Technological Problems in Cultivation of Plant Cells

at High Density
HIDE0 TANAKA, Institute of Applied Biochemistry, The University
of Tsukuba, Ibaraki, Japan

Summary
Using Cudruniu tricuspidutu cells as model plant cells which have high sensitivity to
hydrodynamic stress, technological problems in the cultivation of the plant cells at high
density were investigated. Using shake flasks on a reciprocal shaker and Erlenmeyer
flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell
densities in shaken cultures, particle breakdown and damage to the largest cell aggregate
group (above 1981 pm in diameter) occurred and normal cell growth became impeded. The
mass-transfer coefficient ( K )for a model solid-liquid system @-naphthol particles and water)
in place of a system of plant cells and a liquid medium was proposed as an intensity index
of hydrodynamic stress effects on plant cells in suspension cultures under various conditions
in the bioreactor systems. Normal cell growth was obtained under culture conditions for
K values less than about 4.4 x lo- cm/sec. The characteristics of various bioreactors used
until now were investigated by considering the three main technological factors (capacity
of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of
culture broth mixing and air-bubble dispersion). The most suitable bioreactor for culturing
plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M).
The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight
per liter of medium.

INTRODUCTION
Plant tissue culture has been applied on a wide scale. Concerning the.
application of plant tissue culture to the production of materials, the
materials can be divided into two main classes, cells and metabolites. In
order to produce cells and metabolites associated with cell growth, efficiently and in great quantities, cultural techniques to obtain cells at a high
density in a large-scale suspension culture are required. However, the
maximum final yield of cell mass which has been obtained in large-scale
suspension cultures is no more than 20 g dry weight per liter of medium
in tobacco cells. One of the causes can be considered as follows: plant
cells are rigid and considerably larger than microorganism cells; moreover, they have a tendency to grow in the state of aggregates. The cells,
therefore, are rather sensitive to hydrodynamic stress generated by aeration and agitation (or shaking) and other operations used in supplying
Biotechnology and Bioengineering, Vol. XXIII, Pp. 1203-1218 (1981)
0 1981 John Wiley & Sons, Inc.
CCC ooO6-3592/81/061203-16$01.60

1204

TANAKA

oxygen and mixing culture broth. The result is that the cells are easily
damaged and broken in high oxygen supply conditions and under strong
mixing of the culture broth corresponding respectively to a high oxygen
consumption and high apparent viscosity of culture broth at high density .2-4 So far quantitative considerations on the effects of hydrodynamic
stress on various kinds of living cells in suspension cultures have been
investigated by Midler and Finn, Taguchi et
Tanaka et al. , and many
others. However, reports concerning plant cells as an object of investigation have apparently not been published to date. In addition, an intensity index of the effects of hydrodynamic stress on plant cells has not
been established, and also a method to decide the optimum
aeration-agitation conditions and bioreactor system for culturing plant
cells at high density have not been developed.
In this article an intensity index is proposed and technological problems
which may occur in the cultivation of plant cells at high density are
investigated on the basis of the index, using Cudruniu tricuspiduta cells
as model plant cells which have a high sensitivity of hydrodynamic stress.

MATERIALS AND METHODS

Cell Line

Cudruniu tricuspiduta Bureau callus used in this investigation was derived from stems of the plant by Dr. Ohyama of the Sericultural Experiment Station, Japan. The callus has been maintained on Murashige and
Skoogs medium containing 1 mg of benzyl adenine, 30 g of sucrose, and
10 g of agar per liter at 30C in the dark.
Suspension Culture
The medium used for suspension culture was Murashige and Skoogs
medium which contained 2 mg of benzyl adenine and 14 g of sucrose per
liter. Callus, obtained from 2 1-day-old cultures, was inoculated into the
liquid medium and incubated at 30C in the dark. The inoculum size of
callus was 30 g of wet weight (equivalent to about 1 g of dry weight) per
liter of the medium. Most of the suspension culture studies were made
with 500-ml shake flasks on a reciprocal shaker and 500-ml Erlenmeyer
flasks on a rotary shaker. Culture at a high cell density was carried out
in a 10-literjar fermentor with a modified paddle-type impeller. Schematic
diagrams, dimensions, and experimental conditions of the flasks and jar
fermentor are shown in Figure 1 and Table I. The pressure in the jar
fermentor was kept constant at 0.3 kg/cm. The dissolved oxygen concentration in the culture medium was measured by a galvanic oxygen
analyzer (Oriental Electric Co., Ltd).

