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RESEARCH ARTICLE
Optimization of Chemical Pretreatment and Acid Saccharificationfor Conversion of Sugarcane Bagasse to Ethanol
S. K. Uppal • Ramandeep Kaur • Poonam Sharma
Received: 3 January 2011 / Accepted: 2 August 2011 / Published online: 19 August 2011
� Society for Sugar Research & Promotion 2011
Abstract The production potential of cellulosic ethanol
from sugarcane bagasse was studied. Chemical pretreat-
ments were carried out by shaking bagasse with 1, 2 and
3% H2O2 (pH 10, 11.5 and 13) for 24, 48 and 72 h with
subsequent saccharification of pretreated bagasse with
H2SO4 (0.8 and 1.0 M) for 50 min for optimization of
process. Acid hydrolysates were fermented with Saccha-
romyces cerevisiae var ellipsoideus for ethanol production.
Maximum weight loss in alkaline pretreatment (52.30%),
amount of reducing sugars (520.84 mg/g) and ethanol
produced (27.94 ml/100 g pretreated bagasse) were found
in 2% H2O2 (pH 11.5, 48 h) pretreated bagasse saccharified
with 0.8 M H2SO4 after fermentation for 72 h. Pretreat-
ment followed by acid saccharification decreased the time
interval for ethanol fermentation.
Keywords Sugarcane bagasse � Alkaline pretreatment �Acid saccharification � Fermentation
Introduction
The incessant exhaustion of fossil fuel reserves and con-
sequent upward spiral in their prices has stimulated an
extensive evaluation of alternative technologies and sub-
strates to meet the global energy demand. Ethanol offers
certain attractive features over solid and gaseous fuel
which makes it more imperative to be utilized throughout
the world in overcoming the problems of energy crisis and
hunger. Alcohol fuels burn cleaner than other fossil fuel;
hence overcome the great burden of air pollution. Bioeth-
anol can contribute to a cleaner environment and with the
implementation of environment protection laws in many
countries; demand for this is increasing (Zaldivar et al.
2001). Currently, alcohol fuels have been produced on
industrial scale by fermentation of sugars derived from
wheat, corn, sugar beets, sugarcane juice etc. Presently,
nearly all fuel ethanol is produced by fermentation of corn
glucose in America or sucrose in Brazil, and any countries
advanced in agriculture can use current technology for fuel
ethanol fermentation (Lin and Tanaka 2006). But this
technology consumes a lot of food materials, costs too
much, and under the pressure of world’s food crisis at
present, there is even a serious competition between human
food and fuel ethanol. Thus, there is need to find a better
way to solve this problem, and now it is mostly concen-
trated in the field of utilization of agricultural materials.
Sugarcane bagasse, the major byproduct of the sugar cane
industry, is economically viable for the production of
environmentally friendly ethanol fuel. Sugarcane bagasse
is primarily composed of lignin, cellulose and hemicellu-
loses. Processing of lignocelluloses to ethanol consists of
pretreatment to break the lignin seal and disrupt crystalline
structure of cellulose, saccharification for conversion of
carbohydrate polymers into monomeric sugars followed by
fermentation of sugars to ethanol. So the large scale utili-
zation of byproducts of the sugarcane industry, if effi-
ciently implemented, has the dual and important advantage
of generating reasonable profits, not only for sugar pro-
ducers themselves but also for the national economy at
large, as exemplified by cheap electricity, imports
replacement, the proficient use of local fuels and forest
S. K. Uppal (&) � P. Sharma
Department of Plant Breeding and Genetics,
Punjab Agricultural University, Ludhiana 141004, India
e-mail: satinderuppal11@yahoo.co.in
R. Kaur
Department of Chemistry, Punjab Agricultural University,
Ludhiana 141004, India
123
Sugar Tech (July-Sept 2011) 13(3):214–219
DOI 10.1007/s12355-011-0091-3
preservation. So, the present study was intended with
objectives of pretreating sugarcane bagasse with different
treatment combinations of alkali to increase the accessi-
bility of substrate for production of sugars by acid sac-
charification and subsequent fermentation giving maximum
yields of ethanol.
