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Factors affecting ethanol fermentation using Saccharomycescerevisiae 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 Chinab Program of Environment and Ecology, Faculty of Science and Engineering, Meisei University, Tokyo 191-8506, Japan

a r t i c l e i n f o

Article history:

Received 9 November 2011

Received in revised form

1 February 2012

Accepted 5 September 2012

Available online 6 October 2012

Keywords:

Ethanol

Fermentation

Saccharomyces cerevisiae

Yeast

Thermotolerant ability

Strain optimization

* Corresponding author. School of EnvironmPR China. Tel.: þ86 21 54744540; fax: þ86 21

E-mail address: linyansjtu@126.com (Y. L0961-9534/$ e see front matter ª 2012 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2012.09.

a b s t r a c t

Fermentation of sugar by Saccharomyces cerevisiae BY4742, for production of ethanol in

a batch experiment was conducted to improve the performance of the fermentation

process. The thermotolerant ability of S. cerevisiae to grow and ferment glucose at elevated

temperatures similar to the optima for saccharification was investigated. The influences of

temperature, substrate concentration and pH on ethanol fermentation were observed. The

yield for ethanol production and changes in the fermentation pathway were compared

under different conditions.

When the temperature was increased to 45 �C, the system still showed high cell growth

and ethanol production rates, while it was inhibited at 50 �C. The maximum specific

growth rate and the maximum specific ethanol production rate were observed between 30

and 45 �C with different initial glucose concentrations. The maximum sugar conversion at

30 �C after 72 h incubation was 48.0%, 59.9%, 28.3%, 13.7% and 3.7% for 20, 40, 80, 160 and

300 kg m�3 of glucose concentrations respectively. Increased substrate supply did not

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 kg�1 h�1 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 kg�1 h�1 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

ental Science and Engin54740825.in).ier Ltd. All rights reserved019

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

eering, Shanghai Jiao Tong University, Hino, Shanghai 200240,

.

b i om a s s an d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5e4 0 1396

(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 m�3): bacto-yeast extract 10; bacto-peptone 20; glucose 20,

and bacto-agar 20. The preparedmediawas sterilized at 121 �Cfor 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 m�3): bacto-yeast extract 10; bacto-

peptone 20 and glucose 20while 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 m�3) were carried out in duplicate twice

using 20e300 kg m�3 of glucose of the initial glucose solution

as the sole carbon source for S. cerevisiae. Experiments were

performed in 500ml Erlenmeyer flasks with 250ml 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 andmonitored

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 m�3 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 themediumcontainingdifferentamounts

of glucose. The final yeast concentration was 2 kg m�3 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

2.0

b i om a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5e4 0 1 397

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 kgm�3 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 m�3 and yeast concentration of 2 kg m�3 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 problemswith the fluidity ofmembranes [20,21].

0

5

10

15

20

25

0 24 48 72 96 120 144 168

Incubation time(hours)

Eth

anol

con

cent

ratio

n (k

g m

-3)

10°C 20°C30°C 40°C45°C 50°C

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.

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 �Cwith 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].

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

10 20 30 40 50Temperature (°C)

µt/µ

30

20 kg m-340 kg m-380 kg m-3160 kg m-3300 kg m-3

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).

Fig. 3 e Specific ethanol production rates at different initial

sugar concentrations with different controlled

temperatures after 72 h’ incubation. * Specific ethanol

production rates were calculated as milligrams of ethanol

produced per grams of cell mass per hour (g kgL1 hL1

of SS).

0

20

40

60

80

100

120

140

160

180

200

0 50 100 150 200 250 300

Initial glucose concentration(kg m-3)

Spec

ific

Eth

anol

Pro

duct

ion

Rat

e(g

kg-1

h-1

) an

d

Eth

anol

Con

vers

ion

Rat

e (%

)

48h-SEPR 72h-SEPR48h-ECR 72h-ECR

Fig. 5 e Specific ethanol production rates and ethanol

conversion efficiency at different initial sugar

concentrations after 48 and 72 h incubation at 30 �C.

b i om a s s an d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5e4 0 1398

3.2. Influence of substrate concentration

The batch experiment was performed with various glucose

concentrations to develop ethanol production. The initial

glucose concentrations in the batch experiments were 20, 40,

80, 160 and 300 kg m�3 tested at 30 �C. The experimental

conditions and the results summarized in Fig. 4 show the

changes in ethanol concentrations at different initial glucose

concentrations over a oneweek incubation period, while Fig. 5

demonstrates the specific ethanol production rate and

ethanol conversion rate at different initial glucose concen-

trations after 48 and 72 hour’s incubation.

