acid and ethanol and a UV
detector was used for furfural and HMF.
2.6. Central composite design
A central composite design was employed to reduce the total
number of experiments needed to determine the best combination
of parameters for optimization of the process. The statistical soft-
ware Design-expert, version 7.1.4 was used for the central com-
posite design and to analyze the experimental data obtained. The
conditions for each experiment are shown in Table 1.
2.7. Scanning electron microscopy of rapeseed straw surface
characteristics
Samples of rapeseed straw before and after pretreatment were
mounted on stubs and sputter-coated with C prior to imaging with
FEI QUANTA 200 FEG scanning electron microscope (SEM) using
20 kV accelerating voltage.
3. Results and discussion
3.1. Raw material composition
The initial composition of rapeseed straw was determined as:
37.0% cellulose, 19.6% hemicellulose, 18.0% Klason lignin, 5.7%
X. Lu et al. / Bioresource Technology 100 (2009) 30483053 3049
ash and 19.7% others. Carbohydrates accounted for about 60% of
the dry material which makes rapeseed straw a very promising
substrate for ethanol production. The hemicellulose part consisted
of 93% xylose and small amounts of arabinose. The lignin content
of rapeseed straw biomass is in the range of that reported for other
agricultural residues such as corn stover (1719%) (Kim et al.,
2005). The high ash content of rapeseed straw is consistent with
the presence of silica as a major mineral component of straws
and the great inuence of fertilizer application in herbaceous bio-
mass crops (Perez et al., 2007). There were also 19.7% other compo-
nents which are chemically bound water and ethanol soluble
materials and proteins.
3.2. Effect of pretreatment on hydrolysate composition
After pretreatment, the slurry was separated into liquid phase
(hydrolysate) and solid phase. Changes in the composition of the
solid phase and the composition in sugars and other compounds
of the liquid phase, compared to the composition of the raw mate-
rial, as a result of the pretreatment were measured and shown in
Table 2. The variable considered in the analysis of the liquid phase
was xylose yield. Furfural, HMF and acetic acid were considered as
the main inhibitors in this process. In biorenery process, the
hydrolysate (mainly xylose) from pretreatment can be fermented
to e.g. biohydrogen through dark fermentation (Hawkes et al.,
2008; Chen et al., 2008) or fermented to ethanol by ethanol pro-
ducers utilizing pentoses (Qian et al., 2006; Chandel et al., 2007).
Therefore, the optimal combination of pretreatment conditions
was selected according to the high xylose yield and low inhibitor
concentrations (below inhibitory level of the fermentation
process).
The xylose yield values were dependent on the acid concentra-
tion and treatment time applied. An increase in acid concentration
from 0.5% to 2% when experiments carried out at a treatment time
of 5 minutes caused an increase in the xylose yield from 36.7% to
67.2% of the theoretical xylose content (20.3 g/100 g DM). How-
ever, when the acid concentration increased from 1% to 2%, only
a slight increase (1.5%) of the xylose yield was observed. The max-
imum yield achieved was approx. 70%, which is higher than what
has previously been reported with other lignocellulose materials
subjected to liquid hot water treatment. Perez et al. (2007)
achieved 53% xylose yield from wheat straw with liquid hot water
pretreatment at 200 C for 0 min (considered to be the time at
which target temperature in reactor was reached). Sreenath et al.
(1999) obtained a liquid phase with a hemicellulose-derived sug-
ars yield of 50% after treating alfalfa bers at 220 C for 2 min.
Table 2 shows that an increase in the treatment time in the
reactor from 5 to 20 min at a sulfuric acid concentration of 1%, also
produced a noticeable improvement in xylose yield (from 39.1% to
68.4%). However, at an acid concentration of 2%, the increase of
time (from 10 min to 20 min) resulted in decrease in the xylose
recovery (from 67.2% to 27.3%) which was probably due to the
decomposition of the released xylose to degradation products such
as volatile fatty acids.
