Production of ethanol from cassava pulp via fermentation with a surface-

engineered yeast strain displaying glucoamylase

Akihiko Kosugi
a
, Akihiko Kondo
b
, Mitsuyoshi Ueda
c
, Yoshinori Murata
a
, Pilanee Vaithanomsat
d
,
Warunee Thanapase
d
, Takamitsu Arai
a
, Yutaka Mori
a,
*
a
Post-harvest Science and Technology Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan
b
Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Nada-ku, Kobe, 657-8501, Japan
c
Department of Applied Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
d
Nanotechnology and Biotechnology Division, Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, 50 Chatuchak, Ladyao, Bangkok
10900, Thailand
a r t i c l e i n f o
Article history:
Received 6 February 2008
Accepted 6 September 2008
Available online 26 October 2008
Keywords:
Cassava pulp
Glucoamylase
Saccharomycescerevisiae
Arming yeasts
Ethanol
Surface-engineering
a b s t r a c t
Cassava (Manihot esculenta Crantz) pulp, produced in large amounts as a by-product of starch
manufacturing, is a major biomass resource in Southeast Asian countries. It contains abundant starch
(approximately 60%) and cellulose ber (approximately 20%). To effectively utilize the cassava pulp, an
attempt was made to convert its components to ethanol using a sake-brewing yeast displaying glu-
coamylase on the cell surface. Saccharomyces cerevisiae Kyokai no. 7 (strain K7) displaying Rhizopus
oryzae glucoamylase, designated strain K7G, was constructed using the C-terminal-half region of
a-agglutinin. A sample of cassava pulp was pretreated with a hydrothermal reaction (140

C for 1 h),
followed by treatment with a Trichoderma reesei cellulase to hydrolyze the cellulose in the sample. The
K7G strain fermented starch and glucose in pretreated samples without addition of amylolytic enzymes,
and produced ethanol in 91% and 80% of theoretical yield from 5% and 10% cassava pulp, respectively.
2008 Elsevier Ltd. All rights reserved.
1. Introduction
Cassava (Manihot esculenta Crantz) is widely cultivated in
tropical areas and used as food and animal fodder. In Thailand,
approximately 10 million tons of fresh cassava tubers are consumed
annually as a starch staple. When starch is extracted from cassava
tubers during manufacturing, grated cassava tubers are separated
into starch granules and brous residual materials by water
extraction followed by centrifugation. The brous residual material,
called cassava pulp, accounts for approximately 1030% by weight
(wet) of the original tubers. Therefore, the tapioca starch industry
in Thailand is estimated to generate at least one million ton of
cassava pulp annually from 10 million tons of fresh tubers.
According to reports [1] and processing practices in Thailand,
a large amount of starch (up to 60%, on a dry weight basis) together
with cellulosic ber is contained in the cassava pulp.
Ethanol is increasingly used as an alternative fuel in the trans-
portation sector. In general, fuel ethanol is produced mainly from
sugar cane, corn, and, in Thailand, cassava. However, a dramatic
increase in ethanol production using the crops mentioned earlier
may not be practical, because these same crops are important
sources of food and feed, and expansion of fuel ethanol production
using these crops could lead to shortages and price increase in food
and feed. Using agricultural wastes as a source for ethanol
production is an effective alternative. In this context, cassava pulp
has great potential as a raw material for ethanol production
because it contains large amount of starch and cellulosic substances
that can be hydrolyzed and fermented to make ethanol.
Ethanol production from starchy materials by conventional
fermentation requires saccharication with amylolytic enzymes
and subsequent fermentation using the yeast Saccharomyces cer-
evisiae, because this yeast cannot utilize starchy materials. The
two-step process results in high production costs and low
productivity of ethanol. Many reports have been published on the
development of S. cerevisiae strains capable of secreting amylo-
lytic enzymes [2]. Although yeast strains capable of utilizing
starch have been developed, their ethanol-producing ability
remains unsatisfactory. Recently, yeast strains displaying various
proteins on their cell surface have been developed using genetic
engineering [3]. Laboratory yeast strains displaying Rhizopus
oryzae glucoamylase (EC 3.2.1.3) [4], which cleaves glucose from
a-1,4-linked and a-1,6-linked polysaccharides, have produced
ethanol directly from soluble and cooked corn starch [5,6].
* Corresponding author. Tel./fax: 81 29 838 6623.
E-mail address: ymori@affrc.go.jp (Y. Mori).
Contents lists available at ScienceDirect
Renewable Energy
j ournal homepage: www. el sevi er. com/ l ocat e/ renene
0960-1481/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.renene.2008.09.002
Renewable Energy 34 (2009) 13541358
However, these surface-engineered yeast stains, because of the
nature of the parent laboratory strain, cannot produce and accu-
mulate high concentrations of ethanol at a rapid rate. Therefore, it
is necessary to apply the surface-engineering technology to
a high-ethanol-yielding and ethanol-tolerant industrial strain to
produce a practically useful yeast strain for ethanol production.
This report describes the development of a surface-engineered
yeast strain displaying R. oryzae glucoamylase using an industrial
ethanol-producing yeast, S. cerevisiae Kyokai no. 7, and its
fermentation properties on cassava pulp.
2. Materials and methods
2.1. Substrates, enzymes, media, and strains
Cassava pulp was obtained from Sanguan Wongse Starch
Industries (Thailand). Dry pulp was prepared by heating 1 kg wet
cassava pulp at 90

