fed batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the...
TRANSCRIPT
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
1/16
BIOTECHNOLOGY AND BIOENGINEERING,
VOL. XVIII, PAGES 1001-1016 1976)
Fed Batch Culture of
Saccharomyces cerevisiae:
A Perspective of Computer Control to Enhance
the Productivity
in
Bakers Yeast Cultivation
SHUICHI AIBA, SHIRO NAGAI,* and YOSHINORI NISHI-
ZAWA,
Institute
of
Applied Microbiology, University of Tokyo,
T o ky o , J a p a n
Summary
A means t o avoid the glucose effect in the production of bakers yeast from
glucose and/or molasses in a fed batch culture by controlling the feed rate of
fresh medium with an ad hoc measurement of the respiratory quotient, RQ, is
presented. The feed ra te is changed stepwise here such that the value of RQ
ranges from 1.0 to 1.2 throughout the cultivation. Thus far, the specific growth
rate based on the total cell mass and the growth yield obtained throughout
are 0.24 hr-l and 0.55 g cell/g glucose.
Prior to the experimental run mentioned a,bove, equations to predetermine
the feed rate and concentration of glucose in the feed are derived from the mass
balance of limiting substrates (glucose). Since values of either RQ
or l o ,
(Qo2
2
oxygen consumption rate with respect to the total cell mass in the fer-
menter) can be measured quite easily and reliably, computer control of the fer-
mentation in light of this information is discussed.
INTRODUCTION
The well-known phenomenon termed glucose effect cannot be
prevented in the aerobic cultivation of Xaccharom yces cerevisiae in a
glucose medium. This phenomenon is the so-called aerobic fermen-
tation. When glucose concentration in an aerobic culture medium
reaches
70
mg/liter,2 glucose tends to be partly metabolized during
the fermentation to ethanol and COz; the fermentation is claimed to
cease if glucose concentration in the medium is less than the specific
level of
70
mg/liter.
Whereas a larger than expected yield of cells in the absence of aero-
bic fermentation deteriorates the specific growth rate, the fact that the
Present address: Department of Fermentation Technology, Hiroshima
University, Hiroshima, Japan.
1001
@
1976 by John Wiley Sons, Inc.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
2/16
1002 AI BA, NAGAI , AND NI SHI ZAW A
increase of the specific growth rate is accompanied by necessity by a
lower yield of cells is a characteristic of the aerobic cultivation of
baker's yeast. From the viewpoint
of
securing the most favorable
productivity of a n aerobic cultivation of the yeast, there should be a
mediation in uncompromising phenomena between fermentation and
respiration.
Maxon and Johnson3 reported earlier a diauxic growth of the
X
cerevisiae on glucose in an aerobic and batch culture; the maximum
value of the specific growth rate and growth yield reported therein
was
0.41
hr-1 and
0.14
g cell/g glucose when fermentation prevailed.3
Von Meyenburg' published his work on
a
chemostat culture with
S.
cerevisiae using glucose as the limiting substrate, in which the value
of the growth yield deteriorated from 0.50 to 0.145 'g cell/g glucose,
depending on the dilution rates ranging from 0.24 to 0.45 hr-1.
However, the data on the residual concentrations of glucose in the
culture medium are not described in the original paper;' it could
easily be envisaged that the residual concentration of glucose in-
creased when the dilution rate was enhanced.
It is considered significant at this point to minimize the glucose
effect n order to produce a higher yield of cells and, in fact, empiricism
has contributed to the development of the fed batch culture in the
production of baker's yeast. I n addition, Pirt presented some dis-
cussion on the theoretical aspect of the fed batch culture,4 although
a formula to define the feed rate required for negating the glucose
effect has not been established.
In this connection, either the ethanol concentration in exit gas from
the aerobic cultivation or the respiratory quotient RQ)value with
respect to the exhaust gas may be employed as parameters in the fed
batch culture.
If
the concentration of ethanol in the exit line be-
comes detectable, the feed rate of fresh medium into the culture might
be squeezed down and vice versa; indeed, this idea has already been
materialized.5 However, the installation of gas chromatograph on
the exit line is not always allowable from the viewpoint of economy.
