b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5 e4 0 1

Available online at www.sciencedirect.com

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

Factors affecting ethanol fermentation using Saccharomyces

cerevisiae BY4742

Yan Lin a,b,*, Wei Zhang a, Chunjie Li a, Kei Sakakibara b, Shuzo Tanaka b, Hainan Kong a
a
School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China
b
Program of Environment and Ecology, Faculty of Science and Engineering, Meisei University, Tokyo 191-8506, Japan

article info abstract

Article history: Fermentation of sugar by Saccharomyces cerevisiae BY4742, for production of ethanol in
Received 9 November 2011 a batch experiment was conducted to improve the performance of the fermentation
Received in revised form process. The thermotolerant ability of S. cerevisiae to grow and ferment glucose at elevated
1 February 2012 temperatures similar to the optima for saccharification was investigated. The influences of
Accepted 5 September 2012 temperature, substrate concentration and pH on ethanol fermentation were observed. The
Available online 6 October 2012 yield for ethanol production and changes in the fermentation pathway were compared
under different conditions.
Keywords: When the temperature was increased to 45
C, the system still showed high cell growth
Ethanol and ethanol production rates, while it was inhibited at 50
C. The maximum specific
Fermentation growth rate and the maximum specific ethanol production rate were observed between 30
Saccharomyces cerevisiae and 45
C with different initial glucose concentrations. The maximum sugar conversion at
Yeast 30
C after 72 h incubation was 48.0%, 59.9%, 28.3%, 13.7% and 3.7% for 20, 40, 80, 160 and
Thermotolerant ability 300 kg m3 of glucose concentrations respectively. Increased substrate supply did not
Strain optimization improve the specific ethanol production rate when the pH value was not controlled. pH 4.0
e5.0 was the optimal range for the ethanol production process. The highest specific
ethanol production rate for all the batch experiments was achieved at pH5.0 which is
410 g kg1 h1 of suspended solids (SS) which gave an ethanol conversion efficiency of
61.93%. The highest specific ethanol production rate at 4.0 was 310 g kg1 h1 of SS. A
change in the main fermentation pathway was observed with various pH ranges. Forma-
tion of acetic acid was increased when the pH was below 4.0, while butyric acid was
produced when the pH was higher than 5.0. In the presence of oxygen, the ethanol could be
utilized by the yeast as the carbon source after other nutrients became depleted, this could
not occur however under anaerobic conditions.
ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction a means of providing modern energy to the billions who lack

Growing attention has been devoted in the past years to the
conversion of biomass into fuel ethanol, considered the
cleanest liquid fuel alternative to fossil fuels. It is now
understood that it is important to use biomass energy as
it, and it may also be a viable alternative energy source to the
worlds ever depleting natural reserves [1].
There are several kinds of raw materials for ethanol
fermentation. For the utilization of cellulose as the raw
material, simultaneous saccharification and fermentation

