Potential of Taiwan Sorghum Spirits Lees for the Production of

Bioethanol

Mei-Ying Su and Yuan-Tay Shyu*

Department of Horticulture, Nation Taiwan University, No.1, Sec. 4, Roosevelt Rd.,

Taipei City 106, Taiwan, R. O. C.

ABSTRACT
Background: In order to evaluate the availability of using Taiwan sorghum spirits lees
for bioethanol production. Methods: The series of physical treatment, microwave
radiation pretreatment, enzymatic hydrolysis and fermentation processes was
investigated in this study. Two kinds of samples are analyzed, respectively is comes
from Taiwan different production area Kinmen and Chyayi. Results: Composition
analysis suggested that the Kinmen sorghum spirits lees (KS) and Chyayi sorghum
spirits lees (CS) after mashing, contains on dry weight basis approximately 17.2±0.7
and 18.2±0.6% cellulose, 19.0±0.6 and 21.6±1.0% hemicellulose, 18.5±0.8 and
20.6±1.7% acid detergent lignin (ADL), 22.1±0.7 and 23.3±0.4% starch respectively.
Reducing sugars obtained by microwave radiation pretreatment and enzymatic
hydrolysis were achieved an almost 341.3 mg/g dry weight basis (CS). The hydrolyzate
from KS and CS were inoculated with Saccharomyces cerevisiae. The ethanol yields
ranges between 0.36 and 0.42 g/g reducing sugar. Conclusion: These results suggest
that Taiwan sorghum spirits lees are economically suitable biomass resource for
bioethanol production for an alternative to fossil fuels.

Keywords: Sorghum spirits lees; Bioethanol; Microwave radiation pretreatment;

Fermentation

1. Introduction

Sorghum liquor is a kind of popular white wines in the world. After separation of the
liquor, the voluminous residue is called sorghum spirits lees. In the making of Taiwan
sorghum liquor, the main fermentation ingredient is sorghum rice. The rice is processed
by botulinum cooking, gelatinization, and then mixed with rice hull for cooling. During
the fermentation process, the rice is not crushed, so the vinasse still contains a large
amount of starch and lignocellulose. The sorghum spirits lees are generally used as

1
animal feed, fertilizer or additive of skin care products (Lodge et al., 1997; Silva et al.,
2007; Al-Suwaiegh et al., 2002). The wastes produced in the making of sorghum liquor
are approximately 200t/day (Kinmen country government public announcement) in
Kinmen. The above-mentioned consumption of the wastes is very few. Nowadays the
fossil fuels are not sufficient, full utilization of agricultural wastes to produce bioethanol
can increase the economic value of brewing industry and reduce our dependence on
fossil fuels as a source of energy.
Microwave radiation is widely used in food and chemical synthesis industries
(Majetich and Hicks, 1995; Wan et al., 1990). It can conduct heat quickly and make the
object heated evenly from the inside to outside. Microwave heating applied to
pretreatment of lignocellulose, which is able to remove lignin and reduce crystallinity of
cellulose. Past study indicated that microwave technology applied to the degradation
lignocellulose of rice straw and increase the hydrolysis rate (Zhu et al., 2006). The
microwave radiation combined with acid or alkali liquid is used in treatment of brewer's
spend grains, and the reducing sugar yield is several times higher than the treatment
where only microwave radiation is used (Macheiner et al., 2003). A report in 2007
indicated that microwave combined with alkali is a promising pretreatment method to
enhance enzymatic hydrolysis of switchgrass (Keshwani et al., 2007). In comparison to
the traditional pretreatment methods such as acid (alkali) liquid, ammonia fiber
explosion, CO2 explosion, oxidative delignification, and physical steam explosion
(Alizadeh et al., 2005; Sun and Cheng, 2002), the microwave radiation combined with
diluted acid (alkali) not only can have higher heating efficiency and better destructive
effect on the fiber crystal zone but also reduce the byproduct and carbohydrate pyrolysis
which can hinder the alcoholic fermentation (Azuma et al., 1984; Ooshima et al., 1984;
Kitchaiya et al., 2003).
Most research has been focused on the category and concentration of acid (alkali)
when lignocellulose is pretreated by microwave radiation combined with diluted acid
(alkali) (Bjerre et al., 1996; Chosdu et al., 1993). However, the primary pH of every
kind of lignocelluloic feedstock is different, which would have varying degree of
neutralization to affect the microwave radiation pretreatment. This study compared the
efficiency of microwave radiation pretreatment under the different pH to find out the
influence of actual pH which has never been considered and discussed in previous
studies. Among various lignocelluloses, the Taiwan sorghum spirits lees contain a large
amount of starch and lignocellulose. Therefore, it is an extremely good fermentation
material. In the previous study, there is no finding about the treatment condition
affecting the fermentable carbohydrates production. In this experiment, the microwave
radiation combined with different pH is carried out in pretreatment. Then again by way

