b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 7 8 e1 8 8 4

Available at www.sciencedirect.com

http://www.elsevier.com/locate/biombioe

Pretreatment and hydrolysis of cellulosic agricultural wastes

with a cellulase-producing Streptomyces for bioethanol
production
Chuan-Liang Hsu a, Ku-Shang Chang b, Ming-Zhang Lai c, Tsan-Chang Chang d,
Yi-Huang Chang b,*, Hung-Der Jang b,**
a

Department of Food Science, Tunghai University, Taichung 407, Taiwan

Department of Food Science, Yuanpei University, Hsinchu 300, Taiwan
c
Institute of Biotechnology, Yuanpei University, Hsinchu 300, Taiwan
d
Department of Food Science, Mackay Medicine, Nursing and Management College, Taipei 112, Taiwan
b

article info

abstract

Article history:

Production of reducing sugar by hydrolysis of corncob material with Streptomyces sp.

Received 20 February 2010

cellulase and ethanol fermentation of cellulosic hydrolysate was investigated. Cultures of

Received in revised form

Streptomyces sp. T3-1 improved reducing sugar yields with the production of CMCase,

9 January 2011

Avicelase and b-glucosidase activity of 3.8, 3.9 and 3.8 IU/ml, respectively. CMCase,

Accepted 10 January 2011

Avicelase, and b-glucosidase produced by the Streptomyces sp. T3-1 favored the conversion

Available online 2 February 2011

of cellulose to glucose. It was recognized that the synergistic interaction of endoglucanase,

exoglucanase and b-glucosidase resulted in efficient hydrolysis of cellulosic substrate.

Keywords:

After 5 d of incubation, the overall reducing sugar yield reached 53.1 g/100 g dried

Corncob

substrate. Further fermentation of cellulosic hydrolysate containing 40.5 g/l glucose was

Cellulosic hydrolysates

performed using Saccharomyces cerevisiae BCRC 21812, 14.6 g/l biomass and 24.6 g/l ethanol

Streptomyces sp.

was obtained within 3 d. The results have significant implications and future applications

Reducing sugar

regarding to production of fuel ethanol from agricultural cellulosic waste.

2011 Elsevier Ltd. All rights reserved.

Bioethanol

1.

Introduction

Biofuels such as bioethanol are becoming a viable alternative

to fossil fuels. Utilizing agricultural biomass for the production of biofuel has drawn much interest in many science and
engineering disciplines. The current commercial bioethanol is
predominantly produced from sugarcanes or starch. However,
a dramatic increase in ethanol production using the current
starch-based technology may not be practical because it will
compete for the limited agricultural land needed for food and
feed production. Cellulosic biomass (or lignocellulosic

biomass), which has been predicted to be the alternative raw

material for bioethanol production, is an ideal source of
energy because it is both renewable and available in large
quantities throughout the world [1]. Conversion of cellulosic
biomass to glucose and other fermentable sugars has been
considered in the last few decades, which is an attractive
route for ethanol production [2,3]. Bioethanol produced from
cellulosic biomass is already used in several countries, for
example, in Brazil, USA and Sweden [4], and also Taiwan [5],
either pure or as a blend with gasoline. However, the process
for the production of bioethanol from cellulosic materials,

* Corresponding author. Tel.: 886 3 5381183x8481; fax: 886 3 6102342.

** Corresponding author. Tel.: 886 3 5381183x8482; fax: 886 3 6102342.
E-mail address: hungder@mail.ypu.edu.tw (H.-D. Jang).
0961-9534/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2011.01.031