I n this work, the
RQ value which can be determined by measur-
ing the partial pressures of oxygen and carbon dioxide in the exhaust
gas will be used as
a
parameter to control and minimize the aerobic
fermentation. The purpose of this work is, first, to demonstrate
the controllability of this fermentation via
RQ
values after formulat-
ing the feed rate and concentration of glucose in the feed and second,
to discuss a perspective of computer control of baker's yeast produc-
tion which guarantees the maximal yield and productivity.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
3/16
F E D BATCH CULTURE O F S. CEREVISIAE
1003
THEORETICAL CONSIDERATION
In line with the experimental setup appearing later on, suppose
that a fresh feed is charged into a culture vessel
at
a constant rate of
Fi
during
Ati (=
ti+*
i ) .
The subdivision of the cultivation period
of time from 1
= to
to
t =
t p into
At; (i =
0 to
i = n ,
culminating in
the increase of broth volume from V
=
Vo to V
=
V p as shown in
Figure
1,
is only for the convenience of experimentation as well as
the derivation of some equations which follow.
Assuming a complete mixing of the medium in a well-agitated and
aerated fermenter, the rate of change in concentrations of the cell
mass and growth-limiting subst,rate (glucose) are
:
A X ;
At;
= p i x i
F i
) X i
Vo;+ Fi Ati
1
d x ;
1
d ( X ; V ; )
1
d X ;
1
d V ;
- -
I , = ~~
___
X i
dt Vi dt
;
dt
X i V ;
dt
-
1
Voi+ F ; At ;
p i
Fi
t o
tl
72
t l
tl+l
t n t n t i
kAto+-Atq 4 ------
bat14
--- C A t n
t
Fig.
1.
Schematic diagram of parameters in each time interval in t he fed batch
culture.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
4/16
1004 AIBA, NAGAI, AND NISHIZAWA
where X
is
the concentration of cell mass in culture medium (g/liter),
J: is the total cell mass in fermenter (g),
V
is the broth volume (liter),
P is the feed rate of fresh medium (liter/hr), p is the specific growth
rate based on cell mass concentration (hr-l),
p
is the specific growth
rate based on total cell mass (hr-l),
t
is the time (hr).
The subscript i is the ith interval of time (see Fig.
1
and the sub-
script 0 is the initial of the ith interval.
The mass balance for the limiting substrate during
Ato
(cf. Fig.
1)
yields,
Si { Vo
Fo(ti o
Sovo
=
S~Po(ti o
(eccumuhtion) (input)
1
Yo
X i { vo +
Fo t1
o 1 XoVoI
(3)
(consumptiondue to cell
growth
Rearranging eq. (3),
(Si
So)
{
Vo Fo (t i
o
=
Po(ti
o ) S ~ So
provided
1
d X
1 ( X , XO)
P o =
- -=-
X o dt X o ( t l o
Where
S
is the concentration
of
glucose in the culture medium
(g/liter),
SR
is the concentration of glucose in fresh feed (g/liter),
Y
is the growth yield (g cell/g glucose).
Modifying eq.
4) so
that SI SO= A S 0 and
tl
o
= Ato,
Fo
( S R SO)
S0
Ate Vo Po Ato
F o
xo
5)
1 1
Yo
P O X 0
Yo
Vo
Po Ato
A
general expression
for
eq.
( 5 )
is:
Now
the concentration
of glucose,
S R in a fresh feed and the feed
In quasi-steady
rate,
Fi
of the fresh medium are predetermined.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
5/16
FED BATCH CULTURE OF
S.
CEREVISIAE
1005
state, where A S i / A t ;
v 0
and assuming that SR >> S;, the following
equation is derived from eq. (6).
Fix;
1
A X .
