Biotechnology and Bioprocess Engineering 2009, 14: 496-502

DOI/10.1007/s12257-008-0305-z





Low Temperature Pre-treatment of New
Cultivar of Corn for Ethanol Production and
Nutrient Value of Its Distillers Dried Grains
with Soluble

Gi-wook Choi
1
*, Yule Kim
1
, Keun and Kim
2

1
Changhae Institute of Cassava and Ethanol Research, Jeonju 561-203, Korea
2
Department of Bioscience and Biotechnology, The University of Suwon, Hwaseong 445-743, Korea


^~ The objective of this work was to evaluate the production of bioethanol from a new Korean variety of corn (Gangdaok)
and to assess low temperature pre-treatment of corn mashes before simultaneous saccharification and fermentation.
Corn mashes containing 178 g/L of total sugar were fermented with Saccharomyces cerevisiae CHY 1011(KCTC
11250BP) at 35C. Fermentation of mash supplemented with solid glucoamylase was completed within 48 h, and the
ethanol produced was 474.0 and 473.1 L/ton as dry base with low temperature pre-treatment and pressure pre-
treatment, respectively. Furthermore, the DDGS of Gangdaok cultivar contained more essential amino acids (21.1 mg/g)
than did Ambrosia cultivar (USA corn), which is a widely used feedstock. In addition, there were no significant differences
in ethanol yield or amino acid concentration in DDGS between low temperature pre-treatment and pressure pre-
treatment. The results show that Gangdaok holds potential economic advantages if applied to the bioethanol and feed
industries. KSBB

hW bioethanol, new variety of corn, low temperature pre-treatment, amino acid contents of DDGS




INTRODUCTION

Bioethanol as the most promising biofuel today has re-
cently experienced an increased demands as a fuel additive
and starting material for the production of various chemicals.
An increased oil price is the predominant factor stimulating
higher demand of biofuel, which in turn causes higher corn
prices [1-3].
The desire to reduce corn imports from abroad has driven
the research focus on the development of a new cultivar of
corn. Gangdaok is a single cross, yellowish-white, flint
maize hybrid (Zea mays L.) cultivar developed by the maize
breeding team at the National Institute of Crop Science, Ru-
ral Development Administration of Korea in 2005. The dry
matter, total digestible nutrient, and grain yields of Gang-
daok were 18.76 ton/ha, 13.24 ton/ha, and 8.56 ton/ha, re-
spectively, which corresponded to 11% increase in both dry

G` ~
Tel: +82-63-214-7800 Fax: +82-63-214-7805
e-mail: changrd@chethanol.com
matter and total digestible nutrient, and 7% increase in grain
yield compared with those of a check hybrid [4]. Gangdaok
has been released to the public as an early maturing corn,
with high quality and improved yield from 2006. The grain
of the Gangdaok is composed of 69.4% starch, 11.1% mois-
ture, 12.3% crude protein, 3.0% crude fat, 2.6% crude fiber,
and 1.3% ash [5].
During ethanol production, pre-treatment must be preceded
by gelatinization and liquefaction of starch while pressure pre-
treatment has increased the speed by which saccharification of
starch occurs and improved the high fermentation efficiency
of starch containing high levels of amylose. However, produc-
tion costs remain high due to high energy consumption [6].
Research into low temperature pre-treatment has reduced the
energy for ethanol production by about 40% while maintain-
ing starch hydrolysis and fermentation efficiencies equal to
that of the conventional process [1,6,7].
The government policy towards renewable fuels reflects
prevailing attitudes around the world as large investments in
research and facilities for ethanol production have been
slated. Most of this increase in ethanol production capacity is
Biotechnol. Bioprocess Eng. QVT