C for 24 h followed by grinding and sieving
through a 0.5-mm mesh screen (ZM-100; Retsch, Haan, Germany).
The dry pulp was stored in plastic bags at 4

C. The enzymes used
for starch hydrolysis, a-amylase from Aspergillus oryzae and glu-
coamylase from Aspergillus niger, were obtained from Megazyme
Co, Ltd. Cellulase from Trichoderma reesei (Sigma) was used to
hydrolyze the cellulosic ber in the cassava pulp. The sake-brewing
yeast, S. cerevisiae Kyokai no. 7 (K7), was obtained fromthe National
Research Institute of Brewing (NRIB). S. cerevisiae MT8-1/pGA11 [5]
displaying R. oryzae glucoamylase was used as a source for plasmid
extraction. The yeasts were grown aerobically at 30

C with
complete medium(YPD) containing 20 g peptone, 10 g yeast extract
(Difco Laboratories, Detroit, Mich.), and 20 g glucose per liter.
Escherichia coli DH5a (TaKaRa) was used for genetic manipulation.
E. coli was grown at 37

C in LuriaBertani medium containing
100 mg of ampicillin per millilitre.
2.2. Hydrothermal treatment of cassava pulp
Cassava pulp suspended in water at concentrations of 530%
was put in a pressure tube (Ace Glass Inc. USA) and heated at
temperatures from 120 to 180

C for 1 h. To examine the effects of
acid, H
2
SO
4
was added to the pulp suspensions at nal concentra-
tions of 0.1 or 2.0% before heating.
2.3. Enzymatic hydrolysis
The pretreated pulp slurries were enzymatically hydrolyzed to
determine the maximum obtainable sugar. The pH of the slurries
was preadjusted to 5.0 with sodium hydroxide, and 3 M sodium
acetate buffer at pH 5.0 was added at a nal concentration of
50 mM. Cellulase was used in the ratio of 3 U/g dry pulp. Hydrolysis
of cellulosic ber in the slurries was conducted at 50

C for 72 h
before subsequent starch hydrolysis. Samples were collected after
24, 48, and 72 h for analysis of released glucose. After the slurry was
treated with cellulase, a-amylase (300 U/g dry pulp) and glucoa-
mylase (100 U/g dry pulp) were added to hydrolyze the starch
completely. Starch hydrolysis in the slurry was conducted at 50

C
for 48 h. The amount of released glucose was determined using the
Wako glucose CII test (Wako Pure Chemicals Co., Osaka, Japan).
Clear supernatants were obtained from the hydrolyzed slurries by
centrifugation at 7000 rpm for 20 min.
2.4. Fermentation by strain K7
The hydrolyzed slurries (pulp hydrolysates) were supplemented
with yeast extract and yeast nitrogen base with amino acids (Difco
Laboratories, Detroit, MI) at nal concentrations of 5 g/L and 7 g/L,
respectively. The pH was adjusted to 6.0 with calcium hydroxide
and the media were ltered with aseptic lter discs (0.22-mm,
Advantec, Tokyo, Japan). Precultured strain K7 was inoculated into
the medium at 5% (v/v) and incubated at 30