The model used tted the experimental data well (Fig. 1A,
P < 0.05) which proves that the interpretation, based on signi-
cance of the effects of factors in the experimental interval and their
possible interactions, was correct. From the analysis of variances, it
could be concluded that the interaction of acid concentration and
treatment time was more signicant (P < 0.05) than solid content,
which means that acid concentration and treatment time have a
strong inuence on the treatment process. Details of the experi-
mental results on xylose yield in the liquid phase through response
surface analysis are shown in Fig. 1B. Due to economic advantages
of the high solids process, the solid content of the material to be
Table 1
Central composite design conditions for H
2
SO
4
-catalyzed hydrothermal pretreatment
of rapeseed straw.
Run Acid concentration (% w/w) Treatment time (min) Solid content (% w/w)
1 1 20 15
2 1 10 15
3 1 10 20
4 0.5 10 15
5 1 10 5
6 1 10 10
7 2 5 5
8 1 5 10
9 0.5 5 20
10 0.5 5 5
11 2 5 20
12 1 10 15
13 2 20 20
14 0.5 20 5
15 0.5 20 20
16 2 10 15
17 2 20 5
Table 2
The composition of solid and liquid phase (hydrolysate) after H
2
SO
4
-catalyzed hydrothermal pretreatment of rapeseed straw.
Run Solid recovery g/100 g
untreated material
Liquid phase (hydrolysate) Xylose
recovery
Solid phase (g/
100 g DM)
Glucose recovery in
WIS 100%
Xylose
(g/L)
Xylose %
theoretical
Furfural + HMF (g/
100 gDM)
Acetic acid (g/
100 gDM)
Xylan Cellulose
1 59.4 21.0 70.1 0.78 4.23 88.3 5.8 59.4 87.0
2 71.6 19.7 65.7 0.76 3.82 91.8 7.5 56.6 99.4
3 73.6 27.3 68.4 0.57 2.81 93.7 7.8 56.9 102.3
4 82.4 11.0 36.7 0.51 1.68 93.2 14.1 48.2 96.3
5 60.0 14.0 70.0 1.08 4.40 89.4 5.4 62.8 92.1
6 71.1 20.0 66.8 0.71 3.39 96.7 8.6 56.3 97.2
7 52.6 12.1 60.6 1.06 4.12 73.1 3.8 61.9 79.4
8 83.7 11.7 39.1 0.56 1.85 95.5 13.7 47.1 95.8
9 88.7 6.4 15.9 0.25 0.66 90.1 17.2 41.0 88.6
10 81.3 9.1 45.7 0.75 2.52 95.0 12.0 44.0 87.2
11 59.1 21.8 54.5 0.53 2.60 78.2 8.3 59.5 85.0
12 74.4 19.3 64.2 0.65 3.22 94.0 8.3 57.6 105.1
13 79.3 22.8 56.9 0.56 2.50 90.3 9.3 59.1 114.2
14 84.9 9.5 47.7 0.77 2.16 99.3 11.9 48.6 101.7
15 76.7 20.0 50.1 0.50 2.77 90.7 11.3 50.7 95.5
16 60.0 20.2 67.2 0.81 4.21 77.9 3.2 60.4 87.8
17 57.0 5.5 27.3 1.21 4.16 31.9 1.4 35.8 50.2
3050 X. Lu et al. / Bioresource Technology 100 (2009) 30483053
treated was set at 20% (w/w). The surface response chart shows
that the optimal values of xylose yield can be found when the acid
concentration and treatment time were both xed at central
points.
The concentrations of inhibitors are also shown in Table 2. In
our experiments, the range of the total concentration of furfural
and HMF was from 0.5 g/L to 1.2 g/L which was lower than the con-
centration which is considered to inhibit fermentation (Nichols
et al., 2008). It has been reported that acetic acid concentration be-
low 6 g/L was not inhibitory for ethanol fermentation (Larsson
et al., 1999). In our experiments, in most cases the acetic acid con-
centration was below 6 g/L, and at the optimal condition it was
5.6 g/L.
3.3. Effect of pretreatment on solid phase composition
In order to evaluate the efciency of the pretreatment process
the solids recovery in the solid phase was determined as percent-
age of the dry weight of solid remaining after pretreatment, rela-
tive to the dry weight of the raw material. The recoveries
achieved are shown in Table 2, and ranged from 57% to 88%,
depending on the pretreatment conditions. The recoveries
achieved in this study are in the same range as previously reported
(Perez et al., 2007). Lowest recovery was obtained at 2% acid con-
centration and 5% solid loading for 20 min.
