Biomass and Bioenergy 81 (2015) 265e272

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Biomass and Bioenergy

journal homepage: http://www.elsevier.com/locate/biombioe

Research paper

Production of ethanol from raw juice and thick juice of sugar beet by
continuous ethanol fermentation with occulating yeast strain KF-7
Li Tan a, Zhao-Yong Sun a, *, Shinpei Okamoto b, Masatoshi Takaki b, Yue-Qin Tang a,
Shigeru Morimura b, Kenji Kida a, b
a
b

College of Architecture and Environment, Sichuan University, No. 24 South Section 1, First Ring Road, Chengdu 610065, China
Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 5 February 2015
Received in revised form
27 June 2015
Accepted 15 July 2015
Available online 25 July 2015

Sugar beet juice can serve as feedstock for ethanol product due to its high content of fermentable sugars
and high energy output/input ratio. Batch ethanol fermentation of raw juice and thick juice proved that
addition of mineral nutrients could not improve ethanol concentration, but could accelerate the
fermentation rate. Fermentation of thick juice with an initial pH of 9.1 did not affect the fermentation
process. The continuous ethanol fermentation of raw juice was performed at 35
C with a dilution rate of
0.3 h1, resulting in ethanol concentration, ethanol yield and productivity of 70.7 g L1, 89.8% and
21.2 g L1 h1, respectively. A two-stage reactor was used in the continuous ethanol fermentation of thick
juice by feeding fresh yeast cells into the second reactor. This process was stable at a total process
dilution rate of 0.11 h1 with an overall sugar concentration of 190 g L1 in the inuent. The ethanol
concentration was kept at approximately 80 g L1, corresponding to ethanol yield of 82.5% and productivity of 8.8 g L1 h1.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Beta vulgaris
Continuous fermentation
Flocculating yeast
Two-stage fermentation with fresh yeast
cells feed
High ethanol concentration

1. Introduction
Ethanol is an attractive renewable alternative fuel. It is an
oxygenated fuel that contains 35% oxygen, which reduces particulate, hydrocarbon, carbon monoxide and oxynitride emission [1,2].
Using an ethanol/gasoline blend as an alternative transportation
fuel is a valid approach to alleviate the oil crisis and reduce
greenhouse gas emission [3].
Intermediates of sugar beet processing (raw, thin and thick
juice) are attractive materials for ethanol product, due to their high
content of fermentable sugars and high energy output/input ratio
and net energy gain [4e7]. The only preparations required before
fermentation are dilution to a suitable sugar concentration,
adjustment of the pH and addition of fermentation nutrients [7].
Currently in Europe, ethanol is commercially produced from the
intermediates as a supplemental process in crystal sugar production [8,9]. The disadvantage of the intermediates is the poor storability of raw juice and thin juice, which are easily decomposed by

Abbreviation: HPLC, high-performance liquid chromatography; VFA, volatile

fatty acid; GC, gas chromatography.
* Corresponding author.
E-mail address: szy198306@hotmail.com (Z.-Y. Sun).
http://dx.doi.org/10.1016/j.biombioe.2015.07.019
0961-9534/ 2015 Elsevier Ltd. All rights reserved.

microorganisms. In contrast, thick juice is suitable for long-term

storage due to the high sugar concentration that inhibits microbial growth [5,10].
In 2007, the Japanese government launched a national project
for ethanol product from biomass produced in Hokkaido [4]. Hokkaido is the main crop production area in Japan, producing more
than 35 Mt of sugar beet, annually [11]. According to Koga's report,
sugar beet is the most promising feedstock crop for ethanol product
in this area because of its high energy output/input ratio of 7.65 and
net energy gain of 219.3 GJ ha1 year1 for yield mass [4].
In order to reduce the ethanol production cost, it is critical to
devise an efcient process for ethanol fermentation of the intermediates [4,9,12]. Several studies have attempted to optimize
ethanol fermentation of the intermediates via batch fermentation.
Dodic et al. [5] and Grahovac et al. [12] reported that based on the
fermentation rate, ethanol concentration, and sugar consumption,
an initial sugar concentration of approximately 200 g L1 is the
optimal for the fermentation of thick juice. Additionally, Dziugan
et al. [7] and Kawa-Rygielska et al. [13] reported that supplementing the thick juice or thin juice with fermentation nutrients
accelerated ethanol fermentation and improved the nal ethanol
concentration. Previous studies have found that occulating yeast
strain was suitable for developing efcient bioprocesses to produce