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 7 8 e1 8 8 4

which requires pretreatments such as liquefaction and

saccharification, is more complicated than its production
from sugar or starch-based ones. Over the past few decades,
for the production of bioethanol from cellulosic materials, the
focus of previous researchers has been on the enzymatic
hydrolysis of cellulosic biomass to fermentable sugar. Nevertheless, there are still several technical and economical
impediments with regards to the development of commercial
processes that utilize cellulosic biomass [6]. Technologies that
will offer a new cost-effective alternative to overcome those
impediments are being developed.
Many different types of agricultural by-products (or agricultural wastes), such as rice straws, wheat straws, and
corncobs, have been proposed for enzymatic hydrolysis of
cellulosic biomass and ethanol production. Like other agricultural by-products, corncob consists of polymers of mainly
two types of sugars: glucose and xylose [7,8]. Both sugars can
be obtained, in monomeric forms, with high yield rates from
the break-down of polysaccharide chains using steam
pretreatment and subsequent enzymatic hydrolysis [9]. The
monomeric glucose can then be fermented to ethanol with the
help of Saccharomyces cerevisiae. Furthermore, appropriate
pretreatment is a key step for the effective utilization of
cellulosic biomass, due to its recalcitrant nature. Since the
cost of raw materials contributes substantially to the cost of
ethanol production [10], a lower raw material price, together
with a high ethanol yield and efficient enzymes, will decrease
the production cost significantly. Accordingly, several
different pretreatment methods have been used to facilitate
the enzymatic hydrolysis of cellulosic material [11,12] and it
can be concluded from these methods that an efficient
process for obtaining reducing sugars from cellulosic material
is to use proper combination of chemical and physical
pretreatments, followed by enzymatic hydrolysis.
Prior to ethanol fermentation an enzymatic prehydrolysis
can be carried out. An enzymatic prehydrolysis of the cellulosic material decreases the viscosity of the hydrolysates,
which enables easier pumping and stirring. The decrease in
viscosity during prehydrolysis makes the subsequent
submerged fermentation possible even with very high
concentration of hydrolysate, which is crucial to reach high
ethanol concentration. The aim of the prehydrolysis is to
partly hydrolyze the cellulose to oligomeric and monomeric
sugars prior to the yeast addition, which would increase the
ethanol production rate during the fermentation process.
The hydrolysis of natural cellulose to glucose depends on the
synergistic action of three enzymes in cellulase system,
including b-1,4-endoglucanase (EC 3.2.1.4), b-1,4-exoglucanase (EC 3.2.1.91), and b-glucosidase (EC 3.2.1.21) [13]. It has
been reported that these cellulolytic enzymes increase the
hydrolysis efficiency of cellulosic materials in some studies
[14,15]; however, the cost of commercial enzymes will be
a concern in the industrial scaled-up process.
In our previous work, we have successfully demonstrated
that the production of the thermostable cellulase by Streptomyces sp. T3-1 in a lab-scale fermenter [16]. In this study, the
effect of prehydrolysis on reducing sugar concentrations was
investigated using acid and autoclaving pretreated corncob.
The hydrolysis efficiency towards corncob with cellulaseproducing strain Streptomyces sp. T3-1 was examined. The

1879

hydrolysis efficiency of commercial and microbial cellulase

was compared following the prehydrolysis treatment. Further
fermentation of the hydrolysate for ethanol production was
also investigated using S. cerevisiae BCRC 21812.

2.

Materials and methods

2.1.

Microorganisms and cultivation

The cellulase-gene cloning transformant Streptomyces sp. T3-1

was used for hydrolysis of cellulosic materials. Cellulase-gene
cloning was performed using the methods published in our
previous study [17]. The microorganism was grown in culture
medium based on that of Mandels and Reese [18]. It contained
(g/l distilled water): carboxymethyl cellulose sodium salt (10);
peptone (1); (NH4)2SO4 (2); KH2PO4 (2); urea (1); MgSO4$7H2O
(0.5); CaCl2$2H2O (0.4); and 1 ml trace element solution (mg/l):
FeSO4$7H2O (5); MnSO4$H2O (1.6); ZnSO4$7H2O (2); CoCl2$6H2O
(2). The initial pH of the medium was adjusted to 6.5 prior to
sterilization at 121  C for 20 min. The strain of S. cerevisiae
BCRC 21812, purchased from Bioresources Collection &
Research Center, Hsinchu, Taiwan, was used for ethanol
fermentation in this study. Yeast cultures were maintain in
DPY medium containing 2% (w/v) Dextrose, 1% (w/v) Peptone
and 0.5% (w/v) Yeast extract and incubated at 25  C for 48 h.

2.2.

