Bioresource Technology 100 (2009) 41764182

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Bioresource Technology
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Ethanol production from sweet sorghum juice using very high gravity
technology: Effects of carbon and nitrogen supplementations
Lakkana Laopaiboon a,b,*, Sunan Nuanpeng c, Penjit Srinophakun d, Preekamol Klanrit a, Pattana Laopaiboon a
a

Department of Biotechnology, Faculty of Technology, Khon Kaen University, 123 Mittraparp Road, Khon Kaen 40002, Thailand
Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand
c
Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand
d
Department of Chemical Engineering, Faculty of Engineering, Kasertsart University, Bangkok 10900, Thailand
b

a r t i c l e

i n f o

Article history:
Received 13 November 2008
Received in revised form 12 March 2009
Accepted 13 March 2009
Available online 17 April 2009
Keywords:
S. cerevisiae
Ethanol production
VHG fermentation
Sweet sorghum juice
Sugarcane molasses

a b s t r a c t
Ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 was investigated under
very high gravity (VHG) fermentation and various carbon adjuncts and nitrogen sources. When sucrose
was used as an adjunct, the sweet sorghum juice containing total sugar of 280 g l1, 3 g yeast extract l1
and 5 g peptone l1 gave the maximum ethanol production efciency with concentration, productivity
and yield of 120.68 0.54 g l1, 2.01 0.01 g l1 h1 and 0.51 0.00 g g1, respectively. When sugarcane
molasses was used as an adjunct, the juice under the same conditions gave the maximum ethanol concentration, productivity and yield with the values of 109.34 0.78 g l1, 1.52 0.01 g l1 h1 and
0.45 0.01 g g1, respectively. In addition, ammonium sulphate was not suitable for use as a nitrogen
supplement in the sweet sorghum juice for ethanol production since it caused the reduction in ethanol
concentration and yield for approximately 14% when compared to those of the unsupplemented juices.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Ethanol production as an alternative to petroleum-based fuels
can be produced from biomass, a plentiful renewable resource.
To increase the productivity and cost effectiveness of ethanol production, many process improvements including very high gravity
(VHG) technology have been studied. VHG fermentation technology is dened as the preparation and fermentation to completion
of mashes containing 270 or more grams of dissolved solids per litre (Bayrock and Ingledew, 2001). It has several advantages for
industrial applications such as the increase in both the ethanol
concentration and the rate of fermentation, which reduce capital
costs, energy costs per litre of alcohol and the risk of bacterial contamination (Thomas et al., 1996; Bvochora et al., 2000; Bayrock
and Ingledew, 2001; Bai et al., 2004).
Apart from sugarcane (in Brazil), corn grain (in USA), tapioca
starch and sugarcane molasses (in Thailand), other agricultural
raw materials rich in fermentable carbohydrates, including sweet
sorghum, have been of particular interest for biological transformation into ethanol for use as fuel or fuel additive (Schaffert,
1995; Gksungur and Zorlu, 2001). Sweet sorghum has been
promised as a large scale energy crop because its stalks contain

* Corresponding author. Address: Department of Biotechnology, Faculty of

Technology, Khon Kaen University, 123 Mittraparp Road, Khon Kaen 40002,
Thailand. Tel./fax: +66 43 362121.
E-mail address: lakcha@kku.ac.th (L. Laopaiboon).
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.03.046

high fermentable sugar and it can be cultivated in nearly all temperatures and tropical climate areas (Sree et al., 1999). It is also
one of the most drought resistant agricultural crops because of
its capacity to remain dormant during the driest periods (Woods,
2000).
It was reported that under appropriate environmental and
nutritional conditions, Saccharomyces cerevisiae can produce
and tolerate high ethanol concentrations (Thomas et al., 1996;
Bafrncova et al., 1999). VHG fermentation process exploits the
observation that the growth of S. cerevisiae is promoted and prolonged when very low but adequate levels of oxygen are present
and assimilable nitrogen levels are not limiting (Casey and
Ingledew, 1986). Several investigators have observed that yeast extract (Casey et al., 1984; Thomas and Ingledew, 1990; Jones et al.,
1994; Bafrncova et al., 1999), ammonium (Jones et al., 1994), urea
(Jones and Ingledew, 1994a), calcium and magnesium (Dombek
and Ingram, 1986) have protective effects either on growth and fermentation or viability, which stimulate the fermentation rate and
ethanol production. Our previous study showed that S. cerevisiae
NP01 isolated from Long-pang (Chinese yeast cake) for Sato (Thai
rice wine) making was a high-ethanol-producing strain under
VHG condition (Laopaiboon et al., 2008). As total soluble solids in
the sweet sorghum juice cv. KKU 40 has only 18Bx (grams per
100 ml), ethanol fermentation under VHG supplemented with
other carbon sources in order to raise the sugar content in the juice
needs to be investigated. Characteristics of raw sweet sorghum
juice cv. KKU 40 are shown in Table 1. The aim of this study was

