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Feb 22, 2013 (4 years and 4 months ago)

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A
chemostat

approach to analyze the distribution of metabolic
fluxes in wine yeasts during alcoholic fermentation

Quirós
,

M
.
1
,

Martínez
-
Moreno,

R
.
1
,

Barreiro
-
Vázquez,

A
.
2
,

Adelantado,

N
.
2
,

Vázquez
-
Lima,

F
.

3
,

Morales,

P
.
1
,

Albiol
,

J
.
2
,

Ferrer,

P
.
2

and

Gonzalez
,

R
.
1

1

Instituto de Ciencias de la Vid y del Vino (CSIC
-
UR
-
CAR), Logroño,
Spain
.

2

Instituto de Biotecnología y Biomedicina and
3

Departamento de Ingeniería Química, Universidad Autónoma de Barcelona, Barcelona,
Spain
.

Introduction


Saccharomyces

cerevisiae

is

the

most

relevant

yeast

species

conducting

the

alcoholic

fermentation

that

takes

place

during

winemaking
.

This

biological

process

can

significantly

vary

depending

on

a

group

of

variables

that

include

the

yeast

strain

employed

and,

among

others,

the

concentration

of

sugar

and

nitrogen

present

in

the

must,

parameters

that

are

closely

altered

by

the

global

warming

phenomenon
.

Unraveling

the

physiological

behaviour

and

the

distribution

of

the

metabolic

fluxes

of

S
.

cerevisiae

under

winemaking

conditions

would

allow

the

development

of

a

metabolic

model

that

would

help

us

to

predict

the

physiological

behaviour

of

yeast

under

different

circumstances
.

The

main

goal

of

the

present

work

is

to

achieve

different

steady

states

using

a

chemostat

approach

that

mimick

the

main

phases

ocurring

during

winemaking

in

order

to

analyze

the

metabolic

state

of

yeast

during

alcoholic

fermentation
.

Material and
methods


Strains

and
culture

media

S
.

cerevisiae

strain

EC
1118
,

an

industrial

wine

yeast

commercialized

by

Lallemand

Inc
.
,

was

the

strain

used

in

this

study
.

Batch

and

continuous

anaerobic

cultures

were

performed

in

a

Dasgip

parallel

fermentation

system

equipped

with

four

SR
0400
SS

bioreactors

containing

200

mL

of

a

synthetic

must

(pH

3
.
5
)

described

elsewhere

(
Quirós

et

al
.
,

2010
)

at

28

°
C,

100

rpm

and

10

L/h

of

N
2

for

headspace

gassing
.

This

synthetic

must

was

used

as

feed

for

the

performance

of

continuous

cultures

mimicking

the

exponential

growth

phase

during

alcoholic

fermentation

(D=
0
.
25

h
-
1
)

while

the

same

must

lacking

ammonia

and

presenting

75
%

of

the

aminoacids

of

the

original

must

was

used

for

mimicking

the

transition

to

stationary

phase

(D=
0
.
04
-
1
)
.


Metabolic

flux

analysis

(MFA)

The

stoichiometric

model

used

for

MFA

was

adapted

from

Varela

et

al
.

(
2004
)
.

Data

consistency

analysis

was

performed

prior

to

MFA
.

Experimental

data

was

found

to

be

consistent

within

95
%

significance

level

according

to

the

test

proposed

by

Wang

and

Stephanopoulos

(
1983
)
.

