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 Distiller’s Dried Grains

with Soluble

Gi-wook Choi1*, Yule Kim1, Keun and Kim2 1Changhae Institute of Cassava and Ethanol Research, Jeonju 561-203, Korea

2Department of Bioscience and Biotechnology, The University of Suwon, Hwaseong 445-743, Korea=

^Äëíê~Åí= 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 p~ÅÅÜ~êçãóÅÉë= ÅÉêÉîáëá~É CHY 1011(KCTC

11250BP) at 35°C. 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

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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

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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 CO2. 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

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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 α-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 β-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 MgSO4∙7H2O, and 1.0 g/L K2HPO4) [16].

pí~êÅÜ=s~äìÉ

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].

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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 120oC). Sam-ples were first hydrolyzed with α-amylase 0.8 g/kg (en-zyme/material) at 75oC 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].

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Before fermentation, liquefaction of corns before mashing

was performed by low temperature pre-treatment (98oC, 1.5 h) and pressure pre-treatment (120oC, 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 35oC 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 30oC 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 35oC 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.

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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 60oC with a flow rate of 1.0 mL/min and a sample volume of 20 μL. 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, 100oC; injection temperature, 200oC; flame ionization detector tem-perature, 250oC (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=

cáÖK=NK Starch value of Gangdaok and Ambrosia determined by

different methods of saccharification. , Acidic; , GA; ,

SGA.

of the theoretical yield [20].

^ãáåç=̂ ÅáÇ=`çåíÉåí One liter mash was concentrated into wet grain, crude pro-

tein, yeast culture, and ethanol using a rotary evaporator (NE-2001, Eyela, Japan) at 90oC 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 105oC for 2.5 h. DDGS were subjected to 6 N HCl in duplicate and hydrolyzed for 24 h at 110oC. 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

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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

cáÖK=OK Effect of temperature and reaction time on liquefaction value

of Gangdaok.

q~ÄäÉ=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

α-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.

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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 (120oC) for 1 h and low temperature pre-treatment (98°C) 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.

cÉêãÉåí~íáçå=oÉëìäí

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=

cáÖK=PK Change of carbohydrate, glycerol, and ethanol content during fermentation of Gangdaok mash treated with SGA and GA. ●, Dextrin; ○,

Maltose; ▼, Glucose; ▽, Glycerol; ■, Ethanol.

^= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = _=

`= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = a

cáÖK=QK Change of carbohydrate, glycerol and ethanol content during fermentation of Ambrosia mash treated with SGA and GA. ●, Dextrin; ○,

Maltose; ▼, Glucose; ▽, Glycerol; ■, Ethanol.

RMM=

cáÖK=RK Decreasing total sugar content in Gangdaok mashes treated

at different temperatures and by different saccharifying en-

zymes. ●, SGA 98°C; ○, SGA 120°C; ▼, GA 98°C; ▽, GA

120°C.

cáÖK=SK Decreasing total sugar content in Ambrosia mashes treated

at different temperatures and by different saccharifying en-

zymes. ●, SGA 98°C; ○, SGA 120°C; ▼, GA 98°C; ▽, GA

120°C.

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~ÄäÉ=OK Ethanol yield, fermentation rate, and percent of theoretical

yield of ethanol for two corn cultivars

Gangdaok Ambrosia

98°C 120°C 98°C 120°C

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 88.75Fermentation

rate (%) GA 84.93 84.36 83.12 86.77

SGA 94.03 93.61 94.01 94.14Percent 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.

^ëëÉëëãÉåí=çÑ=̂ ãáåç=~ÅáÇë

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~ÄäÉ=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

98°C 120°C 98°C 120°C 98°C 120°C 98°C 120°C 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. It’s ethanol yield and amino acid content of DDGS were also assessed. The optimal condition for pre-treatment at low temperature was 98oC 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.

^ÅâåçïäÉÇÖÉãÉåí 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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