2
of enzymatic hydrolysis, analysis of hydrolysate quality of sorghum spirits lees is
performed, which is used in subsequent alcoholic fermentation with Saccharomyces
cerevisiae.

2. Methods

All experiments were done in triplicates and average values are reported.

2.1. Materials and analysis of biomass components

Chyayi sorghum spirits lees (CS) and Kinmen sorghum spirits lees (KS) were
obtained from Taiwan Tobacco and Liquor Corporation Chyayi Distillery, Chyayi
County, and Kinmen Kaoliang Liquor INC., Kinmen County, Taiwan, respectively.
Before pretreatment sorghum spirits lees were dried to constant weight at 60°C by oven
and reduced to a powder of 60 mesh using a milling machine. All results were
calculated on a dry weight basis. The determination of starch in lees according to
AOAC official method 996.11 amyloglucosidase-α-amylase method (AOAC, 1996).
Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin
(ADL) were determined by van Soest methods (1991) using FiberCap™ 2021/2023
system (FOSS analytical AB, Höganäs, Sweden). NDF is an estimate of the content of
hemicellulose, cellulose and lignin in samples. ADF includes cellulose and lignin as the
major components. Hemicellulose was calculated by the difference between NDF and
ADF. Cellulose was calculated by the difference between ADF and ADL.

2.2. Microwave radiation pretreatment

Before microwave radiation pretreatment, powdered sorghum spirits lees were done
in different treatments of washing. (i) Wash group: Washing with deionized water until
neutral pH and then dried to constant weight at 60°C. (ii) Unwashed group. Those
samples were mixed in deionized water at concentrations 10% (w/v), and then adjusted
to pH 5 , 6, 7, 8, and 9 with 1 N sodium hydroxide (NaOH) or 1 N hydrochloric acid
(HCl) before microwave radiation pretreatment. The biomass mixtures of 100 ml were
placed in 250 ml sealed serum bottles and heated by microwave reactor (NE-R30A,
National Co., Taiwan) at 800 w for 3 min. The pretreated solid residue were washed
with distilled water and then dried for enzymatic hydrolysis.

2.3. Enzymatic hydrolysis

3
 In enzymatic hydrolysis, pretreated samples of 1 g was performed in 250 ml
Erlenmeyer flasks and mixed in 30 ml of 50 mM sodium citrate buffer to control a pH
of 4.8. The enzymes used for enzymatic hydrolysis of cellulose were cellulase from
Trichoderma reesei (E.C. 3.2.1.4) (Sigma-Aldrich Co., USA) at the dosage of 25 FPU/g
dry basis and cellobiase from Aspergillus niger (Sigma-Aldrich Co., USA) at the dosage
of 30 IU/g. The function of cellobiase was to avoid cellobiose inhibition of cellulose
(Ryu and Mandels, 1980). Microbial growth was inhibited by the addition of 0.3% (w/w)
sodium azide. The samples were incubated in an orbital shaking incubator with a
stirring speed of 160 rpm for 48 h at 50 °C, and then the resulting slurry was collected
for starch degradation.
The enzyme used for starch degradation was α-amylase (E.C. 3.2.1.1,
Sigma-Aldrich Co., USA). The hydrolysis solutions were performed at pH of 6.9 by 1N
NaOH and then the dosage of 0.05% (w/v) α-amylase was added. The mixture was
incubated in an orbital shaking incubator with a stirring speed of 160 rpm for 12 h at 20
°C. After hydrolysis, the hydrolysate was heated to 100°C for 3 min to inactivate the
enzyme, and then cool to room temperature. The hydrolysate was centrifuged at 8,000
rpm for 10 min and filtered through a 0.22 μm filter. Liquid was collected for sugar
analysis. The sediment was dried to constant weight at 60°C by oven for analysis of
starch, NDF, ADF, and ADL as previously described.