266

L. Tan et al. / Biomass and Bioenergy 81 (2015) 265e272

ethanol from sugar beet [9,14]. Studies on the continuous ethanol

fermentation of these intermediates are still rare, although
Vucurovic et al. [15] reported a continuous ethanol fermentation
process of thick juice by immobilizing yeast cells onto sugar beet
pulp. However, to the best of our knowledge, there is still no report
on the continuous ethanol fermentation of the raw juice and thick
juice using occulating yeast strain.
In this study, a occulating yeast Saccharomyces cerevisiae strain
KF-7 was used to establish the continuous ethanol fermentation
processes to convert the raw juice and thick juice of sugar beet to
ethanol and to achieve high ethanol yield, ethanol concentration
and productivity.

2. Materials and methods

2.1. Feedstock and microorganism
The feedstock used in this study, sugar beet raw juice and thick
juice, were provided by Hokkaido Bioethanol Co., Ltd. (Sapporo City,
Japan). The harvested and washed sugar beet roots were mechanically sliced into cossettes; these were then used for sugar extraction using hot water at 70
C. The obtained solution was raw juice.
The raw juice was claried by liming followed by saturation with
CO2. The generated solid was ltrated, and the clear liquid portion
was then concentrated in a multi-effect evaporator into thick juice.
The raw juice was stored at 20
C, and it was thawed at 60
C
before use. The thick juice was stored at 4
C. In total, ve plastic
containers of the raw juice and two plastic containers of the thick
juice were sent to our laboratory via frozen chain. Table 1 shows the
representative values of the components of raw juice and thick
juice. Although sucrose was partly enzymatically hydrolyzed to
glucose and fructose, it was acceptable because all of them were
fermentable.
The occulating yeast S. cerevisiae strain KF-7, which was constructed by protoplast fusion of the occulating yeast strain IR-2
and the thermotolerant yeast strain EP-1 [16], was used in this
study.

2.2. Preservation of raw juice from bacterial contamination

After thawing, the sugar beet raw juice was poured into a sterilized ask and pasteurized at 70
C for (0, 1, 3 or 5) h, and it was
stored at room temperature for 10 days. Samples were taken at
specic time intervals to measure the concentrations of lactic acid
and acetic acid.

Table 1
Representative components of raw juice and thick juice. The presenting data were
the mean of triplicate analysis standard deviation.
Components

Content
Raw juice

pH
Lactic acid (mg L1)
Acetic acid (mg L1)
Sucrose (g L1)
Glucose (g L1)
Fructose (g L1)
1
NH
4 (mg L )
Ca2 (mg L1)
Mg2 (mg L1)
K (mg L1)
a

ND, not detected.

6.30
7748.0
1812.0
32.5
56.0
63.8
32.1
165.7
171.2
916.3

0.14
45.3
87.7
2.4
3.7
1.6
2.4
2.1
2.4
6.9

Thick juice
9.24 0.01
13 586.0 545.9
2176.0 117.4
640.3 23.8
87.9 6.4
NDa
104.6 3.8
1568.0 73.5
110.6 7.4
5432.0 63.6

2.3. Preparation of inoculum

A 300 cm3 conical ask containing 100 cm3 of 5% YPD medium
(1% yeast extract, 2% peptone and 5% dextrose) was sterilized at
121
C for 15 min. A loopful of yeast cells was inoculated into the
sterilized medium and was cultivated at 30
C for 16 h with shaking
at 2.7 Hz in a rotary shaker (TB-9R-2F; Takasaki Kagaku, Saitama,
Japan).