Materials and Methods
The sugarcane bagasse used in present study was obtained
from sugarcane varieties CoJ 88 and CoS 8436.
Chemical Composition of Sugarcane Bagasse
Fine bagasse samples were prepared in the laboratory by
method standardized by Uppal et al. (2008). Then the
chemical composition of bagasse was determined by
detergent system method (Goering and Soest 1970).
Pretreatment of Sugarcane Bagasse
Fine and dried sugarcane bagasse was treated with 1, 2 and
3% H2O2 solutions each with pH 10, 11.5 and 13 for 24, 48
and 72 h. The pretreatment process was carried out as
reported in Dawson and Boopathy (2008).
Acid Saccharification of Pretreated Sugarcane Bagasse
The bagasse residue obtained from each of pretreatment
combination was treated with 0.8 and 1.0 M H2SO4 with
solid: liquid ratio of 1: 12 and the mixture was autoclaved for
50 min at 110�C temperature and 1.05 kg/cm2 steam pres-
sure. The mixture was filtered and analyzed for estimation of
reducing sugars by the method given by Nelson (1944).
Fermentation of Bagasse Hydrolysate to Ethanol
Acid hydrolysates of sugarcane bagasse were neutralized to
pH 5.0 and fermented with Saccharomyces cerevisiae var
ellipsoideus in glucose yeast extract (GYE) broth. Fer-
mentation was progressed at 28�C. The samples were taken
after 24, 48 and 72 h and were analyzed for estimation of
ethanol by the method of Caputi et al. (1968).
Results and Discussion
Chemical Composition of Sugarcane Bagasse
The Chemical composition of sugarcane bagasse of two
varieties is given in Table 1. It was found that cellulose,
hemicellulose and lignin, structural components of cell
wall of sugarcane ranged between 45.77–47.33%,
31.70–36.17% and 8.33–13.56% respectively. Similar
results have been reported by Saha (2003) reported that
lignocellulosic biomass had cellulose as its major compo-
nent (35–50%) followed by hemicellulose (20–35%) and
lignin (10–25%). Peng et al. (2009) also reported that
sugarcane bagasse was primarily composed of cellulose
(40–45%), hemicellulose (30–35%) and lignin (20–30%).
Pretreatment of Sugarcane Bagasse
The hydrolysis of cellulose is restricted by crystalline
structure of cellulose microfibrils that are aggregated and
embedded within the lignified cell wall matrix. Due to this
complex structure, lignocellulosics are less susceptible to
acid attack. Therefore to increase the susceptibility of
cellulosic materials to acids, it is essential to expose and
separate elementary cellulose microfibrils by various pre-
treatment methods. Chemical pretreatments have received
more attention because physical pretreatments are rela-
tively inefficient, biological pretreatments are time con-
suming and the combined treatments rarely have improved
digestibility when compared with simple treatments.
Alkaline pretreatment was selected as it is expected to
cause less sugar degradation than acid process. Hydrogen
peroxide, which is a well-known reagent in paper and
cellulose industry, has the great advantage of not leaving
residues in the biomass, as it degrades into oxygen and
water. Moreover, the formation of secondary products with
H2O2 is practically inexistent. Krishna and Chowdary
(2000) also concluded that alkaline peroxide pretreatments
were effective in providing fractionation of hemicellulose
and lignin components and resulted in efficient hydrolysis
in linn leaves. So, alkaline H2O2 was chosen for occurring
under mild conditions i.e. temperature, pressure and
absence of acids (Gould 1984). The per cent weight losses
reported by alkaline pretreatments performed by soaking
sugarcane bagasse of different varieties in various con-
centrations (1, 2 and 3%) of hydrogen peroxide at different
pH (10, 11.5 and 13) and shaking for various time intervals
(24, 48 and 72 h) are shown in Fig. 1.
