Journal of Bioscience and Bioengineering

VOL. 109 No. 1, 41 46, 2010

www.elsevier.com/locate/jbiosc

Continuous ethanol fermentation from non-sulfuric acid-washed molasses using

traditional stirred tank reactors and the flocculating yeast strain KF-7

Yue-Qin Tang,1 Ming-Zhe An,2 Ya-Ling Zhong,3 Morimura Shigeru,2

Xiao-Lei Wu,1 and Kenji Kida2,

Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing 100871, China 1
Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto City, Kumamoto 860-8555, Japan 2
and Sichuan Yalian Technology, Co., Ltd., Gaoxin District, Chengdu 610065, China 3

Received 29 April 2009; accepted 8 July 2009

Available online 28 July 2009

Waste molasses is one of the most important feedstock for ethanol production in Brazil as well as in many Southeast Asian
countries, including China. Sulfuric acid pretreatment is employed in most ethanol distilleries in China to control bacterial
contamination, which results in difficulties in the treatment of wastewater containing high levels of sulfate ions. In this study,
a high efficiency, non-sterilized, continuous ethanol fermentation process without sulfuric acid pretreatment was developed
using the flocculating yeast strain KF-7 and the widely utilized, traditional, stirred tank reactors. An alternative molasses
medium feeding method, which differs from traditional methods, is proposed that effectively controls bacterial
contamination. Separate feeding of 1.2-fold diluted molasses and tap water into the reactor proved to be effective against
bacterial contamination during long-term continuous fermentation. By feeding yeast cells with high metabolic activity to the
second reactor, a two-stage continuous fermentation process that yielded a high ethanol concentration of 80 g/l as well as
high ethanol productivity of 6.6 g/l/h was successfully operated for more than one month. This fermentation process can be
applied to ethanol distilleries in which traditional tank reactors are used.
2009, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Molasses; Stirred tank reactor; Continuous ethanol fermentation; Flocculating yeast; Bioethanol production]

Sugar cane molasses is an abundant agro-industrial byproduct in
Brazil and other tropical countries, including the southern part of
China. Molasses is an important and economical substrate for ethanol
distilleries due to its easily fermentable sugars and low cost.
We have developed non-sterile continuous fermentation processes
using tower-type reactors (1, 2). Since flocculating yeast was used in
these processes, and the yeast cells effectively accumulated in the
reactor, it was possible to operate the system at a high dilution rate,
resulting in a high ethanol concentration and high productivity. Most
importantly, due to the high dilution rate and the large quantity of
cells with high activity, bacterial contamination was successfully
repressed and long-term non-sterile continuous fermentation was
easily realized. However, the general application of these processes is
difficult, since it is not feasible for traditional ethanol distilleries to
construct new tower-type reactors.
Almost all traditional ethanol distilleries in China use tank reactors.
Since the process currently employed by many distilleries is not very
efficient, the continuous fermentation system is always operated at a
relatively low dilution rate of approximately 0.02 h 1 and, in addition,
cell numbers in the reactors are low, generally around 12 108 cells/ml.
Furthermore, unless anti-contamination measures are taken, contam-
ination is very likely to occur. In China, most distilleries employ sulfuric
acid pretreatment as a sterilization method, resulting in an ethanol
concentration of over 80 g/l and an ethanol productivity of about 1.5
2.0 g/l/h. Although this treatment is effective for contamination
control, it results in the accumulation of about 5000 mg/l of sulfate
ions in distillation wastewater, making it difficult to treat the waste-
water (35). Although, previously, some large distilleries effectively
treated wastewater by high-cost evaporation followed by combustion,
most of the smaller distilleries could not, and, ultimately, most of these
small distilleries were closed down. A continuous ethanol fermenta-
tion process without sulfuric acid pretreatment is of great importance
for these types of distilleries.
In this study, which used the flocculating yeast strain KF-7 (6), a
highly efficient, continuous ethanol fermentation process without
sulfuric acid pretreatment was developed for ethanol distilleries in
which traditional stirred tank reactors are utilized. A new molasses
medium feeding method was proposed to control bacterial contam-
ination and was verified to be effective. Several continuous fermenta-
tion processes, including or excluding feeding cells with high activity,
were then investigated with the aim of achieving a high ethanol
concentration as well as high ethanol productivity.
MATERIALS AND METHODS

Yeast strain used The flocculating yeast Saccharomyces cerevisiae KF-7 was
Corresponding author. Tel.: +81 96 342 3667; fax: +81 96 342 3668. used in this study. This yeast was constructed by protoplast fusion of the flocculating
E-mail address: kida@gpo.kumamoto-u.ac.jp (K. Kida). yeast strain IR-2 (7) and the thermotolerant yeast strain EP-1 (6).

