Fuel 97 (2012) 166173

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Fuel
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Comparison of batch and fed-batch fermentations using corncob hydrolysate

for bioethanol production
Yi-Huang Chang a, Ku-Shang Chang a, Cheng-Wei Huang b, Chuan-Liang Hsu c,, Hung-Der Jang a,
a

Department of Food Science, Yuanpei University, Hsinchu 300, Taiwan

Institute of Food Science, Yuanpei University, Hsinchu 300, Taiwan
c
Department of Food Science, Tunghai University, Taichung 407, Taiwan
b

a r t i c l e

i n f o

Article history:
Received 31 January 2011
Received in revised form 5 February 2012
Accepted 6 February 2012
Available online 18 February 2012
Keywords:
Cellulosic hydrolysates
Batch fermentation
Fed-batch fermentation
Bioethanol

a b s t r a c t
The optimal conditions for the maximum production of ethanol from cellulosic hydrolysate in batch and
fed-batch cultures were investigated and compared. The pretreated corncob could be converted into
reducing sugar with maximal yields after the enzyme mixtures were fed. After 48 h of hydrolytic reaction,
overall reducing sugar and glucose concentrations reached 0.61 and 0.36 g/g dried substrate, respectively.
Further batch fermentation of cellulosic hydrolysate was performed using batch cultures of Saccharomyces cerevisiae BCRC 21812, 23.341.1 g/l biomass and 6.923.0 g/l ethanol was obtained. For the fed-batch
fermentation, the effects of feeding glucose concentrations on ethanol fermentation were studied. The
feeding glucose concentration of 30 g/l resulted in a higher ethanol yield than that of 20 g/l and 10 g/l did.
The cell biomass, ethanol yields, and ethanol conversion rate for the fed-batch fermentation, feeding at
30 g/l glucose concentration, were 44.5 g/l, 32.3 g/l and 0.64 g ethanol/g glucose, respectively. The results
indicate that the fed-batch fermentation had a higher ethanol yield than that of the batch fermentation.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
To lessen the worlds dependence on non-renewable resources,
use of agricultural biomass for the production of biofuels such as
bioethanol has drawn much attention to many researchers in the
past few decades. Cellulosic biomass is an ideal source of energy
because it is both renewable and available in large quantities
around the world. However, the process for the production of
ethanol from cellulosic materials is more complicated than its
production from sugar or starch-based ones. Specically, there
are technical and economical impediments in regards to the development of commercial processes that utilize cellulosic biomass.
Technologies that will allow for the cost-effective conversion of
cellulosic biomass into fuels and other chemicals are being developed. These technologies include low-cost thermo or chemical pretreatment, highly effective cellulases and hemicellulases, and
efcient and robust fermentative microorganisms, have made the
commercialization of biofuel production much more promising
[13].
For production of bioethanol, a lower raw material price,
together with a high ethanol yield and efcient enzymes, will decrease the production cost signicantly. Several different pretreatment methods have been used to facilitate the enzymatic
Corresponding authors. Tel.: +886 4 23590121x37306; fax: +886 4 23599059
(C.-L. Hsu), tel.: +886 3 5381183x8482; fax: +886 3 6102342 (H.-D. Jang).
E-mail address: hungder@mail.ypu.edu.tw (H.-D. Jang).
0016-2361/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fuel.2012.02.006

hydrolysis of lignocellulosic material [4]. An efcient process for

obtaining reducing sugars from lignocellulosic material is to use
chemical/physical pretreatment, followed by enzymatic hydrolysis. The hydrolysis of natural lignocellulose to glucose depends
on the synergy of enzymes system, i.e., b-1,4-endoglucanase, b1,4-exoglucanase and b-glucosidase [5], and b-1,4-endoxylanase.
These cellulolytic enzymes have been applied to increase the
hydrolysis efciency of cellulosic materials [6].
Many different types of processes for ethanol fermentation have
been proposed, including batch fermentation, continuous fermentation, continuous fermentation with cell recycling, fed-batch cultures and repeated-batch cultures [7]. Batch fermentation process
is used extensively to convert sugars to ethanol for the production
of beverages and biofuels. As for fed-batch fermentation, the intermittent addition of glucose, without the removal of the fermentation broth, into the fed-batch culture is one of the most common
methods for the production of ethanol in the industry. The advantages of this process include the reduction of substrate and endproduct inhibition, higher productivity of ethanol, higher dissolved
oxygen rate, decreased fermentation time, and higher saccharication rate [8]. Fed-batch fermentation has been reported as a good
process for ethanol production when performed on different raw
materials such as corn stover [9] and recycled paper-derived
material [10].
Even conversion of corncob hydrolysate to bioethanol either by
batch fermentation [1,11] or by fed-batch fermentation [12,13]
separately has been studied in the past, the simultaneous