2.4. Batch ethanol fermentation

The following mineral nutrients (g L1 juice) were added to
90 cm3 of raw juice: CaCl2$2H2O, 1.0; KH2PO4, 0.5; MgSO4$7H2O,
0.5; (NH4)2SO4, 0.5. After sterilization, 10 cm3 of inoculum was
added to the juice to initiate fermentation. Batch ethanol fermentation was performed in a 300 cm3 conical ask. The ask was
immersed in a water bath at 30
C and the fermentation medium
was stirred using a magnetic stirrer for 48 h. Batch fermentation
without addition of mineral nutrients was performed as a control.
Batch ethanol fermentation of thick juice was similar to that of raw
juice, except that the thick juice was diluted to a sugar concentration (glucose fructose sucrose  360/342, where 360/342 is the
conversion factor for sucrose to monosaccharides.) of 200 g L1
according to the reports of Dodic et al. [5] and Grahovac et al. [12],
and the fermentation was performed for 72 h.

2.5. Continuous ethanol fermentation

2.5.1. Single-stage fermentation
The continuous ethanol fermentation of raw juice was carried
out using a single-stage tower shaped reactor with a working volume of 450 cm3, as previously described [17]. Before fermentation,
the reactor was sterilized by circulating a NaClO solution of
20 mg L1 overnight and then was washed with sterilized tap water
continuously for one day. The reactor was lled by 450 cm3 of
inoculum, and then the raw juice was fed to start the continuous
ethanol fermentation. Mineral nutrients (g L1 juice) were added
into the raw juice before feeding: KH2PO4, 0.5; MgSO4$7H2O, 0.5;
(NH4)2SO4, 0.5. To enhance the occulability of the yeast cells,
CaCl2$7H2O was added to the raw juice to increase the Ca2 concentration to 1 g L1; this was done according to the method
described in our previous study [18]. The effects of the dilution rate
and temperature on the continuous ethanol fermentation were
investigated.

2.5.2. Two-stage fermentation

The continuous ethanol fermentation of thick juice was carried
out using a two-stage tower shaped reactor (Fig. 1). The reactors
were sterilized with NaClO solution (20 mg L1, overnight) and
washed with sterilized tap water for one day. Each of the reactors
was lled by 450 cm3 of inoculum, and then the thick juice and
water were fed to initiate the continuous ethanol fermentation [17].
Thick juice and tap water containing mineral nutrients were
continuously and separately fed into the rst reactor (R1) at feeding
rates of f11 and f12, respectively. The fermented mash overowed
from the top of R1 into the bottom of the second reactor (R2), and
thick juice was simultaneously fed into R2 at a feeding rate of f2. The
sugar concentration in R1 (S1) and R2 (S2) were kept at 140 g L1
and 190 g L1, respectively, assuming no sugar was consumed. The
dilution rate of R1 (D1) was kept at 0.2 h1. S1, S2 and D1 were
calculated as follows:

L. Tan et al. / Biomass and Bioenergy 81 (2015) 265e272

pH sensor

267

pH sensor
f13

Fermented
mash

Thermostat
water
Circulation
Circulation

Water containing
mineral nutrients
Thick juice

Thermostat
water

f12
Air

Air
f2

f11

Thick juice

R1

R2

Fig. 1. Schematic ow diagram of the continuous ethanol fermentation using a two-stage tower shaped reactor.

S0  f11
f11 f12

(1)

S2

S0  f11 f2
f11 f12 f2

(2)

D1

f11 f12
V1

(3)

S1 S01

where S0 the concentration of glucose and fructose in thick

juice the concentration of sucrose in thick juice  360/342, and
V1 is working volume of R1, which was 450 cm3. By solving the Eqs.
(1)e(3), f11, f12, and f2 were 16.5 cm3 h1, 73.5 cm3 h1, and
8.4 cm3 h1, respectively. The dilution rates of R2 (D2) and the total
process (DT) were then calculated as follows:

f f12 f2
D2 11
V2
DT

f11 f12 f2
V1 V2

in Fig. 1 with a third complete stirred tank reactor (R3), which is

used for the cultivation of yeast cells with high activity. The
continuous ethanol fermentation was started in the same manner
as described in Section 2.5.2. R10 was performed under the same
conditions as R1 in Section 2.5.2. Thick juice and tap water containing mineral nutrients were continuously and separately fed into
R3 at feeding rates of f31 and f32, respectively. Fermented mash in R3
was pumped into R20 , and thick juice was fed into R20 at a feeding
rate of f20 at the same time. The sugar concentration in R10 (S10 ), R20
(S20 ) and R3 (S3) were kept at 140 g L1, 190 g L1 and 90 g L1,
respectively, assuming no sugar was consumed. The dilution rate of
R10 (D1) and R3 (D3) were kept at 0.2 h1 and 0.1 h1, respectively.
S10 was calculated according to Eq. (1). S20 , S3 and D3 were calculated
as follows:

S02

S0  f11 f20 f31
f11 f12 f20 f31 f 32

(6)

S3

S0  f31
f31 f32

(7)

D3

f31 f32
V3

(8)

(4)

(5)

where V2 is the working volume of R2, which was 450 cm3. By

substituting the relevant values into Eqs. (4) and (5), D2 and DT
were calculated to be 0.22 h1 and 0.11 h1.
2.5.3. Two-stage fermentation with fresh yeast cells feed
Fig. 2 shows the schematic ow diagram of two-stage fermentation with fresh yeast feed. This apparatus is the same one as seen

where V3 is the working volume of R3, which was 270 cm3. By

solving the Eqs. (1), (3) and (6)e(8), f20 , f31 and f32 were
12.6 cm3 h1, 3.2 cm3 h1 and 23.8 cm3 h1, respectively. The
dilution rates of R20 (D20 ) and the total process (DT0 ) were then
calculated as follows:

Fig. 2. Schematic ow diagram of the continuous ethanol fermentation using a two-stage tower shaped reactor with a fresh yeast cells feed.

L. Tan et al. / Biomass and Bioenergy 81 (2015) 265e272

0 f0 f0 f0
f13
2
31
32
V2

D0T

f11 f12 f20 f31 f 32

V1 V2 V3

100

(9)

(10)

By substituting the relevant values into Eqs. (9) and (10), D20 and
DT were calculated to be 0.29 h1 and 0.11 h1.
0

2.6. Calculation of ethanol yield

The ethanol yield was calculated by assuming that 1 g glucose or
fructose yields 0.511 g ethanol. The ethanol yields based on the
fermentable sugars in the sugar beet raw juice and thick juice were
calculated as follow:

Yethanol

Pethanol
 100%
G F S  360=342  0:511

where Yethanol is ethanol yield; Pethanol is the quantity of ethanol

after fermentation; G, F and S are the quantities of glucose, fructose
and sucrose that were used for fermentation, respectively.
2.7. Analytical methods
The sugar concentrations were analyzed using a highperformance liquid chromatography (HPLC) reducing sugar analysis system (Shimadzu; Kyoto, Japan) equipped with a Shim-pack
ISA-07/S2504 column (4 mm i.d.  25 cm length) (Shimadzu) and
a uorescence detector (RF-10AXL) [19]. Additionally, the concentration of volatile fatty acid (VFA) was analyzed on another HPLC
system (Shimadzu) equipped with a Shim-pack SCR-101H column
(7.9 mm i.d.  30 cm length) (Shimadzu) and a UV/VIS detector
(SPD-10AV) [20]. The concentration of ethanol was analyzed by an
internal standard method (with isopropanol) using a gas chromatography system (GC 353B; GL sciences, Tokyo, Japan) equipped
with a TC-1 capillary column (0.25 mm i.d.  60 m length; d.f.:
0.25 mm) and a ame ionization detector [19]. The concentration of
cation was analyzed on an ion chromatography system (Dionex ICS1100; CA, USA) [18]. The yeast number and their viability were
calculated by the methylene blue method using a hemocytometer.
Yeast cell activity reected as the respiration rate was analyzed by
measuring the CO2 production rate using a Warburg manometer
(TAITEC Co., Ltd., Saitama, Japan), as previously described [21].
2.8. Statistical analysis
Analysis of variance and separation of means were implemented
using SPSS version 18.0 (IBM, Armonk, NY, USA). The least signicant difference method was used to compare the means. Signicance of all statistical analysis was declared at P < 0.05.
3. Results and discussion

