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Organosolvent pretreatment and enzymatic hydrolysis of rice straw for the

production of bioethanol

Article in World Journal of Microbiology and Biotechnology · February 2012

DOI: 10.1007/s11274-011-0838-8

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World J Microbiol Biotechnol (2012) 28:473–483
DOI 10.1007/s11274-011-0838-8

ORIGINAL PAPER

Organosolvent pretreatment and enzymatic hydrolysis of rice

straw for the production of bioethanol
Raveendran Sindhu • Parameswaran Binod •
Kanakambaran Usha Janu • Rajeev K. Sukumaran •

Ashok Pandey

Received: 23 February 2011 / Accepted: 30 June 2011 / Published online: 10 July 2011
Ó Springer Science+Business Media B.V. 2011

Abstract The present study investigates the operational Introduction

conditions for organosolvent pretreatment and hydrolysis
of rice straw. Among the different organic acids and Driven by increasing environmental concerns, high oil
organic solvents tested, acetone was found to be most prices, and the uncertainty of petroleum reserves, the pro-
effective based on the fermentable sugar yield. Optimiza- duction of bioethanol from lignocellulose is receiving
tion of process parameters for acetone pretreatment were increasing attention. The conversion of lignocellulosic
carried out. The structural changes before and after pre- biomass to ethanol has many technical and economical
treatment were investigated by scanning electron micros- challenges that delayed its commercialization. To make the
copy, X-ray diffraction and Fourier transform infrared biomass conversion economical, it is necessary to lower the
(FTIR) analysis. The X-ray diffraction profile showed that cost of the pretreatment and enzymatic hydrolysis.
the degree of crystallinity was higher for acetone pretreated Pretreatment is essential to make biomass digestible by
biomass than that of the native. FTIR spectrum also altering biomass structural features (Zhu et al. 2010). Struc-
exhibited significant difference between the native and tural modifications of lignocelluloses are highly dependent on
pretreated samples. Under optimum pretreatment condi- the type of pretreatment employed and have a great effect on
tions 0.458 g of reducing sugar was produced per gram of enzymatic hydrolysis (Kumar and Wyman 2009).
pretreated biomass with a fermentation efficiency of 39%. Rice straw is an attractive lignocellulosic material for
Optimization of process parameters for hydrolysis such as bioethanol production since it is one of the most abundant
biomass loading, enzyme loading, surfactant concentration renewable resources. It has several characteristics that
and incubation time was done using Box–Benhken design. make it a potential feedstock for fuel ethanol production. It
The results indicate that acetone pretreated rice straw can has high cellulose and hemicellulose content that can be
be used as a good feed stock for bioethanol production. readily hydrolysed into fermentable sugars (Binod et al.
2010).
Keywords Rice straw  Bioethanol  Acetone  For the conversion of lignocellulosic biomass into fer-
Pretreatment  Lignocellulosic biomass  mentable sugars, it is necessary to have a pretreatment step
Response surface methodology to break the lignin and to expose cellulose and hemicel-
luloses for enzymatic saccharification. There are several
reports on pretreatment of rice straw using electron beam
irradiation (Jin and Chen 2006), microwave (Zhu et al.
2005), alkali (Zhang and Cai 2008), ammonia (Kim and
Lee 2007), acid (Sumphanwanich et al. 2008), wet oxida-
tion (Wei and Cheng 1985), organosolvent (Jamshid et al.
R. Sindhu  P. Binod  K. U. Janu  R. K. Sukumaran  2005; Pan et al. 2005; Pan et al. 2006), biological using
A. Pandey (&)
white—rot fungi (Taniguchi et al. 2005), organic acids
Centre for Biofuels, National Institute for Interdisciplinary
Science and Technology, CSIR, Trivandrum 695 019, India (Jahan et al. 2006), ionic liquids (Nguyen et al. 2010) and a
e-mail: [email protected]; [email protected] combined process (Kun et al. 2009).

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474 World J Microbiol Biotechnol (2012) 28:473–483

