Bioethanol Production from Enzymatically Saccharified Sunflower Stalks Using Steam Explosion as Pretreatment

World Academy of Science, Engineering and Technology 49 2009
Bioethanol Production from Enzymatically
Saccharified Sunflower Stalks Using Steam
Explosion as Pretreatment
Pilanee Vaithanomsat, Sinsupha Chuichulcherm, and Waraporn Apiwatanapiwat
4.4% was found in stalk. Sharma et al. (2002) also showed
Abstract—Sunflower stalks were analysed for chemical that sunflower stalk was lignocellulosics, like other
compositions: pentosan 15.84%, holocellulose 70.69%, agricultural residues such as wheat straw or corn cob, which
alphacellulose 45.74%, glucose 27.10% and xylose 7.69% based on
contained 33.5% hemicellulose and 38.5% cellulose.
dry weight of 100-g raw material. The most optimum condition for
However, little attention has been focused on utilisation of
steam explosion pretreatment was as follows. Sunflower stalks were
sunflower wastes as substrates for bioethanol production.
cut into small pieces and soaked in 0.02 M H2SO4 for overnight.
After that, they were steam exploded at 207 C and 21 kg/cm2 for 3
Sharma et al (2004) experimented that the cellulose from
minutes to fractionate cellulose, hemicellulose and lignin. The
sunflower stalks and hulls could be biotechnologically
resulting hydrolysate, containing hemicellulose, and cellulose pulp
converted into bioethanol using Saccharomyces cerevisiae
contained xylose sugar at 2.53% and 7.00%, respectively. The pulp
var. ellipsoideus under optimum condition of time 24 h, pH
was further subjected to enzymatic saccharification at 50 C pH 4.8
5.0, 30 C with maximum ethanol yield 12.03 g/ 100 g
citrate buffer) with pulp/buffer 6% (w/w) and Celluclast 1.5L/pulp
sunflower stalk [3].
2.67% (w/w) to obtain single glucose with maximum yield 11.97%.
The present study was therefore undertaken to optimise
After fixed-bed fermentation under optimum condition using
conventional yeast mixtures to produce bioethanol, it indicated
fractionation of main chemical components from sunflower
maximum ethanol yield of 0.028 g/100 g sunflower stalk.
stalks using steam explosion as well as to optimise
enzymatically saccharification of resulting cellulose for
Keywords—Enzymatic, steam explosion, sunflower stalk, bioethanol production.
ethanol production.
II. MATERIALS AND METHODS
I. INTRODUCTION
A. Materials
UNFLOWER seed is the third largest source of vegetable
Soil worldwide, following soybean and palm. Sunflower The sunflower stalks obtained locally in Thailand were
oil, extracted from the commercially available sunflower used as raw material in this study. They were washed,
varieties containing 39-49% oil in the seed, is used for chopped into small pieces and dried at 60 C to constant
cooking, as a carrier oil and for production of biodiesel. In
weight and ground to 40-mesh size using a Wiley mill
Thailand, sunflower plantation areas mostly are located in
(Kinematica AG Co. Ltd., Tokyo, Japan).
Lopburi and Saraburi provinces. Its cultivation volumes
The chemical composition of sunflower stalks was
increase by 10-20% annually as demand for vegetable oil
determined according to the Tappi standards, T204 om-88 for
production is raised, thus also increase huge accumulation of
extractives; T222 om-98 for lignin; T203 om-88 for -
sunflower stalks. These wastes are generally used for cellulose; T223 cm-84 for pentosan; T211 om-93 for ash.
medicinal and cosmetic purposes or burnt in the fields causing
Holocellulose and total phenolic compounds were analysed
environmental pollution [1]. Based on previous studies by
according to the standard methods [4]. Contents of glucose
Marechal and Rigal, chemical composition of sunflower stalk
and xylose was analysed by quantitative saccharification with
and head were analysed [2]. The 16.8% of polysaccharides
H2SO4, followed by high performance liquid chromatographic
(mainly xylose) was detected in the head part whereas only
analysis. After saccharification, an aliquot of sample was
neutralised by solid Ba(OH)2 and the mixture was
homogenised using a magnetic stirrer. Final pH was adjusted
P. Vaithanomsat is with Kasetsart Agricultural and Agro-Industrial Product
to 7 with a solution of saturated Ba(OH)
Improvement Institute, Kasetsart University, 50 Chatuchak, Bangkok,
2. The sample was
Thailand (corresponding author to provide phone: 00-66-2942-8599; fax: 00-
centrifuged at 8000 rpm to remove barium sulfate, and an
66-2562-0338; e-mail: aappln@ku.ac.th; p_vaithanomsat@yahoo.com).
aliquot of 5 ml was filtered through a 0.45 m filter and
S. Chuichulcherm, is with Department of Chemical Engineering, Faculty of
analysed by a high performance liquid chromatography
Engineering, Srinakharinwirot University, Nakhornnayok, Thailand.