The model used to evaluate the cellulose content in solid phase
tted with the experimental data well (Fig. 2A, P < 0.05). The re-
sponse surface showed that the highest cellulose content appeared
at the highest acid concentration and shortest treatment time.
However, when the acid concentration was xed at 1%; 56.9
61.0% of the cellulose content was obtained within the time 10
to 20 min. Considering both a high xylose yield in the hydrolysate
and a high cellulose content in solid phase, the optimal pretreat-
ment condition was selected as 1% H
2
SO
4
at 20% solid content for
10 min. Optimized xylose yields in the hydrolysate were obtained
at the optimal pretreatment condition, resulting in a xylose yield of
68.4% in the hydrolysate and 56.9% cellulose content in solid phase.
The cellulose content in the raw material was 37% (Table 1) and
there was a 20% increase (56.9%) in the solid phase after pretreat-
ment because of the hemicellulose degradation which reduced the
total amount of sugars.
3.4. Effect of pretreatment on enzymatic hydrolysis and fermentation
Fig. 3 shows the glucose, xylose and ethanol concentrations
during the enzymatic hydrolysis and fermentation process. The
concentration of glucose increased with time during the hydrolysis
process. After 24 h hydrolysis 28.0 g/L glucose could be obtained
with a productivity 1.16 g/L h. During the rst 6 h, the productivity
was much higher which was 3.08 g glucose/L h. The decrease of the
glucose production rate with time was due to inhibition of the
enzymatic hydrolysis process by the released glucose (Cara et al.,
2007a,b). Therefore, simultaneous saccharication and fermenta-
tion could be an advantageous method to decrease this hydrolysis
inhibition. The xylose production during the enzymatic hydrolysis
increased the rst 6 h and then was kept stable. The nal concen-
tration of xylose was about 3.0 g/L.
In the fermentation process, the concentration of glucose de-
creased from 28 g/L to nearly zero while the concentration of eth-
anol increased from zero to about 17 g/L during the rst stage and
then the ethanol concentration kept stable. This is similar with
what have been achieved by other researchers (Mosier et al.,
Fig. 1. (A) Predicted versus experimental values for xylose yield in hydrolysate, (B) 3D response surface for xylose yield in relation to acid concentration and treatment time.
Fig. 2. (A) Predicted versus experimental values for cellulose content in solid phase, (B) 3D response surface for cellulose content in relation to acid concentration and
treatment time.
X. Lu et al. / Bioresource Technology 100 (2009) 30483053 3051
2005; Kim et al., 2008a,b). The productivity of ethanol was 2.13 g/
L h during the rst 8 h which is a promising productivity for etha-
nol production. Ohgren et al. (2007) obtained an ethanol produc-
tivity of 0.96 g/L h the rst 24 h of fermentation. The material
had been subjected to 16 h enzymatic hydrolysis with 10% solid
loading. Petersson et al. (2007) got an ethanol productivity from
pretreated oilseed rape straw of 0.91 g/L h in liquid phase and as
low as 0.16 g/L h in solid phase after enzymatic hydrolysis. The -
nal ethanol concentration was 19 g/L, while the concentration of
glucose after hydrolysis was 28 g/L which theoretically can only
produce 14.3 g/L ethanol. However, the nal ethanol concentration
was higher than 14.3 g/L in this study (19 g/L) which shows that
the hydrolysis was still going on along with the fermentation,
although the rate was very slow due to the lower temperature
optimum of the fermentation (37 C) compared to hydrolysis
(50 C) The xylose concentration was stable during the fermenta-
tion, which showed that this yeast could not use xylose.
Yeast and other ethanol producers are known to suffer from
ethanol inhibition. Kadar et al. (2007) reported that S. cerevisiae
ATCC 26602 could be adapted to higher concentration of inhibitors
on spruce matrix with 5.2 vol% (about 50 g/L) nal ethanol concen-
tration. Cazetta et al. (2007) used Z. mobilis to ferment molasses
and a high ethanol concentration of 55.8 g/L could be obtained.
