Biotechnology and Bioprocess Engineering 2009, 14: 828-834

DOI/10.1007/s12257-009-0029-8

Pretreatment and Saccharification of Rice

Hulls for the Production of Fermentable
Sugars
=
Gong-Yuan Wei1,4, Wa Gao2,4, II-Hyuck Jin3, Su-Young Yoo5, Jai-Heon Lee4, Chung-Han Chung3,4,
and Jin-Woo Lee3,4*
1
School of Life Science, Soochow University, Suzhou 215-123, China
2
Department of Medical Bioscience, Dong-A University, Busan 604-714, Korea
3
Department of Biotechnology, Graduate School of Dong-A University, Busan 604-714, Korea
4
BK21 Bio-Silver Project of Dong-A University, Busan 604-714, Korea
5
Research Facility Center of Dong-A University, Busan 604-714, Korea

Abstract Hydrolytic conditions of rice hulls by acid and alkaline treatments before enzymatic saccharification were optimized in
this study. Based on the results of single-factor experiments and an orthogonal array experiment, reaction time was
found to be the most important factor for the acidic hydrolysis of rice hulls. Maximum yield of sugars from 1 g of rice
hulls by acidic treatment under optimized conditions was 213.6 mg. The yield of lignin removal from acidic pretreated
rice hulls by alkaline treatment increased with increase in reaction temperature and time. The amount of sugars ob-
tained from 1 g of pretreated rice hulls by enzymatic saccharification was 307.7 mg, and the conversion rate of sugars
from crude fibers in pretreated rice hulls was about 72%. Instrumental analyses with FTIR and SEM indicated that lig-
nin in rice hulls was partially removed by alkaline treatment, and the structure of rice hulls became deformed and
more fibers were exposed to cellulases after acidic treatment. © KSBB

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INTRODUCTION
Lignocellulosic biomass comprised of cellulose, hemicel-
lulose, and lignin, which has been recognized as a major
renewable resource, and also the most abundant source of
organic components in high amounts on the earth [1,2]. The
cellulose and hemicellulose fractions in biomass can be hy-
drolyzed to fermentable sugars, such as glucose and xylose,
which can then be utilized as substrates for the production of
ethanol and other commercial chemicals [3]. Acids or cellu-
lolytic enzymes can catalyze hydrolysis of lignocellulosic
polysaccharides. Due to its process efficiency and environ-
mental protection, enzymatic hydrolysis is a very promising
method for the saccharification of cellulose at present. How-
ever, pretreatment of lignocellulosic materials is needed be-
fore enzymatic hydrolysis in order to reduce cellulose crys-
tallinity and make the cellulose macromolecules accessible
*Corresponding author
Tel: +82-51-200-7593 Fax: +82-51-200-7505
e-mail: jwlee@dau.ac.kr
to cellulases [4]. Comminution, steam explosion, ammonia
fiber explosion, and acid or alkaline pretreatment processes
have been extensively investigated [5,6]. Among all methods,
dilute acid and alkaline pretreatments have been widely used
to recover sugars from hemicellulose and remove lignin. The
most commonly used acid and alkali are sulphuric acid and
sodium hydrate, respectively [7,8].
Every year billions of pounds of rice hulls are generated
from rice producing countries and most of them are thrown
away as a waste byproduct, which will undoubtedly have too
many negative influences on the global environment [9].
Rice hull, the outer coat of paddy, is one of the most abun-
dant lignocellulosic materials in China, Korea, and USA.
When paddy is processed into edible rice, about 20% (w/w)
of the dry weight is rice hull. Rice hulls can be further util-
ized in the downstream process, although it contains more
than 20% (w/w) lignin. In many places, rice hulls are only
considered as combustible waste to recover energy because
of its low value as animal feed, which is due to its character-
istics such as low digestibility, low bulk density, and pecu-
liar size. Rice hulls are occasionally used as feedstock for the