HIGH DENSITY PLANT CELL CULTIVATION

Jar fermentor

(J-T )

Jar fermentor
(J-M)

t
Air

Air lift
reactor

Shake

COlUmn

flask

ErlenmPyer

tlask

AIT

Fig. 1.

Bubble

1205

t
Air

Schematic diagram of various bioreactor systems.

Apparatus

Schematic diagrams of the bioreactor systems used for the various

physical experiments are shown in Figure 1. For the aeration-agitationtype apparatus, the jar fermentor with a flat-blade-turbine-type impeller
(J-T) and the jar fermentor with a modified paddle-type impeller (J-M),
which was developed for the culture of filamentous microorganism cells
by Tanaka and Ueda, were used in this study. For the aeration-type
apparatus, an air-lift reactor and a bubble column were used. The working
volume of the above apparatus was 1 liter. In addition, two kinds of
flasks, a shake flask and an Erlenmeyer flask, were used. The dimensions and experimental conditions for their bioreactor systems are given
in Table I.
Capacity of Oxygen Supply

As an index to the capacity of oxygen supply to water under various

aeration and agitation conditions in a bioreactor system, the initial volumetric oxygen-transfer coefficient kLa was employed. The kLa value
was determined by a modified gassing-out method reported by Kato et
al. In place of CoC12, however, CuSO4-SH20was used as the catalyst
of the deoxygenation reaction (by sodium sulfite). The dissolved oxygen
concentration was measured by a polarographic oxygen analyzer (Beckman, Model 777). The temperature of water in the bioreactor systems
used was kept constant at 30C.
Intensity of Hydrodynamic Stress Effects on Plant Cells

The solid-liquid mass-transfer coefficient K in the mechanical agitation

vessels, aeration-agitation vessels, bubble columns, etc., is known to be
a function of the operating conditions, the construction and scale of the
apparatus used, the physical properties of the solids and solutions, the
shape and size of the solid particles, the temperature of the solutions, and
other factors.
Hixson et al.* made it clear that K was useful for comparing agitation
efficiency and for designing and determining the operating characteristics
of mixing apparatus.

Oscillations/min.

diameter (cm)
width (cm)
height (cm)
Condition
agitation (rpm)
aeration (vvm)
stroke (cm)

150-630
1 .o

1
11.3
10.0
8 holes
ring type
4
6-flat-bladeturbine type
6.4
I .4
3.3

Working volume (liter)

Diameter, D (cm)
Height of liquid, Hl. (cm)
Sparger

Baffle plate no. (-)

Impeller

Jar fermentor
(J-T)

Bioreactor
system

225-630
I .o

60
0.5-1.5

13.3
8.0
8.0

5
20.0
16.0
8 holes
ring type
0

Modified paddle type

7.0
5.0
5.0

I
11.3
10.0
8 holes
ring type
0

Jar fermentor
(J-M)