W. Apiwatanapiwat is with Kasetsart Agricultural and Agro-Industrial
(HPLC) with an LC10A HPLC (Shimadzu Co. Ltd., Kyoto,
Product Improvement Institute, Kasetsart University, 50 Chatuchak, Bangkok,
Japan) using inositol as an internal standard. The HPLC
Thailand.
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World Academy of Science, Engineering and Technology 49 2009
analysis was performed with an Aminex column HPX-87C
pentosan, respectively, which actually was the property of
(300 x 7.8 mm., Bio-Rad, USA) in connection with a softwood (pentosan <15%). However, this amount was rather
refractive index detector at 80 C using deionised water as
lower when compared with those found in corn cob, bagasse
solvent at a flow rate of 0.6 ml·min-1.
and oil palm trunk of about 35-40% [6, 7] in which they
belonged to the hardwood family. Pentosan content also
B. Steam Explosion Fractionation
indicates the condition used for composition separation by
Air dry sunflower stalk sample (100g) was soaked in 0.02
steam explosion. This is due to the working mechanism that
M H2SO4 for overnight. After that, they were steam exploded
high temperature steam converts acetyl groups in
at 207 C and 21 kg/cm2 for 3 minutes in a stainless steel
hemicellulose molecules into acetic acid which
then
batch digester of 2.5 l capacity (Nitto Koatsu Co. Ltd., Tokyo,
hydrolyses xylan polymer into xylose and xylose oligomers
Japan). Heating was accomplished by direct steam injection
dissolved in hemicellulose solution. Thus, in case of low
into the digester and the auto hydrolysis temperature from 203
pentosan content in sunflower stalk, steam explosion under
to 223 C was reached for 2-5 min. Explosive discharge of the
mild acidic condition and/or stronger steaming condition was
digester contents into a collecting tank was actuated by extremely necessary. Alphacellulose content indicates the
sudden opening a valve. The combined pulp slurry was content of cellulose in form of glucose polymer. The results
collected and extracted with hot water (80 C) of a total also showed that sunflower stalks contained the same
volume of 2 l for 30 minutes. The pulp was filtered, dried at
cellulose content (45.74%) as that in bagasse (45.57%) which
room temperature to determine pulp yield and to analyze was enough to produce for ethanol or other value-added
chemical components with the methods described above. The
products, but much higher than that in oil palm (37.14%) [7].
pulp was stored for enzymatic hydrolysis to glucose and
After steam explosion pretreatment, the steamed fiber was
further fermentation to bioethanol.
washed with hot water. The mixture of oligosaccharides and
monosaccharides resulting from depolymerisation of
C. Optimisation of Enzymatic Saccharification
hemicellulose were easily extracted from the exploded fiber
After steaming process, enzymatic saccharification of the
by washing with hot water. The resulting hydrolysate and
steam exploded pulp into glucose was optimised using multi-
cellulose pulp contained xylose sugar at 1.68% and 3.31%
spectrum cellulose (Celluclast 1.5L, Novozyme A/S, based on raw material dry weight, respectively. It indicated
Denmark) in final volume 250 ml. The pulp was suspended in
that part of obtained xylose were released from sunflower
sodium acetate buffer, pH 4.8, with different ratios of stalks after steam explosion and dissolved in hydrolysate as
pulp:buffer at 2, 4, 5 and 6% (w/v), and incubated for 96 h at
monosaccharides which were ready to be used as substrate for
50 C with continuous stirring. The ratio between enzyme and
further application. However, the combination of xylose
pulp was also varied at 0.33, 0.4, 0.5 and 1% (w/w). A portion
contents as mentioned above was significantly different to the
of reaction mixture was taken off every 2 h for quantitative
xylose content in raw material (7.69% based on raw material
analysis of monomeric glucose using HPLC as mentioned dry weight). This could be due to that part of xylose was in
above.