Few reports are available on organosolvent pretreatment and organic acids like acetic acid (30% v/v) and formic
of lignocellulosic biomass. Pretreatment of hybrid poplar acid (30% v/v) in presence and absence of catalysts (0.2%
(Zhang et al. 2007), switch grass (Moxley et al. 2008) and H2SO4). The pretreatment was carried out in an autoclave
rape seed (Li et al. 2009) with phosphoric acid–acetone at 121°C, 15 lb pressure for 60 min. Neutralization of
were reported in the literature. Others include bioorganic pretreated sample was carried out by washing with tap
solvent pretreatment of beech wood (Itoh et al. 2003); water and dried at room temperature. The pretreatment
crude glycerol pretreatment of wheat straw (Sun and Chen efficiency was checked by measuring hydrolysis efficiency
2008); acetone pretreatment of sugarcane bagasse (Araque by reducing sugar estimation. After primary screening of
et al. 2008); and formic acid pretreatment of sugarcane various organic solvents and organic acids, the most
bagasse (Sindhu et al. 2010). Organosolvent process yield effective pretreatment agent was chosen for further opti-
dry lignin, an aqueous hemicelluloses stream and pure mization of process conditions.
cellulose fraction (Duff and Murray 1996).
The aim of the present study was to optimize process Optimization of process parameters on pretreatment
parameters for acetone pretreatment of rice straw and to
study the effect of various parameters for hydrolysis of The various process parameters involved in the pretreat-
acetone pretreated rice straw. The structural features of ment was optimized in a step wise manner, i.e. selecting
native as well as pretreated rice straw were investigated by one parameter at a time approach. Effects of different
scanning electron microscopy (SEM), X-ray diffraction catalysts like mineral acids, organic acids and alkali on
(XRD) and Fourier transform infrared (FTIR) analysis. acetone pretreatment were carried out. Different concen-
trations (0.2–0.8%) of H2SO4 were added along with ace-
tone (60% v/v) during pretreatment to find the effect of
Materials and methods catalyst concentration. Solid loading during pretreatment
was optimized by adjusting various solid–liquid ratios such
Feed stock as 5, 10, 15, 20, 25 and 30% (w/w). Effect of particle size
on acetone pretreatment of rice straw was carried out with
Rice straw received from Hyderabad, Andhra Pradesh, different particle sizes ([600, 600–1,000 and \1,000 l).
India was used in this study. The raw materials were air Effect of incubation temperature on acetone pretreatment
dried and milled to a size less than 1 mm. The dried was carried out by keeping the flasks in autoclave at dif-
materials were stored at room temperature until further use. ferent temperatures such as 80, 100 and 121°C. Pretreat-
Their dry weight content was measured at 105°C for 24 h. ment was performed at different time points (30–120 min)
The compositional analysis of native rice straw was carried to find out the optimum time.
out by two stage acid hydrolysis protocol developed by
National Renewable Energy Laboratory (NREL) and the Characterization of native and pretreated biomass
result is shown in Table 1.
XRD analysis
Pretreatment
The crystallinity of native and pretreated samples was
Two gram of biomass was taken in a 250 ml stoppered measured by X-ray diffraction using a PANalytical
conical flasks and mixed with different organic solvents (Netherlands), X-pert pro diffractometer set at 40 kV,
like glycerol, methanol and acetone with water in 1:1 ratio 30 mA; radiation was Cu Ka (k = 1.54 Å). The samples
were scanned in a 2h range from 10 to 30° and a step size
of 0.03° was used for the analysis. The crystallinity index
of each sample was expressed using the following equation
Table 1 Composition of native rice straw (Kim and Holtzapple 2006).
Component % w/w of rice straw  
CrI ð%Þ ¼ ðI002  Iam Þ=I002  100
Cellulosea 34.12 ± 3.5
I002 is the intensity of 002 peak at 2h = 22.4° and Iam is the
Hemicelluloseb 28.45 ± 3.2
intensity of the background scatter at 2h = 18.0°.
Total lignin 18.12 ± 3.02
Ash 7.45 ± 0.318
FTIR analysis
Extractives 11.93 ± 0.113
a
Based on total glucan Fourier Transform Infrared (FTIR) spectroscopic analysis
b
Based on total xylan and other C5 sugars was carried out to detect changes in functional groups that

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World J Microbiol Biotechnol (2012) 28:473–483 475