1389-1723/$ - see front matter 2009, The Society for Biotechnology, Japan. All rights reserved.
doi:10.1016/j.jbiosc.2009.07.002
42 TANG ET AL.
Medium for yeast growth YPD medium containing 5% glucose, 1% yeast extract
and 2% peptone was used for seed cultivation. The molasses used in this study was from
Indonesia and the average total sugar concentration was 0.54 g/g. As a nitrogen source,
0.01 g of (NH4)2SO4 was added per g molasses. Tap water was used to dilute the
molasses. The only media that was sterilized was the YPD.
Preparation of the inoculum Stored yeast cells were inoculated into 100 ml of
5% YPD medium in a 500 ml flask. Pre-cultivation was performed aerobically at 30 C for
16 h with mixing using a rotary shaker and a speed of 160 rpm. The resulting pre-
cultivation broth was used as the inoculum for continuous fermentation.
Determination of the molasses medium feeding method The molasses was
diluted 1.2-, 1.5-, 2- or 3-fold with tap water. Each dilution of the molasses was assayed
as to its feeding potential by feeding via a peristaltic pump. Concurrently, each dilution
of molasses was incubated at room temperature to investigate the extent of
contamination. The concentration of volatile fatty acids (VFA) was measured at 0 h,
12 h and 48 h. Ethanol fermentation of each dilution of molasses was simultaneously
performed by batch fermentation using 300 ml Erlenmeyer flasks. The concentration of
ethanol was determined every 12 h.
Continuous ethanol fermentation Five continuous fermentation processes
were studied and compared to determine which process for ethanol production
yielded the highest ethanol concentration and had the highest ethanol productivity.
All the magnetic stirred tank reactors used in the study were manufactured by
Marubishi (Tokyo, Japan). The temperature, the aeration rate and the stirring rate for
each reactor were controlled at 30 C, 0.15 vvm and 150 rpm, respectively, unless
otherwise specified. The concentration of ethanol, residual sugar and volatile fatty
acids, as well as the total cell number in each reactor, was measured every day
during the operation.
Process 1 was a one-stage fermentation process consisting of one stirred tank
reactor (working volume 2.0 l) and one settler for yeast cell sedimentation, as shown in
Fig. 1. Tap water and 1.2-fold diluted molasses were separately fed by peristaltic pumps
to the reactor (hereafter named R1) at rates of 133 ml/h and 67 ml/h, respectively, so
that the sugar concentration in the influent was 150 g/l. The dilution rate of R1 was
0.1 h 1. The fermented broth was pumped to the settler and the yeast cells were
returned to the reactor at a rate of 50 ml/h. Fermented broth was overflowed from the
settler.
Process 2 was a two-stage fermentation process consisting of two stirred tank
reactors and one settler, as shown in Fig. 2. Tap water and the 1.2-fold dilution of
molasses were separately fed by peristaltic pumps to the first reactor (hereafter named
R1, working volume 2.0 l) at rates of 133 ml/h and 67 ml/h, respectively, so that the
sugar concentration in the influent was 150 g/l. The fermented broth from R1 was
pumped to the second reactor (R2, working volume 3.0 l), and 1.2-fold dilutions of
molasses were simultaneously fed to R2 at a rate of 22 ml/h, so that the total sugar fed
to R2 reached 180 g/l, assuming that no sugar was consumed in R1. The fermented
broth from R2 was pumped to the settler and the yeast cells were recycled to R1 at a rate
of 50 ml/h. Fermented broth was overflowed from the settler. The dilution rates of R1
and R2 were 0.1 h 1 and 0.074 h 1, respectively. The dilution rate of the total process
was calculated to be 0.044 h 1.
Process 3 was a two-stage fermentation process consisting of two stirred tank
reactors and two settlers. The process was similar to Process 2, except that a settler
was attached after both R1 and R2, and the yeast cells from the fermented broth of
R1 and R2 that had settled were separately recycled back to R1 and R2, respectively,
at rates of 50 ml/h. Other operation parameters were the same as in Process 2.
Process 4 was a two-stage fermentation process comprised of three stirred tank
reactors and two settlers, as shown in Fig. 3. R1 was operated under the same
FIG. 1. Schematic diagram of the continuous fermentation process, Process 1.
J. BIOSCI. BIOENG.,
FIG. 2. Schematic diagram of the continuous fermentation process, Process 2.
conditions as for Processes 1 and 3. The fermented broth that overflowed from settler-1
was fed to R2. At the same time, the fermented broth from R3, a reactor for the