L. Laopaiboon et al. / Bioresource Technology 100 (2009) 41764182

Table 1
Characteristics of raw sweet sorghum juice cv. KKU40 and sugarcane molasses from
Mitr Phu Viang Sugar Mill, Nongrua, Khon Kaen, Thailand.
Constituents

pH
Total soluble solid (Bx)
Total sugar (g l-1)
Glucosea (g l1)
Fructosea (g l1)
Sucrosea (g l1)
b
NH
4 N (ppm)
b
NO
3 N (ppm)
Total Pc (ppm)
Total Kd (ppm)
Total Nad (ppm)
Total Se (ppm)
Total Caf (ppm)
Total Mgf (ppm)
Total Fef (ppm)
Total Mnf (ppm)
Total Cuf (ppm)
Total Znf (ppm)
a
b
c
d
e
f

Contents
Sweet sorghum juice

Molasses

4.9
18
173.02
20.85
16.80
124.05
21.4
4.4
20
1790
170
120
166
194
2
3
0.3
1.4

4.9
85
696.04
95.42
169.79
387.53

1602
2985
150
56
2.8
7.4

By HPLC.
MgO-Devarda alloy distillation method.
Vanado-molybdate method.
Flame photometry method.
Turbidimetry method.
Atomic absorption.

to compare the efciency of ethanol production from sweet sorghum juice supplemented with sucrose or sugarcane molasses under VHG fermentation using S. cerevisiae NP01. The inuences of
yeast extract and peptone (YEP) or ammonium sulphate
[(NH4)2SO4] as nitrogen sources for ethanol production were also
studied.
2. Methods
2.1. Microorganism and inoculum preparation
S. cerevisiae NP01 isolated from Long-pang (Chinese yeast cake)
from Nakorn Pranom province, Thailand, was inoculated into a
250-ml Erlenmeyer ask containing 150 ml of yeast and malt extract (YM) medium. The medium contained (in g l1) yeast extract
3, peptone 5, malt extract 3 and glucose 10. The ask was incubated on a rotating shaker at 100 rpm, 30 C for 15 h. To increase
cell concentration, the yeast (approximately 3%) was transferred
into a 500-ml Erlenmeyer ask with 360 ml of the YM medium
containing 150 g l1 of glucose to give the initial cell concentration
of 1  106 cells ml1. The asks were further incubated under the
conditions previous mentioned. After 15 h, the cells were harvested and used as an inoculum for ethanol production.
2.2. Raw materials
Sweet sorghum juice cv. KKU40 modied from cv. Keller was
obtained from Department of Agronomy, Faculty of Agriculture,
Khon Kaen University, Thailand. After extraction, the juice was
kept at 18 C until use. Sugarcane molasses obtained from Mitr
Phu Viang Sugar Mill, Nongrua, Khon Kaen, Thailand was kept at
4 C until use.
2.3. Ethanol production medium
Sweet sorghum juice (pH 4.9) containing total soluble solids of
18Bx was adjusted with sucrose or molasses. The total soluble
solids in the juice were adjusted to 24, 28 and 32Bx by sucrose