may have been caused by the pretreatment (Sindhu et al. as hydrolyzate) was used for fermentation. Ethanol fer-
2010). mentation was carried out in 250 ml stoppered conical
flasks containing 150 ml hydrolysate with reducing sugar
SEM analysis concentration of 34.7 mg/ml. The flasks were inoculated
with seed culture (2% v/v) of 12 h old Saccharomyces
Scanning electron micrographs (SEM) were taken at cerevisiae and incubated at room temperature (28 ± 2°C)
magnification 1,5009 for both native and acetone pre- under static conditions. After 72 h of incubation the entire
treated rice straw (particle size [600 lm) using a JEOL fermented broth was centrifuged at 10,000 rpm for 5 min
JSM-5600 scanning electron microscope (Sindhu et al. at 4°C. The supernatant was filtered using 0.4 lm filters
2010). and the ethanol content was analyzed using gas chroma-
tography set at an oven temperature 150°C, injector tem-
Enzymatic hydrolysis perature 175°C and detector temperature 250°C. Nitrogen
with flow rate 30 ml per min was used as carrier gas. The
Enzymatic hydrolysis of the acetone pretreated rice straw ethanol concentrations of the fermented samples were
was carried out in 150 ml stoppered conical flasks by calculated based on the calibration curve drawn with
incubating 2 g of biomass with commercial cellulase (Zy- known concentrations as external standard. The analyses
tex India Private Limited, Mumbai, India). The enzyme were done in triplicate and the mean values were presented.
loading was 30 FPU/g of pretreated dried biomass and the
total reaction volume was made up to 30 ml with 0.1 M
citrate buffer (pH 4.8). The samples were incubated at Results
50°C for 72 h in a shaking water bath (120 rpm). After
incubation, samples were centrifuged to remove the un- Effect of different process parameters on the acetone
hydrolyzed residue. The hydrolyzate was used for reducing pretreatment of rice straw
sugar analysis by 2, 5 dinitrosalicylic acid method (Miller
1959; Aswathy et al. 2010). The result presented in Fig. 1 indicates that among the
different reagent (with and without catalyst) tested, acetone
Optimization of enzymatic saccharification with 0.2% H2SO4 as catalyst was found to be more
by Box–Benhken design effective followed by methanol (with 0.2% H2SO4) in
terms of reducing sugar yield. Formic acid, acetic acid and
The process parameters such as biomass loading, enzyme glycerol were not effective. Hence acetone along with
loading, surfactant concentration and incubation time that H2SO4 as catalyst was selected as pretreatment agent for
affects the enzymatic hydrolysis of acetone pretreated rice further studies. The optimization of different concentration
straw were optimized using a response surface methodol- of acetone from 20 to 100% (v/v) in presence of 0.2%
ogy. A Box and Behnken (1960) design with four variables H2SO4 as catalyst showed that 60% (v/v) was optimum
at three levels and a total of 27 runs were used for the with a reducing sugar yield of 0.347 g/g dry biomass.
study. The three levels of each variable were coded as -1, Further increase from 60% in acetone concentration leads
0 and ?1, which corresponded to the lower, middle and to slight decrease in reducing sugar formation. Acetone
higher values, respectively. For individual parameters these loading above 80% leads to low reducing sugar yield.
were, biomass loading (7.5, 11.25, 15% w/w), enzyme Control experiments were carried out by pretreatment with
concentration (20, 50, 80 FPU/g substrate), Tween 80 water, 0.2% H2SO4 and acetone alone. Pretreatment with
concentration (0.05, 0.125, 0.2% w/w), and incubation time water, acetone and 0.2% H2SO4 yielded 0.02, 0.029 and
(24, 42, 60 h). The software Minitab 15 (Minitab Inc, 0.045 g/g of reducing sugar, respectively. The results
USA) was used for experimental design, data analysis and indicate that pretreatment was effective when acetone was
quadratic model building. The response surface graphs pretreated in the presence of 0.2% H2SO4 as catalyst.
were obtained using the software to understand the effect Among the catalysts used for acetone pretreatment like
of variables individually and in combination, and to mineral acids (HCl, H2SO4 and H3PO4), organic acids
determine their optimum levels. The experimental setup of (CH3COOH and formic acid) and alkali (NaOH, KOH and
Box–Benhken Design is shown in Table 2. Ca (OH) 2), H2SO4 was found to be effective in terms of
reducing sugar yield (0.347 g/g) followed by KOH
Ethanol fermentation (0.296 g/g), NaOH (0.294 g/g) and HCl (0.284 g/g). The
optimization of concentration of H2SO4 as catalyst
After enzymatic hydrolysis, solids and liquids were sepa- (0.2–0.8%, v/v) showed that 0.6% was found to be good for
rated by centrifugation and the liquid portion (designated better reducing sugar yield (0.405 g/g).

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Table 2 Reducing sugar yields

Run Biomass Enzyme Surfactant Incubation Reducing
for individual runs of the RSM
loading (% w/w) loading (FPU/g) (% w/w) time (h) sugar (g/g)
design
1 15 50 0.125 24 0.285
2 15 50 0.125 60 0.295
3 7.5 50 0.2 42 0.215
4 7.5 50 0.125 24 0.519
5 15 20 0.125 42 0.45
6 11.25 50 0.125 42 0.619
7 11.25 50 0.2 60 0.655
8 11.25 50 0.2 24 0.291
9 7.5 20 0.125 42 0.394
10 7.5 50 0.05 42 0.452
11 11.25 80 0.05 42 0.61
12 15 80 0.125 42 0.555
13 11.25 20 0.05 42 0.48
14 11.25 50 0.125 42 0.391
15 7.5 50 0.125 60 0.599
16 11.25 50 0.125 42 0.522
17 11.25 80 0.125 24 0.501
18 15 50 0.2 42 0.399
19 15 50 0.05 42 0.439
20 11.25 80 0.2 42 0.537
21 11.25 50 0.05 24 0.545
22 11.25 80 0.125 60 0.536
23 11.25 20 0.125 60 0.594
24 11.25 50 0.05 60 0.439
25 11.25 20 0.125 24 0.253
26 11.25 20 0.2 42 0.195
27 7.5 80 0.125 42 0.189