Ethanol concentration (g L1)

D02

80
60
40
20
0
0
100

Ethanol concentration (g L1)

268

10

20

30
40
50
Fermentation time (h)

60

70

80

20

30
40
50
Fermentation time (h)

60

70

80

80
60
40
20
0
0

10

Fig. 3. Batch ethanol fermentation of raw juice and thick juice. (A) The effect of adding
mineral nutrients on fermentation. Solid lines, thick juice; dotted lines; raw juice;
diamonds, adding mineral nutrients; squares, without adding mineral nutrients. (B)
The effect of initial pH of thick juice on fermentation. Solid lines, adding mineral nutrients; dotted lines, without adding mineral nutrients; diamonds, initial pH of 9.1;
squares, initial pH of 8.5; triangles, initial pH of 8.0.

accelerated by adding mineral nutrients. Fermentation time was

shortened from 48 h to 24 h and from 72 h to 48 h for batch ethanol
fermentation of raw juice and thick juice, respectively. From an
economic viewpoint, it is crucial to enhance the fermentation rate
so as to improve productivity.
Since the thick juice had a high pH of 9.24, the effect of initial pH
on the fermentation of thick juice was investigated. As shown in
Fig. 3B, the nal ethanol concentrations at various initial pHs were
similar and ranged from 82.7 g L1 to 88.5 g L1, corresponding to
ethanol yields of 80.9 %e86.6 %. Without addition of mineral nutrients, fermentation was only accelerated at an initial pH of 8.0. On
the other hand, with addition of mineral nutrients, performance of
fermentation process was similar at various initial pHs, and
fermentation nished within 48 h. The acceleration of fermentation
was more signicant with the addition of mineral nutrients than
with pH adjustment. The nal pH of the fermented broth was
approximately 4.5, regardless of the initial pH. The results
demonstrated that thick juice exhibited excellent fermentation
performance at the high initial pH of 9.1 with the addition of
mineral nutrients. Therefore, in the continuous ethanol fermentation, pH adjustment could be omitted.

3.1. Batch ethanol fermentation

3.2. Continuous ethanol fermentation of raw juice
The effects of adding mineral nutrients were evaluated by
conducting batch ethanol fermentation of raw juice and thick juice.
As shown in Fig. 3A, the nal ethanol concentration was not
enhanced by adding mineral nutrients. This indicated that the
fermentation nutrients in the raw juice and thick juice were sufcient to support ethanol fermentation, which was in accordance
with the results obtained by Zhang et al. [9], Grahovac et al. [12] and
Ogbonna et al., [14]. In contrast, fermentation was signicantly

Bacteria show a preference for thawed raw juice [5,10]. The

result of a preliminary experiment showed that pasteurization of
the thawed raw juice at 70
C for 3 h was sufcient prevent to
bacterial contamination for 10 days because of no signicant increase in concentrations of lactic acid and acetic acid. In the
continuous ethanol fermentation, the frozen fresh raw juice was
thawed and pasteurized every (2e3) days to minimize the

L. Tan et al. / Biomass and Bioenergy 81 (2015) 265e272

VFAs concentration (mg

5
4
3

2
1
0
30

50
60
70
Ethanol concentration (g L1)

80

90

However, a decreasing trend in ethanol concentration appeared at

37
C. When the fermentation temperature was further increased
to 39
C, ethanol concentration and yeast cell viability decreased to
approximately 40 g L1 and 53%, respectively. As shown in Fig. 5,
temperature negatively inuenced cell activity, and lack of activity
at 37
C led to decrease in ethanol concentration. The cell activity at
39
C was slight higher than that at 37
C due to lower ethanol
concentration that alleviated the inhibition effect on yeast cells.
Performing the continuous ethanol fermentation at 35
C, an

0.3 h1
30 C

30 C

40

Fig. 5. Yeast cell activity in the continuous ethanol fermentation of raw juice at various
temperatures. Cross, 30
C; triangle, 33
C; square, 35
C; diamond, 37
C; circle, 39
C.

