Bioresource Technology 152 (2014) 450–456

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

Organosolv pretreatment of rice straw for efficient acetone, butanol,

and ethanol production
Hamid Amiri a, Keikhosro Karimi a,b,⇑, Hamid Zilouei a
a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
b
Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Production of ABE was significantly

improved by organosolv
pretreatment.
 The pretreatment resulted in 60%
lignin removal.
 After pretreatment, more than 311 g
sugar was produced from each kg of
rice straw.
 More than 80 g butanol and 21 g
acetone were produced from each kg
of rice straw.

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

Article history: Acetone–butanol–ethanol (ABE) was produced from rice straw using a process containing ethanol
Received 14 September 2013 organosolv pretreatment, enzymatic hydrolysis, and fermentation by Clostridium acetobutylicum bacte-
Received in revised form 13 November 2013 rium. Pretreatment of the straw with 75% (v/v) aqueous ethanol containing 1% w/w sulfuric acid at
Accepted 16 November 2013
150 °C for 60 min resulted in the highest total sugar concentration of 31 g/L in the enzymatic hydrolysis.
Available online 23 November 2013
However, the highest ABE concentration and productivity (10.5 g/L and 0.20 g/L h, respectively) were
obtained from the straw pretreated at 180 °C for 30 min. Enzymatic hydrolysis of the straw pretreated
Keywords:
at 180 °C for 30 min with 5% solid loading resulted in glucose yield of 46.2%, which was then fermented
ABE fermentation
Organosolv
to 80.3 g butanol, 21.1 g acetone, and 22.5 g ethanol, the highest overall yield of ABE production. Thus, the
Lignocellulose organosolv pretreatment can be applied for efficient production of the solvents from rice straw.
Pretreatment Ó 2013 Elsevier Ltd. All rights reserved.
Rice straw

1. Introduction shifted from biological to petrochemical based process due to

Acetone–butanol–ethanol (ABE) fermentation is one of the first
large-scale industrial fermentation processes to be developed
(Jones and Woods, 1986). Up to the 1950s, about two thirds of
butanol and 10% of acetone in the world were produced by this
process from corn starch and molasses (Bahl and Dürre, 2001).
However, during the 1960s, production of butanol and acetone
⇑ Corresponding author at: Department of Chemical Engineering, Isfahan Uni-
versity of Technology, Isfahan 84156-83111, Iran. Tel.: +98 3113915623; fax: +98
3113912677.
E-mail address: karimi@cc.iut.ac.ir (K. Karimi).
increasing the price of corn and molasses as well as availability
of cheaper petrochemical derived butanol and acetone (García
et al., 2011). In recent years, by increasing concerns about unstable
crude oil supplies and dramatic environmental impact of fossil
fuels, there is a high interest in production of green butanol from
biomass (Kumar and Gayen, 2011).
Even though economically production of acetone, butanol, and
ethanol as bulk chemicals from renewable sources is considered
as a breakthrough toward reduction of dependency on fossil re-
sources, it is the unique characteristics of butanol as a biofuel that
renewed the interest in ABE fermentation (Kumar and Gayen,
2011). In comparison to ethanol, butanol has a higher energy