2.4. Ethanol fermentation

Saccharomyces cerevisiae YPH-499 [MAT a ura3-52 lys2-801 ade2-101 trp1-△63

his3-△200 leu2-△1] was used as fermentation strain. Precultures, 50 ml of YPD
medium (yeast extract 1%, peptone 2%, and dextrose 2%) in 250 ml Erlenmeyer flasks,
were incubated overnight at 30°C and were shaken at 160 rpm in an orbital shaking
incubator. Cells were harvested by centrifugation (8,000 rpm, 5 min, and 4°C) and were
washed once with 0.9% (w/v) NaCl solution before inoculation. The defined mineral
medium (Verduyn et al., 1992) of 100 ml with enzymatic hydrolysates of 30 ml was
performed in 100 ml Erlenmeyer flasks for fermentation. The pH was adjusted to 7.0
with 1N NaOH, and then inoculated with S. cerevisiae YPH-499 at 30°C for 72 h.
Oxygen-limited conditions of the medium were controlled by water seal. After
fermentation, liquid were filtered through a 0.22 μm filter and collected for ethanol
content and residual reducing sugars analysis.

2.5. Analytical methods

4
 Monosaccharides and ethanol were detected by high performance liquid
chromatography (HPLC) (Jasco PU-1580, Japan spectroscopic Co., LTD, Japan) with a
refractive index detector (Waters 410, Milford, MA, USA). Glucose, xylose, and
ethanol were separated using an SUPELCOGEL C-610H HPLC column, 30 cm × 7.8
mm ID (Supelco, Inc., USA) at 30°C and a flow rate of 0.3 ml/min with 0.1%
phosphoric acid as eluent. Acetic acid was separated on the same column at 40°C using
0.1% phosphoric acid as eluent at a flow rate of 0.5 mL/min, and detected using a Jasco
870-UV (Japan spectroscopic CO., LTD. Japan). All samples were filtered through a
0.22 µm filter before analysis. The reducing sugar based on the method using
3,5-dinitrosalicylic acid reagent (DNS method) (Miller, 1959).

3. Results and discussion

3.1 Biomass components

The main compositions of Sorghum spirits lees on dry-weight basis are shown in
Table 1. Water content of KS and CS were 74.9±0.8 and 73.2±1.3% respectively. The
sorghum rice is directly processed by botulinum cooking and then gelatinization in the
sorghum liquor manufacture. Therefore, sorghum rice remains nearly intact after
fermentation. A large amount of starch is left, which is 22-23% of dry weight basis. In
addition, the sorghum bran and the mixed rice hull can generate cellulose and
hemicellulose, which is the good source of carbohydrate. According to the experimental
results, their contents are 17.2±0.7 to 18.2±0.6% and 19.0±0.6 to 21.6±1.0% of dry
weight basis. In comparison to the lignocellulosic feedstocks, such as rice straw,
switchgrass, barley straw, wheat straw, and corn stove used to produce the biomass
energy (Zhu et al., 2006; Badger, 2002; McDonald et al., 1995; Belkacemi et al., 1998;
Sun and Cheng, 2002; Wiselogel et al., 1996; Saha et al., 2005), the sorghum spirits lees
contain higher percentage of starch. Moreover, unlike the starchy materials for ethanol
production will compete for the limited land against food and feed production (Sun and
Cheng, 2002).

3.2 Sugar yields after microwave radiation pretreatment and enzymatic hydrolysis

The sorghum spirits lees was dried and crushed to dissolve in the deionized water,
which pH is 3.7. The acidic sources were the fermented products of liquor process, such
as amino acid, fatty acid, acid from aldehyde oxidation, acid from ester hydrolysis, and

5
tannin (Zou and Shi, 2003). In order to discuss the wash and unwashed treatment of
sorghum spirits lees affecting the sugar yield, a part of sorghum spirits lees powder was
washed in deionized water for neutralization, and dried to reach constant weight before
microwave radiation pretreatment.