C. Samples were
collected every 24 h from fermentation broths and analyzed for
ethanol. A gas chromatograph (model GC-2014: Shimadzu, Kyoto,
Japan) with a ame-ionization detector (FID) was used to measure
ethanol concentration in samples under the following conditions:
glass column (8.0 mm by 3.2 m) packed with Chromosorb 103 (60/
80 mesh); temperatures of column, injector, and detector, 185, 175,
and 250

C, respectively; heliumcarrier gas owrate, 20 ml/min. n-
Propanol was used as an internal standard.
2.5. Construction of plasmids for displaying R. oryzae glucoamylase
on strain K7
Plasmid pGA11 for cell surface display of R. oryzae glucoamylase
was constructed as described previously in Ref. [5]. To amplify
a DNA fragment between glyceraldehydes-3-phosphate dehydro-
genase (GAPDH) promoter and GAPDH terminator containing the
coding region of the glucoamylase with the 3
0
half of a-agglutinin
gene, two primers containing articial SphI and Sse837 I sites
(underlined) (5
0
-ACATGCATGCACCAGTTCTCACACGGAACA-3
0
and
5
0
-TCCTGCAGGTCAATCAATGAATCGAAAATG-3
0
) were designed and
a 4.3-kbp fragment was amplied by PCR using Ex Taq polymerase
(TaKaRa). The amplied fragments were digested with the cognate
restriction enzymes and inserted between the SphI and Sse837 I
sites of pAUR101 (TaKaRa) to generate pAU101RGA. To integrate
pAU101RGA into the S. cerevisiae K7 genomic DNA, the plasmid was
digested with StuI to generate a linear DNA fragment. This DNA
fragment was transformed into the strain K7 by the lithium acetate
method following manufacturers instructions (TaKaRa). Trans-
formants were obtained on YPD plates containing 1.0 mg/ml Aur-
eobasidin A (TaKaRa).
2.6. Glucoamylase assay
After aerobic cultivation of transformed strains in YPD con-
taining Aureobasidin A at 30

C for 24 h, cells were collected by
centrifugation for 5 min at 8000 rpm, resuspended in distilled
water, and used for the enzyme assay. The substrate solution for
glucoamylase assay was prepared by adding soluble starch to the
boiling 20 mM sodium acetate buffer (pH 5.0) to give a concentra-
tion of 1%. The sample was mixed with the substrate solution and
incubated at 30

C for 30 min. Reaction was stopped by boiling the
mixture for 10 min. Released glucose was determined using the
Wako glucose CII test. One unit of glucoamylase was dened as
the amount of enzyme required to release 1 mmol of glucose per
minute from starch [11].
2.7. Fermentation by stain K7G
Slurries of pulp pretreated with hydrothermal reaction and
cellulase were supplemented with yeast extract and yeast nitrogen
base with amino acids at nal concentrations of 5 g/L and 7 g/L,
respectively. The pH was adjusted to 6.0 with calcium hydroxide.
The slurries were inoculated with the strain K7G and fermented at
30

C. Samples were collected every 24 h from fermentation broths
and analyzed for ethanol by gas chromatography.
2.8. Composition of cassava pulp
Starch content in dry cassava pulp was estimated using the total
starch assay kit following manufacturers instructions (Megazyme,
Ireland). Reducing sugar and glucose concentrations in dry cassava
pulp were measured by the Somogyi Nelson method and the Wako
glucose CII test, respectively, using 70% ethanol extracts. The
A. Kosugi et al. / Renewable Energy 34 (2009) 13541358 1355
starch-free ber was obtained by centrifugation at 10,000 rpm for
20 min after starch hydrolysis using a-amylase and amyloglucosi-
dase for 2 days at 50

C. Monosaccharide analysis of the ber was
performed with high performance anion-exchange chromatog-
raphy using CarboPac PA (Dionex Corporation, Sunnyvale, CA, USA)
with pulsed amperometric detection (HPAEC-PAD). The mobile
phase was 2% NaOH at a ow rate of 0.6 ml/min at 28