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Y.-H. Chang et al. / Fuel 97 (2012) 166173

comparison of the efciencies of both batch and fed-fermentations

by the same cellulosic hydrolysate were rare. Additionally, the impact of different fermentation treatments, i.e. batch and fed-batch,
of cellulosic hydrolysate on the dynamics of microbial growth and
ethanol production rate in the respective fermentors were seldom
evaluated. In this work, the hydrolysis process of the corncob substrate using pretreatment with acid, autoclaving and then hydrolysis with the enzyme mixtures was examined. The fermentation
processes and kinetics of the corncob hydrolysate in the batch
and fed-batch cultures of Saccharomyces cerevisiae BCRC 21812
were also compared.

2. Materials and methods

2.1. Cellulosic material and pretreatment
The corncob materials, purchased from a local market, were
oven-dried for 24 h at 50 C, grounded into particles (diameter
210 mm) and stored in pill vials at 25 C. The corncob materials
consisted mainly of 42% (w/w) cellulose and 28% (w/w) hemicellulose, which could be hydrolyzed to reducing sugar. In addition,
there was 20% of lignin in the corncob materials. The rest were
ashes and minor components. The structural carbohydrate and lignin in biomass were determined according to Standard Biomass
Analytical Procedures of National Renewable Energy Laboratory
(NREL).

Acid pretreatment was performed with 1% (v/v) sulfuric acid for

30 min at a solid-to-liquid ratio of 1:10. The mixture was ltered.
Then the ltrate was further hydrolyzed by autoclaving at 121 C
for 60 min, according to the procedures described in our previous
report [14]. After the pretreatment, the cellulosic residue was
soaked in distilled water and incubated in water bath at 50 C for
30 min, and then ltered.
2.2. Microorganisms and cultivation
The strain of S. cerevisiae BCRC 21812, purchased from Bioresources Collection and Research Center, FIRDI (Hsinchu, Taiwan),
was used as an inoculum for ethanol fermentation. S. cerevisiae
BCRC 21812 is traditionally used for alcoholic beverage and bioethanol production. It had been found that S. cerevisiae BCRC 21812
could grow well in YPD media in the presence of 8% (v/v) ethanol
according to our previous preliminary study. Yeast cultures were
maintained in a YPD medium containing 2% (w/v) dextrose, 1%
(w/v) peptone and 0.5% (w/v) yeast extract at 25 C for 48 h. The
initial pH was adjusted to 6.5 either 1 N HCl or NaOH prior to sterilization at 121 C for 20 min.
2.3. Hydrolysis of cellulosic residue by treatment with the enzyme
mixture
The mixture of prehydrolysate obtained from the acid and autoclaving pretreatments was collected and ltered with Whatman

1.0

Sugar concentrations (g/ g DS)

(a)

Cellobiose
Glucose
Xylose
Other sugars
Total sugars

0.8

0.6

0.4

0.2

0.0
Sample 1

Sample 2

Sample 3

Treatments
1.0

Total reducing sugar (g/ g DS)

(b)
0.8

0.6

0.4

0.2

0.0
0

20

40

60

80

Hydrolysis time (Hour)

Fig. 1. (A) Comparison of reducing sugar, glucose, xylose and cellobiose produced from various treatments. Sample 1: autoclaving at 121 C for 60 min. Sample 2: autoclaving
and acid treatment for 15 min. Sample 3: autoclaving, acid, and enzymatic hydrolysis. (B) Change of total reducing sugar produced of sample 3 for 72 h of enzymatic
hydrolysis. The values represent the average of three measurements standard deviation.