0

5

10

15

20

25

30

35

0 24 48 72 96 120 144Incubation time(hours)

Eth

anol

con

cent

rati

on (

kg m-3

)

20 kg m-340 kg m-380 kg m-3160 kg m-3300 kg m-3

Fig. 4 e Changes in ethanol concentration under different

glucose concentrations after 6 days’ incubation at 30 �C.

The production of ethanol was affected by the substrate

concentration between 20 and 300 kg m�3. As shown in Fig. 4,

higher substrate concentrations may achieve higher ethanol

production, but a longer incubation time was required for

higher initial glucose concentrations above 80 kg m�3 at

a temperature of 30 �C when the pH was not controlled. More-

over, higher initial glucose concentrations, such as 300 kgm�3,

may have actually decreased the ethanol conversion efficiency

when the pH value was not controlled, since the higher

substrate and production concentrations may have inhibited

the process of ethanol fermentation (as shown in Fig. 5).

Fig. 5 shows the specific ethanol production rates and

ethanol conversion efficiency at different initial sugar

concentrations after 48 and 72 hour’s incubation at 30 �C. Thedata above illustrates that higher initial glucose concentration

may decrease the ethanol conversion efficiency. The

maximum sugar conversion after 72 hour’s incubation was

observed at 48.0%, 59.9%, 28.3%, 13.7%, and 3.7% for 20, 40, 80,

160 and 300 kgm�3 of glucose, respectively. More substrate did

not improve the specific ethanol production rate when the pH

value was not controlled.

3.3. Influence of pH

Improved ethanol fermentation activity can be achieved by

controlling various parameters. In addition to temperature

and substrate concentration, pH is also a key factor that

affects ethanol fermentation [13]. In this study changes in

ethanol and VFAs were investigated to estimate the activity of

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

Fermentor.

Fig. 6 shows the results of the batch test used to investigate

the effect of pH on ethanol production. When the pH was

lower than 4.0, the incubation time for maximum ethanol

0

5

10

15

20

25

0 24 48 72 96 120 144 168

Incubation time(hours)

Eth

anol

con

cent

ratio

n (k

g m-3

)

pH=5.0 pH not controlledpH=4.0 pH=6.0pH=5.5 pH=3.0

Fig. 6 e Changes in ethanol concentration with an initial

glucose concentration of 40 kg mL3 over one week’s

incubation with different pH values.

0

50

100

150

200

250

300

350

400

450

500

0 24 48 72 96 120 144 168Incubation time(hours)

Spec

ific

eth

anol

pro

duct

ion

rate

(g

kg -

1 h -1

) 300kg m-3-pH not controlled300kg m-3-pH4160kg m-3-pH not controlled160kg m-3-pH4

Fig. 7 e Comparison of the specific ethanol production

rates between pH values set at 4.0 and uncontrolled pH

with initial sugar concentrations of 160 and 300 kg mL3

after one week’s incubation at 30 �C.

b i om a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5e4 0 1 399

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 kg�1 h�1 of SS, with an ethanol conversion efficiency of

61.93%. The specific ethanol production rate at pH4.0 was

310 g kg�1 h�1 of SS, which is not significantly lower than the

value obtained at pH5.0. Therefore, considering the chemical

requirement for pH adjustment, pH 4.0may 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

Table 1 e The ratio of ethanol and VFAs to total productsa afte

pH value Ratio of ethanol and VFAs after 48 h incubation (%

Ethanol Acetic acid Butyric acid

No control 65.36 1.41 0.15

3.0 65.15 2.21 0.07

4.0 65.54 1.32 0.09

5.0 65.54 1.63 0.02

5.5 56.49 6.01 9.18

6.0 48.80 9.00 17.05

a Total products include ethanol, VFAs, CO2 and glucose in terms of carb

microorganisms, and may suggest a change in the main

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 m�3. 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:

4C6H12O6/2CH3COOHþ 3CH3ðCH2Þ2COOHþ 8H2 þ 8CO2 (1)

C6H12O6 þH2O/C2H5OHþ CH3COOHþ 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

r 48 and 72 h incubation at 30 �C with different pH values.