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.11.038
 H. Amiri et al. / Bioresource Technology 152 (2014) 450–456
content, lower solubility in water, lower vapor pressure, and a sim-
ilar air-to-fuel ratio to gasoline. More importantly, butanol is com-
patible with the current automobile engines and the transportation
pipelines. These facts make the biobutanol as an ideal candidate to
replace gasoline (Fortman et al., 2008; Kumar and Gayen, 2011).
The bioconversion of lignocellulosic materials to monomeric
sugars and its consequent fermentation has been suggested for
economical production of ABE (Jurgens et al., 2012). Since 1898,
when the first cellulosic ethanol production was industrialized in
Germany, different technologies have been suggested and devel-
oped for economical conversion of lignocelluloses to fermentable
sugars (Mosier et al., 2005). However, most of these technologies
have been developed for ethanol production, while they do not
match well with requisites of ABE fermentation (Fischer et al.,
2008). In addition, the negative effects of inhibitors are more seri-
ous problems in ABE fermentation compared with ethanolic fer-
mentation (Jang et al., 2012). Therefore, in the production of
fermentable sugars, not only the yield of conversion but also the
concentration of sugars and inhibitors in the hydrolysate are
among the factors affecting the possibility and economy of ABE
production from lignocelluloses.
Enzymatic hydrolysis is a widely used process for conversion of
lignocellulosic materials to monomeric sugars. However, the recal-
citrant structure of the lignocelluloses makes the hydrolysis ineffi-
cient, and it is essential to open up the structure through a
pretreatment (Karimi et al., 2013). The role of pretreatment is even
more important in ABE production from lignocelluloses, since the
process is more sensitive to inhibitors. Diluted sulfuric acid pre-
treatment has been commonly used for ABE production from ligno-
cellulosic resources (Gottumukkala et al., 2013; Qureshi et al.,
2007, 2010a). However, different inhibitors which are generated
in this process severely affect the growth and fermentation ABE
producing microorganism; thus, an additional detoxification pro-
cess is necessary to obtain a high yield which is accompanied with
some sugar loss and additional capital investment (Qureshi et al.,
2010b). Recently, concentrated alkaline and phosphoric acid pre-
treatments were evaluated for production of ABE from rice straw
(Moradi et al., 2013). However, the recovery of the chemical agents
in these processes accompanied with high energy consumption
and production of environmental pollutant wastewaters. Thus,
the organosolv pretreatment can be applied for efficient produc-
tion of the solvents from rice straw.
Among the pretreatment technologies, ethanol organosolv pro-
cess has been considered as one of the most promising methods for
biorefining of lignocelluloses (Alvira et al., 2010). Ethanol organo-
solv pretreatment involves the use of ethanol and water to par-
tially hydrolyze lignin and lignin–carbohydrate bonds to remove
lignin from lignocelluloses (Hu et al., 2011), the major barrier to
the enzymatic hydrolysis (McMillan, 1994). Lignin hinders hydro-
lysis by blocking access of cellulases to cellulose due to irreversibly
binding to the hydrolytic enzymes. Therefore, removal of lignin can
considerably improve the hydrolysis rate (McMillan, 1994). The
organosolv pretreatment is usually carried out using a strong inor-
ganic acid catalyst, e.g., sulfuric acid, which takes a role to hydro-
lyze the lignin bonds in biomass (Zhao et al., 2009). Compared to
other chemical pretreatments, one of the main and unique advan-
tages of organosolv process is recovery of lignin as a valuable
by-product, which is relatively pure, primarily unaltered, and less
condensed than Kraft lignins (Mesa et al., 2011; Zhao et al.,
2009). Furthermore, the recovery of solvent is typically performed
with minimal energy consumption. From the environmental point
of view, separation of lignin as a product could reduce the problem
with wastewater treatment.
Considering the special requirements of ABE fermentation,
organosolv pretreatment may offer several advantages over the
other pretreatments. In addition, lignin and its derivatives were
451
found to be the main inhibitors affecting the ABE fermentation
(Wang and Chen, 2011). Moreover, enrichment of cellulose content
by partial removal of lignin is a possible approach to increase the
maximum obtainable glucose concentration. As a result, removal
of lignin as a purified product through the organosolv pretreatment
can improve the economy of ABE process by improving the ABE pro-
duction and addition of ethanol organosolv lignin as a value-added
product. To our knowledge, there is no publication on evaluation of
organosolv pretreatment for improvement of ABE production.
The main drawbacks and limitations of the previous studies on
ABE production from lignocelluloses are production of hydroly-
sates containing different inhibitors, necessity of detoxification