0.2 h1

0.1 h1
30 C

35 C

33 C

37 C

39 C

80
60
40
20
0
0

14000
L1)

10

15

20
25
Fermentation time (days)

30

35

40

45
7

12000

10000

8000

6000

pH

Ethanol and sugar concentration (g L1)

Yeast number (108 cm3)
Yeast cell viability (%)

Dilution rate
Temperature
100

7
Cell activity (mm3 min1)

contamination risk.
The effect of the dilution rate on the continuous ethanol
fermentation of raw juice was carried out at 30
C by using singlestage fermentation. As shown in Fig. 4A, there were no signicant
changes in ethanol concentration, yeast cell viability and residual
sugar concentration by increasing dilution rate from 0.1 h1 to
0.3 h1. The concentration of acetic acid was below 1500 mg L1,
and the concentration of lactic acid was between 4500 mg L1 and
11 000 mg L1 in the reactor due to its abundance in the feedstock
as shown by horizontal lines in Fig. 4B. Neither acetic acid concentration nor lactic acid concentration signicantly increased
comparing with these in feedstock. Judging from the changes of
VFAs concentrations and residual sugar concentration, there was no
serious bacterial contamination. Although the lactic acid concentration reached approximately 9 g L1 in the reactor, ethanol
fermentation was not inhibited at all, indicating that the yeast
strain KF-7 is lactic acid-tolerant. By increasing the dilution rate
from 0.1 h1 to 0.3 h1, the productivity markedly increased from
(6.2 0.2) g L1 h1 to (19.9 0.5) g L1 h1. This result was in
accordance with the results of our previous studies, in which
kitchen garbage [17], sweet sorghum juice [18], acid hydrolysate of
wood biomass [19] and molasses [22] were the feedstock.
Heat from fermentation will increase the fermentation temperature, while fermentation at high temperature can save the cost
to cool the fermenters. Therefore, at the dilution rate of 0.3 h1, the
effect of temperature on the continuous ethanol fermentation was
investigated. As shown in Fig. 4A, the yeast number reduced at
33
C, but it did not affect the ethanol concentration at 35
C.

269

4000

2000
0

10

15

20
25
Fermentation time (days)

30

35

40

45

Fig. 4. Effect of dilution rate and temperature on the continuous ethanol fermentation of raw juice. (A) Changes of ethanol and sugar concentration, number of yeast and their
viability. Diamonds, ethanol concentration; squares, sugar concentration; crosses, number of yeast; asterisks, yeast cell viability. (B) Changes of VFAs concentration. The horizontal
lines in B indicate the concentrations of lactic acid (solid lines) and acetic acid (dotted lines) in the feedstock. Triangles, lactic acid; circles, acetic acid; pluses, pH.

270

L. Tan et al. / Biomass and Bioenergy 81 (2015) 265e272

ethanol concentration of (70.7 0.8) g L1 was obtained, corresponding to ethanol yield of 89.8% and productivity of
21.2 g L1 h1.
3.3. Continuous ethanol fermentation of thick juice
The continuous ethanol fermentation of thick juice was carried
out by two-stage fermentation at 30
C. As shown in Fig. 6A,
ethanol concentration in R1 stabilized at (59.8 1.6) g L1. The
yeast number and their viability were relatively constant (Fig. 6B).
However, R2 did not perform as expected. Whenever the ethanol
concentration in R2 reached 80 g L1, decrease trends in ethanol
concentration subsequently followed (Fig. 6A), and the yeast
number was 109 cm3, with 50% viable (Fig. 6B). In our previous
study, it has been estimated that the number of viable yeast must
be retained at 1.3  109 cm3 in the reactor to achieve an ethanol
concentration of 85 g L1 [22]. Moreover, the cell activity as reected in the respiration rate as CO2 was very low in R2, being