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Y.-H. Chang et al. / Fuel 97 (2012) 166173

No. 4 lter paper. A commercial cellulase mixture, 1.5 ml (1000 IU/

ml) Cellulase (Sigma, St Louis, MO, USA) supplemented with
0.52 ml (250 IU/ml) Novozyme 188 (Sigma, St Louis, MO, USA),
was used to hydrolyze the cellulosic residue. Enzymatic hydrolysis
was performed with a 100 ml prehydrolysate and the commercial
cellulase solution. The protocols of enzymatic hydrolysis of prehydrolysate to produce reducing sugar were also according to our
previous report [14]. The mixtures were incubated at 50 C in an
orbital shaker with a speed of 160 rpm for 72 h. Samples were
withdrawn and analyzed for levels of total reducing sugar, glucose,
xylose, and cellobiose concentration.
2.4. Batch and fed-batch fermentation of cellulosic hydrolysate
Inoculum was prepared by transferring 5% (v/v) of the cells
(108/ml) of S. cerevisiae BCRC 21812 into fermentation media.
The medium in the batch ethanol fermentation was (%, w/v): cellulosic hydrolysate, 14; peptone 0.5; yeast extract 0.25 at pH 6.0.
The medium for fed-batch fermentation was 2% cellulosic hydrolysate, 0.5% peptone and 0.25% yeast extract. After 24 h, the cellulosic
hydrolysate containing 13% (w/w) glucose was fed. The cultures
were shaken at 150 rpm for 2 d, then adjusted to 100 rpm at
25 C. Samples were collected regularly and ltered through a
0.45 lm Millipore membrane. Glucose, xylose, cellobiose and ethanol concentrations were analyzed by HPLC (Waters Co., MA, USA).
2.5. Analysis methods
The dry weight content of the raw materials was determined by
drying samples for 24 h at 110 C. Samples were withdrawn from
the fermentation broth, and yeast biomass was determined by
measuring cell optical density recorded with a Ultrospec 2100

pro spectrophotometer set at 600 nm (GE Healthcare Co., IL,

USA). The reducing sugars liberated by these reactions were measured using the 3,5-dinitrosalicylic acid method [15], with glucose
as standard. Reducing sugar was calculated as g/g dried substrate
(DS).
Glucose, xylose, cellobiose and ethanol were analyzed by HPLC
(Waters Co., MA, USA) with a cation exchanger Sugarpak column
(300  6.5 mm i.d.). Secondary de-ionized water, at a ow rate of
0.5 ml/min, was used as the mobile phase. The injection volume
was 20 ll and the column temperature was maintained at 90 C.
All samples were ltered through a 0.22 lm lter before undergoing HPLC analysis. The eluate out of HPLC was detected by a refractive index detector at 50 C.

3. Results and discussion

3.1. Pretreatment and enzymatic hydrolysis of cellulosic material
The corncob substrate samples were pretreated with autoclave
at 121 C for 60 min and either with 1.0% (w/v) sulfuric acid for
15 min (Sample 2 in Fig. 1A) or without sulfuric acid (Sample 1).
For Sample 2, after the pretreatment, approximately 0.43 g/g DS
of reducing sugars was recovered (Fig. 1A). The results show that
61.4% (w/w) of the cellulosic substrate was converted to reducing
sugar after pretreatment with autoclave and acid. No toxic effects
from furfurals and HMFs were observed during the fermentation
studies as conrmed by Sumphanwanich et al. [16]. Their results
indicated the corncob waste with acid-treatment generated nontoxic levels of furfurals (0.7 g/l) and HMFs (0.8 g/l) in the hydrolysates for fermentation.
The prehydrolysate was further hydrolyzed with the reaction
of enzyme mixtures at pH 6.0 and 50 C for 72 h (Sample 3).

6.0

(a)

pH values

5.5

5.0

Hydrolysate w/ 1% glucose
Hydrolysate w/ 2% glucose
Hydrolysate w/ 3% glucose
Hydrolysate w/ 4% glucose

4.5

Biomass concentration (g/l)

4.0
50

(b)

40

30

20
Hydrolysate w/ 1% glucose
Hydrolysate w/ 2% glucose
Hydrolysate w/ 3% glucose

10

Hydrolysate w/ 4% glucose
0
0

Fermentation time (days)

Fig. 2. Change of (A) pH values, and (B) concentration of biomass, during the time course of batch fermentation in the hydrolysate medium with 14% (w/w) glucose.