3.2.1 Reducing sugar

The reducing sugar yields from enzymatic hydrolysis of microwave pretreatment

combined with varying pH, were shown in Fig. 1.
In the wash group, the reducing sugar yields were higher when the pH of KS and CS
is adjusted to 5 in microwave radiation pretreatment, CS can reach 341.3 mg/g dry
weight basis. When the pH was increased to neutral value and the reducing sugar yields
were reduced to 134.6 (KS) and 155.2 mg/g dry weight basis (CS). If pH were adjusted
by NaOH to the alkali, the reducing sugar yields increased with the strong alkalinity.
The reducing sugar was 193.4(KS) and 211.3 mg/g dry weight basis (CS) when the pH
reaches a value of 9. Thus in this experiment, HCl treatment has a greater affect on the
production of reducing sugar than NaOH.
An unwashed group, the primary pH of KS and CS were 3.7 before pretreatment.
Thus pH was adjusted with NaOH in pretreatment. According to the experimental result,
the reducing sugar yields of KS or CS would increase with increase of pH value. When
the pH reaches a value of 9, the reducing sugar yields were most 142.7(KS) and 139.5
mg/g dry weight basis (CS). The addition of NaOH is the positive factor for hydrolysis
of sorghum spirits lees.
Comparison of the wash group and unwashed group, the overall reducing sugar yields
was higher in the wash group. It can be inferred the original acidic component in
sorghum spirits lees may impede the hydrolysis. A report in 2003 indicated that tannin
may affect the activity of amylase (Zou and Shi, 2003). Thus the removal of acidic
component from the raw material can increase the reducing sugar yields in the
hydrolysate.
Comparison of reducing sugar yield of KS and CS, under the pretreatment conditions
with different pH values, the reducing sugar yields of CS was more than that of KS
because the cultivated sorghum varieties planted in Taiwan was Taichung No. 5 and
Kinmen No. 9. The Taichung No. 5 was cultivated in Taiwan Jianan area and supplied
for Taiwan Tobacco and Liquor Corporation Chyayi Distillery. The Taichung No. 5 and
Kinmen No. 9 were cultivated in Kinmen area and supplied for Kinmen Kaoliang
Liquor INC. to produce sorghum liquor. The Taichung No. 5 belongs to the yellow
series which seed was non- glutinous sorghum, and the Kinmen No. 9 belongs to the

6
brown series which seed was glutinous sorghum with higher content of tannin (Sung et
al., 2004), so the hydrolysis of KS was more difficult.

3.2.2 Monosaccharides

After pretreatment and enzymatic hydrolysis of KS and CS, the starch and cellulose
hydrolysis can produce glucose (Fig. 2). The treatment affects the glucose yield, (i) for
the wash group where microwave radiation pretreatment was carried out under the acid
or alkali condition, the glucose yields was greater than that of neutral one; the maximum
glucose yield was 214.5(KS) and 287.5 mg/g dry weight basis (CS) at pH 5; (ii) for the
unwashed group where microwave radiation pretreatment was carried out when NaOH
was increased, the glucose yield was increased; (iii) the glucose yield of wash group
was higher than unwashed, and the glucose yield of CS was obviously greater than that
of KS. The factors were the same with the above-mentioned one affecting the reducing
sugar.
After pretreatment and enzymatic hydrolysis of KS and CS to destroy the fiber crystal
zone, the pentose fraction in hemicellulose consists mainly of xylose (Fig. 3). The
maximum yield was 37.6 mg/g dry weight basis (CS). The yield was higher in the wash
group under the acidic condition, and the yield of CS was higher than KS. But, in the
unwashed group, the increase of pH value with NaOH has no effect on the xylose yield.
Obviously, HCl combined with microwave radiation pretreatment could improve the
efficiency of xylose yield. In comparison to steam explosion of straws, sweet sorghum
residue and other wastes (Ballesteros et al., 2004), more xylose could be obtained under
the pretreatment condition in this experiment. In addition, the water washing could
remove the original acidic substance from the sorghum spirits lees, which was helpful
for xylose yield.