Optimization of particle size on acetone pretreatment of

rice straw showed that particle size less than 600 l gave
maximum reducing sugar (0.436 g/g).
Effect of temperature on acetone pretreatment showed
that maximum reducing sugar yield (0.431 g/g) was
achieved when the rice straw was pretreated at 121°C.
Total reducing sugar produced was less than 57% when
carried out at 100°C (0.246 g/g). Effect of incubation time
on acetone pretreatment showed that maximum reducing
sugar was produced at 90 min (0.458 g/g). The pretreat-
Fig. 1 Selection of pretreatment reagents. A Glycerol: water (1:1). ment beyond 90 min showed reduction in sugar yield and
B Acetone: water (1:1). C Acetone: water (1:1) ? 0.2% H2SO4.
D Methanol: water (1:1). E Methanol: water (1:1) ? 0.2% H2SO4. at 120 min the reducing sugar yield was 0.440 g/g.
F Acetic acid (30%). G Acetic acid (30%) ? 0.2% H2SO4. H Formic
acid (30%). I Formic acid (30%) ? 0.2% H2SO4. J Water. K 0.2% Characterization of native and pretreated biomass
H2SO4
FTIR spectroscopy was used to detect changes in cellulose
structure during pretreatment. There is difference in the
The solid loading for pretreatment was carried out from FTIR spectra of native and pretreated sample. Figure 2
5 to 30%, w/w and 15% was found to be optimum with a shows the FTIR spectra of native rice straw, rice straw
reducing sugar yield of 0.433 g/g. Further increase in pretreated with acetone, rice straw pretreated with 0.6%
biomass loading decreased the total sugar yield. H2SO4 and rice straw pretreated with acetone in presence

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World J Microbiol Biotechnol (2012) 28:473–483 477

of 0.6% H2SO4 as catalyst. The most representative bands uniform surface. The acetone pretreated rice straw has a
can be summarized as follows. rough surface. This indicates that pretreatment removed
The band absorption in the 3,250 cm-1 region was external fibers which in turn increase surface area so that
related to stretching of H-bonded OH groups and the two cellulose becomes more accessible to enzymes. Similar
absorption bands 1,100 and 900 cm-1 arose from C–O–C structural changes were earlier reported for rice straw
stretching at the b-1,4-glycosidic linkages (Cao and Tan pretreated with electron beam irradiation (Bak et al. 2009)
2004; Hinterstoisser et al. 2001). The peak of CH2 and for rice straw pretreated with aqueous ammonia
stretching near 2,850 cm-1 region is distinguished feature soaking pretreatment (Ko et al. 2009). Rice straw pre-
of cellulose (Sun et al. 2007). During acetone pretreatment, treated by acetone in presence of 0.6% H2SO4 as catalyst
the rice straw absorbs some materials containing –OH became rough and porous with a number of visible fibrous
(H2SO4 added as catalyst) and this results in a peak at structures were formed.
1,600 cm-1. The bands at 1,350 cm-1 region corresponds Crystallinity index was calculated according to the
to symmetric CH2 bending and wagging (Cao and Tan results of XRD analysis. The X-ray diffraction profile of
2004). A small peak at 670 cm-1 region corresponds to the native and acetone pretreated rice straw is shown in Fig. 4.
out of plane bending of C–O–H (Liu et al. 2006). Com- The crystallinity index of native rice straw was less
pared to control sample the peak at 1,700 cm-1 region is (49.03%) compared to other pretreated samples. Pretreat-
stronger, which arose due to intra molecular hydrogen bond ment with acetone in presence of 0.6% H2SO4 as catalyst
formation (Sun et al. 2007). The peak at 1,030 cm-1 cor- gave the highest crystallinity index (58.59%). If the pre-
responds to C–O-C–O-C bonds in cellulose. treatment is effective the amorphous components like
The FTIR spectra were different for native and pre- hemicelluloses and lignin were removed and the biomass
treated biomass. The intensity of intermolecular hydrogen become more crystalline. In the present study the pre-
bonds is responsible for the shift of –OH vibration in the treated sample gave the highest crystallinity index, indi-
FTIR spectra. The peak shifts to a higher wave number if cating that the pretreatment is effective.
the intensity of intermolecular hydrogen bond is weak (Oh Cellulose crystallinity has been considered as an
et al. 2005). important factor in determining the hydrolysis rate (Alvira
SEM observations of native and acetone pretreated rice et al. 2010). Mansfield et al. (1999) reported that pre-
straw were taken at 1,5009 magnification. Figure 3 treatment increases the crystallinity of the cellulose frac-
showed that the pretreatment induced physical changes in tion. This may be due to the removal or reduction of more
the biomass. The native rice straw have smooth and easily available amorphous cellulose after pretreatment.