production of furfural [10] and activated carbon [11]. If ap-
propriate hydrolysis or saccharification of this lignocellu-
losic biomass to sugars is performed, rice hulls can be used
for the production of ethanol as a fuel [12] together with
other important industrial chemicals. In recent years, many
efforts have been made to use rice hulls as feedstock for the
production of fermentable sugars, followed by the fermenta-
tion of their hydrolysates to produce ethanol by recombinant
E. coli or S. cerevisiae [13-15]. Though some researchers
made progress on the utilization of rice hulls, including pre-
treatments and saccharification [16], there is no literature on
the changes in chemical characteristics, physical properties,
or cellulose microstructure for pretreated rice hulls. In our
present work, conditions for the hydrolysis of rice hulls to
fermentable sugars by dilute sulfuric acid and the removal of
lignin by dilute sodium hydroxide were optimized, and the
chemical components and physical properties of pretreated
rice hulls were also compared.
MATERIALS AND METHODS
Rice Hulls and Cellulase
Rice hulls were obtained from a farm belonging to the Na-
tional Horticulture Research Institute of Korea. It was air-
dried in an oven at 70ºC for 12 h before milled in a hammer
mill and particles smaller than 40 meshes were collected for
further use in experiments. Celluclast 1.5 L purchased from
Novozymes (Bagsværd, Denmark) was used in the enzy-
matic saccharification of rice hulls. Its activities of carboxy-
methylcellulase (CMCase) and filter paperase (FPase) were
853 and 18.8 U/mL, respectively. Avicel pH-101, carboxyl-
methylcellulose (CMC), cellulose, glucose, and xylose were
purchased from Sigma-Aldrich Co. (St. Louis, USA). All
other chemicals used were of standard analytical grades.
Pretreatment and Enzymatic Hydrolysis of Rice Hulls
Powdered rice hulls were first pretreated by dilute sulfuric
acid (0.25~1.5%, v/v) at a desired temperature (100~160ºC)
from 10 min to 1 h in a sand bath. The slurry was separated
by filtration, the filtrate was collected to determine sugars,
and the cake (15%, w/v) was suspended again in dilute so-
dium hydroxide solution after being washed with distilled
water and dried. Pretreatment of rice hulls in dilute sodium
hydroxide solution was carried out in a special-made auto-
clave with an inner pressure ranging from 0.0 to 1.0 MPa
(Aid Engineering Co., Korea) at 120~160ºC. Cellulosic resi-
dues were repeatedly washed with distilled water for neu-
tralization and dried. After acid and alkaline pretreatment,
residual rice hulls were used as substrates for enzymatic sac-
charification.
The hydrolysis of pretreated rice hulls by a cellulase was
carried out in 500 mL Erlenmeyer flasks containing 100 mL
of citrate buffer (0.05 mol/L, pH 5.0), 5 mg of sodium azide,
and 2 g of rice hulls. The mixture was incubated in an orbital
shaker at 50ºC for 48 h. The ratio of cellulase to substrate in
Biotechnol. Bioprocess Eng. 829=
this study was maintained at 1,000 U of Celluclast 1.5 L to 1
g of rice hulls. Samples were withdrawn at suitable time
intervals and filtered for analysis of sugars.
Enzyme Assays
CMCase activity was assayed in a reaction mixture con-
taining 1.0% (w/v) CMC, 50 mmol/L citrate buffer (pH 5.0),
and appropriately diluted enzyme solutions. After incubation
at 50ºC for 20 min, the reducing sugar liberated in the reac-
tion mixture was measured by the dinitrosalicylic acid
(DNS) method [17]. One unit of CMCase activity is defined
as the amount of enzyme that produces 1 µmol of reducing
sugar as glucose in the reaction mixture per minute under the
above-specified conditions.
FPase activity was assayed by incubating a reaction mix-
ture containing a strip of Whatman no.1 filter paper (1 × 6
cm) immersed in 1 mL of 0.05 mol/L citrate buffer (pH 5.0)
and 0.5 mL of appropriately diluted enzyme solution at 50ºC
for 30 min. One unit of FPase activity is defined as the
amount of enzyme that produced 1 µmol of glucose per min-
ute under the above conditions.
Orthogonal Design Method
Orthogonal design is a type of experimental design method,
which is based on the Taguchi parameter design methodol-
ogy [18]. The L9(34) orthogonal array experiment in this
study had four factors: sulfuric acid concentration, reaction
time, rice hulls concentration, and reaction temperature and
each factor had three different levels. Intuitive analysis and
statistical calculations were also carried out according to the
above methods.
Analytical Methods
Chemical composition of rice hulls were determined by
the Feed & Food Nutrition Research Center at Pukyong Na-
tional University in Korea, according to the procedures rec-
ommended by the National Exposure Research Laboratory
(NERL, USA). Reducing sugars were determined by the
DNS method [17]. Infrared spectra were measured by a
Bruker IFS88 Fourier Transform Infra Red (FTIR) spec-
trometer (Bruker Co., Germany). Scanning electron micro-
scopic observations were taken by a JEOL JSM-35CF SEM
(Jeol Co., Japan) in the Research Facility Center of Dong-A
University in Korea.
RESULTS AND DISCUSSION
Pretreatment of Rice Hulls by Dilute Sulfuric Acid
The effects of sulfuric acid on hydrolysis of rice hulls
were investigated. The concentration of rice hulls was 10.0%
(w/v) and sulfuric acid concentration ranged from 0.25 to
1.5% (v/v). Reaction temperature and hydrolysis time for the
rice hulls were 120ºC and 30 min. Reducing sugar from rice
830