Ethanol concentration (g L1)

100
80
60
40
20

0
0

10
15
20
Fermentation time (days)

25

30

10
15
20
Fermentation time (days)

25

30

100

Yeast number (108 cm3)

Yeast cell viability (%)

80
60
40
20
0
0

(0.6 0.1) mm3 min1, when the ethanol concentration reached

80 g L1, which was signicantly lower than that of
(5.1 0.1) mm3 min1 in R1, where the ethanol concentration was
approximately 60 g L1 (Fig. 7). Since there was no increasing trend
in lactic acid concentration or acetic acid concentration (Fig. 6C),
the decrease in ethanol concentration and yeast cell viability
should not be caused by bacterial contamination. Therefore, it can
be concluded that the yeast cells suffered from considerable stress
at the high ethanol concentration, resulting in a decrease of cell
growth and activity, thereby causing a decrease in ethanol concentration [6].
In order to keep the ethanol concentration in R2 at approximately 80 g L1, a third reactor was used to produce fresh yeast
cells, and the fresh yeast cells were continuously fed into R2 to
replenish viable cells and enhance the activity. In our previous
study, it had been proven that the sugar concentration between
80 g L1 and 100 g L1 resulted in the highest cells activity at 30
C
[23]. In this study, sugar concentration of 90 g L1 was fed into R3,
and the cultivation was carried out at 30
C. Ethanol concentration
in R3 was (37.1 2.2) g L1 (Fig. 8A). The yeast number and cell
activity in R3 was (9.8 2.8)  108 cm3 and (9.2 1.0) mm3 min1,
respectively (Figs. 8B and 7). In the continuous ethanol fermentation at 30
C, the decreasing trend in ethanol concentration and
yeast cell viability disappeared (Fig. 8A and B) by feeding the fresh
yeast cells from R3 into R2. The yeast cell viability stabilized at 60%
to 70 %, and the number of viable yeast was (1.7 0.4)  109 cm3
(Fig. 8B). Moreover, the cell activity was (3.8 1.0) mm3 min1,
which was markedly higher than the activity without a fresh feed
(Fig. 7). The ethanol concentration in R2 was (80.1 1.8) g L1
(Fig. 8A), corresponding to ethanol yield of 82.5% and productivity
of 8.8 g L1 h1. The concentration of VFA in each reactor was
relatively constant and at low level, indicating that no bacterial
contamination occurred (Fig. 8C).
Subsequently, the effect of temperature was investigated in the
continuous ethanol fermentation of thick juice. The temperature of
R3 was controlled at 30
C, while the temperatures of R1 and R2
were increased from 30
C to 35
C. As shown in Table 2, the difference of ethanol concentration in R1 was not signicant when the
temperature increased from 30
C to 35
C (P 0.057). The ethanol
concentration in R2 at 33
C was not signicantly different from
that at 30
C (P 0.414), while it signicantly decreased to
75.2 g L1 at 35
C (P 0.016). Thus, the temperature in R2 should
be controlled at 33
C.
The continuous ethanol fermentation of thick juice can be performed year-round, due to the excellent storability [24]. Although
raw juice is not suitable for year-round ethanol fermentation due to

12

4000

Cell activity (mm3 min1)

VFAs concentration (mg L1)

5000

3000
2000
1000
0
0

10
15
20
Fermentation time (days)

25

30

Fig. 6. Performance of the continuous ethanol fermentation of thick juice without

fresh yeast cells feed. (A) Change of ethanol concentration. Diamonds, R1; squares, R2.
(B) Changes of number of yeast and their viability. Solid lines, yeast cell viability;
dotted lines, number of yeast; diamonds, R1; squares, R2. (C) Changes of VFAs concentration. Solid lines, lactic acid; dotted lines, acetic acid; diamonds, R1; squares, R2.

10
8
6
4
2
0
30

40

50
60
70
Ethanol concentration (g L1)

80

90

Fig. 7. Yeast cell activity in the continuous ethanol fermentation of thick juice. Closed,
without fresh yeast cells feed; open, with fresh yeast cells feed; diamonds, R1; squares,
R2; triangle, R3.