Fig. 2 FTIR spectra of native

and pretreated rice straw
a microcrystalline cellulose,
b unpretreated, c acetone (60%)
pretreated, d 0.6% H2SO4
pretreated, e acetone (60%) in
presence of 0.6% H2SO4

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478 World J Microbiol Biotechnol (2012) 28:473–483

Fig. 3 Scanning electron

micrographs of native and a b
acetone pretreated rice straw
a native rice straw, b acetone
pretreated rice straw

1800 time, space and raw material. There were a total of 27 runs
1600 Acetone pretreated for optimizing the four individual parameters in the current
1400
Box–Behnken design. The data were analyzed by multiple
regression analysis using the Minitab software and the
1200
following polynomial equation was derived to represent
Intensity

1000
reducing sugar yield as a function of the variables tested.
800

600
Y ¼ 0:20 þ 0:10X1  6:76X2  6:10X3 þ 0:01X4
400  0:01X21  3:12X22  5:81X23  2:44X24
Native
200 þ 0:01X1 X2 þ 0:18X1 X3  2:59X1 X4 þ 0:02X2 X3
0  1:42X2 X4 þ 0:09X3 X4
10 15 20 25 30
Angle where Y is the predicted reducing sugar yield and X1, X2,
Fig. 4 X-ray diffraction pattern of native and acetone (60%) X3 and X4 are the coded values for biomass loading,
pretreated rice straw enzyme loading, surfactant concentration and incubation
time, respectively.
The experimental data were statistically analyzed using
A contrary observation was reported by Kumar and Wy- the Fischer’s statistical test for analysis of variance
man (2009). High pH pretreatments have been shown to (ANOVA) and the results are shown in Table 3. The
have less effect and even reduced biomass crystallinity. ANOVA of the quadratic Regression model indicated that
the model was moderately significant, as the F value for the
Optimization of enzymatic saccharification by Box– model was 1.4. The Prob [ F value of the model was 0.05,
Benhken design which also confirmed that the model was significant.
Surface plot describing combined effect between pair of
Response surface optimization is more advantageous than factors on hydrolysis of acetone pretreated rice straw were
the traditional single parameter optimization in that it saves given in Figs. 5, 6, 7, 8, and 9, keeping other two variables

Table 3 Analysis of variance

Source Sum of squares Degree of freedom Mean square F value Prob [ F
(ANOVA) for the response
surface model Model 0.23 10 0.02 1.40 0.051
Linear 0.12 4 0.13 1.62 0.218
Interaction 0.12 6 0.02 1.25 0.334
Residual error 0.27 16 0.17
Lack of fit 0.03 2 0.17 1.31 0.515
Pure error 0.03 2 0.01
Total 0.50 26

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World J Microbiol Biotechnol (2012) 28:473–483 479

Reducing sugar (g/g)

0.6
Reducing sugar (g/g)

0.5
0.5

0.4
80 0.4
80

g
in

g
0.3 60

ad

in
60
0.3

ad
lo

lo
40 40

e
m

e
8 30

m
zy
10 20 40

zy
12 50 20

En
14

En
60
Biomass loading Incubation time

Fig. 5 Effect of biomass loading and enzyme loading on reducing Fig. 8 Effect of incubation time and enzyme loading on reducing
sugar yield sugar yield
Reducing Sugar (g/g)

Reducing sugar (g/g)

0.5 0.5

0.4
0.4
0.20
0.20
t

0.15
an

0.3 0.15

nt
0.3
ct

cta
0.10
rfa

8 0.10

rfa
Su

10 20
12 0.05 40

Su
14 60 0.05
Biomass loading 80
Enzyme loading
Fig. 6 Effect of biomass loading and surfactant concentration on
Fig. 9 Effect of enzyme loading and surfactant concentration on
reducing sugar yield
reducing sugar yield

constant at their middle level. Figure 5 indicates reducing

sugar yield as a function of enzyme loading and biomass
loading. It was observed that the average level of biomass
Reducing sugar (g/g)

loading (11.25%) and at the average level of enzyme

0.6
loading (60 FPU) the reducing sugar yield was high.
Figure 6 indicates reducing sugar yield as a function of
0.5 surfactant concentration and biomass loading. The middle
level of biomass loading (11.25%) and low level of sur-
60 factant concentration (0.05%) shows the maximum reduc-
e

0.4
tim

50 ing sugar yield. The interaction effect of incubation time

n
tio

40 and biomass loading was shown in Fig. 7. This surface plot

ba

8 30 explains that at middle value of biomass loading (11.25%)

cu

10
12
In

14 and long duration of incubation time (60 h) gives maxi-

Biomass loading mum reducing sugar. The effect of incubation time and
Fig. 7 Effect of biomass loading and incubation time on reducing enzyme loading on reducing sugar yield was given in
sugar yield Fig. 8. Increased effect on reducing sugar yield was