The alkaline pretreatments 1% (pH 13, 72 h), 2% and
3% H2O2 (pH 11.5, 48 h) removed the most lignin and
hemicellulose in bagasse compared to other treatment
Table 1 Chemical composition of different varieties of sugarcane
bagasse
Parameter (%) CoJ 88 CoS 8436
Bagasse 48.21 46.26
Cellulose 45.77 47.33
Hemicellulose 31.70 36.17
Lignin 13.56 8.33
Sugar Tech (July-Sept 2011) 13(3):214–219 215
123
combinations indicating the maximum delignification and
hemicellulose solubilization. Further increase in pH and
time interval for 2 and 3% H2O2 did not alter much total
amount of lignin and hemicellulose solubilized. Dawson
and Boopathy (2008) also reported that sugarcane bagasse
residue pretreated with 2% H2O2 at pH of 11.5 and soaked
for 48 h removed more lignin than any other pretreatment
option evaluated.
As lignin is partly covalently associated with hemicel-
luloses and celluloses, less lignin content in bagasse of CoS
8436 led to more exposure of hemicellulose and cellulose
to alkali. Loss of weight in alkaline pretreatment was found
to be more from CoS 8436 samples as compared to that in
CoJ 88. This is because along with loss of lignin, hemi-
celluloses were also dissolved by alkali. More hemicellu-
lose and less lignin content in CoS 8436 than in CoJ 88 led
to more weight loss in CoS 8436. During the course of
pretreatment, the integrity of the individual residue parti-
cles was completely lost as the residue disintegrated into
small, highly water absorbent fibers with a pulp-like con-
sistency. This suggested that the degree of crystallinity
within the cellulose fibers had been reduced, yielding a
more hydrated, open structure. Zhao et al. (2008) reported
that enhancement of enzymatic digestibility of sugarcane
bagasse by per acetic acid pretreatment can be achieved
mainly by delignification and an increase in the surface
area and exposure of cellulose fibers. Beardmore et al.
(1980) used Gamma rays irradiation as pretreatment for the
enzymatic hydrolysis of cellulose. Surface area of cellulose
and rate of hydrolysis were drastically increased at higher
doses but the crystallinity remained relatively unaffected.
Acid Saccharification of Pretreated Bagasse
The main purpose of saccharification is hydrolysis of b,
1–4 linkages of cellulose into individual monomer units
which can then be fermented to yield ethanol. The reducing
sugars produced by treating pretreated bagasse with acid
i.e. 0.8 and 1.0 M H2SO4 for 50 min at pressure of 15 psi
have been shown in Fig. 2.
Maximum acid saccharification occurred in samples in
which maximum weight was lost in pretreatment experiment.
With increase in pH of H2O2 solution from 10 to 11.5, pro-
duction of reducing sugars increased significantly and
remained nearly constant with pH 13. From CoS 8436 having
more cellulose content as compared to CoJ 88, more reducing
sugars were produced. The small decrease in rate of hydrolysis
with increase in concentration of sulfuric acid from 0.8 to
10
5
0
15
20
25
30
35
40
45
50
% W
eigh
t Los
s%
Wei
ght L
oss
10
20
30
40
50
60
A B C
Treatments
Treatments
% W
eigh
t Los
s
Treatments
0
A B C
D E F G
D E F G
H I
CoJ 88
CoS 8436
H I
0
10
20
30
40
50
60
A B C D E F G H I
CoJ 88
CoS 8436
CoJ 88
CoS 8436
A
B
C
Fig. 1 Pretreatment of sugarcane bagasse with hydrogen peroxide
solutions of different concentrations and pH values for 24, 48 and
72 h. (A) 1% H2O2, pH 10; (B) 1% H2O2, pH 11.5; (C) 1% H2O2, pH
13; (D) 2% H2O2, pH 10; (E) 2% H2O2, pH 11.5; (F) 2% H2O2, pH
13; (G) 3% H2O2, pH 10; (H) 3% H2O2, pH 11.5; (I) 3% H2O2, pH 13
c
216 Sugar Tech (July-Sept 2011) 13(3):214–219
123
0
100
200
300
400
500
600