cultivation of yeast cells with high activity, was fed to R2 at a rate of 200 ml/h. A 1.2-fold
dilution of molasses was supplied to R2 at a rate of 98.3 ml/h so that the total sugar fed
to R2 reached 180 g/l, assuming that no sugar was consumed in R1 and R3. The
fermented broth from R2 was pumped to settler-2, and the settled yeast cells were
returned to R2 at a rate of 50 ml/h. Fermented broth was overflowed from settler-2. The
working volumes of R1, R2 and R3 were 2.0 l, 3.0 l and 1.0 l, respectively, and the dilution
rates of R1, R2 and R3 were 0.1 h 1, 0.166 h 1 and 0.2 h 1, respectively. The dilution rate
of the total process was calculated to be 0.083 h 1.
Process 5 was a one-stage fermentation process consisting of two stirred tank
reactors and one settler, which could be considered the same as Process 4 without R1.
R3 served as a cell cultivation reactor, and R2 was the ethanol fermentation reactor. By
feeding R2 with a 1.2-fold dilution of molasses at a rate of 60 ml/h, the total sugar fed to
R2 was set at 180 g/l, assuming that no sugar was consumed in R3. The dilution rates of
R2 and R3 were 0.087 h 1 and 0.2 h 1, respectively. The dilution rate of the total
process was calculated to be 0.065 h 1.
Effect of sugar concentration and aeration rate on cell activity The optimum
conditions for the cultivation of cells with high activity were determined for the
operation of R3 in Processes 4 and 5. A stirred reactor with a working volume of 1.0 l was
used for the continuous cultivation of the cells under different conditions. The dilution
rate of the reactor was controlled at 0.2 h 1. The total concentrations of sugar in the
influent were set at 80 g/l, 100 g/l, 120 g/l and 150 g/l by feeding different ratios of tap
water and 1.2-fold dilutions of molasses. Cell activity, as well as cell number and cell
survival rate, was measured at each sugar concentration when the system reached a
steady state. The effect of aeration on cell activity was investigated at a sugar
concentration of 90 g/l. The aeration rate was varied from 0 to 0.15 vvm, and cell activity
and the concentration of intracellular trehalose were measured at each aeration rate.
Analytical methods The total sugar concentration was assayed by the
SomogyiNelson method after hydrolysis of the samples with 1% HCl (8). Ethanol
concentration was measured by gas chromatography using isopropanol as the internal
standard (9). Volatile fatty acids (VFAs) including lactic acid and acetic acid were
analyzed by high performance liquid chromatography (HPLC) (10). The concentration
of intracellular trehalose in yeast cells was measured as previously described (11).
The metabolic activity of yeast cells was assayed by measurement of the CO2
production rate using Warburg's manometric method. The measurement was carried out
at 30 C with 1 ml of 1 M glucose solution and 5 107 cells suspended in 1 ml of 0.01 M
KH2PO4 solution. The volumetric oxygen transfer coefficient (kLa) was determined by the
oxygen balance method. The quantity kLa was calculated from the following equation: kLa
(h 1) = oxygen consumption rate (g/l/h) / (C0 C), where, C0 is the saturated dissolved
oxygen concentration in the broth (g/l) and C is the dissolved oxygen concentration in the
broth (g/l). The oxygen consumption rate was calculated from the following equation:
oxygen consumption rate (g/l/h) = air feeding rate (l/l/h) (oxygen ratio in the influent
air oxygen ratio in the effluent air) 32/22.4.
The total number of yeast cells and viable cells was calculated by the methylene
blue method using a hematitometer.
RESULTS AND DISCUSSION
Determination of the molasses feeding method for continuous
ethanol fermentation The traditional method of feeding molasses
medium for continuous ethanol fermentation in most of the ethanol
distilleries in China is shown in Fig. 4A. In this method, the molasses is
VOL. 109, 2010 ETHANOL PRODUCTION FROM NON-SULFURIC ACID-WASHED MOLASSES
FIG. 3. Schematic diagram of the continuous fermentation process, Process 4.
diluted to the required concentration in a medium preparation tank.
Sulfuric acid is added to decrease the pH and to kill the acid-producing
bacteria that generally contaminate molasses. After neutralization and
the addition of nutrient salt, the medium is fed to the fermentor.
Without sulfuric acid treatment, contamination would definitely
occur in the reactors and also in the medium preparation tank
where the sugar concentration is low enough for bacterial growth.
Indeed, the medium preparation tank actually provides suitable