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480 World J Microbiol Biotechnol (2012) 28:473–483

Literature survey shows that organosolvent pretreatment

is less exploited in the lignocellulosic biomass to ethanol
process. The major drawbacks in this process are the high
cost of the solvents and additional revenue needed for
solvent recycling which otherwise inhibit the fermenting
organisms. The organosolv pretreatment process covers a
broad range of organic solvents such as methanol, ethanol,
acetic acid, peracetic acid, acetone, and so on (Zhao et al.
2009). Pretreatment with low boiling point alcohol such as
methanol and ethanol seems to be the most promising or-
ganosolv process due to the lower chemical costs and easy
recovery of solvents, but the utilization of ethanol for the
Fig. 10 Fermentation efficiency of acetone pretreated rice straw
production of ethanol is absurd. Acetone pretreatment is
similar to low boiling point alcohol pretreatment, but the
observed at high level of incubation time (60 h) and a cost of acetone is higher than those of methanol and eth-
linear increase in reducing sugar yield was observed from anol. Acetone is the most efficient ketone for delignifica-
low to high enzyme concentration. Figure 9 explains the tion (Pan and Sano 1999). Cellulose containing materials,
interaction between surfactant concentration and enzyme such as wood, could be rapidly converted to sugars by an
loading with the reducing sugar yield. Increased effect on acetone–water mixture. An acid catalysed organosolv
reducing sugar yield was observed at low level of surfac- saccharification process was developed by Paszner et al.
tant concentration and a linear increase in reducing sugar (1985), by which wood was hydrolyzed to fermentable
yield from low to high level of enzyme concentration. sugars and lignin. In 2007, Zhang et al. reported a new
process for fractionation of lignocellulosic biomass in
Ethanol fermentation concentrated phosphoric acid-acetone mixture. Li et al.
(2009) reported phosphoric acid–acetone pretreatment of
The results of the fermentation of acetone pretreated and rape seed, bermuda grass and reed for bioethanol produc-
enzymatically saccharified rice straw are given in Fig. 10. tion. In this method phosphoric acid removed hemicellu-
The maximum ethanol concentration (0.954 g of ethanol) loses and partial lignin, and no inhibitors were generated in
was obtained after 72 h using 60% acetone pretreatment this process. In the present study, acetone pretreatment at
followed by enzymatically hydrolysed rice straw. The 121°C in presence of 0.6% H2SO4 improved the sugar
overall efficiency of the process was around 39%. The yield after enzymatic hydrolysis. Identical observation was
calculation of efficiency is based on the theoretical ethanol earlier recorded by Sindhu et al. (2010) for formic acid
yield from glucose. By definition it is the percentage pretreatment of sugarcane bagasse using H2SO4 as catalyst.
conversion of reducing sugar (considered as glucose) ini- Effectiveness of organosolvent treatment depends on the
tially present in the fermentation medium to ethanol. treatment temperature. By the addition of catalysts, the
treatment temperature can be reduced (Duff and Murray
1996). At high temperature, the organic acids generated
Discussion during pretreatment acts as catalyst (Sun and Cheng 2002).
Acetone extracts most of the organic components and
Among the various pretreatment methods as described in produces pure cellulose, which facilitates easy enzymatic
introduction, none of them can be adopted as a universal hydrolysis (Li et al. 2009).
process for recovering cellulose from all types of ligno- Solid loading is an important parameter in biomass
cellulosic biomass. Based on the composition of biomass as pretreatment since it affects the overall energy balance and
well as the product of interest, it is necessary to choose a capital costs for fermentation and downstream recovery
pretreatment process. The most common pretreatment (Eggerman and Elander 2005). Hence maintaining high
methods in commercial use are steam explosion and dilute solid concentration during pretreatment improves the total
acid hydrolysis. The main drawbacks of these processes are sugar recovery. But higher solid loading beyond a certain
the formation of inhibitors which affect the subsequent level has an unfavourable effect. Higher solid–liquid ratio
fermentation. A comparison of various pretreatment may impart less accessibility of biomass by the pretreat-
methods for lignocellulosic biomass shows that carbonic ment reagent which in turn results in incomplete pretreat-
acid, alkaline extraction and organosolv pretreatment are ment. In the present study 15% of solid loading was found
the best process in terms of higher sugar yield and less to be best as the reducing sugar yield decreased beyond this
formation of inhibitors (Gnansounou 2009). value. Studies with solid loading of 5 and 10% gave almost