1 .O-4.0

1
5.2
47.2
Fritted glass
filter plate
0

Bubble column

1 .O-3.0

1
8.9
16.0
Fritted glass
filter plate
0

Air-lift
reactor

TABLE I
Dimensions and Experimental Conditions for Various Bioreactor Systems

7.0

85- 1 20a
-

Shake
flask

70- 160
7.0

0-4
-

Erlenmeyer
flask

HIGH DENSITY PLANT CELL CULTIVATION

1207

Plant cells in suspension cultures are often damaged and broken. The
phenomenon is assumed to be caused by hydrodynamic stress generated
by aeration, agitation, shaking, pumping, and other operations used for
the supply of oxygen and mixing. However, the method to determine
quantitatively the intensity of hydrodynamic stress effects on plant cells
has not established due to the difficulty of direct analysis. In this present
investigation, a new indirect method to determine quantitatively the intensity was proposed from the viewpoint that factors influencing values
of K were taken to be the same as those influencing damage and breakdown of plant cells. Namely, in place of a system of plant cells and a
liquid medium a similar solid-liquid model system for specific gravity
and size was selected, and K obtained in the model system under a given
aeration-agitation condition was employed as an index to the intensity
of hydrodynamic effects on plant cells under the same condition. With
consideration of small differences in the specific gravities of plant cells
and the liquid medium, p-naphthol particles, which are unsoluble in water,
and water were selected as a model system. The specific gravity of pnaphthol particles (1.18 g/ml) is roughly equal to that of plant cells and
also the specific gravity of water is nearly equal to that of the liquid
medium. The shape of the p-naphthol particles is cylindrical (diameter
2.0 mm, length 2.0 mm). The cylindrical particles of p-naphthol were
obtained by melting p-naphthol powder at 125"C, absorbing the melting
p-naphthol into silicone tubes (inner diameter 2.0 mm), cooling the tubes
to room temperature, cutting the tubes at 2.0 mm distances with a cutter,
and removing the particles from the tubes.
In agitation of the solid-liquid system, the solubilization rate of particles can be represented as
A
dC
- = K F ( C * - C)
dt
where t is time (sec), C is the concentration of solution at time t (mg/ml),
C* is the concentration of solution at saturation (mg/ml), K is the masstransfer coefficient (cm/sec), A is the total surface area of solid particles
(cm2), V is the total volume of solution (ml). If the solid particles are
unsoluble, the change of A would be negligible in a short period of time.
Then eqs. (2) and ( 3 ) are obtained from eq. (1):
V
C
K A = -1nt
c*-c
V
C*
K = -In(3)
At C* - C
Each value of K under fixed conditions was calculated from the slope
of the linear relation between ln(C* - C) and t .
The quantity of p-naphthol particles added to 1 liter of water was 3670

1208

TANAKA

mg, equivalent to A of 91.48 cm'. The temperature of the solution in the

bioreactor systems used was kept constant at 30C.
In measuring K for the flasks under various experimental conditions,
the minimum sample solutions as required were collected with injection
in order to minimize the decrease of liquid volume in the flasks.

Intensity of Mixing of Culture Broth and Air-Bubble Dispersion

The viscosity of culture broth increases steadily due to plant cell growth
in the suspension c u l t ~ r e , so
' ~ that mixing of the broth becomes insufficient, and air bubbles cannot be dispersed satisfactorily into the broth
at the high cell densities; consequently, oxygen supply to the culture
broth is impossible. As an intensity index of culture broth mixing and airbubble dispersion to the broth for various bioreactor systems, kLa was
employed for the present plant cells.
The oxygen balance between supply and consumption by cells is

dCldt = kLa (C*

C)

krM

(4)

where C is the dissolved oxygen concentration at t (pI 02/ml), C* is the

dissolved oxygen concentration at saturation (pl OJml), k, is the specific
rate of respiration (pl 02/mg cell hr), and M is the cell mass concentration
(mglml).
Assuming a quasi-steady state, dCldt can be taken to be negligibly small
compared with the value of the right-hand side of eq. (4). Provided that
dCldt = 0, eq. ( 5 ) is obtained from eq. (4):

Prior to kLa determination, plant cells at the logarithmic phase in flask

culture were gathered, a fixed amount were inoculated into the liquid
medium in the culture apparatus kept constant at 30C, and the apparatus
was operated under a given aeration-agitation (or shaking) condition. The
kLa value in the apparatus can be calculated from eq. ( 5 ) using C at quasisteady state and k, for cells at the same phase in flask cultures. The
respiration rate was determined by the method reported by Takahashi
and Yoshida. l 4 Dissolved oxygen concentration in the medium was measured by a oxygen analyzer (Beckman, Model 777).

Analysis
The cell mass concentration was measured as follows: the cell suspension was filtered through a piece of membrane filter cloth (pore size 3.0
pm), and the cells remaining on the filter were immediately washed with
distilled water, dried at 100C for 12 hr, and weighed. The residual sugar
concentration was measured as follows: residual carbohydrate in the filtrate was hydrolyzed by invertase and the carbohydrate hydrolyzed was

HIGH DENSITY PLANT CELL CULTIVATION

1209

measured by the modified method of Somogyi. l5 The value measured was

calculated as sucrose. The P-naphthol concentration was measured as the
optical density at 275 nm of the so1ution.16The cell aggregates were sieved
into appropriate sizes by Tyler standard sieves (stainless steel).
RESULTS

Effect of kLa on Cell Growth in the Shaken Culture

In order to know the influence of shaking conditions on the culture, an
experiment was carried out by changing the speed of the reciprocal
shaker. As shown in Figure 2, the optimum speed for cell growth was 85
oscillations/min. The growth rate of cells and the yield of cell mass for
the 15-day-old culture were observed to decrease with higher speed of
the shaker. Such a phenomenon was observed similarly for cultures using
Erlenmeyer flasks with the rotary shaker. Also, in an experiment where
Erlenmeyer flasks were attached to baffle plates in order to increase the
oxygen supply, the growth rate of cells and the yield of cell mass were
observed to decrease with increasing number of baffle plates for a constant
speed of the shaker.
The yield of cell mass obtained at 15-day-old culture in the shake
flasks and Erlenmeyer flasks with or without baffle plates as a function
of the initial k,a value for their shaking conditions is shown in Figure 3 .
The yield decreased with higher kLa. Therefore, these data show that cell
growth under the shaking conditions used in this experiment was limited
by other factors besides oxygen supply. Also, it is evident that normal