4177

addition and to 28, 32 and 34Bx by molasses addition, which corresponded to total sugar concentrations of approximately 240, 280
and 320 g l1, respectively. Then the juices were supplemented
with 8 g l1 of YEP (3 g yeast extract and 5 g peptone) or 1.3 g l1
of (NH4)2SO4, and used as ethanol production (EP) medium without pH adjustment. The EP medium was transferred into a 500ml air-locked Erlenmeyer ask with a nal working volume of
400 ml and autoclaved at 110 C for 15 min.
2.4. Fermentation conditions
The sterile EP medium containing various sugar and nitrogen
supplements was inoculated to give the initial yeast cell concentration of 1  108 cells ml1. The fermentation was carried out in
batch mode at 30 C under static condition. The samples were
withdrawn at time intervals for analysis.
2.5. Analytical methods
The viable yeast cell numbers and total soluble solids of the fermentation broth were determined by direct counting method
using haemacytometer and hand-held refractometer, respectively.
The fermentation broth was centrifuged at 13,000 rpm for 10 min.
The supernatant was then determined for residual total sugar in
terms of total carbohydrate by phenol sulfuric acid method (Mecozzi, 2005). Ethanol concentration (P, g l1) was analyzed by gas
chromatography (Shimadzu GC-14B, Japan, Solid phase: polyethylene glycol (PEG-20 M), carrier gas: nitrogen, 150 C isothermal
packed column, injection temperature 180 C, ame ionization
detector temperature 250 C; C-R7 Ae plus Chromatopac Data Processor) and 2-propanol was used as an internal standard (modied
from Laopaiboon et al., 2007). The ethanol yield (Y ps ;) was calculated as the actual ethanol produced and expressed as g ethanol
per g total sugar utilized (g g1). The volumetric ethanol productivity (Q p ; g l1 h1) was calculated by ethanol concentration produced (P; g l1) divided by fermentation time giving the highest
ethanol concentration. Fermentable nitrogen or formol nitrogen
in the fermentation broth was analyzed by formol titration method
(Zoecklein et al., 1995).

3. Results and discussion

3.1. VHG fermentation with sucrose as an adjunct and inuences of
various nitrogen sources to ethanol production
Standard ethanol production medium (Melzoch et al., 1994)
contains 3 g l1 of yeast extract and 5 g l1 of peptone which total
fermentable nitrogen equals to 1129 mg l1. Therefore in this study
yeast extract and peptone at those concentrations were supplemented in the sweet sorghum juice as nitrogen sources. To compare the effects of nitrogen source on ethanol production, the
same amount of fermentable nitrogen in (NH4)2SO4 (1.3 g l1)
was supplemented in the juice. A common nitrogen source, urea,
was not studied in the research because it could react with ethanol
yielding ethyl carbamate (urethane) as a product, resulting in lower ethanol concentration (Zoecklein et al., 1995).
pH of the fermentation media was 4.9 which was in the optimum range for yeast growth and ethanol production (Narendranath and Power, 2005). Therefore, pH adjustment of the juice
was not performed. However, proles of pH during ethanol fermentation were monitored (data not shown). The pH of the broth
at all conditions was slightly decreased to 4.5 after 12 h and relatively constant afterwards.
Sugar consumption and ethanol production during batch fermentation of S. cerevisiae NP01 from the sweet sorghum juice sup-

300

Total sugar (g l -1)

120

100

250
80
200
60

150
100

40

50

20

350

120

Total sugar (g l -1)

300

100

250
80
200
60

150
100

40

50

20

350

Total sugar (g l -1)

120

300

100

250
80
200
60

150
100

40

50

20

Ethanol concentration (g l-1)

350

Ethanol concentration (g l-1)

L. Laopaiboon et al. / Bioresource Technology 100 (2009) 41764182

Ethanol concentration (g l-1)

4178

0
0

20

40

60

80

Time (h)
Fig. 1. Sugar consumption and ethanol production during batch ethanol fermentation by S. cerevisiae NP01 from sweet sorghum juice supplemented with sucrose
at various initial soluble solids and nitrogen sources: 24Bx (s, d), 28Bx (5, .) and
32Bx (h, j), total sugar (open symbol) and ethanol (close symbol). (A) No extra
nitrogen source, (B) supplemented with YEP and (C) supplemented with (NH4)2 SO4.