* Corresponding author. School of Environmental Science and Engineering, Shanghai Jiao Tong University, Hino, Shanghai 200240,
PR China. Tel.: þ86 21 54744540; fax: þ86 21 54740825.
E-mail address: linyansjtu@126.com (Y. Lin).
0961-9534/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2012.09.019
396 b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5 e4 0 1
(SSF) combines enzymatic hydrolysis of cellulose with simul-
taneous fermentation of the sugars obtained to ethanol, and
gives higher reported ethanol yields. This therefore requires
lower amounts of enzyme because the yeast fermentation
step helps reduce the end products inhibition from cellobiose
and glucose formed during enzymatic hydrolysis [2e4].
However, SSF requires compatible fermentation and
saccharification conditions with a similar pH, temperature
and optimum substrate concentration [5,6]. The main diffi-
culty with SSF of cellulose is the different temperature optima
required for saccharification (40e50
C) and fermentation
(20e35
C) [5e7]. In order to preserve the viability of the yeast,
the SSF process is typically operated between 30 and 38
C at
which the cellulose is far below its peak operational level [6].
The use of thermotolerant yeasts capable of fermenting
glucose to ethanol at elevated temperatures, which are closer
to the optima for the activity of the cellulolytic complex, is
therefore advisable when employing SSF processes [7].
Moreover, for the existing or converted fermentable
sugars, it is also important to improve the efficiency of the
fermentation system to utilize it with a high ethanol produc-
tion ability. On the other hand, since the distillation cost per
unit amount of ethanol produced is substantially higher at low
ethanol concentrations [8], several investigators have dealt
with the idea of concentrating sugar solutions prior to
fermentation [8e11]. Clearly it is necessary to improve the
ethanol fermentation performance and to solve the problem
between the concentration of ethanol produced and sugar
added if an economically sustainable system is to be created
using this method. During batch fermentation, many influ-
ential parameters, such as pH, pO2 and temperature, can
greatly influence the specific rate of growth, and inhibition
can be caused either by product or substrate concentration
[12,13]. The viability of cell populations, the specific rate of
fermentation and the sugar uptake rate are all directly related
to the desired medium condition [14e16].
The purpose of this research was to obtain high ethanol
production with high productivity and to test the thermoto-
lerant ability of Saccharomyces cerevisiae to grow and ferment
glucose at elevated temperatures similar to the optima for
saccharification. The effect of temperature, pH value and
initial glucose concentration on the production of ethanol by
S. cerevisiae was evaluated to improve the ethanol fermenta-
tion performance.
2. Materials and methods
2.1. Yeast strain and culture conditions
S. cerevisiae BY4742 (originally from EUROSCARF, Germany)
[17,18], a strain preserved at the laboratory (Department of
Chemistry, Meisei University, Japan), was used in this study. S.
cerevisiae was maintained at 4
C on agar slants containing (in
kg m3): bacto-yeast extract 10; bacto-peptone 20; glucose 20,
and bacto-agar 20. The prepared media was sterilized at 121
C
for 20 min. Pre-cultures were inoculated from agar slants and
grown at 30
C overnight in 250 ml shake-flasks with mineral
medium containing (in kg m3): bacto-yeast extract 10; bacto-
peptone 20 and glucose 20 while stirring at 1.8 Hz. Pre-cultures
of (10 ml) were used to inoculate 500 ml baffled shake-flasks
containing 250 ml of the above media. The inoculum was
grown for approximately 24 h and used to further inoculation.
2.2. Batch fermentation
Batch fermentation experiments on the effects of temperature
and initial substrate concentrations under microaerobic
conditions (stirring at 1.8 Hz with a final dissolved oxygen
concentration of 1.0 g m3) were carried out in duplicate twice
using 20e300 kg m3 of glucose of the initial glucose solution
as the sole carbon source for S. cerevisiae. Experiments were
performed in 500 ml Erlenmeyer flasks with 250 ml total liquid
volume and were initiated by transferring 5% of the starter
culture to the prepared media. The experiments were carried
out for 7 days in isothermal conditions at 30
C and monitored
by harvesting 15 ml samples every 24 h for analyses.
Experiments on the effect of pH on ethanol fermentation
were run in a fermentor (MBF-500ME P.A., EYELA) with
a 0.003 m3 working volume, stirred at 1.3 Hz, and the tempera-
ture was controlled at 30
C. The pH value was controlled by
automatic addition of 2 kmol m3 NaOH or HCl. The fermentor
was sealed and equipped with a syringe for sample removal. It
was also fitted with a CO2 exhaustion port to allow venting of
carbon dioxide which is a byproduct of the reaction. The
experiments were started by adding the specific amount of
yeast inoculated into the medium containing different amounts
of glucose. The final yeast concentration was 2 kg m3 of SS.
2.3. Analytical methods
Ethanol and Volatile Fatty Acid (VFA) were measured using
a gas chromatograph (GC-8APF, SHIMADZU) equipped with
a 3 m  2.6 mm glass column packed with polyethyleneglycol
(PEG) (Chromosob W 80/100 AW-DMCS, SHIMDZU) (80e100
mesh). The column temperatures for ethanol and VFA
analyzing were 80
C and 140
C, respectively. The injection
port and flame ionization detector temperature for ethanol
was 100
C, while for VFA it was 180
C. Nitrogen, used as the
carrier gas for both ethanol and VFA, was set at gas flow