Yi Yi
At;
F;SR=
Vo;
+ F i
At;)
7)
If
Y ;
s constant throughout the fed batch culture,
(9)
1
Y
OSR t i o)
=
x i X O )
1
Y
i
=
1, F ~ S Rtz i = x Z x l )
(
10a)
1
i
=
n, FnSR ( +I
n)
=
7
Xn+1 X n )
(lob)
1
i =O Y
i AtiSR
=
Xn+l 2 0 )
therefore
(11)
X n + 1
Z X F
XO
S R
=
y
( V F VO)
Y
2
F i
At;
i =O
where X F is the final value of the total cell mass, xn+l n the fermenter
(g), V s the final value of the broth volume (liter).
Equation (11) suggests that the value of
S R
n the fresh medium
can be estimated once the target of production is established, i.e.:
X F
starting from
zo
in the total cell mass and the broth volume in-
creases, VF
V o
each expected values in the fed batch culture, and
the value of
Y
is given, respectively. The feed rate F ; for the ith
time interval can also be assessed as shown below from eqs. (2) and
(6)
when the quasi-steady state and the specific case in which
SR >>
are dealt with.
MATERIALS AND METHODS
Organism and Medium Composition
The strain used in this work was bakers yeast 8.
ereerisiae)
from
The two kinds of media used,riental Yeast
Co.,
Ltd., Tokyo.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
6/16
1006
AIBA, NAGAI, AND NISHIZAWA
semisynthetic and molasses, were composed of the following. Semi-
synthetic medium: glucose, 20 g; (NH2)&O, 2.15 g; NaHP04.2Hz0,
1.0
g; MgS04-7Hz0,0.38g; KC1, 0.22 g; sodium citrate, 2.5 g; yeast
extract,
0.5
g;
1
ml of vitamin solution (biotin, 0.04 g; vitamin
B1,
0.08 g; vitamin B6, 2.0 g; calcium pantothenate, 1.0 g and inositol,
20
g/liter), 1 ml of mineral solution (CuS04.5H@,
0.05
g, ZnS04.7Hz0,
0.8
g; and Fe(S04)z(NH4)2-6Hz0 ,
.3
g/liter), tap water, 1,000 ml;
and pH
=
5.0 adjusted with an aqueous solution of HzS04 (2N).
Molasses medium: molasses was treated with steam for
1
hr a t about
80C and centrifuged for 1 min
at
4,000 x
g
to be free from solid
ingredients and then diluted to about
a 30
sugar content (as
glucose). Urea was supplemented to the medium with a ratio of
0.5 g (urea) to 30 g (glucose).
Fed
Batch
Culture
A cell suspension obtained from the Oriental Yeast Co. was inocu-
lated into a bench-scale fermenter (nominal volume =
10
liters,
initial working volume
=
3.5 liters, L. E. Marubishi Co., Ltd.,
Tokyo, Model MD-500) in order to have 4 g/liter in the cell concen-
tration just prior to the star t of the fed batch culture. After
0.5
hr
in the batch culture (initial glucose concentration
=
1 g/liter), the
feeding of the fresh medium into the fermenter was begun at an
adequate rate using two peristaltic pumps
A
and B, Taiyo Kagaku
Co., Tokyo), where the feed rate of Pump
B
was about 10 to 20 of
that of Pump A. Pump A, as a staple control, was operated con-
tinuously t o feed the fresh medium a t a feed rate which was changed
stepwise periodically to achieve a cell growth such tha t the RQ value
did not fluctuate too much from 1.0. Pump
B,
as a fine control,
was operated intermittently ; the time of operation depended on the
value of RQ, i.e., this latter pump was switched on when the RQ
value was below the datum of 1.0 and/or turned off when the
RQ
value exceeded another datum of 1.10. Usually, about 60 sec was
needed to change the feed rate after confirming the varying RQ values.
Obviously, using two pumps made it convenient to substi tute for
a
single one which could vary the feed rate continuously in light
of
information originating from the experimentation (see Figs. 2 and 3 .
As was mentioned earlier, the improvised use of this pump (or pumps)
necessitated the change in the feed rate stepwise rather than con-
tinuously.
The rotation speed
of
an impeller and the temperature of the culti-
vation were kept at
600
rpm and 30C, respectively. The air flow
rate was controlled with a specific miniflow valve set a t
2.45
and/or
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
7/16
FED BATCH CULTURE OF S . CEREVISIAE 1007
3.27 liters/min with respect to the cell growth; the value of pH was
also controlled at
4.5 with an aqueous solution of NaOH 2 N ) .