which is accompanied with some sugar loss (Qureshi et al.,
2013), and environmental problems pertaining to wastewater con-
taining different degraded materials produced during the pretreat-
ment. In this study, ethanol organosolv pretreatment was
evaluated for production of ABE from rice straw. This pretreatment
does not have the mentioned drawbacks and separate a lignin with
high purity, which is a value added product. The straw is one of the
most favorable substrates for biological products in terms of quan-
tity and world wide availability (Kim and Dale, 2004). The pre-
treated rice straw was hydrolyzed by two commercial hydrolytic
enzymes and fermented by Clostridium acetobutylicum. One of the
goals was obtaining higher yield of ABE from the straw compared
to the results obtained by other pretreatment methods.
2. Methods
2.1. Raw materials and enzymes
Rice straw, used as a substrate in all experiments, was obtained
from Lenjan field with a cultivar named ‘‘Sazandegi’’ (Isfahan, Iran).
It was partly ball-milled and screened to achieve a size of between
833 lm (20 mesh) and 177 lm (80 mesh). Dry weight content of
the sample was measured by drying at 105 °C in a convection oven.
The materials were stored at room temperature in resealable plas-
tic bags.
Two commercial enzymes of cellulase (Celluclast 1.5L,
Novozyme, Denmark) and b-glucosidase (Novozyme 188,
Novozyme, Denmark) were used for the hydrolysis. The cellulase
activity was 65 FPU/ml according to the method presented by
Adney and Baker (1996), and the protein content of the enzyme
was 42 mg/ml as measured by Bradford assay. The b-glucosidase
activity was 210 IU/ml, measured by the method presented by
Ximenes et al. (1996).
2.2. Pretreatment methods
Fifty grams (dry weight) of the straw was mixed with 400 g
(solid-to-liquid ratio of 1:8) of 75% (v/v) aqueous ethanol contain-
ing 1% w/w (based on the straw dried mass) sulfuric acid as a cat-
alyst. Treatments were carried out in a high-pressure stainless
steel vessel as a batch reactor with a working volume of 500 ml
(Amiri et al., 2010). The reaction mixture was heated at a rate of
3 °C/min. After reaching the desired temperature, i.e., 150 or
180 °C, the mixture was maintained at the temperature for 30 or
60 min. Afterwards, it was cooled in an ice bath. The pretreated
materials were then washed with 60 °C aqueous ethanol (75% (v/
v), 3  100 ml) and air dried overnight. Finally, the pretreated
materials were stored in resealable plastic bags at 5 °C until use.
2.3. Enzymatic hydrolysis
The untreated and pretreated materials were subjected to enzy-
matic hydrolysis by cellulase and b-glucosidase in 50 mM sodium
452 H. Amiri et al. / Bioresource Technology 152 (2014) 450–456
citrate buffer (pH 4.8). The hydrolysis was conducted at 45 °C and
140 rpm for 72 h using 25 FPU cellulase and 40 IU b-glucosidase
per gram of either treated or untreated biomass at two solid load-
ings of 5% and 8%. The cellulase loadings were 33 mg protein per
gram of cellulose for untreated rice straw, whereas it was varied
from 26 to 30 mg protein per gram of cellulose for the pretreated
straw. The hydrolysates were separated from the remaining solid
fraction by centrifugation (5 min, 5000 rpm) and then fermented.
2.4. Microorganism and inoculum preparation
C. acetobutylicum NRRL B-591, obtained from Persian type cul-
ture collection (PTCC) (Tehran, Iran), was used in all fermentations.
In order to prepare the preculture, 1.5 g cooked meat medium
(Sigma–Aldrich) was added to 25 ml distilled water. After addition
of 0.27 g glucose, the mixture was autoclaved (at 121 °C for
20 min) and cooled to 75 °C. Spore suspension (0.5 ml) was heat
shocked at 75 °C for 2 min after addition to the medium (Moradi
et al., 2013). Subsequently, the medium containing spores was
cooled in ice-cold water for 1 min. The heat shocked spores were
then incubated at 37 °C for 16–18 h (Moradi et al., 2013). For bio-
mass preparation, a 100 ml medium containing (g/L): 30 glucose, 5
yeast extract, 2 (NH4)2SO4, 1 NaCl, 0.75 KH2PO4, 0.75 K2HPO4, 0.2
MgSO4, 0.01 MnSO47H2O, and 0.01 FeSO47H2O was prepared.
Then, the medium was autoclaved (at 121 °C for 20 min) and
cooled to 35 °C (Moradi et al., 2013). Afterwards, 5 ml of the pre-
culture was added to the medium, and the growth was conducted
at 37 °C for 16–18 h.
2.5. ABE fermentation
After addition of 0.05 g yeast extract to 50 ml of the hydrolysates
in 118 ml serum bottles, pH of the solutions was adjusted to 6.5
using 5 M NaOH. The solution was autoclaved (at 120 °C for
10 min) and cooled to room temperature. Subsequently, 0.5 ml of
a buffer (50 g/L KH2PO4, 50 g/L K2HPO4, and 220 g/L CH3COONH4),
a vitamin (0.1 g/L para-aminobenzoic acid, 0.1 g/L thiamin, and
0.001 g/L biotin), and a mineral (20 g/L MgSO47H2O, 1 g/L
MnSO4H2O, 1 g/L FeSO47H2O, 1 g/L NaCl) solutions, was filter ster-
ilized (Millipore filter; 0.22 lm) and added to each bottle (Moradi
et al., 2013). The bottle was then purged with pure nitrogen passed
over a heated reduced copper column to remove trace oxygen and