pressures of 200 and 150 kPa, respectively. Before injection,
analyzed samples were filtered through a 0.20 mm membrane
filter to allow determination of the VFA and ethanol concen-
trations in supernatants. Cell growth was determined by
measuring the optical density (OD) at 600 nm using a spec-
trophotometer (UV-1600, SHIMADZU). The cell dry weight was
obtained using a calibration curve. The cell dry weight was
proportional to cell turbidity and absorbance at 600 nm [19].
Biomass concentration was determined by use of the dry-
weight method for SS [13]. Accordingly, samples were centri-
fuged, and then the settled solids were washed with distilled
water and dried for 2 h at 105
C.
3. Results and discussion
3.1. Influence of temperature
Competition during ethanol fermentation carried out at
different temperatures may be a way of testing the endurance
 b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5 e4 0 1
of the strain used in this system. This could then be used as
a method for determining the optimal condition for ethanol
fermentation and also a criterion for rapidly selecting one of
several strains while at the same time studying resistance to
temperature in a controlled situation, i.e. under laboratory
conditions.
In this study, the influence of temperature on the ethanol
fermentation by S. cerevisiae BY4742 was studied with regard
to biomass and ethanol production. Batch fermentation in
shake flasks for ethanol production was carried out in dupli-
cate for one week at various initial glucose concentrations
from 20 to 300 kg m3 and controlled at constant temperatures
of 10, 20, 30, 40, 45 and 50
C. Experimental results revealed the
cells increased exponentially at the beginning of incubation,
then entered a stationary phase after several days’ incubation,
for all operating temperatures. Higher temperatures made the
exponential growth of the cells shorter (data not shown).
Fig. 1 shows the changes in ethanol concentration at
different temperatures with the initial glucose concentration
of 40 kg m3 and yeast concentration of 2 kg m3 of SS over
a one week incubation period. For general ethanol production
by yeast, the maximum fermentation time in batch process
was 72 h [20].
Experimental data in Fig. 1 showed that when the
temperature increased, the maximum fermentation time was
shortened, but a much higher temperature inhibited the
growth of cells and then the fermentation significantly
declined. In this study, cell growth and ethanol production
declined considerably at 50
C, which showed the inhibition
effect on cell growth at higher temperatures. This phenom-
enon may be explained because the higher temperature
results in changing the transport activity or saturation level of
soluble compounds and solvents in the cells, which might
increase the accumulation of toxins including ethanol inside
cells [20,21]. Moreover, the indirect effect of high temperature
might also be ascribed to the denaturation of ribosomes and
enzymes and problems with the fluidity of membranes [20,21].
25
10°C
30°C
Ethanol concentration (kg m )
45°C
-3
20
15
10
5
0
0 24 48 72 96
Incubation time(hours)
Fig. 1 e Changes in ethanol concentration at different
temperatures with an initial glucose concentration of
40 kg mL3 over a one week incubation period.
397
However, at lower temperatures the cells showed lower
specific growth rates which may be attributed to their low
tolerance to ethanol at lower temperatures [22,23]. The
maximum specific growth rate and the maximum specific
ethanol production rate were observed between 30 and 45
C
with different initial glucose concentrations as shown in Fig. 3.
It is commonly believed that 20e35
C is the ideal range for
fermentation and at higher temperatures almost all fermen-
tation would be problematic [6,7,12,20,22]. However, as shown
in Figs. 2 and 3 in this study, when the temperature was
increased to 45
C, the system still showed a high cell growth
and ethanol production rates and the lowest mt/m30 at different
glucose concentrations was around 0.8. We also observed
a higher specific ethanol production rate at higher glucose
concentrations when tested at 45
C. With a higher tolerant
fermentation temperature, similar to the optimal temperature
for cellulolytic activity, it may be possible for the SSF process
to improve the final efficiency. Moreover, as shown in Fig. 1,
ethanol yields may further be improved at elevated temper-
atures for a shorter culture time.
In addition, the ethanol concentration was found to peak
and then decline at temperatures above 20
C, and the lower
glucose concentration made the decline time occur earlier.
These biochemical changes may indicate that cells originally
growing on glucose switched from a fermentative metabo-
lism using mainly glycolysis and forming ethanol, to a respi-
ratory metabolism in which the ethanol formed in the earlier
stages of growth was consumed using the tricarboxylic acid,
glyoxylate cycles and mitochondrial electron transport chain
[24]. Results in Fig. 1 also show that ethanol concentration
rose steadily at low temperatures and won’t decline within
168 h, possibly because at these lower temperatures the yeast
was not active which is because of the low tolerance to
ethanol [22,23].
2.0
20 kg m-3
1.8 40 kg m-3
80 kg m-3
1.6 160 kg m-3
20°C 300 kg m-3
40°C
1.4
50°C
1.2
µ t /µ 30
1.0
0.8
0.6
0.4
0.2
10 20 30 40 50
120 144 168 Temperature (°C)
Fig. 2 e Ratio of specific growth rate (m) at different
controlled temperatures to that at 30
C with different
initial sugar concentrations after 72 h incubation (mt/m30 is
a ratio of specific growth rate at t
C to m at 30
C).
398 b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5 e4 0 1