Cellulosic material and pretreatment

Corncob, purchased from a local traditional market, was used

as cellulosic substrate for this study. The corncob materials
were oven-dried at 50  C for more than 24 h, grounded into
particles of about 2e10 mm in diameter using a stainless-steel
grinder, and stored in pill vials at room temperature. Acid
pretreatment was performed with 0.25 or 0.5% (v/v) sulfuric
acid for 10e30 min at a solid-to-liquid ratio of 1:10. The
mixture was filtered and then the filtrate was further hydrolyzed by autoclaving at 121  C for 15, 30, and 60 min, respectively. After the pretreatment, the cellulosic residue was
soaked in distilled water and incubated in water bath at 50  C
for 30 min, and then filtered. The filtrate collected from the
acid and autoclave pretreatment were used to determine the
reducing sugar contents. The cellulosic residue was collected
and then hydrolyzed with commercial cellulase.

2.3.
Hydrolysis of cellulosic residue with commercial
cellulase
After the cellulosic substrate was autoclaved for 60 min, prehydrolysis with the commercial enzyme mixture was performed at 50  C. 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 of corncob cellulosic residue was performed in 500 ml serum bottles, containing 10 g solid substrate
and commercial enzyme mixtures by adding 0.1 M sodium
acetate buffer (pH 6.0) to make a solution of 500 ml in volume.
The total cellulase activity of the developmental mixture
was 3.3 IU/ml and it consisted of b-1,4-glucano-hydrolase and

1880

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 7 8 e1 8 8 4

b-1,4-glucosidase. The serum bottles were incubated in a water

bath at 40  C and the reaction mixtures were sampled periodically and filtered through 0.45 mm Millipore membrane. The
total reducing sugar, and contents of glucose, xylose and
cellobiose of the filtrate was then determined as described in
section 2.5.

2.4.
Hydrolysis of cellulosic residue by Streptomyces sp.
T3-1 cultures
Corncob substrate was mixed with 0.1 M sodium acetate
solution at a solid-to-liquid ratio of 1:10. The mixture was then
pretreated by autoclaving at 121  C for 60 min and the pyrolysates were filtered with Whatman No. 4 filter paper. The
filtrate substituting CMC was added to the culture medium of
Mandels and Reese [18], and then autoclaved for 15 min. Ten
percent (v/v) of Streptomyces sp. T3-1 at inocula of 108 spores/
ml was inoculated into the culture medium and incubated at
37  C in an orbital shaker with speed of 150 rpm. Samples were
withdrawn periodically from the cultures, and the filtrate was
analyzed for cellulase activity and the concentrations of total
reducing sugar, glucose, xylose and cellobiose.

2.5.

Ethanol fermentation of cellulosic hydrolysate

Inoculum was prepared by transferring 5% (v/v) of cells of

S. cerevisiae BCRC 21812 into a 500 ml flask containing 100 ml of
fermentation medium. Each ml of inocula contained 108 yeast
cells. The fermentation medium for ethanol production contained (%, w/v): cellulosic hydrolysate, 4; peptone 0.5; yeast
extract 0.25 at pH 6.0. After inoculation the cultures were
shaken at 150 rpm for 2 d and 100 rpm thereafter and incubated at 25  C thereafter. Samples were regularly collected
from the fermentation media and filtered through 0.45 mm
Millipore membrane. Glucose, xylose, cellobiose and ethanol
of the filtrate were analyzed on an HPLC system.

2.6.

Analysis methods

The Streptomyces culture was centrifuged at 5,000 g for 20 min

and the clear supernatant used for assay of enzyme activities.
All enzyme activities were determined by incubating the
crude enzyme solution with its respective substrate which
was dissolved in sodium phosphate buffer (100 mM, pH 7.0) at
50  C using the modified methods by Ghose [19]. The b-1,4endoglucanase (CMCase) activities were measured by reacting
1 ml 1% (w/v) CMC with 1 ml crude enzyme solution for 60 min
in a shaking water bath. In addition, b-1,4-exoglucanase
(Avicelase) and b-glucosidase activity was measured with the
same conditions as with CMCase except for the substitution of
the substrate for Avicel or Salicin, respectively. One international unit (IU) of enzyme activity was defined as the amount
of enzyme that forms 1 mmol glucose/min.
The dry material content of the samples was determined by
drying samples for 24 h in an oven at 110  C. Samples were
withdrawn from the fermentation broth and the yeast biomass
was determined by measuring cell optical density recorded at
600 nm with a Ultrospec 2100 pro spectrophotometer (GE
Healthcare Co., IL, USA). The reducing sugars liberated by these
reactions were measured using the 3, 5-dinitrosalicylic acid