C. The total
nitrogen content of cassava pulp was estimated using a modied
Dumas method (Sumigraph NC-900, Shimadzu, Kyoto, Japan).
Klason lignin was determined using the Ha gglund method [7].
3. Results and discussion
3.1. Components of cassava pulp
Table 1 shows the composition of cassava pulp produced after
starch extraction at a starch factory in Thailand. Signicant
amounts of starch (60.6%) and non-starch polysaccharide (29% as
ber) were detected in the pulp. Results are similar to those
reported by Srioth et al. [1]. Monosaccharide analysis of the non-
starch polysaccharides indicated that glucans, such as cellulose,
were the major polysaccharide. The analyzed compounds accoun-
ted for 94.7% of the weight of the total dry pulp.
3.2. Optimization of pretreatment conditions for cassava pulp
To determine optimal pretreatment methods for ethanol
production from cassava pulp, hydrothermal reaction with and
without dilute sulfuric acid was examined. The experimental
procedure is summarized in Fig. 1. First, to test the effects of
a combination of hydrothermal reaction (120180

C for 60 min)
and addition of H
2
SO
4
(0.1% and 2%), glucose yield in the pulp
hydrolysates was measured after enzyme treatments of the cassava
pulp (Fig. 1).
The greatest glucose yield (0.75 g/g pulp) was obtained under
hydrothermal conditions (140

C for 1 h) without addition of H
2
SO
4
(Fig. 2). Yields were equivalent to approximately 90% of the theo-
retical value according to the composition shown in Table 1. No
major differences in glucose yields between 120

C and 160

C were
found with or without addition of 0.1% H
2
SO
4
(Fig 2). Glucose yields
from hydrothermal reaction with 2% H
2
SO
4
were lower than those
with or without 0.1% H
2
SO
4
(Fig. 2), suggesting formation of by-
products with increased H
2
SO
4
concentration. Reports have indi-
cated that by-products, such as hydroxylmethyl-furfural (HMF),
furfural, furoic acid, and phenol, are produced from irreversible
conversion of pentoses, hexoses, and lignin during pretreatment
with acid [8]. Based on these results, hydrothermal reaction at
140

C without H
2
SO
4
was used to pretreat cassava pulp.
However, these conditions are not optimal for energy balance.
Assuming that thermal efciency is 100% and the specic heat for
cassava pulp is 1.25 kJ/kg k [9], the energy consumed in heating
1 ml of 5% pulp suspension from 25

C to 140

C and keeping it at
140

C was 488.6 J. Because the glucose generated (0.744 g/g dry
pulp) provides 580.3 J, the energy balance was 91.7 J. Similarly,
the energy balance with no heating and at 120, 160, and 180

C
without H
2
SO
4
was 241.8, 154.1, 4.1, and 143.7 J, respectively,
indicating lowering treatment temperature is important to improve
energy balance. In practice, where complete insulation is impos-
sible, it is also necessary to shorten treatment time to reduce the
energy required to maintain reaction temperature.
To examine the effects of pulp concentration on glucose yield,
5%, 10%, 20%, and 30% suspensions of pulp were hydrolyzed with
enzymes after hydrothermal reaction at 140

C for 1 h. Glucose
yield was highest with 5% pulp, and it decreased as pulp concen-
tration increased (Fig. 3). The low hydrolysis efciencies with
higher concentrations of pulp probably are due to inhibition of
enzymes by increasing amounts of end-products, by-products,
and/or insufciency of hydrothermal reaction by substrate over-
load [10]. The energy balance with 5, 10, 20, and 30% cassava pulp
Table 1
Composition of cassava pulp
Components g/100 g Dry pulp
Starch 60.6
Reducing sugars (glucose)
a
4.7 (0.09)
a
Nitrogen 0.4
Non-starch polysaccharides
Glucan 19.1
Xylan 4.2
Arabinan 1.4
Galactan 0.5
Mannan 0.7
Others 0.9
Klason lignin 2.2
Total 94.7
Analyses were conducted according to Section 2.
a
Free glucose in the dry pulp.
Cassava pulp
Hydrothermal reaction
Temp: 120C to 180C
Time: 60 min
H
2
SO
4
: 0% to 2%
Hydrolysis of fiber
Cellulase: 3U/g pulp
Temp: 50C
Time: 72 h
Hydrolysis of starch
-Amylase: 300U/g pulp
Glucoamylase: 100U/g pulp
Temp: 50C
Time: 48 h
Fermentation test
Yeast strain: K7
Temp: 30C
Time: 2 to 7 days
Fermentation test
Yeast strain: K7G
Temp: 30C
Time: 5 to 7 days
Pulp hydrolysates
Pretreated
pulp
Fig. 1. Experimental procedures used to evaluate the hydrolysis of cassava pulp.
0.0
0.2
0.4
0.6
0.8
1.0
No thermal
treatment
120C
60 min
140C
60 min
160C
60 min
180C
60 min
G
l
u
c
o
s
e