200
48h-SEPR 72h-SEPR

Specific Ethanol Production Rate(g kg h ) and

180 48h-ECR 72h-ECR

-1
160

-1

Ethanol Conversion Rate (%)

140

120

100

80

60

40

20

0
0 50 100 150 200 250 300
-3
Initial glucose concentration(kg m )

Fig. 3 e Specific ethanol production rates at different initial Fig. 5 e Specific ethanol production rates and ethanol
sugar concentrations with different controlled conversion efficiency at different initial sugar
temperatures after 72 h’ incubation. * Specific ethanol concentrations after 48 and 72 h incubation at 30
C.
production rates were calculated as milligrams of ethanol
produced per grams of cell mass per hour (g kgL1 hL1
of SS).
The production of ethanol was affected by the substrate
concentration between 20 and 300 kg m3. As shown in Fig. 4,
higher substrate concentrations may achieve higher ethanol
3.2. Influence of substrate concentration
production, but a longer incubation time was required for
higher initial glucose concentrations above 80 kg m3 at
The batch experiment was performed with various glucose
a temperature of 30
C when the pH was not controlled. More-
concentrations to develop ethanol production. The initial
over, higher initial glucose concentrations, such as 300 kg m3,
glucose concentrations in the batch experiments were 20, 40,
may have actually decreased the ethanol conversion efficiency
80, 160 and 300 kg m3 tested at 30
C. The experimental
when the pH value was not controlled, since the higher
conditions and the results summarized in Fig. 4 show the
substrate and production concentrations may have inhibited
changes in ethanol concentrations at different initial glucose
the process of ethanol fermentation (as shown in Fig. 5).
concentrations over a one week incubation period, while Fig. 5
Fig. 5 shows the specific ethanol production rates and
demonstrates the specific ethanol production rate and
ethanol conversion efficiency at different initial sugar
ethanol conversion rate at different initial glucose concen-
concentrations after 48 and 72 hour’s incubation at 30
C. The
trations after 48 and 72 hour’s incubation.
data above illustrates that higher initial glucose concentration
may decrease the ethanol conversion efficiency. The
35 maximum sugar conversion after 72 hour’s incubation was
20 kg m-3 observed at 48.0%, 59.9%, 28.3%, 13.7%, and 3.7% for 20, 40, 80,
40 kg m-3
30 80 kg m-3 160 and 300 kg m3 of glucose, respectively. More substrate did
160 kg m-3 not improve the specific ethanol production rate when the pH
Ethanol concentration (kg m)
-3

25 300 kg m-3
value was not controlled.