In this study, the nal ethanol concentration was lower than the
one that can inhibit the yeast.
The nal ethanol concentration was 19 g/L in this study which
is about 2% vol% of alcohol. For an economically feasible process,
ethanol yields need to be as high as possible which can decrease
the energy input of the following distillation (Kim et al.,
2008a,b). Based on the cellulose content in the solid phase after
pretreatment, it can be calculated that the maximum theoretical
ethanol concentration was 32.2 g/L. The sugars released fromenzy-
matic hydrolysis were efciently and quickly fermented to ethanol
(Fig. 3). So, if the sugar concentration from the enzymatic hydroly-
sis could further be increased, the nal ethanol concentration
would also be increased. After 24 h enzymatic hydrolysis, 28 g/L
glucose was released. The sugar concentration was still increasing
after 24 h fermentation and it could be higher if longer time was
given (Fig. 3). In other studies, the enzymatic hydrolysis performed
for 48 h to 72 h and even for 7 d (Ewanick et al., 2007; Zhao et al.,
2008). In this study, 60% of the cellulose was converted to glucose
after 24 h enzymatic hydrolysis. It proves that the H
2
SO
4
-catalyzed
hydrothermal pretreatment had improved the enzymatic hydroly-
sis of the straw which can shorten the time needed for complete
hydrolysis.
3.5. Physical surface changes to rapeseed straw due to pretreatment
Besides affecting the chemical composition of rapeseed straw,
pretreatment also affected the physical appearance of the rapeseed
straw at the microscopic level. SEM was used to observe the struc-
ture change of rapeseed straw before and after pretreatment with
1% H
2
SO
4
at 20% solid content for 10 min. The vascular structures
of raw rapeseed straw can be clearly seen in the rawmaterial with-
out pretreatment. The surface of the raw rapeseed straw that is
undamaged by the grinding is more-or-less smooth and continu-
ous. The structure of cellulose is a linear polysaccharide polymer
and the long chain of cellulose can be seen clearly by SEM. Hemi-
cellulose has a random, amorphous structure with little strength
and was shown as small irregular clumps on the surface and en-
twined with cellulose and lignin. After pretreatment, the smooth,
continuous surface of untreated rapeseed straw has been de-
stroyed and perforated by the pretreatment process. The long
chain of cellulose was cut and most of the hemicellulose was re-
moved. This is also supported by the analysis of the hydrolysate
composition, showing that 68.4% of hemicellulose was converted
to pentose (Table 2). It can be seen that a lot of pores are distrib-
uted on the surface of the rapeseed straw. Oblong pores on the sur-
face were approximately 5 lm by 1 lm in size. The pores inicted
by the pretreatment process, may increase the enzyme-accessible
surface area which increases the enzyme digestibility of the rape-
seed straw and presumably shorten the time of enzymatic hydro-
lysis process. In this study, 60% of cellulose was converted to
glucose within 24 h which proved that the pretreatment increased
the enzymatic ability of the rapeseed straw. Similar results were
also obtained by other researchers using other materials such as
corn stover (Mosier et al., 2005; Kim and Lee, 2007).
3.6. Overall mass balance
A detailed mass balance was made for each step and is shown in
Fig. 4. The pretreatment carried out with 1% sulfuric acid at 20% so-
lid content for 10 min, was the most optimal combination of
parameters, resulting in the highest yields. After pretreatment,
26.4% of the solid was dissolved and 27.6 g xylose/200 g raw mate-
rial and 10.1 g glucose/200 g rawmaterial were released during the
pretreatment process. Following pretreatment, a 24 h enzymatic
hydrolysis process, released 41.7 g glucose and 2.9 g xylose. In
the two processes, there were 51.8 g glucose and 30.5 g xylose
released which meant 63.7% of the glucan was hydrolyzed to
monomeric glucose and 75.1% of the xylan was converted to mono-
meric xylose through pretreatment and cellulase hydrolysis. Fig. 3
shows that the concentration of glucose and xylose are kept
increasing during the rst 24 h and it would had increased more
0
5
10
15
20
25
30
0 8 16 24 32 40 48 56 64 72
Time (h)
C
o
n
c
.
