Fed Batch culture of saccharomzces cerevisiae: a perspective of computer control to enhance the...

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.


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.


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.


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.


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.


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


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


8/16

1008


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.


9/16


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.


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

=


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.


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).


14/16

1014


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


15/16


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.


16/16

1016


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

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