20
3.3. Influence of pH
15
Improved ethanol fermentation activity can be achieved by
10 controlling various parameters. In addition to temperature
and substrate concentration, pH is also a key factor that
5 affects ethanol fermentation [13]. In this study changes in
ethanol and VFAs were investigated to estimate the activity of
0 the ethanol production ability with changes in pH. This was
examined at pHs 3.0, 4.0, 5.0, 5.5 and 6.0 in an anaerobic Jar
0 24 48 72 96 120 144
Fermentor.
Incubation time(hours)
Fig. 6 shows the results of the batch test used to investigate
Fig. 4 e Changes in ethanol concentration under different the effect of pH on ethanol production. When the pH was
glucose concentrations after 6 days’ incubation at 30
C. lower than 4.0, the incubation time for maximum ethanol
 b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5 e4 0 1 399

25 500
pH=5.0 pH not controlled 300kg m-3-pH not controlled

Specific ethanol production rate (g kg h )

-1
pH=4.0 pH=6.0 450 300kg m-3-pH4
pH=5.5 pH=3.0 160kg m-3-pH not controlled

-1
400 160kg m-3-pH4
20
Ethanol concentration (kg m )
-3

350

300
15
250

200
10 150

100

5 50

0
0 24 48 72 96 120 144 168
0 Incubation time(hours)
0 24 48 72 96 120 144 168
Fig. 7 e Comparison of the specific ethanol production
Incubation time(hours)
rates between pH values set at 4.0 and uncontrolled pH
Fig. 6 e Changes in ethanol concentration with an initial with initial sugar concentrations of 160 and 300 kg mL3
glucose concentration of 40 kg mL3 over one week’s after one week’s incubation at 30
C.
incubation with different pH values.

microorganisms, and may suggest a change in the main

concentration was prolonged, but the maximum concentra-
tion was not very low. When the pH value was above 5.0, the
quantity of ethanol produced substantially decreased.
Therefore a pH range of 4.0e5.0 may be regarded as the
operational limit for the anaerobic ethanol production
process. The highest specific ethanol production rate for all
the batch experiments was achieved at pH 5.0 which is
410 g kg1 h1 of SS, with an ethanol conversion efficiency of
61.93%. The specific ethanol production rate at pH4.0 was
310 g kg1 h1 of SS, which is not significantly lower than the
value obtained at pH5.0. Therefore, considering the chemical
requirement for pH adjustment, pH 4.0 may be regarded as the
operational limit for the ethanol production process.
In addition, in Fig. 6 the ethanol concentration did not
decrease after the nutrient was consumed as in Figs. 1 and 4.
This may indicate that the ethanol could not be utilized as the
carbon source under anaerobic condition.
Our experimental results could indicate that pH plays
an important role in determining the fermentation pathway
used in anaerobic ethanol production processes. Table 1
shows competition for the substrate, glucose, by the
fermentation pathway at various pH ranges. The above results
show that the main products were ethanol and butyrate
between pH 5.5e6.0 at 30
C with the initial glucose concen-
tration of 40 kg m3. When the pH value was lower than 5.0,
acetic acid was the main product. These results suggest that
the mechanism of ethanol production may include the reac-
tions as follows:
4C6 H12 O6 /2CH3 COOH þ 3CH3 ðCH2 Þ2 COOH þ 8H2 þ 8CO2 (1)
C6 H12 O6 þ H2 O/C2 H5 OH þ CH3 COOH þ 2H2 þ 2CO2 (2)
Eqs. (1) and (2) refer to Moat and Gaudy and Gaudy,
respectively [25,26]. As indicated in Eq. (1), 4 mol of glucose
were converted to 2 mol of acetic acid and 3 mol of butyrate
acid. Here, although the butyrate acid formation is not popu-
larly discussed in yeast metabolism, this phenomena was
observed in this study and also agreed with the results of
Teresa and Carmen [27].
There are also many examples of an alteration of the fatty
acid profile in the yeast [28,29]. During this process, with the
pH value higher than 5.0, much glucose was consumed and