(
g
/
L
)
glucose in hydrolysis
xylose in hydrolysis
glucose in fermentation
xylose in fermentation
ethanol
Fig. 3. The time course of glucose, xylose and ethanol concentrations during the
hydrolysis and fermentation process.
s d i l o s d e v l o s s i d n u g 2 . 7 4 1 g 0 0 8 r e t a W
s d i l o s d e v l o s s i d g 8 . 2 5
e s o c u l g g 1 . 0 1 , e s o l y x g 6 . 7 2 g 0 0 2 w a r t s d e e s e p a R
82.2g glucose 147.2g solids 800g water
40.6g xylose
Cellulase 1.5L
11g (0.11g/g cellulose)
e t a s y l o r d y H e s a d i s o c y l g -
e s o c u l g g 7 . 1 4 d i u q i L ) e s o l u l l e c g / g 5 0 . 0 ( g 5
e s o l y x g 9 . 2
Residual solid 93.7g Ethanol 28.0g
Pretreatment
Hydrolysis (24h)
Fermentation
Fig. 4. Mass balance for pretreatment, hydrolysis and fermentation for rapeseed
straw.
3052 X. Lu et al. / Bioresource Technology 100 (2009) 30483053
if fermentation to ethanol was not initiated. Finally, 28 g ethanol
was obtained after 48 h fermentation which corresponds to 67%
of theoretical yield.
In this study, H
2
SO
4
-catalyzed hydrothermal pretreatment was
carried out at higher solid content (20%) compared to the solids
content in other studies (510%). Pretreatment at high solid con-
tent can reduce the energy input for heating the water which can
increase the economic feasibility of the biorenery process. The
high sugars concentrations obtained from the pretreatment at high
solid content are advantageous for producing biofuels such as bio-
hydrogen or bioethanol.
4. Conclusions
Rapeseed straw is an attractive raw material for fuel ethanol
production due to its high content of carbohydrates (more than
60% carbohydrates). This study has demonstrated that hemicellu-
lose in the rapeseed straw can be removed efciently by H
2
SO
4
-
catalyzed hydrothermal pretreatment at a high solid loading
(20%). The optimal combination of pretreatment conditions was
found to be 20% solids content with addition of 1% sulfuric acid
and treatment time of 10 min at 180 C. 27.3 g/L xylose was re-
leased corresponding to 68.4% of the theoretical yield. Furfural,
HMF and acetic acid which are inhibitors for the fermentation pro-
cess, were 0.57 g/100 g DM (furfural + HMF) and 2.81 g/100 g DM,
respectively, which are both below level that can cause inhibition.
After enzymatic hydrolysis and fermentation, 19 g/L ethanol was
produced corresponding to 67% of the theoretical yield.
Acknowledgements
The authors acknowledge the nancial support from the Re-
search and Innovation Council under the strategic research pro-
gram of Bio. REF. Project No. 2104-06-0004. Additionally, the
China scholarship council is acknowledged for its nancial support.
We thank Lars Ellegaard and Birgir Norddahl for their help on the
reactor and Ramona Valentina Mateiu for her work on SEM.
References
Angelidaki, I., Kongjan, P., Thomsen, M.H., Thomsen, A., 2007. Biorenery for
sustainable biofuel production from energy crops; conversion of lignocellulose
to bioethanol, biohydrogen and biomethane. AD 11 Conference in Australia, 22
September.
Ballesteros, M., Oliva, J.M., Negro, M.J., 2004. Ethanol from lignocellulosic materials
by a simultaneous saccharication and fermentation process with
Kluyveromyces marxianus CECT 10875. Process Biochem. 39, 18431848.
Bothast, R.J., Schlicher, M.A., 2005. Biotechnological processes for conversion of corn
into ethanol. Appl. Microbiol. Biotechnol. 67, 1925.