(DNS) method [20], with glucose as standard. Total reducing

sugar was calculated as g/g dried substrate (DS).
Glucose, xylose, cellobiose and ethanol were analyzed by
HPLC (Waters model Isocratic HPLC pump) with a cation
exchanger Sugarpak column (300  6.5 mm, Waters, Milford, MA,
USA). Secondary de-ionized water was used as the mobile phase
at a flow rate of 0.5 ml/min. The injection volume was 20 ml and
the column temperature was maintained at 90  C. All samples
were filtered through a 0.22 mm filter before subjecting to HPLC
analysis. The eluate was detected by a refractive index detector at
50  C. The known standards were used as controls. All chemicals
were of reagent grade and obtained from commercial sources.

3.

Results and discussion

3.1.
Acid and autoclave pretreatment of cellulosic
residue
The effects of pretreatments were investigated first. The
corncob substrate was treated with 0.25 or 0.5% sulfuric acid for
10e30 min and then autoclaved at 121  C for 15, 30, 60 min,
respectively. The results are shown in Fig. 1. By treating with
0.25 or 0.5% sulfuric acid for 20 min, approximately 27.1 g/100 g
DS of reducing sugar was produced (Fig. 1a). No significant
difference was found between the substrates treated with 0.25
and 0.5% sulfuric acid. Although acid pretreatment helps the
hydrolysis of cellulosic material, however, it has been reported
that after acid treatment some by-products, e.g., furfural and 5hydroxyfurfural, will be produced where these degradation
compounds are known to inhibit the fermentation step [21]. To
avoid formation of the by-products in the hydrolysis process, an
alternative pretreatment method without acid addition should
be performed. In Fig. 1b, after being autoclaved at 121  C for
60 min, highest yields of reducing sugars were observed (58.1 g/
100 g DS and higher), compared to the yields of autoclaving for
only 15e30 min. This implies that most of the cellulosic
components in the corncob substrate were converted to
reducing sugar after treatment with heating by autoclaving. In
addition, the higher yields of reducing sugars from 60 min of
autoclaving could be contributed by the glucose converted from
the oligomers in the pyrolysate due to extensive autoclaving.
Existence of small amounts of oligomers such as cellulobiosan,
a dimeric form of levoglucosan in cellulosic pyrolysate was also
reported by Bonn [22]. Therefore, the hydrolysis efficiency of the
pyrolysate by prolonged autoclaving time to 60 min can be
increased. Several pretreatment batches were performed at the
same condition whereupon these batches of pryolysates were
collected and mixed thoroughly. This thoroughly mixed pretreated material was used throughout the whole study and
stored at 18  C prior to the experiments.

3.2.
Hydrolysis of cellulosic substrate with commercial
cellulase for reducing sugar production
After being autoclaved for 60 min, the cellulosic substrate was
hydrolyzed with the commercial enzyme mixture. Fig. 2 shows
data from these experiments. The reducing sugar yield was
only 17.0 g/100 g DS after the commercial enzyme mixture
was reacted for 48 h (Fig. 2a), whereas the glucose, xylose and

1881

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 7 8 e1 8 8 4

20

15

5.8
15
5.6
10
5.4
5
5.2

0
0.25% sulfuric acid

0.5% sulfuric acid

10 min
20 min
30 min

80

CMCase
Avicelase
-glucosidase

Sugar concentration (g/l)

14
60

40

5.0

10

6.0

pH value

Reducing sugar yield (g/100 g DS)

25

Reducing sugar yield (g/100 g DS)

10 min
20 min
30 min

30

Reducing sugar yield (g/100 g DS)

20

35

Enzyme activity (IU/ml)

Glucose
Xylose
Cellobiose

12
10
8
6
4
2

20

10

20

30

40

50

60

70

80

Hydrolysis time (hour)

0
15 min

30 min

60 min

Autoclaving time

Fig. 1 e Concentration of reducing sugar after pretreated

with (a) diluted sulfuric acid, and (b) pretreated with 0.5%
sulfuric acid followed by autoclaving. Values represent the
average of three measurements standard deviation.

cellobiose concentrations reached 7.5, 10.9, 0.7 g/l (Fig. 2c),

respectively, after prehydrolysis. Generally, concentrations of
reducing sugar, glucose, xylose and cellobiose reached their
respective maxima and stayed unchanged after 2 h of enzymatic hydrolysis. However, the amounts of resulting reducing
sugar were slightly higher compared to those of acid and
autoclave pretreatment process. During the course of hydrolysis, activities of CMCase, Avicelase and b-glucosidase were
maintained at 1.7, 1.0 and 1.3 IU/ml, respectively (Fig. 2b). Since
the total initial cellulase activity of the commercial enzyme
mixture was only 3.3 IU/ml, the reducing sugar produced was
comparably lower. Furthermore, the activity of b-1,4-exoglucanase in the commercial enzyme mixture was low as a result
of low hydrolysis efficiency.