plemented with sucrose at the initial soluble solids of 24, 28 and

32Bx and various nitrogen sources are shown in Fig. 1. The initial
and nal total sugar concentrations at the various conditions are
shown in Table 2. The sugars were almost completely consumed

under high gravity (HG) fermentation (the initial soluble solids of

24Bx). Under VHG conditions at the initial soluble solids of 28
and 32Bx, stuck fermentation was observed with approximately
4864 and 120146 g l1 of total sugar remaining in the fermentation broth, respectively. The amount of sugar remaining was also
dependent on supplemented nitrogen sources. Not all sugars in
the media were completely utilized by the yeast. This might be
due to thermal stress as described by Jones and Ingledew
(1994a). They found that the amount of sugar that could be fermented decreased when fermentation temperature was above
25 C. However, lower temperature might cause lower ethanol productivity. This was supported by Bai et al. (2008) who reviewed
that the negative impact of high temperature on the ethanol fermentation performance was much worse under the VHG conditions than the regular fermentation.
Table 2 also summarizes the important kinetic parameters (P;
Q p and Y ps ) of the ethanol fermentation under various conditions.
The results showed that initial total soluble solids or initial sugar
concentration had signicant effects on the main kinetic parameters. The juice containing initial total soluble solids of 28Bx, corresponding to total sugar of approximately 280 g l1, and YEP gave
the maximum ethanol concentration with the value of
120.68 0.54 g l1. At the initial total soluble solids of 24 and
28Bx, YEP enhanced the nal ethanol concentrations approximately 3% compared to those of the control (no extra nitrogen supplement). Although the fermentation time was extended under the
medium containing YEP (Table 2), this result might be explained
that the yeast was able to produce ethanol at very high ethanol
concentrations (more than 15% by volume), and thus, achieved a
goal of VHG fermentation (Bai et al., 2008). The advantage of having higher ethanol concentration was that it could reduce energy
consumption in distillation (Bai et al., 2004). Similar results were
reported by Jones et al. (1994), who found that addition of yeast
extract failed to enhance the ethanol productivity at 30 C from
sugarcane juice fortied with freeze-dried wheat hydrolysate or
molasses adjuncts. However, because of lower ethanol productivity
due to longer fermentation time in the presence of YEP, further
studies will be carried out to improve the productivity.
When (NH4)2SO4 at the same amount of fermentable nitrogen
detected in YEP was used as a nitrogen supplement, P; Q p and Y ps
obtained were signicantly lower than those of the control and
the juice supplemented with YEP, respectively. Negative effect
of ammonium sulphate on nal concentration might be due to
more byproducts occurred. The amount of total sugar utilized
was similar between the control media and the media supplemented with ammonium sulphate at all gravities, but Y ps of the
latter was markedly lower (Table 2). There were some studies reported that excessive ammonium addition might cause the in-

Table 2
Kinetic parameters of ethanol production from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01.
Extra nitrogen sources

None

YEP

(NH4)2SO4

a
b
c

Bxa

24
28
32
24
28
32
24
28
32

Initial total sugar (g l1)

248.16 1.39
285.10 5.11
336.01 2.38
236.06 3.14
286.06 4.71
336.89 0.39
246.72 1.21
287.17 3.93
333.28 4.71

Final total sugarb (g l1)

12.80 3.05
63.76 2.14
134.63 2.34
5.96 1.23
47.71 2.67
120.30 3.12
8.48 0.59
59.98 1.57
145.50 3.18

Parameters (mean SD)c

P (g l1)

Q p (g l1 h1)

Y ps (g g1)

t (h)

113.20 0.81
117.28 0.14
107.39 1.07
116.71 0.85
120.68 0.54
108.23 0.16
96.58 0.73
101.48 0.06
83.31 0.06

2.83 0.02
2.44 0.00
2.24 0.02
2.43 0.02
2.01 0.01
1.50 0.00
2.01 0.02
2.11 0.00
1.74 0.00

0.48 0.03
0.53 0.01
0.53 0.00
0.51 0.01
0.51 0.00
0.50 0.01
0.41 0.00
0.45 0.02
0.44 0.02

40
48
48
48
60
72
48
48
48

Total soluble solids in Bx.

Total sugar at fermentation time.
P; ethanol concentration; Q p , volumetric ethanol productivity; Y ps , ethanol yield and t, fermentation time. The experiments were performed in duplicate.