) Ratio of ethanol and VFAs after 72 h incubation (%)

Ethanol Acetic acid Butyric acid

65.55 1.48 0.10

65.15 2.51 0.07

65.54 1.28 0.05

65.54 0.86 1.51

56.49 3.01 15.74

48.80 6.05 14.80

on.

0

10

20

30

40

50

60

70

80

90

100

0 24 48 72 96 120 144 168

Incubation time(hours)

Eth

anol

con

vers

ion

effi

cien

cy (

%)

300 kg m-3-no control300 kg m-3-pH4160 kg m-3-no control160 kg m-3-pH4Theoretical value

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

week’s incubation at 30 �C. * The maximum theoretical

ethanol fermentation yield was expressed by carbon and

calculated according to the equation of

C6H12O6 / 2C2H5OH D 2CO2.

b i om a s s an d b i o e n e r g y 4 7 ( 2 0 1 2 ) 3 9 5e4 0 1400

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

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

when the pH was 4.0 and 5.0, the quantity of by-products was

less than that observed under other conditions.

Results in section 3.2 show that a higher substrate

concentration may prevent the ethanol fermentation process

occurring. One of the reasons may be the accumulation of

high concentrations of ethanol and by-products which make

the pH change. So if the pH was set at a suitable value, the

efficiency might be somewhat increased. Compared with

fermentation where the pH was not controlled, when the pH

was controlled at 4.0, the VFAs in the final product were

reduced and the specific ethanol production rate and the

ethanol fermentation efficiency were significantly improved

(as shown in Figs. 7 and 8). After pH adjustment, the ethanol

fermentation yields were increased to the range of 99.8% and

97.4% of the maximum theoretical value with the initial

glucose concentrations of 160 and 300 kg m�3, respectively.

The highest specific ethanol production rates at pH 4.0 were

340 and 260 g kg�1 h�1 of SS, respectively.

4. Conclusions

The present work tested the thermotolerant ability of S. cer-

evisiae to grow and ferment glucose at elevated temperatures

and examined the influences of temperature, initial substrate

concentration and pH value on ethanol fermentation.

The maximum specific ethanol production rates were

observed between 30 and 45 �C with different initial glucose

concentrations.When the temperaturewas increased to 45 �C,the system still showed higher cell growth and ethanol

production rates and the lowest mt/m30 at different initial

glucose concentrations was 0.8.

Higher substrate concentration could achieve higher

ethanol production, but a longer incubation timewas required

for initial glucose concentrations above 80 kg m�3 at 30 �Cwhen the pH was not controlled.

The changes in the operational pH in the ethanol produc-

tion process may have induced a change in the main

fermentation pathway. Thus it is important to control pH

value in the range of 4.0e5.0. Beyond this range, the formation

of by-products, such as acetic acid and butyric acid may have

consumed some of the substrate and reduced the efficiency of

ethanol fermentation.

Acknowledgments

The research project was sponsored by Major Science and

Technology Program for Water Pollution Control and Treat-

ment (2009ZX07101-015-003) and State Key Laboratory of

Pollution Control and Resource Reuse Foundation

(PCRRF09002).

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Yan Lin holds a Ph.D. (2005) in environmental engineering fromShanghai Jiao Tong University, China. She has worked as a post-doctoral researcher in the Asian Center for EnvironmentalResearch at Meisei University, Japan since 2005. She is now anassociate professor in Shanghai Jiao Tong University. Her currentproject is “Environmental conservation technology and utilizationof biomass in Asia (environmental technology) e ethanolfermentation”, financed by the Ministry of Education, Culture,Sports, Science and Technology, Japan.