sealed using a butyl rubber stopper fastened with an aluminum
crimp. After inoculation with 6 ml of actively growing culture
(optical density of 1.2–1.6 at 610 nm), fermentation was conducted
at 37 °C for 72 h. During fermentation, liquid samples were period-
ically withdrawn using a sterile syringe through the rubber stopper.
The samples were centrifuged at 9000 rpm for 25 min and stored at
18 °C before sugar and ABE content analyses.
2.6. Analytical methods
Moisture and total solids (Sluiter et al., 2008a), structural carbo-
hydrates, lignin, and ash contents (Sluiter et al., 2008b) of the un-
treated and pretreated straw were determined. The cell
concentration was estimated from optical density (OD) using a pre-
determined correlation between OD at 610 nm and the dry cell
weight (at 105 °C until constant weight) (Moradi et al., 2013).
Concentration of the fermentation products as well as sugars
were analyzed by high-performance liquid chromatography
(HPLC), equipped with UV/VIS and RI detectors (Jasco International
Co., Tokyo, Japan). Concentration of acetone, butanol, ethanol, ace-
tic acid, and butyric acid were determined by an Aminex HPX-87H
column (Bio-Rad, Richmond, CA, USA) at 60 °C with 0.6 ml/min elu-
ent of 0.005 M sulfuric acid. Sugars were analyzed on an Aminex
HPX-87P column (Bio-Rad, Richmond, CA, USA) at 80 °C with
0.6 ml/min eluent of deionized water. Concentrations of sugars,
acetone, butanol, and ethanol were determined by RI detector,
while acetic acid and butyric acid were quantified on UV chro-
matograms at 210 nm.
All experiments were performed in duplicates and the averages
of the results are presented.
3. Results and discussion
3.1. Pretreatment
Rice straw was subjected to ethanol organosolv pretreatment
prior to hydrolysis in order to improve the production yield and
concentration of ABE in the subsequent fermentation. The pretreat-
ment temperature and time were selected by considering the pre-
vious studies results (Obama et al., 2012) and preliminary
investigations. The solid recovery and chemical composition of
the pretreated materials are summarized in Table 1. The untreated
straw consisted of 49.2% glucan, 29.2% xylan, 3.5% arabinan, and
16.3% Klason lignin. Different parts of the straw were differently af-
fected by the pretreatment. The organosolv pretreated material
was considered as cellulosic fibers, which depending on the ap-
plied conditions, contained various amounts of hemicellulose and
residual lignin (Zhao et al., 2009). The organosolv pretreatment
at 180 °C for 60 min resulted in the highest lignin removal (60%),
obtaining a pretreated material with 9.8% Klason lignin. In addi-
tion, the pretreatment at 180 °C for 30 min resulted in 56% lignin
removal. Furthermore, the pretreatment at the lower temperature,
i.e., 150 °C, resulted in 41% and 45% lignin removal after 30 and
60 min treatment, respectively. In other words, increasing the tem-
perature from 150 to 180 °C resulted in more than 33% higher lig-
nin removal. It was in agreement with previous studies that
showed that the lignin removal is highly affected by the process
temperature and increased by increasing temperature (Shatalov
and Pereira, 2005).
Through the organosolv pretreatment, in addition to lignin, the
hemicellulosic sugars are partially dissolved in the organic liquor.
After pretreatment, the organic liquor was evaporated to recover
the solvent and diluted with water to precipitate the lignin, as
one of the valuable products (Zhao et al., 2009). The remaining
aqueous solution is rich in hemicellulosic sugars, mainly xylose
in the case of rice straw, and is considered as a product which
can be utilized by pentose fermenting microorganisms or con-
verted to furfural, xylitol, and some other chemicals (Zhao et al.,
2009). In this study, the remaining liquor, after evaporation of eth-
anol, was diluted with water and subjected to ABE fermentation.
However, using different dilution ratios (1:1.5, 1:2, and 1:5 the ini-
tial to final liquor), no detectable butanol was produced through
the fermentation (data not shown).
Through the pretreatment, the xylan content decreased by 40%
and 49% after 30 and 60 min pretreatment at 180 °C, while it was
42% and 45% after 30 and 60 min pretreatment at 150 °C, respec-
tively. Increasing the temperature and retention time of the
organosolv pretreatment, generally, increases the lignin and xylan
removal (Koo et al., 2012). In the case of rice straw, the lignin re-
moval was considerably increased by temperature, whereas the
conditions had a minor effect on xylan removal.
The organosolv pretreatment of rice straw generally resulted in
enrichment of cellulose content as a result of partial solubilisation
of both hemicellulose and lignin. Cellulose content of the untreated
rice straw was 49.2% which was increased up to 62.3% by the pre-
treatment at 180 °C for 60 min, while the pretreatment at 180 °C
for 30 min resulted in the material with 58.9% cellulose.
 H. Amiri et al. / Bioresource Technology 152 (2014) 450–456 453