Analytical Methods
The optical densities measured at 610 nm in wavelength with a
spectrophotometer (Hitachi Works, Model 101) were converted to
dry cell mass concentration after establishing
a
calibration chart.
The calibration was made by filtering the cells through a Millipore
filter (pore size = 1.2 pm) and drying the cake at 105C for 1 hr in a
semisynthetic culture medium of glucose. In the molasses medium,
the cell mass concentration was determined directly by filtering
a
sample broth through the Millipore filter (pore size = 1.2 pm),
followed by drying a t 105C for 1 hr.
The concentration
of
glucose in the glucose medium was deter-
mined with the Glucostat reagent (Fujisawa Medical Supply Co.,
Ltd., Osaka), whereas the molasses was first hydrolyzed with a
concentration of HC1 in a boiling water bath for 40 min and then
analyzed by the Somogyi method to determine the concentration of
glucose.
The concentration of ethanol in the culture medium was deter-
mined by the microdiff usion method. The dissolved oxygen concen-
tration was measured occasionally with a membrane electrode (L.
E.
Marubishi Co., Ltd.) ; actually, the oxygen concentration was well
above the level which might have limited the cell growth.
The respiration rate, Q o 2 , he total oxygen consumption rate,
I o 2 ,
the specific rate of carbon dioxide evolution,
con ,
and the respiratory
quotient, RQ, were estimated by calculating the difference of the
partial pressures
of
oxygen and carbon dioxide in air between input
and output through the fermenter, with a Beckman oxygen analyzer
(Type 777) and an infrared gas analyzer (Shimadzu Works, Tokyo,
Model URA-2) , respectively. The response times of these analyzers
were quite short, approximately 30 sec in preliminary experiments.
RESULTS AND DISCUSSION
Fed Batc h Culture of Glucose M edi um
An example of fed batch culture
of
glucose medium is shown in
Figure 2. The total cell mass, 5 the total oxygen consumption rate,
I o 2 ,
and the respiratory quotient, RQ, are on the left-hand side of
the ordinate, while concentrations of glucose, S, and ethanol, P in the
culture medium, the specific rate of respiration, Q o 2 , he specific rate
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
8/16
1008
AIBA, NAGAI, AND NISHIZAWA
U
I
I
I
0
0
0 1
2
3
4
t I h r
Fig. 2.
Growth patterns of bakers yeast in the fed batch culture of the
glucose medium. The differences in partial pressures of oxygen, Ape, and
carbon dioxide,
A p c o ,
between input and output air were recorded continuously.
RQ
values as the ratio of
A ~ c o , / A ~ o ,
nd l o , Ape, times air flow rate) are
then recorded continuously. Since the total cell mass,
x
was determined inter-
mittently, Qo,
(Zo,/z)
and
Q C O , Q o , R Q )
were both observed in discrete rather
than continuous terms. The flow ra te of fresh feed,
F ,
was observed directly
by using a measuring cylinder, but the data in the figure are described sche-
matically. M
l / z . A z / A t ) ,
Y
(FiS~/zi) ,
nd
Y r/180
Y) values were assessed
from the curve drawn through the data points
of
x espectively.
0 )
, A) ,
0)
,
0)o 3)Q c o , .
of carbon dioxide evolution,
Qco,,
the specific ra te of glucose consump-
tion,
v,
the specific rate
of
increase in the total cell mass,
p ,
the feed
rate of the fresh medium,
F ,
and the growth yield,
Y ,
are on the right-
hand side of the ordinate. The data observed are shown in the lower
diagram of the figure, whereas in the upper diagram the characteristic
of the fed batch
culture
is
demonstrated by
respective calculations
from the data given in the lower diagram.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
9/16
FED BATCH CULTURE OF
S.
CEREVISIAE 1
om
Prior to the experimental run, the concentration of glucose, SR n
the feed was calculated from eq.