Cara, C., Moya, M., Ballesteros, I., Negro, M., Gonzalez, A., Ruiz, E., 2007a. Inuence of
solid loading on enzymatic hydrolysis of steam exploded or liquid hot water
pretreated olive tree biomass. Process Biochem. 42, 10031009.
Cara, C., Romero, I., Oliva, J.M., Saez, F., Castro, E., 2007b. Liquid hot water
pretreatment of olive tree pruning residues. Appl. Biochem. Biotechnol. 136
140, 379394.
Cazetta, M.L., Celligoi, M.A.P.C., Buzato, J.B., Scarmino, I.S., 2007. Fermentation of
molasses by Zymomonas mobilis: effects of temperature and sugar
concentration on ethanol production. Bioresour. Technol. 98, 28242828.
Chandel, A.K., Kapoor, R.K., Singh, A., Kuhad, R.C., 2007. Detoxication of sugarcane
bagasse hydrolysate improves ethanol production by Candida Shehatae NCIM
3501. Bioresour. Technol. 98, 19471950.
Chen, S.D., Lee, K.S., Lo, Y.C., Chen, W.M., Wu, J.F., Lin, C.Y., Chang, J.S., 2008. Batch
and continuous biohydrogen production from starch hydrolysate by Clostridium
species. Int. J. Hydrogen Energy 33, 18031812.
Davis, L., Jeon, Y., Svenson, C., Rogers, P., Pearce, J., Peiris, P., 2005. Evaluation of
wheat stillage for ethanol production by recombinant Zymomonas mobilis.
Biomass Bioenergy 29, 4959.
Ewanick, S.M., Bura, R., Saddler, J.N., 2007. Acid-catalyzed steam pretreatment of
lodgepole pine and subsequent enzymatic hydrolysis and fermentation to
ethanol. Biotechnol. Bioenergy 98, 737746.
Galbe, M., Zacchi, G., 2002. A review of the production of ethanol from softwood.
Appl. Microbiol. Biotechnol. 59, 618628.
Gulati, M., Kohlman, K., Ladisch, M.R., Hespell, R., Bothast, R.J., 1996. Assessment of
ethanol production options for corn products. Bioresour. Technol. 58, 253264.
Hawkes, F.R., Forsey, H., Premier, G.C., Dinsdale, R.M., Hawkes, D.L., Guwy, A.J.,
Maddy, J., Cherryman, S., Shine, J., Auty, D., 2008. Fermentative production of
hydrogen from a wheat our industry co-product. Bioresour. Technol. 99, 5020
5029.
Kadar, Z., Maltha, S.F., Szengyel, Z., Reczey, K., Laat, W.D., 2007. Ethanol
fermentation of various pretreated and hydrolyzed substrates at low initial
pH. Appl. Biochem. Biotechnol. 136140, 847858.
Karakashev, D., Thomsen, A.B., Angelidaki, I., 2007. Anaerobic biotechnological
approaches for production of liquid energy carriers from biomass. Biotechnol.
Lett. 29, 10051012.
Kim, S., Holtzapple, M.T., 2005. Lime pretreatment and enzymatic hydrolysis of corn
stover. Bioresour. Technol. 96, 19942006.
Kim, T.H., Lee, Y.Y., 2007. Pretreatment of corn stover by soaking in aqueous
ammonia at moderate temperatures. Appl. Biochem. Biotechnol., 8192
(Biotechnol. Bioenergy, 98, 737746).
Kim, T.H., Taylor, F., Hicks, K.B., 2008a. Bioethanol production from barley hull using
SAA (soaking in aqueous ammonia) pretreatment. Bioresour. Technol. 99, 5694
5702.
Kim, Y., Hendrickson, R., Mosier, N.S., Ladisch, M.R., Bals, B., Balan, V., Dale, B.E.,
2008b. Enzyme hydrolysis and ethanol fermentation of liquid hot water and
AFEX pretreated distillers grains at high-solids loadings. Bioresour. Technol. 99,
52065215.
Larsson, S., Palmqvist, E., Hahn-Hagerdal, B., Tengborg, C., Stenberg, K., Zacchi, G.,
Nilvebrant, N., 1999. The generation of fermentation inhibitors during dilute
acid hydrolysis of softwood. Enzyme Microb. Technol. 24, 151159.