3.3 Residue of starch, cellulose, hemicellulose and ADL after pretreatment and
enzymatic hydrolysis

The Residue of starch, cellulose, hemicellulose and ADL are presented on Fig. 4 and
5. The starch in the sorghum spirits lees could produce maltose and glucose after
pretreatment and enzymatic hydrolysis. Besides, the cellulose hydrolysis could produce
glucose. The results indicated that under the acidic condition, the residuals of starch and
cellulose is lower in the wash group (Fig. 4 and 5). For this reason starch and cellulose
could be almost hydrolyzed in the experiment. The hydrolysis ratio equations are as
follows:

7
( raw content in sorghum liquor wastes% - residue after hrdrolysis %)
× 100% (1)
raw content in sorghum liquor wastes%
Equation (1) describes the hydrolysis ratio of starch and cellulose, which maximum
yield was 89.2% and 70.3% when the pH was 6 and 5 respectively in the wash group
(CS). The hemicellulose could produce xylose after microwave radiation pretreatment
(Fig. 4 and 5). The hydrolysis ratio of hemicellulose was calculated in equation (1),
which could reach 37.9% when the pH was 5 in wash group (CS). The microwave
radiation pretreatment combined with HCl seem to be effective method to obtain xylose
from hemicellulose. ADL was phenolic compound couldn’t be hydrolyzed with
cellulosic enzyme. It could be destructed by chemical agents so as to promote the
digestibility of hemicellulose (Fig. 4 and 5) (Cameron et al., 1991; Kerley et al., 1988).

3.4 Acetic acid yields after pretreatment and enzymatic hydrolysis

Acetic acid is suggested to be common fermentation inhibitor in lignocellulosic

hydrolysates (Baugh et al., 1988), which could be released during pretreatment and
affecting the S. cerevisiae fermentative performance (Larsson et al., 1999). The
potential influence of acetic acid should be considered in relation to its concentration in
the hydrolysate, because ethanol yield could be increased by low level of acetic acid,
whereas ethanol yield decreases at higher concentrations (Olsson and Hahn-Hagerdal,
1996; Larsson et al., 1999; Palmqvist et al., 1999). It was discussed that low
concentration of acids stimulate the production of ATP, and biomass could be form with
less ATP (Larsson et al., 1999). In this study, acetic acid content in the hydrolysate
ranges between 0.07-0.21 mg/g dry weight basis (Fig. 6), which increased with the pH
decreased in pretreatment. Obviously, NaOH has a greater inhibition on the production
of acetic acid than HCl.

3.5 Ethanol yields after fermentation of the hydrolyzate

Hydrolysate of KS and CS could be fermentation with S. cerevisiae to produce

ethanol (Fig. 7). The ethanol concentration ranges between 0.36-0.42 g/g reducing sugar.
The maximum ethanol yield could reach as high as 0.42 g/g reducing sugar when the
pH of CS is adjusted to 5 under the pretreatment conditions, and the yield accounts for
82.4% of the theoretical yield (Chen et al., 2007). Thus the sorghum spirits lees could
produce fermentable carbohydrate after hydrolysis, which certainly be used in
production of ethanol.

8
3.6 Residual reducing sugars after fermentation of the hydrolyzate

After the hydrolysate was fermentation with S. cerevisiae, the residual reducing sugar
and utilization ratio were regarded as the basis to evaluate the sufficiency of
fermentation (Table 2). In this experiment, the reducing sugar utilization ratio equation
is as follow:
( reducing sugar yield after hydrolysis - residual reducing sugar after fermentation)
× 100% (2)
reducing sugar yield after hydrolysis
Equation (2) describes the reducing sugar utilization ratio ranges, which from 71.6 to
83.9%. After fermentation of hydrolysate, the residual of reducing sugar was 55.0 mg/g
dry weight basis and the maximum utilization ratio could reach as high as 83.9% when
the pH was 5 (CS) under the pretreatment conditions. Equally, the ethanol yield was
highest in the same condition.