11) since SR
=
24 g/liter, assuming
x0
= 14
g,
Vo
= 3.5
liters,
XF = 26
g,
V = 4.5
liters, and
Y
=
0 . 5
g
cell/g glucose. The actual da ta of
S R
prepared thus far was S R =
23.2 g/liter.
Yeast cells inoculated into the fermenter were cultivated for 0 .5 hr
in batch as stated earlier, followed by the fed batch culture at t
=
0
(see Fig. 2) . The initial feed rate, F o
=
0.22 liters/hr at t =
0
was
derived from eq. l a ) , assuming
SR
=
23.2 g/liter,
5 0
=
12.8 g,
pa = 0.20 hr-l, and Y = 0.5 g cell/g glucose. The feed rate which
should have been periodically changed was arbitrarily manipulated
stepwise such that the RQ values in
situ
would be within the preset
boundaries 1 .0 - 1.10) (see Materials and Methods). It is clear
from the figure tha t RQ values, though oscillated, could be controlled
within the preset range by changing the feed rate stepwise; the
response of RQ o the feed-rate change was fairly rapid.
Thus far, as concerns this experimental set-up (analyzer per se plus
dead-space above the culture medium in the fermenter, etc.), the
time required for the analyzer to respond to the change in C02
emergence from the yeast was estimated to be of the order of
60
sec.
Since the time required for manipulating the pump(s) was also of the
same order of magnitude as referred to earlier, the change in RQ
values observed could be commensurable with that of the feed rate.
In fact, when RQ
>
1, the change in
F
entails fairly rapid emer-
gence of QCO,see Figs. 2 and 3) . When RQ
>
S) (cf. eq.
l a ) ) ,
and
Y
is p /v . 180 (on a
gram basis) derived from
p
and
v
assessed earlier.
Values of p increased from
0.20
to
0.24
hr-' in Figure
2,
while V
values were between
2.0
and
2.4
mmol glucose/g cell hr, depending
on the feed rate. Accordingly, values of
Y
increased most likely
from
0.48
to
0.55
g cell/g glucose, although they were a bit modulated.
These values of
p r
and Y observed in this fed batch culture are nearly
the same as the maximum values reported for aerobic growth
RQ =
1.0)
of the S . cerevisiae in glucose-limited chemostat cultures.'
Fed Batch Culture
of
Molasses Me dium
The experimental conditions employed in this run were as follows:
xo =
15 g,
V O=
3.5 liters,
XF
= 35 g, and V F
=
5.0 liters. If
Y
is
taken as
0.5
g cell/g glucose, S R is assumed to be
26.7
g/liter from
eq.
11).
However, the actual data of SR prepared and
xo
were
28.4
g/liter and
14.1
g, respectively.
By and large, the results
of
this run in Figure
3
resembled that of
glucose in Figure 2 except for S , which will be elaborated below.
The higher concentration of residual sugar might have originated from
the nonfermentative sugar which accumulated as the fed batch
culture progressed. Values of p , v , and Y were assessed as shown in
the upper portion of Figure
3
exactly by the same procedure men-
tioned earlier in Figure 2.
Incidentally,
p
ranged from
0.20
to
0.22
hr-l, while the growth yield,
Y
fluctuated around
0.48
to 0.52 g cell/g
glucose.
Y,I, and RQ
It
was confirmed from Figures
2
and
3
that the feed rate of the
fresh medium could be controlled solely by the observed value of RQ.
However, it
is
deemed more desirable when considering computer
control to formulate the feed rate in close connection with the meta-
bolic activity of baker's yeast represented by the value of
RQ.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
11/16
FED BATCH CULTURE OF S. CEREVISIAE
1011
0
0
1
2
3
h r )
Fig.
3. Growth patterns of bakers yeast
in
the fed batch culture of the molasses
medium.
0 )
A)
O)P,
0 )
o
3)
Qco,.