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Y.-H. Chang et al. / Fuel 97 (2012) 166173

The reducing sugar concentration reached 0.61 g/g DS after 48 h

of hydrolysis (Fig. 1B). However, extending the hydrolysis time
beyond 48 h did not help further in increasing the reducing sugar concentration. From the reducing sugar concentration after
hydrolysis, it indicates that 87.1% (w/w) of the cellulosic/hemicellulosic components were hydrolyzed and converted to reducing sugar after treatment with the enzyme mixture. Xylose was
detected in the hydrolysate, showing that the presence of b-1,4endoxylanase assisted in the hydrolysis of xylan in the substrate.
Additionally, the b-1,4-endoglucanase and b-1,4-exoglucanase
hydrolyze cellulose chains resulted in the formation of cellobiose, which can be further cleaved into glucose by cellobiase. It
was found that a signicantly low amount of cellobiose existed
in the cellulosic hydrolysate, indicating that cellobiase increased
the hydrolysis of cellobiose in the resulting prehydrolysate. In
addition, a high amount of glucose and a comparatively lower
amount of cellobiose existed in the cellulosic hydrolysate, indicating good activities of b-glucosidase in enzyme mixtures.
Thereby, with the aid of enzymatic hydrolysis, higher yields of
total reducing sugar (0.61 g/g DS), glucose (0.36 g/g DS) and xylose (0.17 g/g DS) in the resulted hydrolysates were achieved.
These ndings indicate that the enzyme mixtures helped to increase the hydrolysis efciency of the cellulosic hydrolysates
and were necessary to produce the monosaccharides for further
ethanol fermentation.

3.2. Batch fermentation of cellulosic hydrolysate for bioethanol

production
Batch fermentation for bioethanol production was performed in
the cellulosic hydrolysate-based media containing various concentrations of glucose as the main carbon source. Due to the concern
that the high concentration of glucose in the hydrolysate would inhibit the growth of yeast, the maximum concentration of glucose
(40 g/l) was used. To determine the effect of glucose concentration
on the growth prole of S. cerevisiae BCRC 21812, batch experiments were performed in conical asks with glucose concentration
in the hydrolysates ranging from 10 to 40 g/l. Fig. 2 shows the plots
of cell biomass and pH values against fermentation time. For the
hydrolysate medium, the pH decreased slowly and remained above
5.6 throughout the rst ve days of the fermentation and decreased rapidly from 5.7 to 5.0 after 6 d of cultivation (Fig. 2A).
As reported by Palmqvist and Hahn-Hagerdal [17], cell growth in
cellulosic hydrolysates strongly depended on pH, due to the large
concentration of dissociated weak acids at low pH. The pH, around
5.0 during the entire fermentation process, did not inuence the
growth of yeast cells and thus favored the ethanol production.
The yeast cell biomass increased from 23.3 to 41.1 g/l with the increased concentration of glucose from 1% to 4% in the hydrolysate
(Fig. 2B). In addition, after the inoculation of yeast cells, the microbial biomass began to increase, reached the maximal values after

30

(a)

(b)
Glucose
Xylose
Cellobiose
Ethanol

25

20

15

Concentrations (g/l)

10

0
50

(c)

(d)

40

30

20

10

0
0

Fermentation time (days)

Fig. 3. Change of glucose, xylose, cellobiose and ethanol during batch culture in the hydrolysate medium with (A) 1%, (B) 2%, (C) 3% and (D) 4% (w/w) glucose.