y
i
e
l
d
(
g

g
l
u
c
o
s
e
/
g

d
r
y

p
u
l
p
)
Treatment
: No acid, : 0.1 %H
2
SO
4
, : 2.0 % H
2
SO
4
,
Fig. 2. Effects of hydrothermal reaction with and without addition of H
2
SO
4
on
enzyme hydrolysis. Concentration of cassava pulp was 5% (w/v) for all conditions.
Enzyme hydrolysis was conducted after the hydrothermal reaction.
A. Kosugi et al. / Renewable Energy 34 (2009) 13541358 1356
was 91.7, 488.6, 1140.3, and 1901.1 J, respectively. Thus, pulp
concentration is signicant for achieving high hydrolysis efciency
and energy gain.
3.3. Fermentation of pulp hydrolysates using strain K7
Strain K7, which is used for industrial brewing of Japanese
sake, is capable of high-ethanol productivity and tolerance
compared with laboratory strains. To determine whether the
pulp hydrolysates inhibit yeast growth, fermentation tests were
performed using strain K7. High-ethanol yields and productivities
were observed when 5% and 10% pulp hydrolysates were fer-
mented with strain K7, while ethanol yield and productivity from
20% and 30% pulp hydrolysates were signicantly lower (Table 2).
In contrast, all reference fermentations, where glucose was used
as substrate instead of cassava pulp hydrolysates, showed high-
ethanol yield and productivity even with high substrate
concentrations (Table 2). Thus, low performance from fermen-
tation with 20% and 30% pulp hydrolysates probably is due to
accumulation of inhibitory by-products from the hydrothermal
treatment [8].
3.4. Fermentation of pretreated pulp using the yeast transformant
K7G displaying R. oryzae glucoamylase
PCR testing conrmed that the linearized plasmid pAU101RGA
was integrated into the genomic DNA of strain K7, and that the
transformant strain K7G successfully integrated one copy of the
R. oryzae glucoamylase gene with a-agglutinin into the AUR1
allele of strain K7 genomic DNA [11]. The glucoamylase activity of
the strain K7G was 60.2 U/g [wet weight] of cells (Table 3). No
activity was detected with wild-type strain K7 and control strain
K7A harboring pAUR112. These results indicate that K7G dis-
played R. oryzae glucoamylase on its cell surface by the anchoring
function of the C-terminal a-agglutinin.
Cassava pulp treated with cellulase following hydrothermal
reaction (140

C for 1 h) was fermented using K7G and K7A
without addition of amylolytic enzymes. As shown in Fig. 4, K7G
produced signicant amounts of ethanol from 5%, 10%, 20%, and
30% pulp, while K7A produced very little ethanol (Fig 4). Ethanol
production rate from cassava pulp by K7G without amylase
treatment was slower, especially during the early phase of
fermentation compared to that by K7 with cassava pulp treated
with a-amylase and glucoamylase. The slow ethanol production by
K7G presumably is due to the time required for saccharication of
starch by the glucoamylase displayed on the cell surface. The
fermentation rate by K7G could be easily improved by increasing
initial cell density [12] and by codisplaying a-amylases with glu-
coamylase [6].
Strain K7G effectively fermented cellulase-pretreated cassava
pulp to produce ethanol in high yields: 91% and 80% of the theo-
retical value with 5% and 10% of cassava pulp, respectively, (Table 4).
The maximum ethanol concentration accumulated by K7G was
comparable to those attained by K7 with cassava pulp pretreated
with amylase in addition to cellulase.
The results obtained in this study show the effectiveness of
surface-engineered yeast displaying amylase on the surface for the
production of ethanol from cassava pulp. Research continues into
the construction of a yeast strain codisplaying cellulases and
amylases, which could fully utilize cassava pulp and directly
produce ethanol without addition of enzymes.
Table 3
Glucoamylase activity of transformed yeast strain K7G
Strains Activity (U/g [wet wt.] of cells)
K7 ND
a
K7A
b
ND
a
K7G 60.2
Values are averages of three independent experiments.
a
Not detectable.
b
K7A is yeast strain K7 harboring pAUR112 as control against K7G.
0
10
20
30
40
50
0 1 2 3 4 5 6 7
E
t
h
a
n
o
l