Time ( d a y )

Fig. 2. Effect of shaking conditions on cell growth of Cudruniu tricuspiduta in suspension

culture. Fifty milliliters of Murashige and Skoogs medium containing benzyl adenine (2
mg/liter) and sucrose (1.4%)in a 500 ml shake flask was used. Cells were incubated at
30C on a reciprocal shaker in the dark. ( 0 , *) 85 oscillations/min, ( 3 , ). 100 oscillations/min,
( A , A) 120 oscillations/min.

TANAKA

1210

O!

5b

lA0

I;O

2b0

2kO

300

kLa ( hr )

Fig. 3. Effect of initial volumetric oxygen-transfer coefficient kLa on the cell mass
concentration at 15-day-old culture. Initial sucrose concentration was 14 g/liter. A rotary
shaker with shake flasks was operated at 85, 100, and 120 oscillationshin. A rotary
shaker with Erlenmeyer flasks was operated at 130 rpm. Numbers in the figure indicate
number of baffle plate in Erlenmeyer flasks. ( 0 ) Shake flask, (4 Erlenmeyer flask.

cell growth becomes impeded under the high oxygen supply conditions
used in order to obtain a high cell density in the culture.
Effect of Hydrodynamic Stress on Cell Growth

Figure 4 shows the size distribution of cellaggregates for 15-day-old

cultures under three conditions of shaking using the shake flasks. Cell
aggregate size was distributed widely in the region of 80 to above 1981
301 85 ( oscillslmin

Fig. 4. Size distribution of cell aggregates at 15-day-old culture. A reciprocal shaker

with shake flasks was operated at 85, 100, and 120 oscillationshin. The culture conditions
were the same as described in Fig. 2.

HIGH DENSITY PLANT CELL CULTIVATION

1211

pm. Typical cells were elliptical in shape, the diameter ranging from about
40 pm to 50-80 pm, so it is evident that there were few single cells in the
culture broths. By comparing three size distributions, it was found that
the ratio of the largest cell aggregate group (above 1981 pm in diameter)
decreased and that of the smallest one (below 80 pm in diameter) increased
with higher speed of the shaker. The smallest cell aggregate group during
filtration plugged the pores of a membrane filter cloth (pore size 3.0 pm)
and almost all cells viewed under the microscope were observed to be
broken and to have died. Also, for the culture using Erlenmeyer flasks,
the ratio of the largest cell aggregate group decreased and that of the
smallest one increased with higher speed of the shaker and increasing
number of baffle plates.
These results suggest that the low growth rate of cells with the higher
shaker speed or greater number of baffle plates is caused by a particle
breakdown and damage of the largest cell aggregate group by hydrodynamic stress. Thus, the intensity of hydrodynamic stress effects on cells
in the shaken culture under various conditions was investigated. The
intensity was measured using cylindrical P-naphthol particles which had
dimensions almost equal to the cell aggregates contained in the largest
group which were apt to be broken and damaged by hydrodynamic stress.
The effect of shaking conditions using the shake flasks and Erlenmeyer
flasks on the mass-transfer coefficient K of the p-naphthol-water system
is shown in Figure 5. The increase of K with higher speed of the shaker
in the shake flasks was remarkable in comparison with that in the
Erlenmeyer flasks. The increase of K by changing from 100 to 110 oscillations/min was especially remarkable. The phenomenon corresponded
to the situation whereby water in the flask collided with the shoulders of
Reciprocal shaker speed ( oxillslmin )

2L

70

100

130

160

Rotary shaker speed ( r p m )

Fig. 5. Effect of shaking conditions on the mass-transfer coefficient of p-naphthol-water

system, K , at 30C. A 500-ml shake flask and a 500-1111 Erlenmeyer flask contained 50 and
100 ml of water, respectively. Cylindrical particles of P-naphthol (diameter = 2.0 mm,
length = 2.0 mm, density = 1.18 g/ml), 3.67 g/liter, were added to their flasks. Numbers
in the figure indicate number of baffle plates in Erlenmeyer flasks. ( 0 ) Shake flask, ( A )
Erlenmeyer flask.