oligosaccharide form and not shown in chromatogram when
D. Bioethanol Fermentation
detected with HPLC. This was in contrast with the contents of
A 24 h suspension of S. cerevisiae at OD
holocellulose and alphacellulose, which were not truly
660 = 0.6 was
inoculated into 100 ml fermentation medium (in 250 ml affected by the steam explosion condition. As shown by the
Erlenmeyer flasks) containing 12 g/l glucose solution from the
glucose contents of 0.19% and 23.62% based on raw material
previous step. Glucose and sucrose were also added into dry weight in the hydrolysate and cellulose pulp, respectively.
samples 2 and 3, respectively, to enhance the growth of yeast.
This indicated that steam explosion condition used in this
All media, except the control media (pH 6.7), were adjusted to
experiment was not strong enough to degrade and release
pH 3.2 to accommodate yeast growth. The fermentation was
glucose from sunflower stalks and almost of glucose still
remained in cellulose pulp (27.10% in raw material). Thus, the
carried out at 30 C with 80 rpm shaking. Ethanol was steam explosion technique was good and suitable for
estimated periodically by gas chromatography (GC) using separation of hemicellulose from cellulose in sunflower stalks
HP5890 SeriesII apparatus equipped with Agilent 6890 Series
for further applications.
injector.
III. RESULTS AND DISCUSSION
B. Enzymatic Saccharification
A. Steam Explosion Fractionation of Sunflower Stalks
Effect of Sample Size on Enzymatic Hydrolysis of Cellulose
The contents of ash, ethanol/benzene extractives, lignin,
The steam exploded sunflower pulp was enzymatically
holocellulose, -cellulose, pentosan, xylose and glucose of
hydrolysed using commercially available Celluclast 1.5L
sunflower stalks were 10.65, 9.03, 21.92, 70.69, 45.74, 15.84,
enzyme (74 FPU/ml). The ground and non-ground steam-
7.69 and 27.10% of dry sunflower stalks, respectively. Such
exploded sunflower pulp as substrates for the hydrolysis was
low content of pentosan was consistent with those shown by
compared for its efficiency. Fig. 1 showed that size of
[5] and [2] that sunflower stalks contained 18.2 and 17%
substrate did not affect the efficiency of enzymatic hydrolysis
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World Academy of Science, Engineering and Technology 49 2009
as observed by that the obtained reducing sugars at time reaction and became stable after 50 h. However, the difference
intervals were not significantly different. Therefore, non-
of reducing sugars amount between each enzyme
ground steam-exploded sunflower pulp was used as substrate
concentration tended to decrease when 2% of enzyme
for next experiment.
concentration based on dry sunflower pulp was reached
indicating the equilibrium of this hydrolysis reaction.
2000
1800
9000
) 1600
8000
1400
7000
)
1200
a
r
s

(
ppm
m
6000
1000
ug
s 800
5000
a
r
s
(
pp
i
ng
ug
600
s
4000
e
duc 400
c
i
ng
R
3000
2%ground
e
du
200
R
0.33%
2%non-ground
2000
0.67%
0
1.33%
1000
2.00%
0
10
20
30
40
50
60
70
80
2.67%
Time (h)
0
0
10
20
30
40
50
60
70
80
Fig. 1 The effect of sample size on enzymatic hydrolysis
Time (h)
Fig. 3 The effect of enzyme concentration on enzymatic hydrolysis
Effect of Pulp Concentration on Enzymatic Hydrolysis of
Cellulose
Fig. 2 showed the efficiency of pulp hydrolysis using
C. Bioethanol Fermentation
commercial enzyme at different concentrations of sunflower
The glucose solution from previous step was used as
pulp. It was obvious that the efficiency of enzymatic
substrate for ethanol fermentation. Figs. 4 and 5 demonstrated
hydrolysis was relevant to the concentration of pulp. When
the decrease in sugar concentration and increase in ethanol
pulp concentration increased from 2% to 6%, the efficiency of
concentration during the fermentation reaction. It indicated a
hydrolysis was also raised and would be able to constantly
sharp decrease in sugar concentration in samples no. 2 and 3
raise as significantly observed by the amount of reducing since the beginning of fermentation. This could be due to that
sugars. However, the pulp concentration used in this the addition of glucose and sucrose in samples 2 and 3,
experiment could not be increased more than 6% since the
respectively, accommodated the yeast growth. The results also
experimented mixture would be too viscous and the reaction
revealed that ethanol could be fermented either from
would not be homogeneously stirred. Thus, in next sunflower stalks glucose (control and sample 1) or the mixture
experiments, 6% pulp concentration was applied.
of that with pure glucose or sucrose (samples 2 and 3,
respectively). However, the higher ethanol was obtained from
4000
samples 2 and 3 since more sugar was put into the reaction.