Table 1 e The ratio of ethanol and VFAs to total productsa after 48 and 72 h incubation at 30
C with different pH values.
pH value Ratio of ethanol and VFAs after 48 h incubation (%) Ratio of ethanol and VFAs after 72 h incubation (%)

Ethanol Acetic acid Butyric acid Ethanol Acetic acid Butyric acid

No control 65.36 1.41 0.15 65.55 1.48 0.10

3.0 65.15 2.21 0.07 65.15 2.51 0.07
4.0 65.54 1.32 0.09 65.54 1.28 0.05
5.0 65.54 1.63 0.02 65.54 0.86 1.51
5.5 56.49 6.01 9.18 56.49 3.01 15.74
6.0 48.80 9.00 17.05 48.80 6.05 14.80

a Total products include ethanol, VFAs, CO2 and glucose in terms of carbon.
400 b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5 e4 0 1

100 The maximum specific ethanol production rates were

300 kg m-3-no control observed between 30 and 45
C with different initial glucose
90 300 kg m-3-pH4
concentrations. When the temperature was increased to 45
C,
Ethanol conversion efficiency (%)

160 kg m-3-no control

80 160 kg m-3-pH4 the system still showed higher cell growth and ethanol
Theoretical value
70 production rates and the lowest mt/m30 at different initial
glucose concentrations was 0.8.
60
Higher substrate concentration could achieve higher
50 ethanol production, but a longer incubation time was required
40 for initial glucose concentrations above 80 kg m3 at 30
C
when the pH was not controlled.
30
The changes in the operational pH in the ethanol produc-
20 tion process may have induced a change in the main
10 fermentation pathway. Thus it is important to control pH
value in the range of 4.0e5.0. Beyond this range, the formation
0 of by-products, such as acetic acid and butyric acid may have
0 24 48 72 96 120 144 168 consumed some of the substrate and reduced the efficiency of
Incubation time(hours) ethanol fermentation.