p
r
o
d
u
c
t
i
o
n
(
g
/
l
i
t
e
r
)
Time (days)
Fig. 4. Time course of ethanol production on pretreated cassava pulp using yeast strain
K7G displaying glucoamylase. Open circles represent 5% pretreated pulp, closed circles
represent 10% pretreated pulp, open squares represent 20% pretreated pulp, closed
squares represent 30% pretreated pulp. Closed triangles indicate the fermentation
prole of control strain K7 harboring pAUR112 (K7A).
0.0
0.2
0.4
0.6
0.8
1.0
5 % 30 %
Cassava pulp concentration (w/v)
G
l
u
c
o
s
e

y
i
e
l
d
(
g

g
l
u
c
o
s
e
/
g

d
r
y

p
u
l
p
)
20 % 10 %
Fig. 3. Effects of increasing pulp concentration on enzyme hydrolysis. The hydro-
thermal reaction was conducted at 140

C for 60 min without addition of H
2
SO
4
.
Enzyme treatment was performed after the hydrothermal reaction.
Table 2
Fermentation of cassava pulp hydrolysates by strain K7
Cassava
pulp
(w/v %)
Initial
substrate
a
(g glucose/L)
Ethanol
produced
(g/L)
Ethanol yield
on substrate
b
(g ethanol/g
substrate)
Ethanol
productivity
c
(g ethanol/L/h)
Yield
(%)
5 37.2 (33) 18.6 (17.1) 0.50 (0.51) 0.77 (0.71) 98 (100)
10 63.1 (65) 29.6 (32.2) 0.47 (0.49) 0.62 (0.67) 92 (96)
20 105.8 (108) 32.9 (48.5) 0.31 (0.45) 0.27 (0.67) 61 (88)
30 155.5 (165) 46.0 (65.6) 0.30 (0.40) 0.27 (0.68) 58 (78)
a
Initial substrate is the amount of glucose contained in the pulp hydrolysates.
b
Ethanol produced [in g/L] divided by initial substrate [in g glucose/L].
c
Ethanol produced [in g/L] divided by total fermentation time [in hours]. The values
in parentheses are the results for reference fermentation, where cassava pulp
hydrolysates were replaced by glucose.
A. Kosugi et al. / Renewable Energy 34 (2009) 13541358 1357
4. Conclusions
To utilize cassava pulp containing abundant starch and cellulosic
ber, its components were converted to ethanol using S. cerevisiae
strain K7G displaying glucoamylase on its cell surface, which was
constructed using a cell surface-engineering system based on a-
agglutinin. The strain K7G effectively produced high yields of
ethanol from cassava pulp pretreated by hydrothermal reaction
(140

C for 1 h) and then by cellulase to hydrolyze the cellulose in
the pulp.
Acknowledgments
The authors are grateful to Mr. Thosapol Tantiwong and Mr.
Achcha Leeungkul (Sanguan Wongse Industries Co., Ltd) for
providing cassava pulp. The authors wish to thank Dr. Shingo
Magara and Dr. Ryohei Tanaka (Forestry and Forest Products
Research Institute) for technical support in carbohydrate analysis.
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Table 4
Fermentation of pretreated cassava pulp by strain K7G
Cassava
pulp
(w/v %)
Total sugar
a
(g glucose/L)
Ethanol
produced
(g/L)
Ethanol yield
on substrate
b
(g ethanol/g
total sugar)
Ethanol
productivity
c
(g ethanol/L/h)
Yield
(%)
5 36.5 16.9 0.46 0.18 91
10 61.1 25.2 0.41 0.26 80
20 116.0 34.9 0.30 0.24 59
30 151.4 42.1 0.28 0.25 55
a
Total sugar is indicated as the amount of glucose plus glucose moiety in the starch
contained in the pretreated pulp.
b
Ethanol produced [in g/L] divided by total sugar [in g glucose/L].
c
Ethanol produced [in g/L] divided by total fermentation time [in hours].
A. Kosugi et al. / Renewable Energy 34 (2009) 13541358 1358