Bioresource Technology xxx (2012) xxx–xxx

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Comparative study of various pretreatment reagents on rice husk

and structural changes assessment of the optimized pretreated rice husk
Teck Nam Ang, Gek Cheng Ngoh ⇑, Adeline Seak May Chua
Department of Chemical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia

h i g h l i g h t s

" Among the pretreatment reagents, HCl hydrolyzed rice husk (RH) the best.
" The optimized pretreatment condition is mild compared with other similar studies.
" The increased pore volume & size of pretreated RH favors fungal fermentation.

a r t i c l e i n f o a b s t r a c t

Article history: The performance of alkalis (NaOH and Ca(OH)2) and acids (H2SO4, HCl, H3PO4, CH3COOH, and HNO3) in
Available online xxxx the pretreatment of rice husk was screened, and a suitable reagent was assessed for subsequent optimi-
zation using response surface methodology. From the assessment, HCl that hydrolysed rice husk well was
Keywords: optimized with three parameters (HCl loading, pretreatment duration, and temperature) using Box–
Response surface methodology Behnken Design. The optimized condition (0.5% (w/v) HCl loading, 125 °C, 1.5 h) is relatively mild, and
Optimization resulted in 22.3 mg TRS/ml hydrolysate. The reduced model developed has good predictability, where
Rice hulls
the predicted and experimental results differ by only 2%. The comprehensive structural characterization
Pretreatment
Structural assessment
studies that involved FT-IR, XRD, SEM, and BET surface area determination showed that the pretreated
rice husk consisted mainly of cellulose and lignin. Compared to untreated rice husk, pretreated rice husk
possessed increased pore size and pore volume, which are expected to be beneficial for fungal growth
during fermentation.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction alkaline pretreatments have been extensively used for pretreating

Rice husk is a by-product of rice milling industry, and it repre-
sents approximately 20% by weight of rough rice (Hashim et al.,
1996). In 2008, there were estimated 125 Mt and 137 Mt rice husk
generated in Asia and worldwide, respectively (FAOSTAT, 2010).
Rice husk contains relatively high cellulose content (40–60%), and
it is widely available at relatively low cost. Attributing to its recal-
citrant nature, direct conversion of untreated rice husk usually re-
sults in low product yield. Therefore, pretreatment is necessary to
partially disrupt the recalcitrant structure to achieve delignification
of the lignocellulosic biomass. This renders the solid substrate more
accessible to enzyme or microorganism during bioconversion.
Prevailing rice husk pretreatments reported in literatures in-
clude acid (Dagnino et al., 2013), alkaline (Saha and Cotta, 2008;
Singh et al., 2011), hydrothermal (Zheng et al., 2007), alkaline per-
oxide (Saha and Cotta, 2007), and ionic liquid dissolution pretreat-
ments (Ang et al., 2011; Lynam et al., 2012). Among them, acid and
lignocellulosic biomass (Kaar and Holtzapple, 2000; Saha and
Cotta, 2008).
To date, no specifically effective reagent in pretreating rice husk
has been reported. To have a greater insight into the pretreatment
of rice husk, alkalis and acids were screened and assessed in the
study, and the best performing reagent was subjected to subse-
quent optimization study. Various structural analyses including
Fourier transform–infrared spectroscopy (FT-IR), X-ray diffraction
(XRD), scanning electron microscopy (SEM), and Brunauer–
Emmett–Teller (BET) surface area determination were conducted
to assess the compositional, chemical, and structural changes of
the biomass that is imparted by the selected pretreatment.
2. Methodology
2.1. Materials

⇑ Corresponding author. Tel.: +60 3 7967 5286; fax: +60 3 7967 5371. Rice husk was collected from Ng Trading Company, Selangor,
E-mail address: ngoh@um.edu.my (G.C. Ngoh). Malaysia. The sample was washed and dried at 55 °C before being