growth conditions for contaminating bacteria. If sulfuric acid treat-
ment of the molasses is to be omitted, then it becomes very important
to consider how to repress the growth of bacteria in the medium
preparation step and in the subsequent reactor steps.
One good way to repress bacterial growth is to feed the
undiluted molasses directly to the reactor. This method would
avoid the problem of bacterial growth before feeding the molasses
to the reactor. Therefore, as long as the yeast cells in the reactor
had high metabolic activity, bacterial growth could be repressed
and bacterial contamination could be controlled. However, one
problem with this method is that, since the viscosity of undiluted
molasses is very high, it cannot be fed by a peristaltic pump on a
laboratory scale unless it is diluted. We therefore determined
whether there was a suitable dilution of molasses that would allow
it to be fed by peristaltic pump, but would still maintain a high
enough sugar concentration to prevent bacterial contamination. Our
experiments showed that molasses could be fed by a peristaltic
pump at a 1.2-fold dilution. The effect of dilution time on bacterial
contamination is shown in Fig. 5A. The concentration of total
volatile fatty acids did not increase after a 48 h incubation at 30 C
with molasses that was diluted 1.2- or 1.5-fold. However, it did
FIG. 4. Schematic diagram of the traditional molasses medium feeding method (A) and the method used in this study (B).
43
increase when the molasses was diluted 3-fold. As shown in Fig.
5B, no ethanol was produced when the molasses was diluted 1.2-
or 1.5-fold. These results suggest that not only was the growth of
bacteria inhibited, but the growth of yeast was also inhibited in
molasses that was diluted 1.2- or 1.5-fold, in which the sugar
concentration was high.
Based on these results, we designed the new molasses feeding
method that is shown in Fig. 4B. In this method, the molasses was first
diluted 1.2-fold with tap water and then fed directly to the reactor. At
the same time, tap water was fed separately to the reactor to adjust
the sugar content in the influent (molasses + tap water) to the desired
concentration. By changing the ratio of the feeding rates of 1.2-fold
dilutions of molasses and tap water, the sugar concentration in the
influent of each reactor could be controlled.
Continuous ethanol fermentation from non-sulfuric acid-
washed molasses Process 1 A one-stage fermentation pro-
cess was first studied to evaluate the molasses feeding method
proposed in the previous section. As shown in Fig. 6A, the
concentrations of lactic acid and acetic acid and the pH of the broth
were maintained at approximately 1900 mg/l, 400 mg/l and 4.5,
respectively, over the four-week operation period. Since the concen-
trations of lactic acid and acetic acid contained in the stored molasses
were approximately 7100 mg/l and 1360 mg/l, respectively, the
detected acids in the fermented broth were likely from the original
molasses and were not produced during the ethanol fermentation.
This result indicates that there was no bacterial contamination over
the entire operation period, and that the medium feeding method was
effective for long-term continuous ethanol fermentation from
molasses.
44 TANG ET AL.
FIG. 5. Effect of molasses dilution on bacterial contamination (A) and ethanol
fermentation (B). Open circles, 1.2-fold dilution of molasses; open squares, 1.5-fold
dilution of molasses; asterisk, 2-fold dilution of molasses; open triangles, 3-fold
dilution of molasses.
The concentration of ethanol, the concentration of residual sugar
and the total cell number in the broth are shown in Fig. 6B. These
parameters were maintained at a steady level over the entire
FIG. 6. Time course of various parameters during the continuous ethanol fermentation
in Process 1. During fermentation, the temperature, the aeration rate and the stirring
rate were controlled at 30 C, 0.15 vvm and 150 rpm, respectively. (A) Open circles, pH;
open squares, acetic acid concentration; open triangles, lactic acid concentration; (B)
open circles, ethanol concentration; open squares, residual sugar concentration; open
triangles, total cell number.
J. BIOSCI. BIOENG.,
operation period, suggesting that the fermentation system was stable.
The concentrations of ethanol and residual sugar were, on average,
67 g/l and 8 g/l, respectively. The total cell number was approximately
2.5 108 cells/ml, and the average percent of viable cells was
approximately 90%. The ethanol yield was 0.45 (88% of theoretical