Table 1
Chemical composition and solid recovery of untreated and organosolv pretreated rice straw.

Pretreatment conditions Composition

T (°C) Time (min) Glucan (%) Xylan (%) Arabinnan (%) Acid soluble lignin (%) Acid insoluble (Klason) lignin (%) Ash (%) Solid recovery (%)
Untreated 49.2 ± 1.2 29.2 ± 1.4 3.5 ± 0.2 0.7 ± 0.1 16.3 ± 1.3 6.7 ± 0.2 –
150 30 55.6 ± 0.8 21.7 ± 0.1 2.4 ± 0.1 1.7 ± 0.1 12.4 ± 0.2 7.9 ± 0.3 78.3 ± 0.9
150 60 54.8 ± 0.7 20.9 ± 0.6 2.7 ± 0.1 1.7 ± 0.2 11.7 ± 0.2 8.4 ± 0.2 76.8 ± 1.5
180 30 58.9 ± 1.2 24.4 ± 0.1 2.3 ± 0.4 1.5 ± 0.1 9.9 ± 0.2 6.6 ± 0.1 72.1 ± 1.3
180 60 62.3 ± 1.2 22.4 ± 0.6 1.9 ± 0.4 1.3 ± 0.1 9.8 ± 0.2 7.5 ± 0.2 66.9 ± 1.0

3.2. Enzymatic hydrolysis
The pretreated and untreated straws were subjected to enzy-
matic hydrolysis at 45 °C for 72 h using 25 FPU cellulase and
40 IU b-glucosidase per gram of substrate with 5% and 8% solid
loadings. Concentration of sugars and the yield of glucose are
shown in Fig. 1. Total concentration of sugars was increased after
the pretreatment in all applied conditions.
After 72 h enzymatic hydrolysis with 5% solid loading, 15.1 g/L
glucose, 5.6 g/L xylose, and 0.9 g/L arabinose were obtained from
the straw pretreated at 180 °C for 30 min; thus, the obtained total
sugar concentration was 76% higher than that obtained from the
untreated straw. In other words, glucose yield of 46.2% was ob-
tained. At 8% solid loading, glucose yield of 32.9% was obtained
from the straw pretreated at 180 °C for 30 min while it was
44.2% from the straw pretreated at 150 °C for 60 min. Despite
18% higher lignin content, the straw pretreated at 150 °C for
60 min was converted to glucose with 34% higher yield. This obser-
vation may be related to the fact that the lignin content is not the
only parameter affecting the hydrolysis. Change in the structure of
lignin and its interaction with other lignocellulosic parts are also
among the important factors. Furthermore, the hemicellulose re-
moval (mainly xylan in the case of rice straw) can improve the
yield of hydrolysis (Karimi et al., 2013). Therefore, without consid-
ering the other affecting parameters, the yield of hydrolysis could
not be correlated with lignin content.
Enzymatic hydrolysis (with 2% solid loading) of alkaline (using
12% w/v NaOH at 0 °C for 180 min) and concentrated phosphoric
acid (85% at 50 °C for 30 min) pretreated rice straw had been pre-
viously evaluated for ABE production in which total sugar concen-
tration of less than 10.8 g/L was obtained (Moradi et al., 2013).
Increasing the solid loading in the hydrolysis process form 5% to
8% increased the total sugar concentration. The highest total
sugar concentration of 31.0 g/L, containing 21.5 g/L glucose, 7.9 g/L
xylose, and 1.5 g/L arabinose, was obtained after 72 h hydrolysis
with 8% solid loading from the rice straw pretreated at 150 °C for
60 min. In this process, the glucose yield of 44.2% was obtained.
3.3. ABE fermentation
Rice straw which was pretreated at different conditions (at 150
and 180 °C for 30 and 60 min) was hydrolyzed using 5% and 8% so-
lid loadings, and the hydrolysates were subsequently subjected to
ABE fermentation by C. acetobutylicum at 37 °C for 72 h. Depending
on the pretreatment conditions and the solid loading in the hydro-
lysis step, different amounts of butanol, acetone, ethanol, acetic
acid, and butyric acid were detected through the fermentation.
Concentrations and yields of the fermentation products are shown
in Fig. 2 and Table 2, respectively. In addition, the results of ABE
production from rice straw using different pretreatment technolo-
gies from the current and previous studies are summarized in
Table 3.
The organosolv pretreatment of the straw increased the concen-
tration of butanol and acetone. Pretreatment at 180 °C for 30 min
and the subsequent hydrolysis using 5% solid loading resulted in
production of 29.2 g/L sugars, and fermentation of the sugars re-
sulted in production of 5.1 g/L butanol, 1.34 g/L acetone, 1.43 g/L
ethanol, 1.18 g/L acetic acid, and 1.23 g/L butyric acid. In compari-
son with acetone and ethanol, butanol concentration in the fer-
mentation product was more affected by the pretreatment
conditions. Even though prolonging the pretreatment at 150 °C
enhanced the concentration of butanol from rice straw, the