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.09.045

Please cite this article in press as: Ang, T.N., et al. Comparative study of various pretreatment reagents on rice husk and structural changes assessment of
the optimized pretreated rice husk. Bioresour. Technol. (2012), http://dx.doi.org/10.1016/j.biortech.2012.09.045
2 T.N. Ang et al. / Bioresource Technology xxx (2012) xxx–xxx
milled to approximately 30 mesh size, and was stored in a dry cab-
inet prior to use.
2.2. Assessment of pretreatment reagents
Screening was conducted on two alkalis and five acids, namely
sodium hydroxide (NaOH, Merck), calcium hydroxide (Ca(OH)2,
Sigma–Aldrich), sulphuric acid (H2SO4, Fisher Scientific), hydro-
chloric acid (HCl, Merck), phosphoric acid (H3PO4, Ajax Chemicals),
acetic acid (CH3COOH, Riedel–de Haen) and nitric acid (HNO3,
Scharlau Chemie). With reference to the literatures (Chang et al.,
1997; Saha and Cotta, 2008), the screening were carried out at
the following conditions: (i) concentration of reagent, 0.5% (w/v);
(ii) rice husk loading, 10.0% (w/v); (iii) water loading, 10.0 ml/g;
(iv) pretreatment temperature, 100 ± 1 °C; (v) pretreatment dura-
tion, 2 h. Total reducing sugars (TRS) content was employed as
an indirect pretreatment indicator, and its content in hydrolysate
was determined by DNS method (Miller, 1959). The main action
of acids and alkalis is to dissolve hemicellulose, to some extent cel-
lulose, and lignin. Thus, TRS released from the hydrolysis of hemi-
cellulose/cellulose can suitably reflect the extent of structure
disruption in rice husk. Pretreatment reagent that releases the
highest TRS in hydrolysate was selected for subsequent optimiza-
tion studies. To investigate the compositional changes imparted
by the pretreatment reagents, cellulose, hemicellulose, lignin, and
ash contents of the pretreated rice husk samples were character-
ized by using the Association Official Analytical Chemists (AOAC,
(2005)) official methods.
2.3. Optimization of pretreatment
The pretreatment reagent selected in Section 2.2 was employed
in the optimization of rice husk pretreatment. Three parameters
known to have effect on pretreatment, namely reagent loading
(X1), heating duration (X2) and heating temperature (X3), were
investigated. To determine the low and high levels of the chosen
parameters, preliminary tests were conducted at: (i) reagent load-
ing, 0–4% (w/v); (ii) heating duration, 1–6 h; (iii) heating tempera-
ture, 60–140 °C. The tests were performed by one-factor-at-a-time
approach, and TRS detected in hydrolysate was measured as re-
sponse for optimization of pretreatment.
The range of each parameter determined from the preliminary
tests was applied in the optimization study using Box–Behnken
experimental design (BBD). TRS detected in the hydrolysate was
determined as response (Y). The maximum release of TRS in hydro-
lysate was determined using response surface methodology (RSM),
and the regression analysis of optimization data was performed
with the aid of Design-Expert Version 6.0.6 (Stat-Ease Inc.,
Minneapolis).
2.4. Analytical techniques
The FT-IR spectra of the rice husk samples between 600 and
4000 cm1 at 4 cm1 nominal resolution were recorded at room
temperature with a FT-IR/FT–FIR spectrometer (Perkin Elmer,
Spectrum 400, USA). The spectra were presented in relative trans-
mittance percentage (%) of wave number (cm1) and the back-
ground was recorded with empty cell.
The crystallinity of the rice husk samples was examined by XRD
measurement performed with a D8 Advanced X-ray diffractometer
(Bruker AXS, USA) using Cu Ka monochromatized radiation at
40 kV and 40 mA at ambient temperature. The samples were
scanned and the intensities were recorded in 2h range from 10°
to 80° with a step size of 0.02°. The crystallinity index (CrI) of
the rice husk samples was calculated by using equation as reported
by Parikh et al. (2007).
Please cite this article in press as: Ang, T.N., et al. Comparative study of various pretreatment reagents on rice husk and structural changes assessment of
the optimized pretreated rice husk. Bioresour. Technol. (2012), http://dx.doi.org/10.1016/j.biortech.2012.09.045
The structural changes of the pretreated rice husk were as-
sessed with scanning electron microscope Quanta™ 200 FESEM
(FEI, USA) operated at 2–5 kV accelerating voltage under low
vacuum.
The surface area and average pore size of rice husk samples
were determined by nitrogen adsorption isotherm at 77 K using a
high-performance six-sample surface area and pore size analyzer
AutosorbÒ-6B (Quantachrome, Florida, USA). The nitrogen adsorp-
tion–desorption isotherm was operated at relative pressure P/P0 of
0.3, where P is the system pressure and P0 is the initial pressure at
1 bar.
3. Results and discussion
3.1. Assessment of pretreatment reagent
Based on the results of screening for suitable pretreatment re-
agent, each alkali and acid had pretreated rice husk to a varying ex-
tent. Low amount of TRS was detected in the hydrolysates of NaOH
and Ca(OH)2 pretreatments. This is because the alkalis only frac-
tionally hydrolysed hemicellulose and cellulose (Weil et al.,
1994), but mainly delignified rice husk (Brannvall, 2004). Generally,
findings from the assessment show that acids were better pretreat-
ment reagents than alkalis (Fig. A1). The highest TRS was detected
in the hydrolysate of HCl pretreatment (15.0 ± 0.6 mg/ml), followed
by HNO3 (12.2 ± 0.1 mg/ml) and H2SO4 (7.2 ± 0.3 mg/ml). Pretreat-
ment with CH3COOH, H3PO4, NaOH, and Ca(OH)2 produced less
than 1 mg TRS/ml hydrolysate, which is similar to pretreatment
using only water.
The characterization of rice husk samples showed that all acid-
pretreated rice husk had reduced hemicellulose content (Fig. 1),
which explains the acids main role were hydrolysing the amor-
phous hemicellulose in the substrate (Orozco et al., 2007). Besides,
cellulose was also partially hydrolysed during the acid pretreat-
ments of rice husk (Weil et al., 1994), particularly pretreatments
with strong acids, such as HCl, HNO3 and H2SO4. The availability
of more reactive protons disrupt hydrogen bonding of cellulose
chain prior to hydrolysis resulting in higher TRS yield (Orozco
et al., 2007).
Among the reagents, HCl hydrolysed rice husk and released the
highest amount of TRS during pretreatment. This signifies the
effectiveness of HCl pretreatment in disrupting rice husk structure,
while retaining significant amount of cellulose (60%) in pre-
treated rice husk. Thus, HCl pretreatment was further optimized.
3.2. Optimization of pretreatment
The preliminary pretreatment study shows that TRS yield in-
creased sharply in the first 2 h of pretreatment and reached plateau
after 3 h (Fig. A2a). In HCl loading study, TRS yield increased shar-
ply with HCl loading range between 0.25 and 0.75% (w/v) as more
protons are available for the hydrolysis of rice husk (Fig. A2b).
However, little increment in TRS yield was observed with HCl load-
ing higher than 1.0% (w/v) signifies that the rate of hydrolysis is
limited by the surface area of rice husk available for reaction. Fur-
thermore, the TRS yield was found to increase proportionally with
the pretreatment temperature (Fig. A2c).
The low and high levels of HCl loading (X1), pretreatment dura-
tion (X2), and temperature (X3) were determined in the preliminary
studies. The design matrix of BBD including the response (Y) is gi-
ven in Table 1, where Y is the TRS detected in the hydrolysate. From
the runs, the highest TRS (23.9 mg/ml) was obtained with pretreat-
ment conditions at 0.75% (w/v) HCl loading, 120 °C for 2 h. The
least TRS (10.9 mg/ml) was detected when pretreatment was con-
ducted at 0.25% (w/v) HCl loading, 100 °C for 2 h. Only minute
 T.N. Ang et al. / Bioresource Technology xxx (2012) xxx–xxx 3