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World J Microbiol Biotechnol (2012) 28:473–483 481

the same sugar yield as that of 15% loading (0.433 g/g). reducing sugar was produced at 90 min (0.458 g/g) and
The decrease in reducing sugar yield above 15% of solid beyond that there was a drastic decrease. Similar obser-
loading may be due to the decreased accessibility of the vation was earlier reported for formic acid pretreatment of
biomass by acetone in presence of H2SO4 as catalyst which sugarcane bagasse (Sindhu et al. 2010).
might cause the incomplete removal of lignin. Presence of The second-order polynomial regression equation
lignin in pretreated biomass irreversibly binds with cellu- obtained from the experimental data can be used to predict
lase during enzymatic hydrolysis resulting less sugar the hydrolysis rate at any enzyme loading, substrate con-
release (Binod et al. in press). As reported by Kootstra centration, surfactant concentration, and hydrolysis time
et al. (2009) and Noureddini and Byun (2010) in their within the range of the experimental design. The optimum
studies on the pretreatment of wheat straw and distiller’s conditions of enzymatic hydrolysis of acetone pretreated
grains, respectively, high solid loading can also promote rice straw were biomass loading (11.25% w/w), enzyme
the formation of inhibitory compounds, such as furfural, loading (60 FPU), surfactant concentration (0.05% w/w)
which will inhibit subsequent cellulose hydrolysis. Most of and incubation time (60 h) with reducing sugar yield of
the reported literatures show that a solid loading of 0.655 g/g. Xu et al. (2007) performed enzymatic hydrolysis
10–15% is good for biomass pretreatment (Li et al. 2009; of ammonia liquor pretreated soybean straw using an
Itoh et al. 2003; Sindhu et al. 2010; Satyanagalakshmi et al. enzyme loading of 50 FPU/g substrate and obtained a
2011). maximum hydrolysis yield of 51.22% at 5% (w/v) substrate
Another important parameter in pretreatment of ligno- concentration for 36 h. Sun and Chen (2008) observed that
cellulosic materials is particle size. As the size of the an enzyme loading of 44 FPU/g substrate was necessary to
particle decrease, the accessible surface area for reagent to have a hydrolysis yield of 92% from enzymatic digestion
act increases which in turn improves the efficiency. Milling of wheat straw pretreated by atmospheric glycerol auto-
and sieving is the easiest way to decrease the particle size. catalysis at 2% (w/w) substrate concentration after 48 h.
In the present work, rice straw was milled and sieved to Mussato et al. (2008) concluded that 45 FPU/g substrate
attain the particle size in the range of \600, 600–1,000 and was an enzyme loading enough to achieve complete con-
[1,000 lm among which smallest particle size gave the version of cellulose from brewer’s spent grain pretreated
best result. This may be due to increased exposure of by a two-step chemical pretreatment process using dilute
biomass materials by the action of reagent which ultimately acid and alkali at 2% (w/v) substrate concentration for
increases accessibility of cellulose by enzymes in the 96 h. Several such studies have evaluated the influence of
subsequent enzymatic hydrolysis stage. the cellulase loading in enzymatic hydrolysis. Chen et al.
Complete removal of lignin from lignocellulosic mate- (2007) demonstrated the requirement of higher cellulase
rial during pretreatment is a necessary prerequisite for concentrations to corncob cellulose hydrolysis, verifying
successful enzymatic hydrolysis of cellulosic fraction. an increase in hydrolysis yield from 50 to 150 FPU/g
Organic solvent pretreatment breaks the internal lignin and substrate, although above 100 FPU/g substrate the raise
hemicellulose bonds and separate the lignin and hemicel- was weaker. Tengborg et al. (2001) studied the hydrolysis
lulose fraction. Temperature plays a major role in this of whole slurry of steam-pretreated softwood with a cel-
reaction. The hard woods and herbaceous materials are lulases loading until 120 FPU/g cellulose do not achieve a
readily delignified in acid-catalysed systems, where as soft complete hydrolysis even after 120 h. Lu et al. (2007)
woods require higher temperature. The present study shows reached to saturation with an increase in enzyme concen-
that at lower temperature (80 and 100°C) the sugar yield tration from 20 to 25 FPU/g with an optimum for cellulase
was substantially low where as at 121°C, 0.431 g/g concentration of 22 FPU/g substrate. These studies show
reducing sugar yield was observed. The results indicate that that different biomass and different conditions of pre-
the temperature had a significant effect on pretreatment. treatment and enzymatic hydrolysis reaction may lead to
Identical observations were earlier reported for acid pre- very different results. In our study it was observed a rising
treatment of water hyacinth by Satyanagalakshmi et al. model response with the increase of cellulase concentration
(2011) and formic acid pretreatment of sugarcane bagasse from 21 to 60 FPU/g dry biomass.
by Sindhu et al. (2010), when pretreatment carried out at Acetone pretreatment of rice straw substantially
three different temperatures 121°C was found to be opti- improved the recovery of sugars with an overall yield of
mum. During acid pretreatment increase in temperature is 0.458 g/g dry biomass. One of the most important advan-
reported to increase delignification (Silverstein et al. 2007). tage of using acetone as a pretreatment agent is that it will
Residence time for pretreatment is also crucial for sugar not generate any inhibitors and the acetone can be recycled.
generation. For acid pretreatment, it has been reported that, Several authors have reported that pretreatment using or-
increase residence time at higher temperature causes the ganosolv process will not generate or produce minimal
degradation of sugars. In the present study maximum level of inhibitors which will not affect subsequent