Fig. 8 e Comparison of ethanol conversion efficiency

between pH set at 4.0 and uncontrolled pH with initial
sugar concentrations of 160 and 300 kg mL3 after one
Acknowledgments
week’s incubation at 30
C. * The maximum theoretical
ethanol fermentation yield was expressed by carbon and
The research project was sponsored by Major Science and
calculated according to the equation of
Technology Program for Water Pollution Control and Treat-
C6H12O6 / 2C2H5OH D 2CO2.
ment (2009ZX07101-015-003) and State Key Laboratory of
Pollution Control and Resource Reuse Foundation
(PCRRF09002).
converted to by-products, so the ethanol conversion efficiency
was greatly decreased (as shown in Figs. 7 and 8).
In the case of Eq. (2), 1 mol of glucose was converted into references
1 mol of ethanol and 1 mol of acetic acid. Although there was
some ethanol produced, the ethanol fermentation yield was
still reduced by the acetic acid production. Table 1 shows that [1] Ueno R, Urano N, Kimura S. Effect of temperature and cell
when the pH was 4.0 and 5.0, the quantity of by-products was density on ethanol fermentation by a thermotolerant aquatic
yeast strain isolated from a hot spring environment. Fish Sci
less than that observed under other conditions.
2002;68:571e8.
Results in section 3.2 show that a higher substrate [2] Banat IM, Nigam P, Singh D, Marchant P, McHale AP. Ethanol
concentration may prevent the ethanol fermentation process production at elevated temperatures and alcohol
occurring. One of the reasons may be the accumulation of concentrations. Part I: Yeasts in general. World J Microbiol
high concentrations of ethanol and by-products which make Biotechnol 1998;14:809e21.
the pH change. So if the pH was set at a suitable value, the [3] Lin Y, Tanaka S. Ethanol fermentation from biomass
resources: current state and prospects. Appl Microbiol
efficiency might be somewhat increased. Compared with
Biotechnol 2006;69:627e42.
fermentation where the pH was not controlled, when the pH
[4] McMillan JD, Newman MM, Templeton DW, Mohagheghi A.
was controlled at 4.0, the VFAs in the final product were Simultaneous saccharification and cofermentation of dilute-
reduced and the specific ethanol production rate and the acid pretreated yellow poplar hardwood to ethanol using
ethanol fermentation efficiency were significantly improved xylose-fermenting Zymomonas mobilis. Appl Biochem
(as shown in Figs. 7 and 8). After pH adjustment, the ethanol Biotechnol 1999;77/79:649e55.
fermentation yields were increased to the range of 99.8% and [5] Krishna SH, Reddy TJ, Chowdary GV. Simultaneous
saccharification and fermentation of lignocellulosic wastes
97.4% of the maximum theoretical value with the initial
to ethanol using a thermotolerant yeast. Bioresour Technol
glucose concentrations of 160 and 300 kg m3, respectively. 2001;77:193e6.
The highest specific ethanol production rates at pH 4.0 were [6] Wu ZW. Bioconversion of lignocellulosic materials into fuel
340 and 260 g kg1 h1 of SS, respectively. ethanol: pretreatment and non-isothermal simultaneous
saccharification and fermentation. 1st ed. US: Auburn
University; 1998. pp. 30e1.
[7] Ballesteros M, Oliva JM, Negro MJ, Manzanares P,
4. Conclusions Ballesteros I. Ethanol from lignocellulosic materials by
a simultaneous saccharification and fermentation process
(SFS) with Kluyveromyces marxianus CECT 10875. Process
The present work tested the thermotolerant ability of S. cer-
Biochem 2004;39:1843e8.
evisiae to grow and ferment glucose at elevated temperatures [8] Zacchi G, Axelsson A. Economic evaluation of
and examined the influences of temperature, initial substrate preconcentration in product & of ethanol from dilute sugar
concentration and pH value on ethanol fermentation. solutions. Biotechnol Bioeng 1989;34(2):223e33.
 b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5 e4 0 1
[9] Cysewski GR, Wilke CR. Utilization of cellulosic materials
through enzymatic hydrolysis. I. Fermentation of
hydrolysate to ethanol and single cell protein. Biotechnol