TANAKA

1212

the flask. By attaching baffle plates to the Erlenmeyer flask also, K increased remarkably.
The yield of cell mass obtained at 15-day-old culture in the shake
flasks and Erlenmeyer flasks with or without baffle plates as a function
of K for shaking conditions is shown in Figure 6. The yield decreased
with higher K. Therefore, these data indicate that the cell growth for the
shaken culture in this experiment was mainly limited by hydrodynamic
stress. Also, it was found that a normal cell growth was obtained under
shaking conditions which yielded K values less than about 4.4 x
cmlsec.
Comparison of Various Bioreactors by kLa and K

In order to compare various bioreactors by kLa and K values, an experiment was carried out by changing the operating conditions. The relation between kLa and K at 30C in various bioreactors is shown in
Figure 7. Under operating conditions which gave a large kLa value in all
bioreactors used, a large K value was obtained, but the quantitative relation between kLa and K in each bioreactor was different. For the bioreactor suitable for culturing plant cells, a system yielding as large a kLa
value as possible under experimental conditions which give a small I(
value is desirable. Therefore, it was considered that the two kinds of
flasks on shakers and aeration-type apparatus were more suitable as
bioreactors than aeration-agitation-type apparatus.
Selection of Bioreactors for Culturing Plant Cells at High Density

In culturing plant cells at high density, the intensity of culture broth

mixing and air-bubble dispersion should be considered in addition to both
the capacity of oxygen supply and the intensity of hydrodynamic stress

-10
E
\

-8

3C 6
._
I

5
gV 4

y
\
u- *

0 2

HIGH DENSITY PLANT CELL CULTIVATION

1213

200

150

>

-I00

50
0

4
5
6
7
~ ~ 1 ( 0cm/sec
)
)

Fig. 7. Relation between initial volumetric oxygen-transfer coefficient k ~ at

a 30C. Experimental conditions of various bioreactor systems used were the same as described in
Table I.

effects on plant cells. Then, in order to select bioreactors for culturing

plant cells at high density, the characteristics of various bioreactors used
until now were investigated by considering the above three factors.
The intensity of culture broth mixing and air-bubble dispersion was
measured by kLa in the presence of plant cells. Under experimental conditions giving K values less than about 4.4 x
cm/sec which was
obtained for normal cell growth, the effect of cell mass concentration on
kLa in various apparatus is shown in Figure 8. However, an experimental
cm/sec) more than the standard value
condition giving K (4.82 x
was used in J-T, because a suitable aeration-agitation condition giving a
K value close to the standard value could not be obtained for the apparatus.
With higher cell mass concentration, the culture broth mixing was insuf-

( )-K~IO(crn/sec )

B b l i e column (4.02)

Ail

reactor
( 3.47 )
6

11.0

21.6

trii mass concentration ( g l i )

Fig. 8. Effect of cell mass concentration on volumetric oxygen-transfer coefficient k L a .

Cell mass at 12-day-old culture (logarithmic growth phase) in shake flasks (85 oscillations/
min) was used. Operation conditions: J-T agitation 225 rpm,aeration 1 vvm; J-M agitation
400 rpm, aeration 1 vvrn; air-lift reactor aeration 2 vvm; bubble column aeration 1 vvm.

TANAKA

1214

ficient, the air bubbles could not be dispersed satisfactorily into the broth,
and kLa decreased. The degree of the decrease if kLa was larger in the
aeration-type apparatus than in the aeration-agitation-type apparatus.
The ratio of the kLa value at a cell mass concentration of 1.1% to the kLa
value in the absence of cell mass was 0.30 for a bubble column, 0.50 for
an air-lift reactor, and about 1.O for an aeration-agitation-type apparatus,
respectively. However, kLa at a cell mass concentration more than 2.0%
decreased considerably not only in the aeration-type apparatus, but also
in the aeration-agitation-type apparatus, J-T. In the bubble column, onethird of the volume of cell suspension from the bottom did not flow due
to accumulation of cells in the part. In the air-lift reactor, only the cell
suspension in the inner pipe of the reactor flowed and that in the other
parts did not. In J-T also, accumulation of cells in the part between the
impeller and bottom and around the baffle plates was found. Therefore,
mixing of the cell suspension and dispersion of air bubbles were insufficient, the true dissolved oxygen concentration C in the cell suspension
could not be obtained and, consequently, kr.a values could not be calculated from eq. (5). From the results, it was found that those apparatus
at cell mass concentrations more than 2.0% were not useful as bioreactors.
For J-M, on the other hand, mixing of the cell suspension and dispersion
of air bubbles were satisfactorily at cell mass concentrations more than
2.0%, and the decrease of kLa value was slight. Therefore, J-M seemed
to be the most suitable bioreactor apparatus for culturing plant cells at
high density. Then, a culture of C. tricuspiduta cells at high density was
carried out in J-M (working volume 5 liter). The growth curve of the cells
under aeration-agitation conditions giving a K value less than 4.4 X lop3
cm/sec is shown in Figure 9. The agitator speed was maintained at 60
I