4179

L. Laopaiboon et al. / Bioresource Technology 100 (2009) 41764182

Table 3
Fermentable nitrogen utilized during ethanol production from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources by S.
cerevisiae NP01.
Extra nitrogen sources

Bxa

Initial total sugar (g l1)

Final total sugarb (g l1)

None

24
28
32
24
28
32
24
28
32

248.16 1.39
285.10 5.11
336.01 2.38
236.06 3.14
286.06 4.71
336.89 0.39
246.72 1.21
287.17 3.93
333.28 4.71

12.80 3.05
63.76 2.14
134.63 2.34
5.96 1.23
47.71 2.67
120.30 3.12
8.48 0.59
59.98 1.57
145.50 3.18

YEP

(NH4)2SO4

Fermentable nitrogen (mg l1)

Initial

Utilized

657.28 7.21
626.91 0.00
594.96 1.80
1138.96 10.81
1149.15 18.02
1098.19 18.02
1114.40 7.92
1094.80 3.96
1061.20 11.88

391.12 1.80
294.29 16.22
215.31 19.82
420.40 21.62
402.60 50.45
287.90 10.81
439.60 3.96
380.80 7.92
313.60 7.92

See Table 1.
See Table 1.
The experiments were performed in duplicate and the results were expressed as mean SD.

9.0

8.5
8.0
7.5
7.0
6.5

production, and hence it would not be used for subsequent

experiments.
Bai et al. (2008) reported that assimilation nitrogen is the most
important component in the fermentation medium. In this study,
utilization of fermentable nitrogen in ethanol fermentation under
various media is shown in Table 3. In the juice without nitrogen
supplementation, the amount of fermentable nitrogen utilized decreased when sugar concentration in the juice increased. The results suggested that yeast nitrogen assimilation might be
repressed under high sugar concentrations. Under HG conditions
(24Bx), the amount of nitrogen utilized in all media were similar.
However, under VHG conditions at the same initial soluble solids,
the yeast utilized fermentable nitrogen in the juice containing extra nitrogen sources more than the juice without nitrogen
supplementation.

6.0

-1

Total sugar (g l )

8.5
8.0
7.5
7.0

120

300

100

250
80
200
60

150
100

40

6.0

50

20

5.5

6.5

5.0
9.0

350

8.5

-1

8.0
7.5
7.0
6.5
6.0
5.5

120

300

100

250
80
200
60

150
100

40

50

20

5.0

0
0

20

40

60

80

20

40

60

-1

350

5.0
9.0

Ethanol concentration (g l-1)

5.5

Total sugar (g l )

Log viable cell concentration

(cells ml-1)

Log viable cell concentration

(cells ml-1)

Log viable cell concentration

(cells ml-1)

crease in higher alcohols (Beltran et al., 2005), acetic acid (Bely et

al., 2003) or hydrogen sulphide (Wang et al., 2003). However, the
byproducts were not monitored in our experiment. The results
obtained indicate that (NH4)2SO4 seems not to be suitable for
use as a nitrogen supplement in sweet sorghum juice for ethanol

Ethanol concentration (g l )

80

Time (h)

Time (h)
Fig. 2. Yeast viability of S. cerevisiae NP01 during batch ethanol fermentation from
sweet sorghum juice supplemented with sucrose at various initial total soluble
solids and nitrogen sources: 24Bx (d), 28Bx (.) and 32Bx (j). (A) No extra
nitrogen source, (B) supplemented with YEP and (C) supplemented with (NH4)2 SO4.

Fig. 3. Sugar consumption and ethanol production during batch ethanol fermentation by S. cerevisiae NP01 from sweet sorghum juice supplemented with molasses
at various initial total soluble solids: 28Bx (s, d), 32Bx (5, .) and 34Bx (h, j),
total sugar (open symbol) and ethanol (close symbol). (A) No extra nutrient, (B)
supplemented with YEP.

4180

L. Laopaiboon et al. / Bioresource Technology 100 (2009) 41764182

Table 4
Kinetic parameters of ethanol production from sweet sorghum juice supplemented with molasses at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01.
Extra nitrogen sources

None

YEP

Bxa

28
32
34
28
32
34

Initial total sugar (g l1)

242.56 4.29
279.78 2.36
315.89 0.00
247.53 4.08
277.02 2.38
315.18 5.56

Final total sugarb (g l1)

18.26 2.12
63.29 0.19
85.29 1.60
23.94 2.36
34.88 1.98
63.57 3.28

Parameters (mean SD)c

P (g l1)

Q p (g l1 h1)

Y ps (g g1)

t (h)

100.54 0.70
102.08 2.63
98.29 2.16
104.99 1.51
109.34 0.78
104.95 2.18

2.51 0.02
2.13 0.05
2.05 0.13
1.46 0.02
1.52 0.01
1.46 0.03

0.45 0.01
0.43 0.00
0.43 0.04
0.47 0.04
0.45 0.01
0.44 0.02

40
48
48
72
72
72

The experiments were performed in duplicate.