With respect to the aerobic fermentation, another yield,
Yp,a
s
defined
as
follows:
Q p
Y P I S
=
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
12/16
1012 AIBA, NAGAI, AN11 NISHIZAWA
provided that Q , is the specific rate of ethanol production (mol
ethanol/g cell hr),
v
is the specific rate of glucose consumption (mol
glucose/g cell hr),
kl
s the stoichiometric constant which correlates
C02evolution with ethanol production in the fermentation of glucose
(1 mol ethanol/mol CO,),
k2
is the stoichiometric constant which
correlates 0 2 consumption with C02evolution in complete oxidation
of glucose (1 mol C02/mol
0 2 ) ,
l o is the total oxygen consumption
rate
(Qo,a)
mol 02/hr).
Equation (13) can be translated to the i th interval of time
as
follows :
k~
( I 0 , ) i
(RQi
1)
( y p / s ) = ( d i X i /
Y
;) (1 180)
Assuming again the quasi-steady state and S R >>
S i ,
the following
equation which assesses the value of
F i
is derived from eqs.
12)
nd
(14).
(15)
Equation
(15)
clearly denotes that the information in I o 2 and
RQ
stemming from the analysis of the exhaust gas from the fermenter
at
each time-interval defines the feed rate, if and only if the values of
Y p l a
nd S R given earlier from
eq. (11)
are made available.
The relationship between Y p l a nd RQ calculated from the experi-
mental data in Icigurcs 2 and 3 is shown in Figure 4. The linear
correlation could be assumed to be in the range of RQ (RQ = 1.0 -
1.2)
which was employed here in each fed batch culture.
(16)
where K is the proportionality constant (empirical) (mol ethanol
mol 02/mol glucose mol C02).
The values of K given from the slope of the lines in Figure 4 were
3 S O for glucosc and 2.65 for molasses media. Substi tuting
eq.
(16)
into cq. (15).
Then
( Y p / a )
=
K ( R Q i
1)
Equation (17) means that the feed rate in order to maintain the
quasi-steady state can be defined exclusively with
I,,,
values
at
that
time.
Procedures to Control
the
Feed Rate
Two ways to control the feed rate in the f(.d batch culture were
On(. is to change the ratelready proposed here in this work.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
13/16
FED BATCH CULTURE
OF S.
CEREVISIAE
1013
O
0.8
0.6
K = 3.80
s e m i
-
s y n t h e t i c
Fig. 4.
The relationship between Y,,, and RQ in the fed batch culture of
baker's yeast in semisynthetic (glucose) and molasses media..
Y,,*
was assessed
from the data of Q C O , Q o , RQ ) , Q o , (ZoJz), and Y F i S ~ / z i )cf. eq. 13));
the data points do not correspond exactly to those of z in the original figures
(Fig. 2
or 3 ) ,
because the interpolated data (see curves drawn through the data
of z)pertaining to
RQ >
1 were ta.ken. 0 ) emisynthetic medium; A) o-
lasses medium.
referring to the values
of
RQ, the other
is
by affecting
l o 2
using eq.
The feed rate, Fc l estimated from l o , (eq. (17)) and another
rate, F,,,, employed in the experimental runs which refer to instan-
taneous values of RQ are sporadically compared in Figure 5a (semi-
systematic medium) and b (molasses medium).
The value of Fcal
should define the feed rate once the value of RQ, approximately be-
tween 1.0 and 1.15 or 1.20 (see Fig. 4) in these examples which
warrant the linearity between
Ypl8
nd
( R Q
1 ) ) is given. In other
words, control based solely on l o z s essentially provided with freedom
to choose an appropriate value of RQ within the specific range.
Conversely, the control of the feed rate based on
l o 2
should be
coupled with a sophisticated mechanism for controlling the feed
pump, keeping the value of RQ as closely as possible around a par-
(17).
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
14/16
1014
AIBA, NAGAI, AND NISHIZAWA
0.6
I
I
I
( 0 ) semi
-
syn the t i c
0.4 -
4
-
LL
rn
Fig.
5.
The feed rate, Fexpeproduced
from
Figs. 2 and 3 is com pared with
the rate,
Fa ,
s assessed from eq. 1 7 ) : a) semisynthetic medium, b) molasses
medium.
In the assessment of Fa, ,
K
values shown in Fig .