A B

C D

Fig. 1. Effect of (A) H2SO4 concentration, (B) rice hull concentration, (C) reaction time, and (D) reaction temperature on hydrolysis of
rice hulls. ●, residual of rice hull after treatment; ◊, reducing sugar; and △, yield of sugar to substrate.

hulls increased with increase in concentration of sulfuric acid,
as shown in Fig. 1A. The amount of sugar from treatment by
1.5% sulfuric acid was about 3 times of that by treatment of
0.5% sulfuric acid. The yield of reducing sugar was more
than 200 mg/g rice hulls and more than 46% (w/w) of carbo-
hydrate in rice hulls was hydrolyzed to sugars, when the
concentration of sulfuric acid was higher than 1.0% (v/v).
Subsequently, the remainder of rice hulls also decreased to
about 70% (w/w) of the substrate with increased concentra-
tion of sulfuric acid.
The effects of the rice hulls on hydrolysis itself were also
investigated. The sulfuric acid concentration was 1.0% and
concentration of rice hulls ranged from 5.0 to 20.0%. Reduc-
ing sugar from rice hulls increased with increased concentra-
tion of rice hulls as a substrate, as shown in Fig. 1B. Amount
of sugar from 20.0% rice hulls was about 4 times of that
from 5.0% rice hulls. Although there was no significant dif-
ference between remains after hydrolysis, some decrease in
the yield of reducing sugar was found.
The effects of reaction time on hydrolysis of rice hulls
were examined. Concentrations of rice hulls and sulfuric
acid were 10.0% and 1.0%, respectively. Reaction time
ranged from 10 to 60 min. Reducing sugar from rice hulls
increased with increased reaction time, as shown in Fig. 1C.
Reducing sugar concentration, as well as the yield of sugars,
was maintained at an almost constant level with more than
20 min of reaction time, even though remains of rice hulls
decreased.
The effects of reaction temperature on hydrolysis of rice
hulls were also examined. Reaction temperature ranged from
100 to 160ºC. Reducing sugar from rice hulls increased with
increased reaction temperature, as shown in Fig. 1D. Optimal
temperature for hydrolysis of rice hulls was found to be
140ºC. The amount of sugar at a reaction temperature of
140ºC was about 3.6 times of that at a reaction temperature
of 100ºC. More carbohydrates can be hydrolyzed to mono-
meric sugars at a higher temperature. However, some sugars
are partially degraded to byproducts in acidic conditions at a
higher temperature such as furfural and hydroxylmethyl fur-
fural [19], which may be the reason why the yield of sugars
from rice hulls at 160ºC was lower than that at 140ºC in this
study.
An L9(34) orthogonal design experiment with four factors
of sulfuric acid concentration, reaction time, rice hulls con-
centration, and reaction temperature, was carried out to op-
timize hydrolytic conditions of rice hulls. Based on intuitive
 Biotechnol. Bioprocess Eng. 831=