expected to come from newly developed plants for dry-
milling of corn, which will result in an excess of distillers
dried grains with solubles (DDGS) [8]. Indeed, production of
bioethanol from 100 kg corn produces approximately 31.6
kg of DDGS, along with 34.4 kg of ethanol, and 34 kg of
CO
2
. DDGS contains higher concentration of nutrients such
as protein, fat, vitamins, minerals, and fiber than its parent
grain [8]. The crude protein content of DDGS is relatively
high, ranging from 30% [10], and helps to provide essential
amino acids in the feed [9] for monogastric animals which
are unable to synthesize them. Corn DDGS are also potential
feed ingredients for the swine industry, although DDGS is
presently not considered an important ingredient [11].
Therefore, it is important to re-evaluate the nutrient con-
tents and availability of those nutrients in DDGS for poultry
and swine, along with their varied digestibility [12]. Much
research work into new processing conditions conducted to
improve the nutritional value of DDGS has found that the
nutritional quality can be affected by yeast growth and raw
materials [13]. However, the effect of saccharifying enzymes
and pre-treatment conditions on the nutritional value of
DDGS has not yet been studied [14].
Therefore, the present study focuses on development of a
low temperature pre-treatment process for Gangdaok and its
effectiveness on ethanol production and nutrient content of
DDGS compared with Ambrosia.


MATERIALS AND METHODS

MateriaIs, Enzymes, and Strain

The experiment was conducted with two cultivars of corn
grain. Gangdaok was developed by and obtained from the
Korean National Institute of Crop Science (NICS) and Am-
brosia (USA) was purchased from domestic starch factories.
Commercial -amylase Termanyl 120L (Novozymes, Den-
mark) and commercial glucoamylase (GA) Spirizyme plus
(Novozymes, Denmark) were used.
Solid glucoamylase (SGA) was produced by solid state fer-
mentation of Aspergillus usamii mut. shiro-usamii (KCTC
6954) using wheat bran [15]. In order to develop the strains
further, screening with a higher enzyme activity following
UV-light mutagenesis was performed. SGA contained cellu-
lase (703 U/g), xylanase (100 U/g), glucoamylase (2,087 U/g),
protease (171 U/g), and -glucanase (573 U/g).
S. cerevisiae CHY 1011(KCTC 11250BP) used for fer-
mentation was isolated from soil samples collected from
ethanol factories. The culture medium used for the yeast was
YPD medium (2.5 g/L yeast extract, 2.5 g/L peptone, 100
g/L glucose, 0.25 g/L MgSO
4
7H
2
O, and 1.0 g/L K
2
HPO
4
)
[16].

Starch VaIue

Total starch in raw materials was analyzed by two meth-
ods of hydrolysis; glucoamylase-hydrolysis and acid-
hydrolysis. The glucoamylase-hydrolysis of starch was done
by either of commercial glucoamylase (GA) or solid glu-
coamylase (SGA) [17].

Pre-treatment

Before determination of the liquefaction value, raw mate-
rials were dry-milled to obtain a particle size less than 1 mm.
A mixture of Gangdaok grain (100 g) and water (343 mL)
was pretreated in 500 mL Erlenmeyer flasks at various tem-
peratures (90, 94, 98, 102, 108, 112, 116, and 120
o
C). Sam-
ples were first hydrolyzed with -amylase 0.8 g/kg (en-
zyme/material) at 75
o
C for 30 min in the water bath and then
heated to the desired hydrolysis temperature for 0.5, 1, 1.5,
or 2 h. Following centrifugation (Hanil SUPRA 22K, Seoul,
Korea) at 3,000 rpm for 5 min the liquefaction value (liquid
volume/total volume) was determined [16,18].