Fig. 1. Chemical composition of rice husk after acid and alkaline pretreatments (based on same initial sample weight).

amount of furfural (5% of TRS) was detected in the hydrolysates, The optimization study shows that HCl loading and pretreat-
whereas 5-(hydroxymethyl)furfural was not detected. ment temperature have greater effect compared to the pretreat-
The Analysis of Variance (ANOVA) of the reduced quadratic ment duration, and both these influencing parameters interacted
model confirmed the significance and goodness of fit of the model in the pretreatment of rice husk as shown in Fig. 2. Higher temper-
(Table 2). The reduced model expressed in actual terms is given in ature is needed to achieve the maximal TRS yield when lower HCl
Eq. (1). The reduced model explained 95.2% of the variability in the loading is used, and vice versa. The TRS yield showed a quadratic
optimization pretreatment of rice husk. dependence on the pretreatment temperature with high effect of
term X32.
Y ¼ 156:32 þ 49:74X 1 þ 9:89X 2 þ 2:36X 3  7:39X 21
Numerical optimization was conducted for the maximization of
 0:008X 23  0:31X 1 X 3  0:08X 2 X 3 ð1Þ TRS release, and the optimum pretreatment conditions were 0.5%
(w/v) HCl loading, 125 °C for 1.5 h with a predicted TRS yield of
22.8 mg/ml. Verification of the optimized pretreatment condition
confirmed the TRS released at 22.3 ± 0.3 mg/ml, which is a mere
Table 1 2% discrepancy from the predicted value that further suggests
Design matrix of BBD and response.
the accuracy of the reduced model.
Run Parameter Response
X1 (%, w/v) X2 (h) X3 (°C) Y (mg/ml) Table 2
1 0.25 1 120 18.3 Analysis of variance (ANOVA) of quadratic model for optimization of rice husk
2 0.25 3 120 21.6 pretreatment.
3 1.25 1 120 21.5
Source Sum of Degree of Mean F-value P-value
4 1.25 3 120 22.3
square freedom square
5 0.25 2 100 10.9
6 0.25 2 140 23.6 Model 173.04 7 24.72 25.44 <0.0001
7 1.25 2 100 19.0 X1 7.88 1 7.88 8.11 0.0192
8 1.25 2 140 19.5 X2 1.85 1 1.85 1.91 0.2007
9 0.75 1 100 16.1 X3 59.46 1 59.46 61.19 <0.0001
10 0.75 1 140 23.5 X12 14.43 1 14.43 14.85 0.0039
11 0.75 3 100 19.1 X32 39.43 1 39.43 40.58 0.0001
12 0.75 3 140 20.2 X1X3 37.33 1 37.33 38.42 0.0002
13 0.75 2 120 23.9 X2X3 9.83 1 9.83 10.11 0.0112
14 0.75 2 120 23.3 Residual 8.75 9 0.97
15 0.75 2 120 23.1 Lack of fit 7.28 3 1.46 3.96 0.1034
16 0.75 2 120 23.1 Pure error 1.47 4 0.37
17 0.75 2 120 22.2 Corrected total 181.79 16