Table 1. Yield of sugars from rice hulls pretreated by dilute Table 2. The mean values of L9-34 experiments at each level
H2SO4 only, and both of dilute H2SO4 and Celluclast 1.5 Yield
a
Yield
b

L under the designed conditions of the L9(34) orthogonal

experiments Rice Tem- Rice Tem-
H2SO4 Time H2SO4 Time
hull perature hull perature
H2SO4 Time Rice hull Tempera- Yield
a
Yield
b (% ) (min) o (% ) (min) o
o
(%) ( C) (%) ( C)
(% ) (min) (%) ture ( C) (mg/g) (mg/g)
M1j 156.3 167.3 153.8 152.0 195.5 204.3 195.3 184.8
1 0.5 20 10 120 109.5 ± 0.2 143.5 ± 1.1
M2j 181.7 168.2 189.1 185.0 218.2 208.4 226.9 233.1
2 0.5 40 15 140 189.4 ± 2.0 227.5 ± 3.2
M3j 181.8 184.4 179.0 172.2 222.6 223.7 214.7 218.5
3 0.5 60 20 160 170.0 ± 5.1 215.5 ± 5.5
R 25.5 17.1 35.3 33.0 27.1 19.4 31.6 38.3
4 1.0 20 15 160 190.5 ± 2.6 231.5 ± 3.9
a
Yield; yield of sugars from rice hulls pretreated by dilute H2SO4.
5 1.0 40 20 120 159.0 ± 0.7 189.2 ± 1.7 b
Yield; yield of sugars from rice hulls pretreated by dilute H2SO4 and
6 1.0 60 10 140 195.6 ± 6.2 234.0 ± 6.5 Celluclast 1.5 L.
7 1.5 20 20 140 201.9 ± 1.9 237.8 ± 2.9
8 1.5 40 10 160 156.2 ± 4.9 208.5 ± 5.5
9 1.5 60 15 120 187.5 ± 2.0 221.6 ± 3.0
rice hulls pretreated dilute sulfuric acid and Celluclast 1.5 L
c
was reaction temperature as shown in Table 2.
10 1.0 60 15 140 213.6 ± 0.2 252.2 ± 1.4
a
Yield; yield of sugars from rice hulls pretreated by dilute H2SO4. Removal of Lignin from Rice Hulls by Dilute Sodium
b
Yield; yield of sugars from rice hulls pretreated by dilute H2SO4 and
Hydroxide
Celluclast 1.5 L.
c
10; an additional experiment for verification of optimal conditions for
hydrolysis of rice hulls. Hemicellulose and lignin, which hinder the enzymatic di-
gestibility of cellulose, should be removed in the process of
pretreatment in order to increase the accessibility of cellulose
to cellulase and improve the rate of enzyme hydrolysis. Pre-
treatment by dilute sulfuric acid is regarded as one of the
most effective methods of hydrolyzing hemicellulose to xy-
lose and other sugars, together with recovering fermentable
sugars from hemicellulose and cellulose [20]. Chemical
compositions of rice hulls pretreated by 1.0% sulfuric acid
and those pretreated by 1.0% sulfuric acid followed by 1.0%
sodium hydroxide were analyzed, as shown in Table 3. Al-
though most hemicelluloses were decomposed to ferment-
able sugars, the content of lignin in rice hulls pretreated by
Fig. 2. Intuitive analysis of orthogonal experiments with four 1.0% sulfuric acid was similar to that in untreated rice hulls.
factors of H2SO4 concentration, reaction time, rice hulls However, the content of lignin in rice hulls pretreated by
concentrations, and reaction temperature. 1.0% sulfuric acid followed by 1.0% sodium hydroxide was
reduced from 23.71 to 7.45%. It seems that treatment of so-
dium hydroxide after pretreatment of sulfuric acid at a high
analysis and statistical calculations using data, the reaction temperature resulted in a great benefit for lignin removal in
temperature was found to be the most important factor for rice hulls and subsequently, saccharification of cellulose.
acid hydrolysis of rice hulls, followed by concentrations of The yield of lignin removal from acidic pretreated rice hulls
rice hulls and sulfuric acid, and reaction time, as shown in by 1.0% sodium hydroxide increased with increased reaction
Table 1. Optimal concentrations of sulfuric acid and rice temperature and time, as shown in Fig. 3. Maximum yield of
hulls for the hydrolysis of rice hulls were found to be 1.0 and sugars from 1 g of rice hulls pretreated by 1.0% sulfuric acid
10.0%, respectively, as shown in Fig. 2. Optimal reaction followed by 1.0% sodium hydroxide was 146.4 mg.
time and temperature were also found to be 60 min and
140ºC. Maximum yield of sugars from 1 g of rice hulls by Enzymatic Hydrolysis of Rice Hulls
acidic treatment under optimized conditions was 213.6 mg,
whereas that by acidic and enzymatic treatments was 252.2 Enzymatic hydrolysis of untreated and pretreated rice
mg. Results from single-factor experiments, as shown in hulls was carried out. The yield of sugars was considerably
Figs. 1A, 1B, 1C, and 1D, showed a similar tendency as improved when rice hulls were pretreated by dilute acid
those from the orthogonal design experiment with four fac- and/or alkali, as shown in Table 4. After the removal of
tors. The result from an L9 (34) orthogonal design experiment hemicellulose and/or lignin, residuals of pretreated rice hulls
showed that the most significant factor for hydrolysis of rice became more accessible to a cellulase. Amounts of sugars
hulls pretreated by dilute sulfuric acid was found to be the from 1 g of pretreated rice hulls by enzymatic saccharifica-
concentration of rice hulls, whereas that for hydrolysis of tion were from 252.2 to 307.7 mg, whereas that from 1 g of
832