value of 0.51), and the ethanol productivity was about 6.7 g/l/h. In
order to improve the reactor utility and to reduce the cost of ethanol
production, we aimed to increase the ethanol concentration in the
fermented both. We reported that, when two-stage tower reactors
replaced the one-stage tower reactor, the concentration of ethanol
increased from 67 g/l to 73 g/l at a sugar concentration of 163 g/l in
the influent (12). Therefore, two-stage fermentation processes using
the stirred tank reactor described below were examined in order to
meet this goal.
Process 2 and Process 3 The two-stage process (Process 2)
shown in Fig. 2 was studied first. Unexpectedly, the fermentation
results from this process were very bad (Fig. 7). The concentrations of
residual sugar in R1 and R2 increased to approximately 43 g/l and
78 g/l, respectively, and the concentrations of ethanol in R1 and R2
were almost the same, at about 50 g/l. Although the total cell number
in both reactors was about 4.0 108 cells/ml due to the recycling of the
cells to R1, the percentage of viable cells was relatively low, being only
about 65%. Because of these results, the system was stopped on the
fifth day and another two-stage process, Process 3, was studied (data
not shown).
Fermentation was obviously improved in Process 3 by recycling of
the cells to their original reactor. The ethanol concentration of the
fermented broth in R2 increased to 67 g/l, which was the same level as
that of R1. However, compared to Process 1, R2 did not contribute to
the fermentation. Furthermore, the ethanol yield from Process 3 was
only 0.37 (73% of theoretical value of 0.51).
The results of Processes 2 and 3 suggest that the most powerful
way to increase the ethanol concentration in R2 and to achieve a
higher ethanol yield would be to enhance the cell activity.
Process 4 and Process 5 Feeding cells with high activity directly
to R2 would be a good approach to improve the metabolic activity of
cells in R2. We therefore determined the optimum conditions for the
cultivation of cells with a high metabolic activity.
The effect of sugar concentration on cell activity is shown in Fig.
8A. Sugar concentrations of 80 g/l and 100 g/l resulted in the highest
cell activity, as assessed by the CO2 evolution rate. When the sugar
concentration was increased to 120 g/l, the CO2 evolution rate
decreased sharply. When the sugar concentration was increased to
150 g/l, the rate was only 4.2 l/min, which was only 24% of the rate
observed when the sugar concentration was 80 g/l or 100 g/l. The
total cell number was about 6.0 108 cells/ml when the sugar
concentration was 80 g/l or 100 g/l. This number decreased with
FIG. 7. Time course of various parameters during continuous ethanol fermentation in
Process 2. During fermentation, the temperature, the aeration rate and the stirring rate
were controlled at 30 C, 0.15 vvm and 150 rpm, respectively. Circles, ethanol
concentration (open, R1; closed, R2); squares, total cell number (open, R1; closed, R2);
triangles, residual sugar concentration (open, R1; closed, R2).
VOL. 109, 2010
FIG. 8. Effect of sugar concentration (A) and aeration rate (B) on various parameters of cell
activity. Cellular activity was assayed by measurement of: (A) the CO2 evolution rate
(open circles); total cell number (open squares); percent of viable cells (open triangles);
(B) intracellular trehalose (open triangles) and CO2 evolution rate (closed triangles).
increasing sugar concentration to about 2.0 108 cells/ml at a sugar
concentration of 150 g/l. The percentage of viable cells also decreased
with increasing sugar concentration, reaching approximately 98% and
90% at sugar concentrations of 80 g/l and 100 g/l, respectively. Hojo et
al. obtained the highest cell yield at a sugar concentration of 100 g/l,
when the yeast cell yield, Yx/s, was investigated under different sugar
concentrations (13).
On the basis of the above results, the effect of variations of the
aeration rate on cell activity was studied under conditions at which the
inlet sugar concentration was 90 g/l (Fig. 8B). Cell activity was
improved by aeration, and aeration rates of 0.1 and 0.15 vvm,
corresponding to a range of kLa from 90 to 130 h 1, resulted in the
highest cell activity.
Since yeast intracellular trehalose can be used to investigate
whether the cells are in unfavorable conditions (11, 14), the trehalose
content of cells was therefore determined under different aeration
rates. The trehalose content was high when no air was supplied. As