Fermentation

Before fermentation, liquefaction of corns before mashing
was performed by low temperature pre-treatment (98
o
C, 1.5
h) and pressure pre-treatment (120
o
C, 1 h). SGA or GA (30
SP) was added to the liquefied mashes and the mixtures were
subjected to simultaneous saccharification and fermentation
(SSF) at 35
o
C for 72 h. Fermentations were performed in a 5
L jar fermenter (BioFlo IIC, New Brunswick, USA) contain-
ing 3 L of corn mashes.
Yeast seed culture was grown at 30
o
C for 24 h in a 500
mL flask containing 150 mL of YPD medium, followed by
inoculation of the resulting culture into a 5 L jar fermenter.
During SSF, the temperature was maintained at 35
o
C and
agitation speed 60 rpm. After 72 h fermentation, the ethanol
and residual sugar concentration of the samples were evalu-
ated. Ethanol production yield was expressed in Liter of 99.5
(v/v) % ethanol/Ton of dried material.

AnaIyticaI Method

Sugar content of the mash-hydrolysate was diluted and its
concentration analyzed by HPLC [18]. An RSpack KC-811
(Shodex, Japan) column was used while the mobile phase
consisted of 0.009N sulfuric acid and 99.75% deionized wa-
ter. Sugars, organic acid, and glycerol fractions were all
separated. The temperature was kept at 60
o
C with a flow rate
of 1.0 mL/min and a sample volume of 20 L. The effluent
from the column was monitored using a Refractive Index
Detector (2414, Waters, USA) [19].
The ethanol concentration of the alcoholic mashes was
measured by gas chromatography (Agilent 4890, USA), col-
umn, 6.6% CARBOWAX 20M; detector, flame ionization
detector; carrier gas, nitrogen; oven temperature, 100
o
C;
injection temperature, 200
o
C; flame ionization detector tem-
perature, 250
o
C (Agilent Chemstation Data Analysis System,
USA) using isopropanol as the internal standard. The ethanol
productivity, which was defined as grams of ethanol per liter
per hour, was calculated from the final ethanol concentration.
The ethanol conversion yield was calculated based on the
initial sugar concentration and was reported as a percentage
QVU


















cK NK Starch value of Gangdaok and Ambrosia determined by
different methods of saccharification. , Acidic; , GA; ,
SGA.


of the theoretical yield [20].

Amino Acid Content

One liter mash was concentrated into wet grain, crude pro-
tein, yeast culture, and ethanol using a rotary evaporator
(NE-2001, Eyela, Japan) at 90
o
C for 6 h, followed by drying
to produce DDGS. The amount of DDGS was determined
and used to calculate the equilibrium moisture content di-
rectly by drying at 105
o
C for 2.5 h. DDGS were subjected to
6 N HCl in duplicate and hydrolyzed for 24 h at 110
o
C. Acid
hydrolysate was subsequently analyzed for amino acid con-
tent using an amino acid auto analyzer (Amino acid analyzer
S-433, Sykam, Germany) [21].


RESULTS AND DISCUSSION

Starch VaIues

The starch values of analyzed corn samples are presented
in Fig. 1. In both Gangdaok and Ambrosia cultivars, starch
values were higher for SGA and acid saccharification than
for GA saccharification. For SGA and acid saccharification,
the starch values of Gangdaok and Ambrosia were 70 and
67%, respectively, while for GA saccharification they were
63 and 65%, respectively. Therefore, strictly according to
SGA and acid saccharification, Gangdaok had higher starch
values than Ambrosia. However the results of GA sacchari-
fication show Ambrosia had slightly higher starch values
than Gangdaok. The factors affecting enzymatic hydrolysis
of starch could include substrates involved, amylolytic en-
zyme activity, and the presence of other plant material-
digesting enzymes such as protease, lipase, cellulase, xy-
lanase, and glucanase etc. SGA is therefore more advanta-
geous than GA since it contains other plant material-
digesting enzymes capable of degrading the plant materials















cK OK Effect of temperature and reaction time on liquefaction value
of Gangdaok.


q~ NK Optimal pre-treatment conditions for ethanol production in
Gangdaok
Low temperature Pressure
Grain weight (kg) 1.0 1.0
Water (L) 3.43 3.43
-Amylase (g) 0.08 0.08
SGA (g) 6.0 6.0
Temperature (C) 98 120
Time (h) 1.5 1.0


surrounding the starch granules, thereby allowing degrada-
tion of the starch by glucoamylase.