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Y.-H. Chang et al. / Fuel 97 (2012) 166173

utilized by the yeast strain after 6 d of fermentation when the glucose concentrations in the hydrolysate were 34% (Fig. 3C and D).
Furthermore, considerable quantities of xylose and cellobiose were
also detected, which could not be utilized by the yeast cells. When
the substrate concentration reached 4%, yeast biomass and ethanol
yield were not signicantly increased, suggesting that a considerable inhibitory effect had occurred. In addition, the ethanol yields
were 18.3 and 23.0 g/l when the initial glucose concentrations in
the hydrolysate were 3% and 4%, respectively. Besides, rate of the
conversion of glucose to ethanol was 0.580.61 g ethanol/g glucose, using 34% glucose in the hydrolysate, a signicant higher
amount than that (0.45 g ethanol/g glucose) of Yu and Zhang [1].
To develop an improved culture method for ethanol production
with S. cerevisiae, batch ask cultures were rstly carried out to
determine the suitable substrate concentration of the initial media.
It was found that S. cerevisiae grew with a similar pattern in glucose concentrations up to 4%, indicating a good ability to deal with
osmotic stress. This made it possible to feed concentrated glucose
solution in a discontinuous way during the fed-batch fermentation.

2 d of incubation, and then remained steady thereafter. These results indicated that the yeast grew well on the cellulosic hydrolysate with glucose at a concentration up to 4%. However, cell growth
was greatly repressed when the glucose concentration reached 4%.
Specically, when the glucose concentration increased from 3 to
4%, the yeast cell biomass did not show obvious increase, i.e. from
40.2 to 41.1 g/l. This fact showed that the concentration of glucose
at 4% would inhibit the growth of yeast, due to the halt in cell
biomass.
Fig. 3 shows the change of glucose, xylose and cellobiose concentration, and ethanol yield by S. cerevisiae culture after 6 days
as compared to the initial glucose concentration in the hydrolysate.
In the fermentation using 12% glucose in the hydrolysate, the glucose was exhausted after 2 d, whereas the ethanol production yield
increased rapidly after the rst day of fermentation. This result
indicates that the glucose consumption was consistent with the
time period of ethanol production. As shown in Figs. 3A and B,
the glucose was rapidly used up by the yeast within 2 d, with 1
2% glucose in the hydrolysate. However, 1.24.1 g/l xylose and
1.73.3 g/l cellobiose were detected and could not be utilized by
the yeast cells after 2 d. The fermentation was completed after
2 d. The maximal concentrations of ethanol were 6.9 and 8.5 g/l
for the cultures of 1% and 2% glucose in the hydrolysate, respectively, when the glucose were used up. The fermentation results
suggest that S. cerevisiae could grow well in the hydrolysate medium and achieve virtually complete conversion to ethanol from
glucose in the hydrolysate. However, 810 g/l of glucose was not

3.3. Fed-batch fermentation of cellulosic hydrolysate for bioethanol

production
It requires a high initial sugar concentration in the cellulosic
hydrolysate to obtain high concentrations of ethanol in the fermentation broth. However, higher sugar concentration in the
hydrolysate often causes mixing and heat transfer problems, due

6.0

30

(a)
5.8

25

5.4
15

5.2
5.0

10

pH value

Biomass concentration (g/l)

5.6
20

4.8

4.6
0

(b)

Glucose
Xylose
Cellobiose
Ethanol

25

Concentration (g/l)

Hydrolysate supplementation
20

15

10

0
0

Fermentation time (day)

Fig. 4. Time course of (a) biomass and pH, and (b) concentration of sugars and ethanol, during the fed-batch fermentation in the hydrolysate medium, which contained 2% (w/
w) glucose and was fed with 1% glucose after 1 d of fermentation.

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Y.-H. Chang et al. / Fuel 97 (2012) 166173

only 15 g/l (Fig. 5B). After the addition of the hydrolysate, the ethanol yields increased and reached the maximum on the second day
of fermentation. Thus, the overall ethanol yields and glucose conversion to ethanol rate were estimated to be 24.0 g/l and
0.60 g ethanol/g glucose. The results of fed-batch culture with initial 2% glucose in hydrolysate and an addition of 3% glucose are
showed in Fig. 6. The cell biomass increased rapidly after the inoculation of S. cerevisiae and reached the maximum (44.5 g/l) on the
second day of fermentation; moreover, the pH of the broth decreased steadily from 5.8 to 5.2 during the fermentation process
(Fig. 6A). After feeding the hydrolysate to the media, the ethanol
yields increased and reached 32.3 g/l at the second day of fermentation. Thus, rate of the conversion of glucose to ethanol was estimated to be 0.64 g ethanol/g glucose. As proposed in this study,
these resulting data were higher than those from the batch culture
system. Furthermore, rate of the conversion of glucose to ethanol
from the fed-batch fermentation in this study was signicantly
higher than that (0.44 g ethanol/g glucose) of the study using the
batch culture of Candida tropicalis [18].

to the rheological properties of a very dense brous suspension [7].