0.25 h
-
1

0.04 h
-
1

Corresponding
batch phase

Chemostat

steady state

Corresponding
batch phase

Chemostat

steady state

Culture time (h)

8
-

17

5.5 RT

(1 RT = 3.70h)

20
-

35

5.5 RT

(1 RT = 25h)

Specific growth rate (h
-
1
)

0.37


0.15

0.27

0.023


0.034

0.04

Specific consumption /
production rates
(
mmols

∙ g DCW
-
1

∙ h
-
1
)


Glucose

-
7.3


-
10.2

-
8.9

-
4.4


-
7.2

-
4.4

Fructose

-
2.2


-
7.7

-
2.8

-
2.2


-
3.6

-
2.5

Ethanol

6.2


22.0

17.0

11.2


18.6

12.3

Glycerol

1.08


3.16

1.68

0.67


1.11

0.78

Acetic acid

0.07


0.19

0.12

0.09


0.15

0.02

Succinic acid

0.04


0.26

0.08

0.03


0.05

0.07

Lactic acid

0.04


0.08

0.09

0.03


0.04

0.02

Results


Two

different

phases

occurring

along

a

batch

fermentation

were

mimicked

using

continuous

cultures
:

-

Stage

1
:

Exponential

growth

phase
.

No

limiting

nutrients

(D=
0
.
25

h
-
1
)

-

Stage

2
:

Transition

to

stationary

phase
.

Depletion

of

ammonia

(D=
0
.
04

h
-
1
)

Figure

1

shows

the

CO
2

transfer

rates

along

the

steady

states

performed

at

the

two

dilution

rates

studied
.

Table

1

shows

the

specific

consumption

and

production

rates

calculated

for

this

two

first

stages

along

the

batch

fermentation

and

those

calculated

for

the

steady

states

mimicking

these

conditions
.


-

Near

µ
max

conditions

(Stage

1
)

79
%

of

the

carbohydrate

carbon

consumed

came

from

glucose

whereas

during

Stage

2

fructose

supplied

36
%
.

-

In

both

phases,

carbon

was

mainly

used

in

enerrgy

production

(etahbol

synthesis)

but

increased

from

79
%

in

the

exponential

phase

to

90
%

in

the

transition

phase
.

The

flux

into

the

TCA

cycle

was

kept

very

low

(near

zero)

in

both

conditions

(Fig
.

2
)
.

-

The

flux

directed

to

the

pentose

phosphate

pathway

was

growth

rate

dependent

(Fig
.

2
)
.

-

The

carbon

flux

distributions

were

similar

to

those

previously

obtained

for

batch

cultures

using

synthetic

musts

(Varela

et

al
.
,

2004
)

and

for

S
.

cerevisiae

lab

strains

grown

anaerobically

in

chemostat

cultures

(Jouhten

et

al
.
,

2008
)

Conclusions

-

The

steady

states

obtained

at

both

dilution

rates

assayed

reflect

the

physiological

state

of

S
.

cerevisiae

EC
1118

during

the

two

phases

of

the

batch

fermentation
.

-

The

medium

used

for

feeding

the

chemostat

culture

performed

at

D=
0
.
04

h
-
1

needs

to

be

improved

as

most

of

the

specific

production

and

consumption

rates

obtained

for

the

steady

states

are

very

close

to

the

range

bounds

calculated

for

the

corresponding

batch

phase
.

-

This

study

proves

that

it

is

possible

to

reproduce

different

phases

of

a

wine

fermentation

by

performing

chemostat

cultures,

providing

a

new

tool

for

the

future

construction

of

a

predictive

model

for

the

global

process
.


References



Jones

et

al
.

(
2005
)

Climate

change

73
:
319
-
343
.



Jouhten

et

al
.

(
2008
)

BMC

Systems

Biology

9
:
60
.




Quirós

et

al
.

(
2010
)

International

Journal

of

Food

Microbiology

139
:
9
-
14




Varela

et

al
.

(
2004
)

Applied

Environmental

Microbiology

70
:
3392
-
3400
.



Wang

NS

and

Stephanopoulos

G
.

(
2003
)

Biotechnology

and

Bioengineering

25
:
2177
-
2208

Figure

1
.

Carbon

dioxide

transfer

rate

(CTR)

and

carbon

dioxide

transfer

(VCT)

dynamic

during

a

continuous

culture

at

0
.
25

h
-
1

dilution

rate
.


Figure

2
.

Comparison

of

metabolic

flux
.

Reconciled

data

(specific

consumption

and

production

rate)

were

used

in

the

model

pathway
.