Table 3. Comparison of chemical compositions of untreated and pretreated rice hulls

Composition Rice hulls
(%) Untreated Pretreated by 1.0% H2SO4 Pretreated by 1.0% H2SO4 followed by 1.0% NaOH
Moisture 18.23 ± 0.11 5.57 ± 0.26 7.19 ± 0.00
a
Crude lipid 0.51 ± 0.12 0.15 ± 0.00 ND
Crude protein 1.92 ± 0.04 0.83 ± 0.05 0.31 ± 0.00
Crude ash 12.95 ± 0.39 10.81 ± 0.32 3.60 ± 0.22
Crude fiber 42.97 ± 0.28 60.75 ± 0.09 71.44 ± 0.03
Salts 0.08 ± 0.00 ND ND
Lignin 23.71 ± 0.24 21.89 ± 0.12 7.45 ± 0.05
a
ND; not detectable.

A B

Fig. 3. Effects of (A) reaction temperature and time on removal of lignin and yield of sugar, (B) from pretreated rice hulls.

Table 4. Yield of sugars after saccharification of untreated and pretreated rice hulls
Reducing sugar after Yield of sugar from fiber Total yield of sugar from raw
Substrate a
saccharification (g/L) (%) material (mg/g)
Untreated rice hull 0.38 ± 0.07 4.4 ± 0.8 19.0 ± 3.5
Pretreated by 1.0%
1.20 ± 0.02 9.9 ± 0.2 252.2 ± 1.1
H2SO4
Pretreated by 1.0% H2SO4 followed
3.37 ± 0.10 23.6 ± 0.7 307.7 ± 2.3
by 1.0% NaOH
Avicel 17.25 ± 0.05 − 862.5 ± 2.5
a
Total yield; total yield of sugar include sugar from pretreatment and saccharification.