Laser, M., 2002. A comparison of liquid hot water and steam pretreatments of sugar
cane bagasse for bioconversion to ethanol. Fuel and Energy Abstracts 43, 243
244.
Liu, Z.L., 2006. Genomic adaptation of ethanologenic yeast to biomass conversion
inhibitors. Appl. Microbiol. Biotechnol. 73, 2736.
Lu, X.B., Zhang, Y.M., Yang, J., Liang, Y., 2007. Enzymatic hydrolysis of corn stover
after pretreatment with diluted sulfuric acid. Chem. Eng. Technol. 30, 938
944.
Lu, X.B., Zhang, Y.M., Liang, Y., Yang, J., Zhang, S.T., Suzuki, E., 2008. Kinetic studies of
hemicellulose hydrolysis of corn stover at atmospheric pressure. Korean J.
Chem. Eng. 25, 302307.
Mosier, N., Hendrickson, R., Ho, N., Sedlak, M., Ladisch, M.R., 2005. Optimization of
pH controlled liquid hot water pretreatment of corn stover. Bioresour. Technol.
96, 19861993.
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/analytical
procedures.html.
Negro, M.J., Manzanares, P., Ballesteros, I., Oliva, J.M., Cabanas, A., Ballesteros, M.,
2003. Hydrothermal pretreatment conditions to enhance ethanol production
from poplar biomass. Appl. Biochem. Biotechnol. 105108, 87100.
Nichols, N.N., Sharma, L.N., Mowery, R.A., Chambliss, C.K., Walsum, G.P.V., Dien, B.S.,
Iten, L.B., 2008. Fungal metabolism of fermentation inhibitors present in corn
stover dilute acid hydrolysate. Enzyme Microb. Technol. 42, 624630.
Ohgren, K., Rudolf, A., Galbe, M., Zacchi, G., 2006. Fuel ethanol production from
steam-pretreated corn stover using SSF at higher dry matter content. Biomass
Bioenergy 30, 863869.
Ohgren, K., Vehmaanpera, J., Siika-Aho, M., Galbe, M., Viikari, L., Zacchi, G., 2007.
High temperature enzymatic prehydrolysis prior to simultaneous
saccharication and fermentation of steam pretreated corn stover for ethanol
production. Enzyme Microb. Technol. 40, 607613.
Perez, J.A., Gonzalez, A., Oliva, J.M., Ballesteros, I., Manzanares, P., 2007. Effect of
process variables on liquid hot water pretreatment of wheat straw for
bioconversion to fuel-ethanol in a batch reactor. J. Chem. Technol. Biotechnol.
82, 929938.
Petersson, A., Thomsen, M.H., Hauggaard-Nielsen, H., Thomsen, A., 2007. Potential
bioethanol and biogas production using lignocellulosic biomass from winter
rye, oilseed rape and faba bean. Biomass Bioenergy 31, 812819.
Qian, M.Y., Tian, S., Li, X.F., Zhang, J., Pan, Y.P., Yang, X.S., 2006. Ethanol production
from dilute-acid softwood hydrolysate by co-culture. Appl. Biochem.
Biotechnol. 134, 273283.
Sreenath, H.K., Koegel, R.G., Moldes, A.B., Jeffries, T.W., Straub, R.J., 1999. Enzymic
saccharication of alfalfa ber after liquid hot water pretreatment. Process
Biochem. 35, 3341.
Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, C.M., Lee, Y.Y., 2005.
Coordinated development of leading biomass pretreatment technologies.
Bioresour. Technol. 96, 19591966.
Zeng, M., Mosier, N.S., Huang, C., Sherman, D.M., Ladisch, M.R., 2007. Microscopic
examination of changes of plant cell structure in corn stover due to hot water
pretreatment and enzymatic hydrolysis. Biotechnol. Bioenergy 97, 265278.
Zhao, Y., Wang, Y., Zhu, J.Y., Ragauskas, A., Deng, Y., 2008. Enhanced enzymatic
hydrolysis of spruce by alkaline pretreatment at low temperature. Biotechnol.
Bioenergy 99, 13201328.
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