Pre-treatment of Gangdaok Grains

Two parameters, temperature (X axis) and reaction time(Y
axis), affecting the liquefaction value were examined. Re-
sults show that temperature significantly affects the liquefac-
tion value; especially in pressure pre-treatment, the greater
the temperature the greater the liquefaction value. The high-
est liquefaction value was obtained from pressure pre-
treatment (120
o
C) for 1 h and low temperature pre-treatment
(98C) for 1.5 h (60.9 and 60.6% of the liquefaction value,
respectively) (Fig. 2). Since low temperature pre-treatment
can save heating energy as much as 40%, this is a more pre-
ferred method compared with pressure pre-treatment. Table
1 shows the pre-treatment optimal conditions for liquefaction
of Gangdaok.

Fermentation ResuIt

Following mashing of corn grains with either low tem-
perature pre-treatment or pressure pre-treatment (Table 1),
SGA or GA was added to the mixture to stimulate simulta-
neous saccharification and fermentation. HPLC analysis
showed that the Gangdaok and Ambrosia mashes immedi-
ately following low temperature and pressure pre-treatments
Biotechnol. Bioprocess Eng. QVV


^ _












` a












cK PK Change of carbohydrate, glycerol, and ethanol content during fermentation of Gangdaok mash treated with SGA and GA. , Dextrin; ,
Maltose; , Glucose; , Glycerol; , Ethanol.


^ _











` a












cK QK Change of carbohydrate, glycerol and ethanol content during fermentation of Ambrosia mash treated with SGA and GA. , Dextrin; ,
Maltose; , Glucose; , Glycerol; , Ethanol.
RMM
















cK RK Decreasing total sugar content in Gangdaok mashes treated
at different temperatures and by different saccharifying en-
zymes. , SGA 98C; , SGA 120C; , GA 98C; , GA
120C.
















cK SK Decreasing total sugar content in Ambrosia mashes treated
at different temperatures and by different saccharifying en-
zymes. , SGA 98C; , SGA 120C; , GA 98C; , GA
120C.


at the beginning of fermentation contained dextrin, 140 and
126 g/L; maltose, 2 and 3 g/L; and glucose, 18 and 37 g/L,
respectively (Figs. 3 and 4). The mash contained approxi-
mately 178 and 182 g of total sugar per 1 L of mash (Figs. 5
and 6). Initial concentrations of sugar (maltose, glucose)
present in mash pressure pre-treated before fermentation
were higher than when low temperature pre-treated. How-
ever, this difference in initial sugar concentration did not
affect the fermentation pattern.
Ethanol production during fermentation of Gangdaok and
Ambrosia along with decreasing dextrin and sugar levels are
shown in Figs. 4 and 3, respectively. The concentration of
residual total sugar (RTS) in the Gangdaok and Ambrosia
mashes after 72 h of fermentation were 10.9 1.4 g/L and
8.9 1.4 g/L, respectively, when subjected to SGA. For GA,
the values were 13.5 0.6 g/L and 14.3 4.1 g/L, respec-
tively (Figs. 5 and 6). Therefore, relatively higher amounts
of RTS were observed at the end of fermentation with GA.
q~ OK Ethanol yield, fermentation rate, and percent of theoretical
yield of ethanol for two corn cultivars
Gangdaok Ambrosia

98C 120C 98C 120C
SGA 474.0 473.1 479.1 479.5 Ethanol yield
(L/Ton)
GA 453.6 450.5 449.1 468.8
SGA 88.76 88.58 88.66 88.75 Fermentation
rate (%)
GA 84.93 84.36 83.12 86.77
SGA 94.03 93.61 94.01 94.14
Percent of
theoretical
yield of ethanol
GA 90.90 90.41 90.76 92.98