3.3.
Cellulases and reducing sugar production from
Streptomyces sp. T3-1 cultures
Time course of a typical batch cultivation of Streptomyces sp.
T3-1 in the 500 ml flask is shown in Fig. 3. Biomass

Fig. 2 e Time courses of (a) pH and concentration of

reducing sugar, (b) cellulase activity, and (c) sugar
concentration, changes in the autoclave pretreatment,
followed by commercial cellulase hydrolysate. Values of
enzyme activity represent the average of three
measurements standard deviation.

concentration reached a maximum value of 8.0 g/1 on the fifth

day, where the yield of total reducing sugar was 53.1 g/100 g
DS (Fig. 3a). In addition, the production of CMCase, Avicelase,
and b-glucosidase that reaches maximum activities on the
fifth day was associated with the growth of Streptomyces sp.
T3-1. Maximum CMCase (3.8 IU ml/1), Avicelase (3.9 IU ml/1)
and b-glucosidase (3.8 IU ml/1), which were obtained in the
shake-flask cultures, are shown in Fig. 3b. Glucose, xylose and
cellobiose produced during hydrolysis were analyzed by HPLC
at the interval of 1 d (Fig. 3c). The synergistic interaction of
endo- and exo- glucanase was observed, which resulted in
efficient hydrolysis of cellulosic substrate. The b-1,4-endoglucanase and b-1,4-exoglucanase produced from Streptomyces sp. T3-1 hydrolyzed cellulose chains and resulted in
formation of cellobiose, which could be further cleaved into
glucose by b-glucosidase. In addition, a high amount of
glucose (40.5 g/l) and a comparably lower amount of cellobiose
(3.0 g/l) were found in the cellulosic hydrolysate, indicating
satisfactory activities of b-1,4-endoglucanase, b-1,4-exoglucanase and b-glucosidase in Streptomyces sp. T3-1 cellulase.

1882

50

6.0

10

100

Yield after autoclave

5.8

6
30
4
20
2

10
0

5.6
5.4
5.2

pH value

40

Biomass concentration (g/l)

5.0
4.8
4.6

CMCase
Avicelase
-glucosidase

4
3

Total reducing sugar yield (g/ 100g DS)

Enzyme activity (IU/ml)

Reducing sugar yield (g/100 g DS)

60

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 7 8 e1 8 8 4

Yield after hydrolysis of commercial cellulase

80

Yield after hydrolysis of Streptomyces cellulase

Yield after acid treatment

60

40

20

Sample 1

Sample 2

Sample 3

Fig. 4 e Comparison of yields of total reducing sugars

produced from different hydrolysis methods at various
stages of respective pretreatments. Sample 1 and 2 were
the hydrolysate obtained from pretreating with autoclave
followed by hydrolyzing with commercial cellulase or
Streptomyces sp., respectively. Sample 3 was pretreated
with acid, autoclave followed by commercial enzymatic
hydrolysis. Values represent the average of three
measurements standard deviation.

Sugar concentration (g/l)

50

Glucose
Xylose
Cellobiose

40
30
20
10
0
0

Incubation time (day)

Fig. 3 e Time courses of (a) biomass, pH and concentration

of reducing sugar, (b) cellulase activity, and (c) sugar
concentration, with the culture of Streptomyces sp. T3-1 in
the autoclaved hydrolysate medium. Values of enzyme
activity represent the average of three
measurements standard deviation.

However, a considerable amount of xylose (19.1 g/l) was

observed during the time course of incubation. It was
proposed that some extent of xylanase activity was exhibited
by Streptomyces sp. T3-1. A further study of utilization of
xylanase produced by Streptomyces sp. is required to be studied
in the future.

3.4.