Please cite this article in press as: Ang, T.N., et al. Comparative study of various pretreatment reagents on rice husk and structural changes assessment of
the optimized pretreated rice husk. Bioresour. Technol. (2012), http://dx.doi.org/10.1016/j.biortech.2012.09.045
4 T.N. Ang et al. / Bioresource Technology xxx (2012) xxx–xxx
Fig. 2. Response surface plot of HCl loading and pretreatment temperature on TRS
yield (pretreatment duration = 2 h).
3.3. Characterization of pretreated rice husk
FT-IR investigation revealed that the pretreated rice husk exhib-
its enhanced cellulosic content as shown by the intensified bands
at 1046 and 2924 cm1 (Fig. A4). Besides, cellulose portion of the
pretreated rice husk has more disordered structure, and lignin
was also observed in the HCl pretreated rice husk. These findings
are complemented by chemical composition analysis of the rice
husk samples. The pretreated rice husk consists of mainly cellulose
(65.1%, w/w) and lignin (26.7%, w/w) which can be seen from the
increased intensity of relative transmittance of cellulose- and lig-
nin-related bands in the FT-IR spectrum, whereas hemicellulose
that is easily hydrolysed was not detected.
Pretreated rice husk was slightly more crystalline than the un-
treated rice husk after HCl pretreatment. The increased in the CrI
from 51.5 of the untreated rice husk to 56.3 of the pretreated rice
husk is due to the removal of amorphous hemicellulose and cellu-
lose from rice husk by HCl during pretreatment. The evidence for
the removal is confirmed by the SEM micrographs, whereby at
1500 magnification, pretreated rice husk exhibited a smoother
surface than the untreated rice husk (Fig. A5).
Furthermore, the BET surface area analysis demonstrated that
the specific surface area of the pretreated rice husk was signifi-
cantly reduced from 21.2 to 9.6 m2/g, which is due to the hydroly-
sis of amorphous hemicellulose/cellulose in rice husk and merging
of smaller pores into larger pores. The total pore volume and pore
size of the rice husk after pretreatment had increased from
1.76  102 to 1.81  102 cc/g and 33.2 to 75.2 Å, respectively.
The pretreated rice husk with higher pore volume and bigger pore
size is of advantageous for fungal growth during fermentation (Hsu
et al., 2010), in spite of its slightly higher CrI and lower specific sur-
face area compared to the untreated rice husk. Nevertheless, the
underlying relationship of structural features and biomass digest-
ibility of the pretreated substrate depends on the size of enzyme
used in the enzymatic hydrolysis, and the accessibility of microbes
to substrate during fermentation.
Please cite this article in press as: Ang, T.N., et al. Comparative study of various pretreatment reagents on rice husk and structural changes assessment of
the optimized pretreated rice husk. Bioresour. Technol. (2012), http://dx.doi.org/10.1016/j.biortech.2012.09.045
4. Conclusion
HCl has been found to be more superior to the other investi-
gated reagents, and its subsequent HCl pretreatment optimization
gives relatively mild optimal condition involving low HCl loading,
low pretreatment temperature and short duration. The reduced
model exhibits 2% discrepancy between the predicted and experi-
mental values that suggests its accurate predictability. Structural
characterization analyses showed that the pretreated rice husk,
which consisted mainly of cellulose and lignin, had increased in
pore size and pore volume, which is favorable for the attachment
and growth of fungus during fermentation.
Acknowledgement
The authors thank University of Malaya for the financial sup-
port (UMRG RG006/09AET and PS161/2010A). The authors also
thank Ng Trading Company, Selangor, Malaysia for providing the
rice husk sample.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2012.
09.045.
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