L. Tan et al. / Biomass and Bioenergy 81 (2015) 265e272

Ethanol concentration (g L1)

100

80
60
40

20
0
0

Yeast number (108 cm3)

Yeast cell viability (%)

100

10
15
20
Fermentation time (days)

25

30

10
15
20
Fermentation time (days)

25

30

80
60
40
20
0
0

VFAs concentration (mg L1)

5000

potentially reduce the cost for the condensation of raw juice.

Considering industrial ethanol fermentation, preservation from
bacterial contamination is quite important, especially the continuous ethanol fermentation [26]. In this study, bacterial contamination did not occur, although sterilization was not conducted.
Using occulating yeast strain, the continuous ethanol fermentation of thick juice was performed at relative high dilution rate of
0.11 h1. Bacteria without occulability were washed out at this
high dilution rate. The relative high ethanol concentration of
80 g L1 in R2 inhibited the growth of bacteria. Besides, the undiluted thick juice could be directly fed into the reactors, preventing
the feedstock from bacteria contamination [23]. Flocculating yeast
strain is superior for developing efcient ethanol fermentation
process [9]. The productivity in this study was 8.8 g L1 h1, while it
was 2.3 g L1 h1 in the continuous ethanol fermentation of thick
juice using immobilized yeast cells, and it could not perform at
dilution rate of 0.05 h1 due to intensive yeast leaching from the
support [15]. Ogbonna et al. [14] has reported that immobilization
of occulating S. cerevisiae strain IR2 (one of the parent strains of
KF-7) onto loofa sponge resulted in ethanol concentration of
approximately 80 g L1 and productivity of (6.2e11.1) g L1 h1 by
using a repeated batch fermentation at 30
C. A carrier for immobilization was used, although the yeast strain was occulating. In
this study, the similar ethanol concentration and productivity were
obtained without using any carrier for immobilization. Moreover, in
this study the continuous ethanol fermentation was performed at a
higher fermentation temperature of 33
C, which was advantageous standing on the viewpoint of cooling the reactor.

4000
4. Conclusion

3000
2000
1000
0
0

10
15
20
Fermentation time (days)

25

30

Fig. 8. Performance of the continuous ethanol fermentation of thick juice with fresh
yeast cells feed. (A) Change of ethanol concentration. Diamonds, R1; squares, R2, triangles, R3. (B) Changes of number of yeast and their viability. Solid lines, yeast cell
viability; dotted lines, number of yeast; diamonds, R1; squares, R2, triangles, R3. C,
changes of VFAs concentration. Solid lines, lactic acid; dotted lines, acetic acid; diamonds, R1; squares, R2; triangles, R3.

poor storability [25], it can serve as feedstock during the harvest

season. In this study, it has been proven that raw juice can be stored
for 10 days after pasteurization at 70
C for 3 h. Besides, the
continuous ethanol fermentation of raw juice using single-stage
fermentation exhibited excellent fermentation performance.
Feeding raw juice into R1 and R3 during the harvest season can

Table 2
Effect of temperate on the continuous ethanol fermentation of sugar beet thick juice
with fresh yeast cells feed. The presenting data were the means at each
temperature standard deviation.
Temperature (
C)

Ethanol concentration (g L1)

R1

R2

30
33
35

65.5 1.4a
63.7 2.8a
63.0 0.9a

81.2 0.8a
79.7 1.6a
75.2 2.6b

a, b

271

The letters indicate statistical difference (P < 0.05) at various temperatures.

This study showed that the occulating S. cerevisiae strain KF-7

is appropriate for establishing continuous ethanol fermentation
process using sugar beet raw juice and thick juice as feed, with the
advantages of improved productivity and thermotolerant fermentation. Single-stage fermentation was sufcient for sugar beet raw
juice. Two-stage fermentation was proper for sugar beet thick juice.
Although ethanol concentration uctuated in the second reactor
(R2) due to stress of high ethanol concentration (80 g L1), it was
overcome by feeding fresh yeast cells.
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