See Table 1.
b
See Table 1.
c
See Table 1.
a

Sugar consumption and ethanol production during batch fermentation from the sweet sorghum juice supplemented with
molasses under the presence and absence of YEP are shown in
Fig. 3. The proles of sugar and ethanol concentrations during fermentation were similar to those when using sucrose as the carbon
adjunct (Fig. 1). Table 4 shows the initial and nal total sugar concentrations at various conditions and summarizes the important
kinetic parameters of the ethanol fermentation. P and Y ps of the
broth containing YEP were higher than those of the control broth
at all gravities. The amount of utilized sugar in the control medium
and the medium supplemented with YEP at 28Bx were similar,
while the utilized sugar in the control media at 32 and 34Bx were
lower than those of the supplemented media at the same gravities
(Table 4). However, the cell numbers at specic fermentation time
throughout the experiments under various conditions were similar
(Fig. 4). These results indicated that the supplemented YEP did not
promote cell growth but it stimulated sugar utilization and ethanol

Log viable cell concentration

(cells ml-1)

3.2. VHG fermentation using molasses as an adjunct and inuences of

various nitrogen sources to ethanol production

production under very high gravities. In contrast, the rate of ethanol fermentation (ethanol productivity) in the presence of YEP was
signicantly lower than that of the unsupplemented medium due
to longer fermentation time (Table 4). These ndings were consistent with those when using the juice supplemented with sucrose
and YEP. The reasons of these results were previously described
in Section 3.1. The juice supplemented with molasses and YEP at
the initial total soluble solids of 32Bx, corresponding to total sugar
of approximately 280 g l1, gave the maximum ethanol concentration with the value of 109.34 0.78 g l1. Under these conditions,
the productivity and yield of ethanol were 1.52 0.01 g l1 h1
and 0.45 0.01 g g1, respectively. Y ps slightly decreased when sugar concentrations were increased (Table 4). Possible reasons were
that some consumed sugar might be used for maintenance and/or
converted to more byproducts. Ozmichi and Kargi (2007) reported
that the ethanol yield decreased when the feed sugar content increased more than 200 g l1 due to high osmotic pressure and
maintenance requirements at high sugar concentrations. Similar
results were also observed by Jones et al. (1994) and Limtong
et al. (2007).

9.0

Log viable cell concentration

(cells ml-1)

In this study, fermentable nitrogen remaining in the control

medium (without nitrogen supplementation) was 4060% depending on the initial total soluble solids in the juice. This clearly indicated that nitrogen was not limited in the control medium, but the
capability of nitrogen utilization or nitrogen requirement by yeast
might depend on other factors such as yeast strain (Jiranek et al.,
1995; Taillandier et al., 2007), sugar concentration, temperature,
presence of oxygen (Valero et al., 2003) and initial fermentable
nitrogen content (Taillandier et al., 2007). Table 3 also indicated
that the amount of nitrogen consumption was not always related
to ethanol production efciency. Nitrogen utilized in the control
medium was lower than that in the juice supplemented with
(NH4)2SO4, but ethanol production from the control medium was
higher. The amount of nitrogen consumption seemed to relate to
type of nitrogen sources supplied (Table 3). Especially, in case of
using ammonium salts to increase the nitrogen content, excessive
ammonium addition might cause the increase in the byproducts
(Beltran et al., 2005; Bely et al., 2003; Wang et al., 2003) as previously mentioned.
Viability of the yeast during ethanol fermentation from sweet
sorghum juice under various conditions is shown in Fig. 2. The results indicated that S. cerevisiae NP01 was a suitable microorganism for ethanol fermentation under VHG levels. It could survive
and retain its metabolism under very high ethanol concentrations
up to 120 g l1 (15% by volume) (Table 2) with high viable cells
remaining in the fermentation broth (Fig. 2). Moreover, Nagashima
(1990) reported that to achieve cell viability and ethanol production, appropriate amount of fermentable nitrogen must also be
available. In our studies, fermentable nitrogen had still been available in the medium until the end of the fermentation.