Conclusions

The sorghum spirits lees were produced in agricultural processing. Through the
analysis in this study, it contains a large amount of carbohydrate and suitable for
production of bioethanol. The optimal factors that affect production of bioethanol from
sorghum spirits lees can be drawn from this study: (i) When the original acidic
substance was removed from the feedstock, the hydrolysate has higher yield of
fermentable carbohydrate; (ii) The hydrolysate could have higher yield of fermentable
carbohydrate after microwave radiation pretreatment combined with HCl and
subsequent enzymatic hydrolysis; (iii) After the hydrolysate was processed by S.
cerevisiae fermentation, the ethanol yield could reach the theory threshold of 82.4%,
and the utilization ratio of reducing sugar could reach 83.9%. Thus, it can be concluded
that only little suppressor inhibit the fermentation. This study evaluates treatment
method and identifies conditions for the sorghum spirits lees transformed to the
bioethanol was the valuable reference information. Further studies should be focused on
simultaneous saccharification and fermentation and find more efficient ways of utilizing
pentose and hexose to the fermentation process.

References
Alizadeh, H., Teymouri, F., Gilbert, T.I., Dale, B.E., 2005. Pretreatment of switchgrass
by ammonia fiber explosion (AFEX). Appl. Biochem. Biotechnol. 121(1-3):
1133-1141.
Azuma, J., Tanaka, F., Koshijima, T., 1984. Enhancement of enzymatic susceptibility of

9
 lignocellulosic wastes by microwave irradiation. J. Ferment. Technol. 62 (4),
377–384.
Al-Suwaiegh, S., Fanning, K.C., Grant, R.J., Milton, C.T., Klopfenstein, T.J., 2002.
Utilization of distillers grains from the fermentation of sorghum or corn in diets for
finishing beef and lactating dairy cattle. J. Anim. Sci. 80, 1105–1111.
Badger, P.C., 2002. Ethanol from cellulose: a general review. Trends in new crops and
new uses. ASHS Press, Alexandria, VA.
Ballesteros, M., Oliva, J.M., Negro, M.J., Manzanares, P., Ballesteros, I., 2003. Ethanol
from lignocellulosic materials by a simultaneous saccharification and fermentation
process (SFS) with Kluyveromyces marxianus CECT 10875. Process Biochemistry
39, 1843–1848.
Baugh, K.D., Levy, J.A., McCarty, P.L. 1988. Thermochemical pretreatment of
lignocellnlose to enhance methane fermentation: II. Evaluation and application of
pretreatment model. Biotechnol. Bioeng. 31, 62-70
Belkacemi, K., Turcotte, G., de Halleux, D., Savoie, P., 1998. Applied Biochemistry
and Biotechnology 70–72, 441–462.
Bjerre, A.B., Olesen, A.B., Fernqvist, T., 1996. Pretreatment of wheat straw using
combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose
and hemicellulose. Biotechnol. Bioeng. 49, 568–577.
Cameron, M.G., Cremin Jr., J.D., Fahey Jr., G.C., Clark, J.H., Berger, L.L., Merchen,
N.R., 1991. Chemically treated oat hulls in diets for dairy heifers and wethers:
effects on intake and digestion. J. Dairy Sci. 74, 190-210.
Chen, Y., Sharma, R.R., Keshwani, D., Chen, C., 2007. Potential of Agricultural
Residues and Hay for Bioethanol Production. Appl Biochem Biotechnol 142,
276–290.
Chosdu, R., Hilmy, N., Erizal, Erlinda, T.B., Abbas, B., 1993. Radiation and chemical
pretreatment of cellulosic waste. Radiat. Phys. Chem. 42, 695–698.
Deepak R. Keshwani, D.R., Cheng, J.J., Burns, J.C., Li, L., Chiang, V., 2007.
Microwave Pretreatment of Switchgrass to Enhance Enzymatic Hydrolysis. An
ASABE Meeting Presentation Paper Number: 077127
Kerley, M.S., Garleb, K.A., Fahey Jr., G.C., Berger, L.L., Moore, K.J., Phillips, G.N.,
Gould, J.M., 1988. Effects of alkaline hydrogen peroxide treatment of cotton and
wheat straw on cellulose crystallinity and on composition and site and extent of
disappearance of wheat straw cell wall phenolic and monosaccharides by sheep. J.
Anim. Sci. 66, 3235-3244.
Kinmen country government public announcement. http://www.kinmen.gov.tw/Layout
/main_ en/index.aspx?frame=18