4
were used, respec-
tively. For ease of discussion, ZZQ values are also reproduced from previous
figures.
ticular value employed; otherwise, the RQ values oscillate greatly
around RQ =
1.0 as
illustrated in the figure. If the above situation
where a precise and instantaneous control of the pump had been
understood, the variations of
RQ
in the figure would have been
minimized.
In carrying out the experiments in this work, thc feed rate, which
was far above the value of
F,,,,
as acceptable
so
far
as
the require-
ment
to
minimize the glucose effect in terms of RQ from
1.0
-
.15
was considered.
Briefly, it goes without saying that the value
of Fcz l
serves as datum
to define the flow rate of the medium, leaving the exact value of
RQ
undefined. I t is shown from Figure
.5
that the upper and lower
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
15/16
FED BATCH CULTURE OF
S.
CEREVISIAE
1015
diagrams dealt with
F,,,
values rather than with the Fcalvalues,
respectively, as
a whole, even in the absence of any automatic con-
trol
of
the feed pump where the deviation between
Fcsl
and
F,,
in
both diagrams in Figure
5
seems to support the appropriate range
of RQ
from the viewpoint of minimizing the glucose effect.
The possibility and advisability of controlling the fed batch
culture using either l o r nd/or RQ coupled with a computer is now
self-evident.
Nomenclature
feed ra te of fresh medium (liters/hr)
total oxygen consumption rate
(QOZ
z) mol Ot/hr)
proportionality constant (empirical) (mol ethanol mol 02/mol glucose mol
COr)
stoichiometric constant to correlate COZ evolution with ethanol produc-
tion in fermentation of glucose (1 mol ethanol/mol COZ)
stoichiometric constant to correlate 0 2 consumption with COZevolution
in complete oxidation of glucose (1 mol COr/mol 0 2
specific ra te of COZevolution (mol CO2/g cell hr)
specific rate of respiration (mol 02/g cell hr)
specific rate of ethanol production (mol ethanol/g cell hr)
respiratory quotient (mol COn/mol
0 2 )
concentration of glucose in culture medium (g/liter)
concentration of glucose in fresh feed (&liter)
time (hr)
broth volume (liter)
final value of broth volume (liter)
concentra.tion of cell mass in culture medium (g/liter)
total cell mass in fermenter (9)
final value of total cell mass in fermenter (g)
growth yield (g cell/g glucose)
yield coefficient of ethanol produced to glucose consumed (mol ethanol/
mol glucose)
Greek Letters
p
specific growth rate based on cell mass concentration (hr-I)
p specific growth rate based on total cell mass (hr-1)
Y specific rate of glucose consumption (mol glucose/g cell hr )
Subscript
i i th interval of time
0
initial
of
ith interval
The authors are indebted to Dr. M. Ohashi, Oriental Yeast Co., Ltd. for the
bakers yeast and molasses used and to Mr. T. Yamagata,
L.
E. Marubishi Co.,
Ltd. for some instruments used throughout this work. They are also grateful
to Mr. T. Karasawa, Oriental Yeast Co., Ltd. and Mr.
H .
Sakuma, L.
E.
Maru-
bishi Co., Ltd. for their technical assistance.
-
8/10/2019 Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the productivity in b
16/16
1016
AIBA, NAGAI, AND NISHIZAWA
References
1.
H .
K.
von Meyenburg,
Arch. Mikrobiol., 66 , 289 1969).
2 . F.
J.
Moss,
P.
A .
D.
Rickard, F. E. Bush, and
P.
Caiger, Biotechnol. Bioe ng.,
3. W. I> Maxon and M . J. Johnson, Znd. En g. Chem., 45,2554 1953) .
4. S.
J.
Pi r t ,
J .
A p pl . C hem. Biotechnol. ,
24,
415 1974).
5 . Aust r ia Paten t
No.
A3194-70.
6 . T. Ozawa, S.
Nagaoka,
and K. Sumino, Hy g. Che m. (Japa n) , 10, 17 1964).
13, 63 1971).
Accepted
for
Publ icat ion February 18, 1976