40 50

45
35
40
30
35
Glucose yield (%)

25
30
Sugar (g/l)

20 25

20
15
15
10
10
5
5

0 0
Untreated 30, 150 60, 150 30, 180 60, 180 Untreated 30, 150 60, 150 30, 180 60, 180
Pretreatment conditions Pretreatment conditions
(Time (min), Temperature (°C)) (Time (min), Temperature (°C))

Fig. 1. Glucose (black), xylose (dark gray), and arabinose (light gray) concentrations, and theoretical glucose yield (s) after 72 h enzymatic hydrolysis of organosolv
pretreated rice straw at 5% (a) and 8% (b) solid loading. Theoretical glucose yield (%) = produced glucose (g/L)  100/(1.111  substrate concentration (g/L)  biomass glucan
fraction).
454 H. Amiri et al. / Bioresource Technology 152 (2014) 450–456

12 10
a b

Acetic acid (g/l); Butyric acid (g/l)

10
8

8
6
ABE (g/l)

4
4

2
2

0 0
Untreated 30, 150 60, 150 30, 180 60, 180 Untreated 30, 150 60, 150 30, 180 60, 180
Pretreatment conditions Pretreatment conditions
(Time (min), Temperature ( C)) (Time (min), Temperature ( C))

Fig. 2. Concentration of butanol (white), acetone (dark gray), ethanol (light gray), acetic acid (), and butyric acid () after 72 h fermentation of hydrolysate of organosolv
pretreated rice straw by hydrolysis at solid loading of 5% (a) and 8% (b).

Table 2
Overall yield of butanol, acetone, ethanol, acetic acid, and butyric acid production from rice straw using different pretreatments.*

Pretreatment conditions Solid loading (%) Overall yields (g/kg rice straw)
Temperature (°C) Time (min) Butanol Acetone Ethanol Acetic acid Butyric acid
Untreated 5 43.6 22.7 23.1 12.8 35.6
150 30 5 57.0 20.5 18.3 15.4 23.6
150 60 5 77.8 18.2 23.1 121.4 37.8
180 30 5 80.3 21.1 22.5 18.6 19.3
180 60 5 61.2 21.9 24.1 11.8 14.7
Untreated 8 7.6 17.4 15.7 <1 <1
150 30 8 27.7 19.4 33.5 37.4 8.2
150 60 8 58.8 24.6 27.1 48.4 4.1
180 30 8 69.8 18.7 14.7 62.2 3.0
180 60 8 60.3 20.3 14.0 18.1 1.9
*
The volume changes through addition of P2 solutions and the culture were considered in the final yields.