However, the Gangdaok and Ambrosia mashes subjected to
SGA showed an accelerated fermentation rate, a reduced
fermentation time of approximately 48 h, and a constant low
residual sugar concentration till 72 h (Figs. 3 and 4).
For Ambrosia, the mashes subjected to pressure pre-
treatment and GA showed a 3.65% higher fermentation rate
and 19.7 L/Ton higher ethanol yield than low temperature
pre-treatment (Table 2). The glycerol content in the mashes
of Gangdaok and Ambrosia was similar to each other regard-
less of the two pre-treatment methods and was 5.1~5.2 g/L
for both cultivars for SGA and 6.3~6.5 g/L for both cultivars
for GA, at the end of fermentation (Figs. 3 and 4). These
glycerol values represent 3~4 (w/w) % of total sugar used. In
addition, Ambrosia mashes showed lower concentrations of
dextrin (approximately 2~3 g/L) than Gangdaok mashes at
the end of fermentation.
The average ethanol yields per ton of raw material were
474 and 479 L/Ton for Gangdaok and Ambrosia, respec-
tively, subjected to low temperature pre-treatment and SGA
(Table 2). The average ethanol yields for Gangdaok and
Ambrosia subjected to pressure pre-treatment with GA were
450 and 468 L/Ton, respectively. This result indicates low
temperature pre-treatment produces higher ethanol yields
than pressure pre-treatment.

Assessment of Amino acids

At the end of the fermentation, Gangdaok and Ambrosia
mashes were dried to DDGS. The mash consisted mainly of
limited dextrin, non-fermentable sugars, yeast, crude fiber,
crude fat, and crude protein. Table 3 shows the essential and
nonessential amino acids in the DDGS of the two corn culti-
vars pre-treated with different temperatures and saccharify-
ing enzymes. The total amino acid concentration of the
DDGS was between 230~310 mg/g, which was also ap-
proximately three times higher than the corn grains. The
average increase of amino acids in all of the DDGS was
260~290%. Amino acid concentration of the Gangdaok
grains was 107 mg/g which was 20.3 mg/g higher than that
of Ambrosia.
The amino acid content of DDGS pressure pre-treated
with SGA was 4.1~31.7 mg/g higher than DDGS subjected
to low temperature pre-treatment. Likewise, the amino acid
Biotechnol. Bioprocess Eng. RMN


q~ PK Amino acid concentration of DDGS and grains of two corn cultivars pre-treated at different temperatures and with different saccharify-
ing enzymes (unit: mg/g)
DDGS Grain
SGA GA
Gangdaok Ambrosia Gangdaok Ambrosia

98C 120C 98C 120C 98C 120C 98C 120C
Gang-daok Ambrosia
Nonessential amino acid
Asp 18.9 19.0 17.0 19.3 18.8 18.4 16.0 15.4 6.6 6.2
Ser 14.5 15.3 13.8 15.4 17.3 16.8 14.3 13.6 5.5 4.7
Glu 66.8 64.2 54.6 60.8 72.5 73.5 59.5 54.4 25.6 19.4
Pro 25.5 26.2 21.0 25.3 30.0 26.7 21.5 19.9 9.6 7.5
Gly 10.3 10.5 10.2 11.4 11.5 11.2 10.6 10.7 3.9 3.6
Ala 12.0 22.2 18.9 21.2 24.9 24.0 19.6 18.5 8.2 6.1
Cys 0.7 0.7 0.4 0.4 0.3 0.6 0.6 0.4 0.2 0.3
NH4 9.6 8.5 6.6 8.0 8.6 7.8 7.3 7.5 5.3 3.5
Sum 158.3 166.6 142.5 161.8 183.9 179.0 149.4 140.4 64.9 51.3
Essential amino acid
Thr 10.2 11.2 10.1 11.3 11.9 11.7 10.1 10.0 3.9 3.2
Val 14.3 14.7 12.9 14.6 15.7 15.0 13.0 13.0 5.1 4.0
Iso 5.1 4.7 4.2 4.8 5.0 4.7 3.2 2.4 1.3 1.3
Met 10.7 10.9 9.3 10.5 11.3 11.0 9.1 8.9 3.7 2.9
Leu 38.8 37.6 30.0 33.8 42.2 41.0 31.9 29.0 13.8 9.7
Phe 14.2 13.5 12.1 13.2 14.9 15.1 12.3 10.5 4.7 3.7
His 10.7 9.1 8.6 10.5 6.2 9.8 5.9 2.3 1.7 3.5
Lys 6.9 7.4 6.8 7.3 5.7 5.0 4.6 4.8 3.3 2.7
Arg 11.8 9.5 9.3 9.7 12.8 12.5 11.5 10.5 4.6 4.4
Sum 122.7 118.6 103.3 115.7 125.7 125.8 101.6 91.4 42.1 35.4
Total 281.0 285.2 245.8 277.5 309.6 304.8 251.0 231.8 107.0 86.7