10
Kitchaiya, P., Intanakul, P., Krairish, M., 2003. Enhancement of enzymatic hydrolysis
of lignocellulosic wastes by microwave pretreatment under atmospheric pressure. J.
Wood Chem. Technol. 23 (2), 217–225.
Larsson, S., Palmqvist, E., Hahn- Hagerdal, B., Tengborg, C., Stenberg, K., Zacchi, G.,
et al., 1999. The generation of fermentation inhibitors during dilute acid hydrolysis
of softwood. Enzyme Microbial Technol. 24, 151-159.
Lodge, S.L., Stock, R.A., Klopfenstein, T.J., Shain, D.H., Herold, D.W., 1997.
Evaluation of corn and sorghum distillers byproducts. J. Anim. Sci. 75, 37-43.
Macheiner, D., Adamitsch, B.F., Karner, F., Hampel, W.A., 2003. Pretreatment and
Hydrolysis of Brewer’s spent grains. Eng. Life Sci. 3 (10), 401-405.
Majetich, G., Hicks, R., 1995. The use of microwave heating to promote organic
reactions. J. Microwave Power Electromagnetic Energy 30 (1), 27–45.
McDonald, P., Edwards, R.A., Greenhalgh, J.F.D., Morgan, C.A., 1995. In 5th (Ed.),
Animal nutrition. Longman Scientific and Technical, England.
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Anal. Chem. 31, 420–428.
Olsson, L., Hahn-Hagerdal, B., 1996. Fermentation of lignocellulosic hydrolysates for
ethanol production. Enzyme and Microbial Technology 18, 312-331.
Ooshima, H., Aso, K., Harano, Y., 1984. Microwave treatment of cellulosic materials
for their enzymatic hydrolysis. Biotechnol. Lett. 6 (5), 289–294.
Palmqvist, E., Grage, H., Meinander, N.Q., Hahn-Hagerdal, B., 1999. Main and
interaction effects of acetic acid, furfural, and phydroxybenzoic acid on growth and
ethanol productivity of yeasts. Biotechnol Bioeng 63, 46–55.
Ryu, D.D.Y., Mandels, M., 1980. Cellulases - biosynthesis and applications. Enzyme
Microb. Technol. 2(2), 91-102.
Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y.V., 2005. Process Biochemistry, 40(12),
3693–3700.
Shih, S., 2009. Sorghum liquor helps build Kinmen's fortunes. Taiwan Journal, 26(1).
Silva, J.C., Cole, N.A., Brown, M.S., Ponce, C.H., Smith, D.R., 2007. Effects of dietary
fat and wet sorghum distiller's grains plus solubles on feedlot performance and
carcass characteristics of finishing heifers. 2007 Beef Cattle Research in Texas. The
Texas A&M University System, The Agriculture Program. p.83-88.
Sun, Y., Cheng, J.J., 2002. Hydrolysis of lignocellulosic materials for ethanol
production: a review. Bioresource Technology 83(1), 1–11.
Sung, W.C., Huang, C.H., Wang, B.S., 2004. Investigation on the change of mochi with
the addition of sorghum. Chia Nan Annual Bulletin 30, 42-52.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Symposium: Carbohydrate

11
 methodology, metabolism, and nutritional implications in diary cattle, Journal of
Diary Science 74(10), 3583-3597.
Verduyn, C., Postma, E., Scheffers, W.A., Van Dijken, J.P., 1992. Effect of benzoic
acid on metabolic fluxes in yeasts: a continuous culture study on the regulation of
respiration and alcoholic fermentation. Yeast 8, 501- 517.
Wan, J., Tse, M., Husby, H., Depew, M., 1990. High-power pulsed microwave catalytic
processes: decomposition of methane. J. Microwave Power Electromagnetic Energy
25 (1), 32–38.
Wang, B., Ge, X.M., Li, N., Bai, F.W., 2006. Continuous Ethanol Fermentation
Coupled with Recycling of Yeast Flocs. Chinese Journal of Biotechnology 22(5),
816-821.
Wiselogel, A., Tyson, S., Johnson, D., 1996. Handbook on bioethanol: Production and
utilization. Taylor and Francis. Washington, DC.
Zhu, S., Wu, Y., Yu, Z., Zhang, X., Li, H., Gao, M., 2006. The effect of microwave
irradiation on enzymatic hydrolysis of rice straw. Bioresource Technology 97,
1964–1968.
Zou, J., Shi, Y., 2003. Chapter VIII: Industrial use of sorghum. Post-harvest
Compendium.