expected, it decreased to about one half when air was supplied at a
rate of 0.025 vvm. However, it was maintained at a stable level when
the aeration rate was increased from 0.025 to 0.15 vvm, corresponding
to a kLa range from 40 to 130 h 1. Judging from the cell activity and
the intracellular trehalose content, enough air to maintain a kLa of
over 40 h 1 should be supplied to the reactor in order to ensure the
presence of cells with a high metabolic activity.
Since a sugar concentration of 100 g/l and an aeration rate of more
than a kLa of 40 h 1 are sufficient to obtain enough cells with high
activity, these conditions were used to operate R3 in Processes 4 and 5.
As shown in Fig. 3, Process 4 was studied to elucidate the effect of
feeding cells with high activity to R2. In this process, cells with high
activity were fed from R3 at a flow rate of 200 ml/h. The system was
operated for more than one month, and the fermentation results are
shown in Figs. 9A and B. The ethanol concentrations in R1, R2 and R3
were maintained at stable levels with average values of 67 g/l, 80 g/l
and 40 g/l, respectively. The total cell numbers in R1, R2 and R3 were
maintained at approximately 2.5 108, 4.3 108, and 6.3 108 cells/
ETHANOL PRODUCTION FROM NON-SULFURIC ACID-WASHED MOLASSES 45
ml, respectively, and the percentages of viable cells were about 90%,
90% and 98%, respectively. The ethanol yield in Process 4 was about
0.44 (87% of theoretical value of 0.51), and the ethanol productivity
was 6.6 g/l/h. This result suggests that fermentation was effectively
improved by increasing the metabolic activity of the cells in R2.
To determine the importance of R1, we further studied Process 5,
which was a modification of Process 4 that lacked R1 (data not
shown). Although Process 5 could be stably maintained, the ethanol
concentration in R2 decreased to 70 g/l, corresponding to an ethanol
yield of 0.39 (76% of the theoretical value of 0.51) and an ethanol
productivity of 4.6 g/l/h.
Based on the results obtained with Processes 1 to 5, Process 4
appeared to be the best process for long-term ethanol fermentation
using non-sulfuric acid-washed molasses as feedstock. When a
traditional stirred tank-type reactor was employed using the condi-
tions of Process 4, an ethanol yield of 0.44 and an ethanol productivity
of 6.6 g/l/h were obtained. These results are excellent and better than,
or at least comparable to, similar studies in which flocculating yeast,
similar to the ones used in this study, were used (13), studies in which
the cells were recycled after centrifugation (15), or studies in which
immobilized cells were used (16, 17).
In conclusion, sulfuric acid is used by most of the ethanol distilleries
in China to treat molasses before it is fed to the ethanol fermentation
reactor. In this manner, it is possible to operate a continuous ethanol
fermentation system that is stable over a long-term period without the
problem of bacterial contamination. However, since the resulting
wastewater contains a high concentration of sulfate ions, it is difficult
to treat it with traditional biological techniques. As an inevitable choice,
large distilleries in China concentrate and combust the wastewater. In
this study, using the flocculating yeast strain KF-7, a non-sterilized
continuous ethanol fermentation without sulfuric acid pretreatment
was studied using traditional stirred tank reactors. A new molasses
medium feeding method that separately supplies molasses and tap
water was proposed. This method was confirmed to be effective for the
control of bacterial contamination during long-term continuous
fermentation. A two-stage fermentation process was studied to achieve
a high ethanol concentration and to make the ethanol production more
economical. In Process 4, by feeding high activity yeast cells to the
FIG. 9. Time course of ethanol concentration (A) and total cell number (B) during
continuous ethanol fermentation of Process 4 over a period of 35 d. Open circles, R1;
open squares, R2; open triangles, R3.
46 TANG ET AL.
second reactor, the ethanol concentration in the fermented broth
reached 80 g/l, and the ethanol productivity was 6.6 g/l/h. These
results are much better than those obtained with the process currently
employed in most distilleries. Process 4 could be used instead of the
fermentation process now employed by most of the ethanol distilleries
in China to make ethanol production from molasses more effective and
more environmentally friendly than with the current methods.
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