Comparison of hydrolysis methods

Fig. 4 shows the comparison of the highest yields obtained

from various hydrolysis methods investigated, i.e., pretreatment by autoclaving followed by commercial enzymatic or
Streptomyces sp. hydrolysis (Sample 1 and 2), as well as
pretreatment with acid, and autoclaving followed by
commercial enzymatic hydrolysis (Sample 3). For Sample 1,
17.0 g of total reducing sugar from 100 g of cellulosic substrate
was produced after the corncob was treated with autoclave at
121  C for 60 min, and then hydrolyzed by the commercial
celluase, without acid pretreatment. For Sample 2, pretreatment by autoclaving with Streptomyces sp. hydrolysis (without
acid pretreatment either) resulted in significantly higher
amount of both total reducing sugar yields (53.1 g/100 g DS and
40.5 g/l of glucose). The amount of reducing sugar from

Sample 1 was approximately only 32% of the total reducing

sugar yielded from Sample 2. The results suggest that samples
treated with the combination of autoclaving and hydrolysis by
Streptomyces sp. is highly effective for the subsequent ethanol
fermentation with complete conversion of monosaccharides
in the mixture. These results also demonstrate that cellulase
activity produced by Streptomyces sp. cultures has the potential to improve the hydrolysis efficiency of cellulosic materials
markedly without further acid pretreatment. In addition, the
amount of the commercial cellulase enzyme used in Sample 1
has to be tripled to reach the same yield of reducing sugar
generated in Sample 2. But the overall cost of the biofuel
production would be driven up due to the increased cost of the
commercial cellulase enzyme.
As for Sample 3, 86.7 g/100 g DS (on the same dried weight
basis) was obtained from the pretreatment of 0.5% (v/v)
sulfuric acid and autoclaving, followed by the commercial
enzymatic hydrolysis. It is also shown that the yield of
reducing sugar already reached 84.2 g/100 g DS after the
pretreatment of acid and autoclave for Sample 3. The effect of
hydrolysis by the commercial enzyme was relatively marginal
compared to the effect of the pretreatment of acid and autoclave. Similar results were found for the biomass samples
processed with the pretreatment of sulfuric acid and autoclaving, followed by the hydrolysis of Streptomyces cellulase
(data not shown). The results also indicate that the cellulase
activities of the commercial enzyme mixture used in Sample 1
and 3 were significantly lower than those of the commercial
hgren et al. [23].
enzymes with a similar ratio reported by O
Though the treatments of Sample 3 resulted in highest yield of
reducing sugars, it has been reported that the acid pretreatment had negative effects on the yield of ethanol of the

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 7 8 e1 8 8 4

16

6.0

14

5.5
10
8
6
5.0

pH v a l u e

Biomass concentration (g/ l)

12

4
2
0

4.5

Glucose
Xylose
Cellobiose
Ethanol

Concentration (g/ l)

40

30

20

10

0
0

Fermentation time (day)

Fig. 5 e Time courses of (a) biomass and pH, and (b)

concentration of sugars and ethanol, changes during the
fermentation process with S. cerevisiae in the hydrolysate
medium.

following fermentation step [24]. To avoid the effects of the

acid pretreatment mentioned above, it would require additional cost for extra pH adjustment of the biomass solution
and raise the overall production cost as a consequence. We
have continued our research to investigate the usage of
cellulase hydrolysis without acid pretreatment for better
yields of both reducing sugar and ethanol as a further study.

3.5.
Fermentation of cellulosic hydrolysate for bioethanol
production
Due to the concern that high concentration of glucose in the
hydrolysate would inhibit the growth of yeast, a high
concentration of glucose (40 g/l) was firstly used for the
investigation during the course of ethanol fermentation by S.
cerevisiae. Furthermore, peptone and yeast extract were used
as nitrogen sources to stimulate the growth of yeast [25]. Fig. 5
shows the time course of ethanol production using the
hydrolysate as the substrate. The time course of yeast
biomass, pH changes, residual sugars and ethanol production
was determined. Fig. 5a illustrates the changes of pH during
the fermentation process of batch culture. For the hydrolysate
medium, the pH decreased slowly and remained above 5.4
throughout the initial 5 d of the fermentation and then
decreased rapidly from 5.4 to 4.7 after 6 d of cultivation. Silva
and Roberto [26] suggested that yeasts had an ability to
maintain a relatively stable pH, which in turn lead to the
inactivation of the toxic compounds in the hydrolysate. As
reported by Palmqvist and Hahn-Hagerdal [27], cell growth in