3500
Moreover, the comparison between control and sample 1
) 3000
showed the effect of pH on ethanol fermentation. It indicated
(
p
p
m
that the better ethanol fermentation was obtained at lower pH
2500
g
a
rs
(pH 3.2).
2000
i
n
g
su 1500
25
1000
2%
sample no.1
R
educ
4%
sample no. 2
500
5%
sample no.3
6%
n
20
control
0
tio
tra
0
10
20
30
40
50
60
70
80
en
Time (h)
c
15
con
r
Fig. 2 The effect of pulp concentration on enzymatic hydrolysis
ga
su
10
Effect of Celluclast1.5L Enzyme Concentration on
cing
)
du
/L
Enzymatic Hydrolysis of Cellulose
re
(g
5
Fig. 3 indicated the effect of Celluclast1.5L enzyme on
hydrolysis of sunflower cellulose. It was clear that the
0
0
2
4
6
8
10
12
14
efficiency of enzymatic hydrolysis of sunflower cellulose also
time (day)
depended on enzyme concentration as shown by the amount
Fig. 4 The decrease in sugar concentration during fermentation
of released reducing sugars in the reaction. The hydrolysis
reaction
efficiency significantly increased at the beginning of the
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World Academy of Science, Engineering and Technology 49 2009
18
[7]
Punsuvon,V., Vaithanomsat, P., Pumiput, P., Janthranurak, S. and
Anpanurak, W. (2005) Fractionation of chemical components of oil palm
16
trunk by steam explosion for xylitol and alcohol production. The
14
Proceedings for 13th International Symposium on Wood, Fibre and
Pulping Chemistry (ISWFPC). Auckland, New Zealand.
t
ion
12
t
ra
10
ncen
l co
8
no
/
L)
g
t
ha
6
e
(m
4
sample no.1
sample no.2
2
sample no.3
control
0
0
2
4
6
8
10
12
14
time (day)
Fig. 5 The increase in ethanol concentration during fermentation
reaction
IV. CONCLUSION
Processing of sunflower stalks by steam explosion
pretreatment allows the fractionation of the main polymers
present in the lignocellulosic matrix. The yields of cellulose
and hemicellulose are strongly dependent on the severity of
the steam pretreatment. Pentosan, recovered by extraction of
the exploded fiber with water, was obtained as a mixture of
oligomeric and monomeric sugars. The content of monomeric
sugars continuously increased with the pretreatment severity
increases. The optimum condition of fractionation was 2.1
MPa, reaction time for 3 minutes at 207 C. In addition,
glucose as a raw material for ethanol production was produced
by the enzymatic hydrolysis of cellulose. It indicated
maximum ethanol yield of 0.028 g/100 g sunflower stalk.
Even though, the yeast mixtures showed to be capable of
converting glucose from sunflower stalks to ethanol, the
ethanol yield was rather lower than the theorethical yield and
therefore the optimal conditions still need to be further
extended.
ACKNOWLEDGMENT
The authors would like to express appreciation for financial
support to the Commission on Higher Education, Thailand.
REFERENCES
[1]
Sharma, S.K. et al. (2002) Enzymatic saccharification of pretreated
sunflower stalks. Biomass and Bioenergy. 23: 237-243.
[2]
Marechal, V. and Rigal, L. (1999) Characterization of by-products of
sunflower culture-commercial applications for stalks and heads.
Industrial Crops and Products. 10: 185-200.
[3]
Sharma, S.K., Kalra, K.L. and Kocher, G.S. (2004) Fermentation of
enzymatic hydrolysate of sunflower hulls for ethanol production and its
scale up. Biomass and Bioenergy. 27: 399-402.
[4]
Schandrel, S.H. (1970) Tannins and related phenolics, in methods in
food analysis. Academic Press, New York.
[5]
Khristova, P., Yossifov, N. and Gabir, S. (1996) Particle board from
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[6]
Hang, Y.D. and Woodams, E.E. (2001) Enzymatic production of
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142.
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