content of DDGS subjected to pressure pre-treatment with
GA was 4.8~19.3 mg/g higher than low temperature pre-
treatment. These results seem to be due to growth differ-
ences of yeast cells caused by different degrees of starch
hydrolysis and the effects of different pre-treatment methods
and saccharifying enzymes. The highest content of total
amino acids was 309.6 mg/g in DDGS of Gangdaok pro-
duced by low temperature pre-treatment with GA, whereas
the highest content in Ambrosia was 277.5 mg/g obtained by
pressure pre-treatment with SGA.
The distribution and levels of essential amino acids of the
DDGS of the two cultivars were similar to those of the
grains. The percentage of lysine in the total amino acid con-
tent can estimate the quality of the lysine in DGGS, since
lysine is usually the first limiting amino acid in diets fed to
swine [22]. The concentration of lysine in the DGGS of the
two cultivars prepared with SGA was in the range of
2.5~2.7%, which is higher than that with GA (1.6~2.0%).
Therefore, SGA was preferred for saccharification over GA
in terms of nutritional quality of DGGS. Gangdaok grains
(42.1 mg) showed a higher content of essential amino acid
than Ambrosia (35.4 mg). The low content of essential
amino acids in the DDGS of Ambrosia seems to result from
the corresponding lower content of essential amino acids in
the grains. The pre-treatment and fermentation of mashes did
not alter the composition of the DDGS, except for the in-
creased level of amino acids derived from yeast cells. The
total essential amino acid content of Gangdaok DDGS was
lower than that of defatted soybean meal (231.3 mg) [23] but
higher than that of Ambrosia corn DDGS (101.6 mg). The
high level of essential amino acids in the DDGS of Gang-
daok in this study indicates the DDGS of Gangdaok can be a
highly valuable feed source.


CONCLUSION

The main objective of this work was to evaluate low tem-
perature pre-treatment of corn mash of the new Korean culti-
var Gangdaok before SSF. Its ethanol yield and amino acid
content of DDGS were also assessed. The optimal condition
for pre-treatment at low temperature was 98
o
C and 1.5 h reac-
tion time. According to the fermentation results, the ethanol
yield produced during low temperature pre-treatment with
SGA was almost identical to the pressure pre-treatment. The
low temperature pre-treatment may save 30~40% energy
compared with pressure pre-treatment with SGA. The DDGS
of Gangdaok contained more essential amino acids (21.1
RMO


mg/g) than did Ambrosia (USA corn), which is a widely used
material for feedstock. There were no distinct differences in
ethanol yield and amino acid concentration of DDGS between
low temperature pre-treatment and pressure pre-treatment.
In conclusion, the results indicate Gangdaok surpasses Am-
brosia in energy-economy and nutritional value of DDGS.
Therefore, Gangdaok can provide economic opportunities for
the bioethanol and feed industries.


AcknowIedgement We acknowledge the financial sup-
port provided by the Korean Rural Development Admini-
stration.


Received December 15, 2008; accepted February 28, 2009


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