9.0

8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0

8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
0

20

40

60

80

Time (h)
Fig. 4. Yeast viability of S. cerevisiae NP01 during batch ethanol fermentation from
sweet sorghum juice supplemented with molasses at various initial total soluble
solids and nitrogen sources: 28Bx (d), 32Bx (.) and 34Bx (j). (A) No extra
nutrient, (B) supplemented with YEP.

L. Laopaiboon et al. / Bioresource Technology 100 (2009) 41764182

When the results of using sucrose and molasses were compared

in terms of sugar utilization, the amount of the utilized total sugar
under both carbon adjuncts were similar when compared at the
same initial total sugar concentrations (Tables 2 and 4). These ndings imply that molasses might contain some inhibitors (e.g.,
hydroxymethylfurfural, hexanol and heptanol) for yeast metabolism as previously reported (Glacet et al., 1985; Fattohi 1994).
However, when the molasses was used as a carbon adjunct to raise
sugar concentration to VHG levels, the inhibitors were diluted to a
level which did not cause adverse effect on sugar utilization.
Regarding to the important kinetic parameters for ethanol production; P, Q p and Y ps of the ethanol fermentation using molasses as an
adjunct were signicantly lower than those using sucrose as an adjunct (Tables 2 and 4). These results imply that some other nutrients in the molasses may promote the formation of byproducts
of the ethanol fermentation.
The advantage of using fermentable sugars (sweet sorghum
juice, cane sugar or molasses) in this study especially under VHG
fermentation is that they can reduce the overall fermentation time.
Other forms of carbon such as starch or dextrins may be used to
raise soluble solids to VHG levels as found in Jones and Ingledew
(1994b). However, there were additional steps for pretreatment
the raw materials before ethanol fermentation process such as
double mashing and saccharication by glucoamylase, which was
a drawback of using complex carbohydrate as the raw materials.
4. Conclusions
This research achieves the goal of VHG fermentation technology that at least 15% (v/v) of ethanol is produced in the fermentation broth (Bai et al., 2008). The results obtained from this
research have demonstrated that sucrose is a good adjunct to
raise total soluble solids or sugar concentration of sweet sorghum
juice to VHG levels for ethanol fermentations, while molasses
causes the lower ethanol production efciency. However, other
methods for raising sugar content in sweet sorghum juice such
as concentrating the juice by evaporation should be considered
to reduce cost of the adjunct. Yeast extract and peptone promote
ethanol fermentation under VHG levels. Other yeast products
such as dried spent yeast may replace yeast extract and peptone
as they are far less expensive. The effects of dried spent yeast on
ethanol production will be investigated. According to the very
high content of fermentable sugars in the sweet sorghum juice
supplemented with cane sugar or molasses (Table 1), the optimum conditions in terms of processing parameters and/or fermentation processes to achieve complete sugar utilization under
VHG levels need to be further studied.
Acknowledgements
This research was nancially supported by the Thailand Research Fund (TRF) and Commission on Higher Education, Thailand.
We would like to thank Assistant Prof. Dr. Paiboon Danviruthai,
Faculty of Technology, Khon Kaen University (KKU) for providing
the NP01 strain and Associate Prof. Dr. Prasit Jaisil, Faculty of
Agriculture, KKU for providing sweet sorghum juice and Ms.
Ratchanee Kortsree, Mitr Phu Viang sugar Mill, Khon Kaen, Thailand for providing molasses and Associate Prof. Dr. Aroonwadee
Chanawong, Faculty of Associated Medical Sciences, KKU for her
internal review of this paper and helpful suggestions. We also
gratefully acknowledge the Royal Bangkok Sports Club (RBSC),
Bangkok, Thailand and the Fermentation Research Center for Value
Added Agricultural Products (FerVAAP) for nancial support for
Mr. Sunan Nuanpeng and Research Center for Environmental and
Hazardous Substance Management, KKU and National Center of

4181

Excellence for Environmental and Hazardous Waste Management,

KKU for some instrumentation supports.
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