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Table 1 Compositions of Sorghum spirits lees a (as percentage on dry weight basis)
Substrate Starch Cellulose Hemicellulose ADL
KS 22.1±0.7 17.2±0.7 19.0±0.6 18.5±0.8
CS 23.3±0.4 18.2±0.6 21.6±1.0 20.6±1.7
a
Data are mean values of triplicate analysis±standard deviation.

13
 350
KS(wash)
Yields of reducing sugar (mg/g dry weight basis)

KS(unwashed)
300 CS(wash)
CS(unwashed)
250

200

150

100

50

0
5 6 7 8 9
Pretreatment condition (pH)

Fig. 1 Yields of reducing sugar from KS and CS after microwave radiation pretreatment
and enzymatic hydrolysis.

14
 300

KS(wash)
Yields of glucose (mg/g dry weight basis)

250 KS(unwashed)
CS(wash)
CS(unwashed)
200

150

100

50

0
5 6 7 8 9
Pretreatment condition (pH)

Fig. 2 Yields of glucose from KS and CS after microwave radiation pretreatment at

different pH and enzymatic hydrolysis.

15
 40
KS(wash)
35
Yields of xylose (mg/g dry weight basis)

KS(unwashed)
CS(wash)
30 CS(unwashed)

25

20

15

10

0
5 6 7 8 9
Pretreatment condition (pH)

Fig. 3 Yields of xylose from KS and CS after microwave radiation pretreatment at

different pH and enzymatic hydrolysis.

16
 Starch(wash)
Starch(unwashed)
Cellulose(wash)
Cellulose(unwashed)
Hemicellulose(wash)
Hemicellulose(unwashed)
ADL(wash)
18 ADL(unwashed)
Residues (as percentage on dry weigh basis)

16

14

12

10

0
5 6 7 8 9
Pretreatment condition (pH)

Fig. 4 Residues of starch, cellulose, hemicellulose and ADL from KS after microwave
radiation pretreatment at different pH and enzymatic hydrolysis

17
 Starch(wash)
Starch(unwashed)
Cellulose(wash)
Cellulose(unwashed)
Hemicellulose(wash)
Hemicellulose(unwashed)
ADL(wash)
ADL(unwashed)
20
Residues (as percentage on dry weight basis)

18

16

14

12

10

0
5 6 7 8 9
Pretreatment condition (pH)

Fig. 5 Residues of starch, cellulose, hemicellulose and ADL from CS after microwave
radiation pretreatment at different pH and enzymatic hydrolysis

18
 0.25
KS(wash)
Acetic acid content (mg/g dry weight basis)

KS(unwashed)
0.20 CS(wash)
CS(unwashd)

0.15

0.10

0.05

0.00
5 6 7 8 9
Pretreatment condition (pH)

Fig. 6 Acetic acid content from KS and CS after microwave radiation pretreatment at
different pH and enzymatic hydrolysis

19
 KS(wash)
0.5 KS(unwashed)
CS(wash)
CS(unwashed)
Ethanol yields (g/g reducing sugar)

0.4

0.3

0.2

0.1

0.0
5 6 7 8 9
Pretreatment condition (pH)

Fig. 7 The ethanol yields after fermentation of the hydrolyzate from KS and CS with S.
cerevisiae.

20
Table 2 Residual reducing sugars and utilization ratio after fermentation of the
hydrolyzate from KS and CS with S. cerevisiae.

Residual reducing sugar Reducing sugar

Pretreatment
Substrate (mg/g dry weight basis) utilization ratio (%)*
condition(pH)
wash wash wash unwashed
KS 5 59.7 23.7 81.9 77.9
6 69.1 29.0 77.9 73.7
7 24.7 26.4 81.6 77.8
8 45.9 36.5 75.8 71.8
9 42.9 40.5 77.8 71.6
CS 5 55.0 26.7 83.9 75.9
6 56.7 25.0 81.8 78.0
7 27.9 30.6 82.0 75.6
8 48.3 37.2 76.1 72.0
9 54.9 36.6 74.0 73.8
* Reducing sugar utilization ratio (%) :
( reducing sugar yield after hydrolysis - residual reducing sugar after fermentation)
× 100%
reducing sugar yield after hydrolysis

21