Bioeng 1976;18:1297e313.
[10] Iraj N, Giti E, Lila A. Isolation of flocculating Saccharomyces
cerevisiae and investigation of its performance in the
fermentation of beet molasses to ethanol. Biomass Bioenergy
2002;23:481e6.
[11] Oh KK, Kim SW, Jeong YS, Hong SI. Bioconversion of cellulose
into ethanol by nonisothermal simultaneous
saccharification and fermentation. Appl Biochem Biotechnol
2000;89:15e30.
[12] Aldiguer AS, Alfenore S, Cameleyer X, Goma G. Synergistic
temperature and ethanol effect on Saccharomyces cerevisiae
dynamic behaviour in ethanol bio-fuel production. Bioproc
Biosyst Eng 2004;26:217e22.
[13] Kasemets K, Nisamedtinov I. Growth characteristics of
Saccharomyces serevisiae S288C in changing environmental
conditions: auxo-accelerostat study. Anton Leeuw 2007;92:
109e28.
[14] Gikas P, Livingston AG. Specific ATP and specific oxygen
uptake rate in immobilized cell aggregates: experimental
results and theoretical analysis using a structured model
of immobilized cell growth. Biotechnol Bioeng 1997;55:
660e72.
[15] Gunasekaran P, Raj KC. Ethanol fermentation
technologyeeZymomonas mobilis. Curr Sci 1999;77:56e68.
[16] Holzberg I, Finn RF, Steinkraus KH. A kinetic study of the
alcoholic fermentation of grape juice. Biotechnol Bioeng
1967:413e23.
[17] Palmer C, Zhou XL, Lin J, Loukin SH, Kung C, Saimi Y. A TRP
homolog Saccharomyces cerevisiae forms anintrace1lular Ca2þ-
Permeable channel in the yeast vacuolar membrane. Proc
Natl Acad Sci USA 2001;98:7801e5.
[18] Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, et al.
Designer deletion strains derived from Saccharomyces cerevisiae
S288C: a useful set of strains and plasmids for PCR-mediated
gene disruption and other applications. Yeast 1998;14:115e32.
[19] Skory CD. Lactic acid production by Saccharomyces serevisiae
expressing a Rhizopus oryzae lactate dehydrogenase gene.
J Ind Microbiol Biotechnol 2003;30:22e7.
401
[20] Phisalaphong M, Srirattana N, Tanthapanichakoon W.
Mathematical modeling to investigate temperature effect on
kinetic parameters of ethanol fermentation. Biochem Eng J
2006;28:36e43.
[21] McMeckin TA, Olley J, Ratkwsky DA, Ross T. Predictive
microbiology: towards the interface and beyond. Int J Food
Microbiol 2002;73:395e407.
[22] Gao C, Fleet GH. The effects of temperature and pH on the
ethanol tolerance of the wine yeasts: Saccharomyces cerevisiae,
Candida stellata and Kloeckera apiculata. J Appl Bacteriol 1988;
65:405e10.
[23] Torija MJ, Rozes N, Poblet M, Guillamon JM, Mas A. Effects of
fermentation temperature on the strain population of
Saccharomyces cerevisiae. Int J Food Microbiol 2003;80:47e53.
[24] Dickinson JR, Schweizer M. The metabolism and molecular
physiology of Saccharomyces cerevisiae. 2nd ed. Florida: CRC
Press; 2004. pp. 13 and 66.
[25] Gaudy AF, Gaudy ET. Microbiology for environmental
Scientists and Engineers. New York: McGraw-Hill; 1980. pp.
519e66.
[26] Moat AG. Microbial physiology. 4th ed. New York: John Wiley
and Sons; 1979. pp. 123e89.
[27] Teresa GC, Carmen AA. Contribution of wild yeasts to the
formation of volatile compounds in inoculated wine
fermentations. Eur Food Res Technol 2006;222:15e25.
[28] Wang C, Xing J, Chin CK, Ho CT, Martin CE. Modification of
fatty acids changes the flavor volatiles in tomato leaves.
Phytochemistry 2001;58:227e32.
[29] Dyer JM, Chapital DC, Kuan JW, Mullen RT, Pepperman AB.
Metabolic engineering of Saccharomyces cerevisiae. The role of
the CDP-choline pathway. J Biol Chem 2001;276:3756e63.
Yan Lin holds a Ph.D. (2005) in environmental engineering from
Shanghai Jiao Tong University, China. She has worked as a post-
doctoral researcher in the Asian Center for Environmental
Research at Meisei University, Japan since 2005. She is now an
associate professor in Shanghai Jiao Tong University. Her current
project is “Environmental conservation technology and utilization
of biomass in Asia (environmental technology) e ethanol
fermentation”, financed by the Ministry of Education, Culture,
Sports, Science and Technology, Japan.