1883

lignocellulosic hydrolysates is strongly dependent on pH due

to the high degree of dissociated weak acids at low pH. In
addition, after the initial inoculation of yeast cells, the
microbial biomass began to increase, reached the maximal
value (14.6 g/l) after 48 h of incubation, and then remained
steady thereafter. The pH stayed steadily around 5.0 during
the entire fermentation process and did not affect the growth
of yeast cells, which favored the ethanol production.
In the fermentation using the hydrolysate, the glucose
concentration decreased rapidly from 40.0 to 9.1 g/l after 2 d
and 5.3 g/l after 6 d (Fig. 5b). However, 12.1 g/l xylose and 0.6 g/l
cellobiose were detected after 2 d, an indication that they were
not utilized by yeast cells during fermentation. Results indicated that S. cerevisiae BCRC 21812 could readily ferment
glucose in hydrolysate to ethanol but hardly metabolize
xylose due to low activities of xylose reductase and xylitol
dehydrogenase. The ethanol production rate in the early
phase of the culture was relatively slow but rapidly increased
after 1 d. Fermentation was completed after 2 d, where
ethanol reached a maximal concentration of 24.6 g/l with
approximately complete depletion of glucose. This indicates
that the consumption of glucose by yeast cells was virtually in
sync with the ethanol production. In spite of the complex
property of the hydrolysate derived from corncob material, no
strong diauxie pattern of sugar utilization was observed here.
In addition, the ethanol yield was 0.67 g ethanol/g glucose,
which is substantially higher than the ethanol yield (0.45 g
ethanol/g glucose) reported by Yu and Zhang [28]. The results
suggest that S. cerevisiae can grow well in the hydrolysate
medium in this study and is able to convert glucose in the
hydrolysate to ethanol completely.

4.

Conclusions

In this study, the hydrolysis efficiency of corncob-based

cellulosic material can be substantial improved with the
technology we developed by culturing of the cellulaseproducing Streptomyces sp. T3-1. The amount of total reducing
sugars and concentration of glucose in the hydrolysate
increased significantly as a result. In search of an economic
way for producing ethanol from cellulosic substrate, the
microbial hydrolysis shown in our study has been demonstrated to be not only an excellent alternative to commercial
cellulase hydrolysis, but also a feasible and economical
process for the future scale-up production of bioethanol. In
order to reduce the total process time for both hydrolysis of
cellulosic substrate and fermentation of hydrolysate to
ethanol, simultaneous saccharification and fermentation
(SSF) may meet the requirement. Further work should be
focused on optimization of the co-culture of Streptomyces sp.
and yeast strain to make the process economically feasible.

Acknowledgment
The authors would like to thank the National Science Council
of the Republic of China (Taiwan) for financially supporting this
research under Contract No. NSC 97-2313-B-264- 001-MY3.