pretreatment at 180 °C for 30 min was more effective than that for
60 min. Increasing the solid loading in the hydrolysis from 5% to 8%
enhanced the total concentration of acetone, butanol, and ethanol.
Using 8% solid loading, enzymatic hydrolysis of rice straw which
was pretreated at 180 °C for 30 min and the subsequent fermenta-
tion resulted in production of 7.1 g/L butanol, 1.9 g/L acetone,
1.5 g/L ethanol, 6.3 g/L acetic acid, and 0.3 g/L butyric acid. Concen-
tration of the acids in the final product was less than 64% of ABE
concentration except in the case of using the straw pretreated at
180 °C for 30 min with 5% solid loading in which acids concentra-
tion was 34% higher than ABE concentration. It should be noted
that the acids mainly form in the exponential (acidogenesis) phase
of growth while the solvents produce at the stationary (solvento-
genesis) phase. Considering the solvents as the desired products,
the fermentation was continued to reach the solventogenesis
phase, and the reported data are belonging to the samples analyzed
at the end of fermentation (72 h). However, the yield of acids is
high at acidogenesis phase, and the fermentation should be inter-
rupted at this phase if the acids are the desired products. ABE fer-
mentation of hydrolysates by C. acetobutylicum using
pretreatments by steam explosion (121 °C; 15 lb; 30 min), dilute-
acid hydrolysis (1% H2SO4; 60 °C; 24 h and 121 °C; 15 min), and en-
zyme assisted hydrolysis (enzyme and 1% HCl; 60 °C; 24 h and
121 °C; 15 min) resulted in production of 2.07, 2.12, and 2.99 g/L
ABE, respectively (Ranjan and Moholkar, 2011). In addition,
through a hydrolysis and fermentation of alkaline (12% w/v NaOH;
at 0 °C; for 180 min) and phosphoric acid (85%; at 50 °C; for
30 min) pretreated rice straw by C. acetobutylicum, ABE concentra-
tion of 2–2.85 g/L had been obtained (Moradi et al., 2013).
Concentration profiles during ABE fermentation of hydrolysates
obtained from pretreated (at 180 °C for 30 min in which the high-
est butanol concentration was obtained) and untreated rice straw
are depicted in Fig. 3. During the first 8 h of fermentation, less than
0.4 g/L ABE was produced (Fig. 3). More than 40% of butanol ob-
tained from the pretreated rice straw was produced during 12 to
24 h of fermentation. Fermentation of the hydrolysate obtained
from the rice straw pretreated at 180 °C for 30 min resulted in
ABE productivity of 0.12 and 0.20 g/L/h at solid loadings of 5%
and 8% in enzymatic hydrolysis, respectively.
In addition to the concentration, the overall production yield of
acetone, butanol, and ethanol as well as acetic acid and butyric acid
based on the initial rice straw utilized were evaluated (Table 2).
The overall yield of ABE production from rice straw was improved
by the organosolv pretreatment. After organosolv pretreatment at
180 °C for 30 min and enzymatic hydrolysis at 5% solid loading,
the ABE fermentation resulted in 80.3 g butanol, 21.1 g acetone,
22.5 g ethanol, 18.6 g acetic acid, and 19.3 g butyric acid from each
kg of initial rice straw. The maximum overall ABE yield (i.e.,
123.9 g/kg rice straw) obtained in this work was about twice as
high as the overall yield that previously obtained through the alka-
line pretreatment (i.e., 64.1 g/kg rice straw) and the phosphoric
acid pretreatment (i.e., 63.0 g/kg rice straw) (Moradi et al., 2013)
 H. Amiri et al. / Bioresource Technology 152 (2014) 450–456 455

Table 3
ABE production form rice straw using different pretreatment technologies.