Such problems could be effectively avoided in the fed-batch fermentation process, where the substrate is added gradually and
the viscosity of the reaction mixture can be kept at a low level.
The glucose concentration in the hydrolysate increased from an
initial 2% to 3% by addition of 1% on the rst day in the fed-batch
process (Fig. 4). It was observed that the cell biomass concentration reached 22.3 g/l on the second day of fermentation and that
the pH of the fermentation broth slightly decreased from 5.8 to
5.5 during the 5 d of fermentation. The residual glucose concentration in the hydrolysate was 1.8 g/l, which was much lower than
that of the batch culture. After the addition of the hydrolysate,
the concentration of xylose increased from 6 to 15.2 g/l. This indicates that S. cerevisiae could readily ferment the glucose in hydrolysate to ethanol but could not metabolize xylose, due to the lack of
xylose-degrading enzymes. In addition, the results show that most
of the glucose in the hydrolysate was used in fed-batch cultures,
therefore, higher concentrations of ethanol (19.0 g/l) were produced than in batch cultures with 3% glucose in the hydrolysate.
Fed-batch cultures shortened the reaction time degrading an
equivalent substrate, therefore enhancing the efciency of utilizing
the cellulosic substrate.
The results of fed-batch culture with initial 2% glucose in cellulosic hydrolysate and an addition of 2% glucose were showed in
Fig. 5. The cell biomass increased rapidly after the inoculation of
S. cerevisiae and reached the maximum (42.5 g/l) on the third day
of fermentation (Fig. 5A). The glucose in the hydrolysate was going
to be used up after 1 d of fermentation, and the ethanol yields were

4. Conclusions
As an economical way to produce ethanol from cellulosic substrate, synergetic hydrolysis of cellulase and xylanase mixtures
creates a feasible process that can be used in the production of bioethanol. Bioethanol production with fed-batch fermentation offers
advantages over that with batch fermentation. The conversion rate

6.0

35
30

5.8

25

5.6
5.4

20

5.2
15
5.0

pH value

Biomass concentration (g/l)

(a)

10
4.8
5
4.6
0

(b)

Hydrolysate supplementation

Glucose
Xylose
Cellobiose
Ethanol

Concentration (g/l)

25

20

15

10

0
0

Fermentation time (day)

Fig. 5. Time courses of (a) biomass and pH, and (b) concentration of sugars and ethanol, during the fed-batch fermentation in the hydrolysate medium, which contained 2%
(w/w) glucose and was fed with 2% glucose after 1 d of fermentation.

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Y.-H. Chang et al. / Fuel 97 (2012) 166173

6.0

50

(a)
5.8
40

5.4

30

5.2
20
5.0

pH value

Biomass concentration (g/l)

5.6

4.8

10

4.6
0

Glucose
Xylose
Cellobiose
Ethanol

Hydrolysate supplementation

(b)

35

Concentration (g/l)

30
25
20
15
10
5
0
0

Fermentation time (day)

Fig. 6. Time courses of (a) biomass and pH, and (b) concentration of sugars and ethanol, during the fed-batch fermentation in the hydrolysate media, which contained 2% (w/
w) glucose and was fed with 3% glucose after 1 d of fermentation.

of ethanol from glucose was higher in fed-batch fermentation than

it was in batch fermentation. Moreover, the substrate inhibition effects on cell biomass and yields of ethanol were less pronounced
for fed-batch fermentation than batch fermentation. Further work
should be focused on scale-up of fed-batch fermentation to make
the process industrially feasible.
Acknowledgment
The authors would like to thank the National Science Council of
the Republic of China (Taiwan) for nancially supporting this research under Contract No. NSC 97-2313-B-264-001-MY3.
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