1884

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 1 8 7 8 e1 8 8 4

references

[1] Farrell AE, Plevin RJ, Turner BT, Jones AD, OHare M,
Kammen DM. Ethanol can contribute to energy and
environmental goals. Science 2006;311:506e8.
[2] Curreli N, Fadda MB, Rescigno A, Rinaldi AC, Soddu G,
Sollai F, et al. Mild alkaline/ oxidative pretreatment of wheat
straw. Process Biochem 1997;32:665e70.
[3] Gaspar M, Juhasz T, Szengyel Z, Reczey K. Fractionation and
utilization of corn fibre carbohydrates. Process Biochem
2005;40:1183e8.
[4] Kann J, Rang H. Bioethanol as a fuel to reduce the greenhouse
effect. Proc Estonian Acad Sciences Chem 2000;49(2):83e104.
[5] Tsai WT, Lan HF, Lin DT. An analysis of bioethanol utilized as
renewable energy in the transportation sector in Taiwan.
Renew Sustain Energ Rev 2008;12:1364e82.
[6] Kumakura M. Preparation of immobilized cellulase beads
and their application to hydrolysis of cellulosic materials.
Process Biochem 1997;32:555e9.
[7] Esteghlalian A, Hashimoto AG, Fenske JJ, Penner MH.
Modelling and optimization of the diluted-sulfuric-acid
pretreatment of corn stover, poplar and switchgrass.
Bioresour Technol 1997;59:129e36.
[8] Kaar WE, Holtzapple MT. Using lime pretreatment to
facilitate the enzymatic hydrolysis of corn stover. Biomass
Bioenergy 2000;18:189e99.
hgren K, Galbe M, Zacchi G. Optimisation of steam
[9] O
pretreatment of SO2-impregnated corn stover for fuel ethanol
production. Appl Biochem Biotechnol 2005;124:1055e67.
[10] Wingren A, Galbe M, Zacchi G. Techno-economic evaluation
of producing ethanol from softwoodda comparison of SSF
and SHF and identification of bottlenecks. Biotechnol Prog
2003;19(4):1109e17.
[11] Mosier N, Wyman C, Dale B, Elander R, Lee YY,
Holtzapple M, et al. Features of promising technologies for
pretreatment of lignocellulosic biomass. Bioresour Technol
2005;96:673e86.
[12] Sun Y, Cheng JY. Hydrolysis of lignocellulosic materials for
ethanol production: a review. Bioresour Technol 2002;83:
1e11.
[13] Tolan JS, Foody B. Cellulase from submerged fermentation.
Adv Biochem Eng Biotechnol 1999;65:41e67.
[14] Chen M, Xia L, Xue P. Enzymatic hydrolysis of corncob and
ethanol production from cellulosic hydrolysate. Int
Biodeterior Biodegradation 2007;59:85e9.

[15] Sreenath HK, Koegel RG, Moldes AB, Jeffries TW, Straub RJ.
Ethanol production from alfalfa fiber fractions by
saccharification and fermentation. Process Biochem 2001;36:
1199e204.
[16] Jang HD, Chang KS. Thermostable cellulases from
Streptomyces sp.: scale-up production in a 50-l fermenter.
Biotechnol Lett 2005;27:239e42.
[17] Jang HD, Chen KS. Production and characterization of
thermostable cellulases from Streptomyces transformant
T3-1. World J Microbiol Biotechnol 2003;19:263e8.
[18] Mandels M, Reese ET. Fungal cellulase and the microbial
decomposition of cellulosic fabric. Dev Ind Microbiol 1964;5:
5e20.
[19] Ghose TK. Measurement of cellulase activities. Pure Appl
Chem 1987;59:257e68.
[20] Miller GL. Use of dinitrosalicylic acid reagent for
determination of reducing sugar. Anal Chem 1959;31:426e8.
borska P, Galbe M, Zacchi G.
[21] Palmarola-Adrados B, Chote
Ethanol production from non-starch carbohydrates of wheat
bran. Bioresour Technol 2005;96:843e50.
[22] Bonn G. Analytical determination of 1, 6-anhydro-b-Dglucopyranose and kinetic studies under hydrothermal
conditions. J Carbohyd Chem 1985;4:405e19.
hgren K, Vehmaanpera J, Siika-Aho M, Galbe M, Viikari L,
[23] O
Zacchi G. High temperature enzymatic prehydrolysis prior to
simultaneous saccharification and fermentation of steam
pretreated corn stover for ethanol production. Enzyme
Microb Tech 2007;40:607e13.
[24] Cara C, Ruiz E, Oliva JM, Saez F, Castro E. Conversion of olive
tree biomass into fermentable sugars by dilute acid
pretreatment and enzymatic saccharification. Bioresour
Technol 2008;99:1869e76.

ova D, Slavikova I, Patpova J,
[25] Bafrncova P, Smogrovi
c
meny Z. Improvement of very high gravity ethanol
DO
fermentation by media supplementation using
Saccharomyces cerevisiae. Biotechnol Lett 1999;21:337e441.
[26] Silva CJSM, Roberto IC. Improvement of xylitol production by
Candida guilliermondii FTI 20037 previously adapted to rice
straw hemicellulosic hydrolysate. Lett Appl Microbiol 2001;
32:248e52.
[27] Palmqvist E, Hahn-Hagerdal B. Fermentation of
lignocellulosic hydrolysates. I. Inhibition and detoxification.
Bioresour Technol 2000;74:17e24.
[28] Yu Z, Zhang H. Ethanol fermentation of acid-hydrolyzed
cellulosic pyrolysate with Saccharomyces cerevisiae. Bioresour
Technol 2004;93:199e204.