Microorganisms Pretreatment Hydrolysis Treatment to Highest Highest Butanol (ABE) Reference

remove butanol productivity yield (g/kg raw
inhibitors (ABE) (g/L) (g/L/h) rice straw)
C. acetobutylicum NRRL Ethanol organosolv (75% EtOH; 1% Enzymatic – 7.10 (10.5) 0.200 80.3 (123.9) This study
B-591 H2SO4 180 °C; 30 min) hydrolysis
C. acetobutylicum NRRL Alkaline (12% NaOH; 0 °C; 180 min) Enzymatic – 1.40 (2.00) 0.030 45.2 (64.1) Moradi et al.
B-591 hydrolysis (2013)
C. acetobutylicum NRRL Phosphoric acid (85%; 50 °C; 30 min) Enzymatic – 2.00 (2.85) 0.030 44.2 (63.0) Moradi et al.
B-591 hydrolysis (2013)
C. acetobutylicum MTCC Steam explosion (121 °C; 15 lb; – – 1.72 (2.07) 0.017 34.4 (41.4)* Ranjan and
481 30 min) Moholkar
(2011)
C. acetobutylicum MTCC Dilute acid (1% H2SO4; 60 °C; 24 h and – – 1.60 (2.12) 0.017 32.0 (42.4)* Ranjan and
481 121 °C; 15 min) Moholkar
(2011)
C. acetobutylicum MTCC Enzyme assisted hydrolysis (enzyme – – 2.10 (2.99) 0.025 42 .0 (59.8)* Ranjan and
481 and 1% HCl; 60 °C; 24 h and 121 °C; Moholkar
15 min) (2011)
Clostridium sporogenes Acid (4% H2SO4; 121 °C; 60 min) Enzymatic Anionic resin 3.43 (5.32) 0.050 34.3 (53.2)* Gottumukkala
BE01 hydrolysis et al. (2013)
Cl. saccharoper Alkaline (2.25% NaOH; 120 °C; Enzymatic – 3.00 (4.5) 0.060 – Soni et al.
butylacetonicum 60 min) hydrolysis (1982)
C. beijerinckii BA101** Dilute acid (1% H2SO4; 121 °C; Enzymatic XAD-4 resin 6.5 (9.3) 0.100 77.4 (110.7)* Qureshi et al.
60 min) hydrolysis (2008)
*
The yields were calculated based on butanol (or ABE) and substrate concentrations, and the solid recovery of pretreatments assumed to be 100%.
**
In this study corn fiber was used for ABE production.

12 12
20
a b
10 10

16

8 8

12
ABE (g/l)

Sugar (g/l)
6 6

8
4 4

4
2 2

0 0
0
12 12
20
cc d
10 10

16

8 8

12
ABE (g/l)

Sugar (g/l)

6 6

8
4 4

4
2 2

0 0
0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Fermentation time (h) Fermentation time (h)

Fig. 3. Concentration profile of ABE (s), butanol (}), acetone (h), ethanol (4), glucose (d), xylose (j), and arabinose (N) by fermentation of hydrolysates obtained after 72 h
enzymatic hydrolysis of untreated (a and b) and organosolv pretreated rice straw at 180 °C for 30 min (c and d) by hydrolysis with solid loading of 5% (a and c) and 8% (b and
d).

(Table 3). Even though the hydrolysis of the pretreated rice straw concentration led to a highly concentrated sugar solution, which in
at 8% solid loading resulted in higher ABE concentration, the over- turn fermented to higher ABE concentration. However, high
all yield of ABE was higher at 5% solid loading. Increasing substrate solid loading was accompanied with difficulties in mixing and
456 H. Amiri et al. / Bioresource Technology 152 (2014) 450–456
end-product enzyme inhibition which was negatively affected the
yield of hydrolysis (Karimi et al., 2013). In addition, butanol has
been shown to have an inhibitory effect on ABE fermentation
(García et al., 2011); thus, the lower yield of ABE production may
be related to increasing the solid loading as a result of butanol inhi-
bition at higher concentrations. Therefore, utilizing pretreated
straw at higher solid loading resulted in production of ABE with
higher concentration but lower overall yield. Therefore, the maxi-
mum ABE concentration of 10.5 g/L was obtained through the
organosolv pretreatment at 180 °C for 30 min, enzymatic hydroly-
sis at 8% solid loading, and the ABE fermentation.
4. Conclusion
The organosolv pretreatment can be applied for efficient pro-
duction of the solvents from rice straw. The yield of ABE produc-
tion from rice straw as well as the concentration of ABE was
improved by organosolv pretreatment without necessity of detox-
ifications. A relatively high yield of 123.9 g ABE was obtained from
each kg of rice straw using the pretreatment followed by enzy-
matic hydrolysis and fermentation. In addition, the concentration
of ABE was increased up to 10.5 g/L through the fermentation of
the organosolv pretreated rice straw hydrolysates.
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