A B C D E F G H IR
educ
ing
suga
rs (
mg/
g ba
gass
e)R
educ
ing
suga
rs (
mg/
g ba
gass
e)R
educ
ing
suga
rs (
mg/
g ba
gass
e)Treatments
A B C D E F G H I
Treatments
Treatments
CoJ 88, 0.8M acid
CoJ 88, 1.0M acid
CoS 8436, 0.8M acid
CoS 8436, 1.0M acid
CoJ 88, 0.8M acid
CoJ 88, 1.0M acid
CoS 8436, 0.8M acid
CoS 8436, 1.0M acid
CoJ 88, 0.8M acid
CoJ 88, 1.0M acid
CoS 8436, 0.8M acid
CoS 8436, 1.0M acid
0
100
200
300
400
500
600
A
B
C
0
100
200
300
400
500
600
A B C D E F G H I
Fig. 2 Production of reducing
sugars by acid saccharification
of sugarcane bagasse pretreated
with different combinations for
24, 48 and 72 h. (A) 1% H2O2,
pH 10; (B) 1% H2O2, pH 11.5;
(C) 1% H2O2, pH 13; (D) 2%
H2O2, pH 10; (E) 2% H2O2, pH
11.5; (F) 2% H2O2, pH 13;
(G) 3% H2O2, pH 10; (H) 3%
H2O2, pH 11.5; (I) 3% H2O2,
pH 13
Sugar Tech (July-Sept 2011) 13(3):214–219 217
123
1.0 M may be due to breakdown of sugars and formation of
hydroxymethylfurfural (Smith et al. 1982). The enzymatic
hydrolysis of waste sugarcane bagasse in water media was
carried out by Zheng et al. (2002). It was concluded that
industrialization of the enzymatic hydrolysis in water medium
is feasible. Acid hydrolysis of cellulosic pyrolysate to glucose
and its fermentation to ethanol were investigated by Yu and
Zhang (2004). The maximum glucose yield (17.4%) was
obtained by the hydrolysis with 0.2 mol/l sulfuric acid using
autoclaving at 121�C for 20 min.
Fermentation of Bagasse Hydrolysate to Ethanol
Hydrolysate samples prepared by three pretreatment com-
binations i.e. 1% (pH 13, 72 h), 2% and 3% H2O2 (pH
11.5, 48 h) and saccharification with 0.8 M H2SO4 were
chosen for further fermentation experiments as maximum
delignification, hemicellulose solubilization and maximum
saccharification were achieved with these treatments. The
yields of ethanol produced by S. cerevisiae var ellipsoideus
are presented in Figs. 3 and 4.
Maximum ethanol production was obtained from 2%
H2O2 pretreated bagasse of CoS 8436 as maximum lignin
and hemicellulose loss and hence maximum reducing
sugars were produced by this treatment. Ethanol production
increased from 24 h of fermentation to 48 h and the
increase after 72 h was less. Similar results were obtained
by Dhillon et al. (1988) by fermenting saccharified
hydrolysate from rice straw containing 7.68% reducing
sugars with S. cerevisiae to produce 2.89% alcohol in 36 h
at 27�C.
Dawson and Boopathy (2008) reported that maximum
ethanol production was achieved on day 18 and 21 of
fermentation from alkali and acid pretreated bagasse
respectively without saccharification. Our studies revealed
that chemical pretreatment followed by acid saccharifica-
tion led to higher ethanol production after 48 h and there
was slight increase in ethanol production after 72 h. These
results suggested that acid saccharification after pretreat-
ment of bagasse reduces fermentation time and hence
hastens the process of ethanol production.
Conclusions
Chemical pretreatments of sugarcane bagasse with 1%
H2O2 (pH 13, 72 h), 2 and 3% H2O2 (pH 11.5, 48 h)
removed the most lignin and hemicellulose. The reducing
sugars produced by acid saccharification (0.8M H2SO4)
were also found to be maximum with above combinations.
The maximum ethanol was produced from 2% H2O2 (pH
11.5, 48 h) pretreated and 0.8 M saccharified bagasse. It
was found that maximum ethanol production by fermen-
tation can be achieved in shorter time intervals from
bagasse which was firstly pretreated followed by sacchar-
ification than directly from pretreated bagasse.
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Eth
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