untreated rice hulls was 1.9 mg. More than 72% of crude
fibers in the rice hulls were transformed to fermentable sug-
ars.
Physical Properties and Cellulosic Microstructure of
Rice Hulls
Chemical composition is one of the factors influencing the
enzymatic hydrolysis of rice hulls, but physical properties
and cellulosic microstructure are also potential factors that
affect the enzymatic hydrolysis. Observation with an infra-
red spectroscopy is always used to investigate changes in
structure and chemical constituents of lignocellulosic materi-
als [21]. Rice hulls, belonging to herbaceous species, also
have two types of lignin, guaiacyl lignin and syringyl lignin
[22]. According to the reference, the lignin characteristic
peaks were observed at 1238 cm−1 (C-O of guaiacyl ring),
1315 cm−1 (C-O of syringyl ring), 1504 cm−1(aromatic skele-
tal vibration), and 1630 cm−1 (aromatic skeletal vibration of
C=O stretching). The Fourier transform infrared (FTIR)
spectra of untreated and pretreated rice hulls are shown in
Fig. 4. It seems that there is no significant difference be-
tween the spectra of untreated and acid pretreated rice hull,
but only one peak disappeared at 1735 cm−1 (C=O stretch,
unconjugated) after acid treatment. However, compared with
the spectrum of alkali treated rice hulls, the band intensities
at all lignin peaks (1504~1630 cm−1) of untreated and acid
pretreated rice hulls were both higher. This result again
 Biotechnol. Bioprocess Eng. 833=

B
Fig. 4. FTIR spectra of (A) untreated rice hulls, (B) rice hulls
pretreated by 1% H2SO4, and (C) rice hulls pretreated by
1% H2SO4 followed by 1% (w/v) NaOH.

proved the efficiency of delignification by alkaline pretreat-

ment. After pretreatment by dilute acid and alkali, a large
fraction of hemicellulose and lignin is removed from the rice
hulls, and consequently, some physical properties and struc-
tures of the material were changed.
Observations of scanning electron microscope (SEM) pic-
tures of untreated and pretreated samples were conducted as
shown in the Fig. 5. Untreated rice hulls exhibited rigid and
highly ordered fibrils, but some fibrils were partially broken
after acid treatment because of the attack by H+ at a high
temperature. It is obvious that fibers in alkali pretreated rice
hulls were distorted. Microfibrils were also separated from
the initial connected structure and fully exposed, thus exter-
nal surface area and porosity of the rice hulls increased. C

CONCLUSION

Hydrolytic conditions of rice hulls by acid treatment were

optimized and the most important factor was found, in this
study, to be reaction temperature. Yield of sugars from rice
hulls by acid treatment under optimized conditions was 252
mg/g, which is about 58% of a theological yield based on the
content of crude fiber in rice hulls. High content of lignin in
cellulosic materials has a negative effect on enzymatic sac-
charification by cellulase [20,23]. Removal rate of lignin
from acidic pretreated rice hulls by 1.0% sodium hydroxide
increased with reaction temperature and time. According to
analysis of chemical compositions of untreated and pre-
treated rice hulls in this study, about 70% of lignin from pre-
treated rice hulls by alkaline treatment was removed. The
amount of sugars from 1 g of pretreated rice hulls by enzy- Fig. 5. Scanning electron microscopic observations of (A) untreated
matic saccharification was 307.7 mg and the conversion rate rice hulls, (B) rice hulls pretreated by 1% H2SO4, and (C)
of sugars from crude fibers in pretreated rice hulls was about rice hulls pretreated by 1% H2SO4 followed by 1% NaOH.
834
72%. Instrumental analyses with FTIR and SEM indicated
that lignin in rice hulls was partly removed by alkaline
treatment, and the structure of rice hulls was deformed and
more fibers were exposed to cellulases after acidic treatment.
The process for mass production of fermentable sugars by
optimized conditions for hydrolysis of rice hulls in this study
will be developed for production of ethanol and other useful
chemicals in future studies.
Acknowledgement This work paper was financially sup-
ported by Dong-A University Research Fund.
Received February 13, 2009; accepted May 19, 2009
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