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fermentation. If pretreatment is carried out at higher tem- Binod P, Janu KU, Sindhu R, Pandey A. In: Pandey A, Larroche C,
peratures, there will be a chance for formation of inhibitors. Ricke SC (eds) Biofuels: alternative feedstocks and conversion
processes. Elsevier, USA (in press)
Therefore, the process temperature should preferably be Box GEP, Behnken DW (1960) Some new three level designs for the
limited (Huijgen et al. 2008). Fermentation experiments study of quantitative variables. Technometrics 2:455–475
using separate hydrolysis and fermentation as well as Cao Y, Tan H (2004) Structural characterization of cellulose with
simultaneous saccharification and fermentation indicates enzymatic treatment. J Mol Struct 705:189–193
Chen M, Xia LM, Xue PJ (2007) Enzymatic hydrolysis of corncob
that there is no significant generation of inhibitors dur- and ethanol production from cellulosic hydrolysate. Int Biodeter
ing the organosolv pulping (Pan et al. 2005; Weil et al. Biodegr 59:85–89
2002). Duff SJB, Murray WD (1996) Bioconversion of forest products
The results indicate that the optimum conditions for industry waste cellulosics to fuel ethanol: a review. Bioresour
Technol 55:1–33
acetone pretreatment of rice straw were 60% (v/v) con- Eggerman T, Elander RT (2005) Process and economic analysis of
centration of acetone, 0.6% concentration of H2SO4 as pretreatment technologies. Bioresour Technol 96:2019–2025
catalyst, 15% (w/w) solid loading, particle size [600 l, Gnansounou E (2009) In: Pandey A (ed) Hand book of plant-based
incubation temperature 121°C and incubation time of biofuels—fuel ethanol current status and outlook. CRC Press,
pp 57–71
90 min. The structural changes had a significant influence Hinterstoisser B, Akerholm M, Salmen L (2001) Effect of fiber
on the capacity of cellulase to adsorb and diffuse along the orientation in dynamic FTIR study on native cellulose. Carbo-
cellulose surface. The X-ray diffraction profile of native hydr Res 334:27–37
and acetone pretreated rice straw showed that the crystal- Huijgen WJJ, Vanderlaan RR, Reith JH (2008) Modified organosolv
as a fractionation process of lignocellulosic biomass for copro-
linity index of native rice straw was less when compared to duction of fuels and chemicals. 16th European Biomass
other pretreated samples. The optimum conditions of Conference and Exhibition, Valencia, Spain, June 2–6
enzymatic hydrolysis of acetone pretreated rice straw were Itoh H, Wada M, Honda Y, Kuwahara M, Watanabe T (2003)
biomass loading (11.25% w/w), enzyme loading (60 FPU), Bioorganosolv pretreatments for simultaneous saccharification
and fermentation of beech wood by ethanolysis and white rot
surfactant concentration (0.05% w/w) and incubation time fungi. J Biotechnol 103:273–280
(60 h). The maximum reducing sugar yield at optimum Jahan MS, Lee ZZ, Jin Y (2006) Organic acid pulping of rice straw I:
conditions was 0.655 g/g. From the results it can conclude cooking. Turk J Agric For 30:231–239
that acetone pretreatment followed by enzymatic sacchar- Jamshid MR, Alireza T, Pejman RC (2005) Pulping of rice straw by
high boiling point solvents in atmospheric pressure. Iran Polym J
ification is an effective method for the production of fer- 14:223–227
mentable sugars which further can be utilized for Jin S, Chen H (2006) Superfine grinding of steam—exploded rice
bioethanol production. straw and its enzymatic hydrolysis. Biochem Eng J 30:225–230
Kim S, Holtzapple MT (2006) Effect of structural features on enzyme
Acknowledgments The authors are grateful to Technology Infor- digestibility of corn stover. Bioresour Technol 97:583–591
mation, Forecasting and Assessment Council (TIFAC), Department of Kim TH, Lee YY (2007) Pretreatment of corn stover by soaking in
Science and Technology, Government of India and Council of Sci- aqueous ammonia at moderate temperatures. Appl Biochem
entific and Industrial Research (CSIR), New Delhi for financial sup- Biotechnol 36:81–92
port to Centre for Biofuels. Ko JK, Bak JS, Jung MW, Lee HJ, Choi IG, Kim TH, Kim KH (2009)
Ethanol production from rice straw using optimized aqueous
ammonia soaking pretreatment and simultaneous saccharification
and fermentation process. Bioresour Technol 100:4374–4380
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