Fig. 9. Growth curve of Cudrunia tricuspidutu suspension cells in 10-literjar fermentor

containing benzyl adenine (2 mg/liter) and sucrose (2.2%) in 10-liter J-Mwas used. Sucrose
(3.0%) was fed at 17 days of culture. Agitation was 60 rpm.

HIGH DENSITY PLANT CELL CULTIVATION

1215

rpm, the air flow rate was changed up to 0.5-1.5 vvm to maintain a
positive dissolved oxygen pressure. Schenk and HildebrandtI7 reported
that increase of sucrose concentration to 4% or higher in plant cell cultures
on agar media slowed the growth rate initially. They estimated the phenomenon might be an osmotic effect. Based on an assumption that there
may be the same phenomenon in suspension cultures of plant cells, the
initial sucrose concentration in the medium was 2.2% and sucrose corresponding to the concentration of 3.0% in the culture broth was fed at
17 days of culture. The yield of cell mass for the 25-day-old culture was
about 30 g as dry weight per liter of medium.
DISCUSSION
In culturing useful plant cells at high density it is necessary to choose
an optimum aeration-agitation (or shaking) condition in a bioreactor by
considering the capacity of oxygen supply, the intensity of hydrodynamic
stress effects on the plant cells, and the intensity of culture broth mixing
and air-bubble dispersion. Bioreactors should also be evaluated by considering the above.
The idea that the mass-transfer coefficient in a model solid-liquid system in place of a system of plant cells and a medium can be employed
as an intensity index of hydrodynamic stress effects on plant cells originates from the study by Bylinkina and Birukov." The writers thought
that one of the conditions to get an equal antibiotic yield for mycelial cells
in bioreactors of different design and scale was to maintain an equal state
at the surfaces of cells in their bioreactors, and the equal state was obtained by selecting aeration-agitation conditions giving an equal masstransfer coefficient in the model system. As a model system of mycelial
cells and a medium, they used a system of granulated urea and a glycerol
solution. In this investigation, the mass-transfer coefficient was employed
as an intensity index of hydrodynamic stress effects from the viewpoint
that the equal state at the surfaces of cells in different bioreactors was
held for physical effects. The model system used by them did not closely
follow a practical culture system in that the liquid was not water, the
treatment for the differences in density between the solid and the liquid
was not quantitative, the solid was not considered in shape and size, etc.
On the other hand, the model system proposed in this investigation has
fully considered such points and it more closely follows a practical system.
The mass-transfer coefficient values obtained in the present system are
considered to reflect more closely that of the real situation. However, in
the suspension cultures of plant cells under certain experimental conditions having small capacity of oxygen supply, part of the cell aggregates
was observed to accumulate or move on the bottom the culture vessel
and flasks. Therefore, the A values for plant cells vary under those conditions, and K A obtained from eq. ( 2 ) can not be separated into K and

1216

TANAKA

A. Nevertheless, K was calculated from eq. (3) based on an assumption

that A is constant under all culture conditions for convenience in this
investigation. Also, the specific gravity and shape of (3-naphthol particles
are not nearly equal to those of plant cell aggregates. As described above,
the new method proposed in this paper to determine quantitatively the
intensity of hydrodynamic stress on plant cells has some problems which
must be solved.
In order to get large amounts of useful plant cells, it is more desirable
to culture the cells in the state of single cells than in the state of aggregates
from the viewpoints of hydrodynamic stress effects on cells, uptake of
nutrients by cells, mixing of culture broth, etc. C. tricuspidata cells used
in this investigation are almost in the state of aggregates at a slow speed,
so that the utility value of these cells in the industrial viewpoint may be
low even if these cells are useful. However, many plant cells grow in the
state of aggregates and are very sensitive to physical stress. From such
considerations, it may be valid to use C. tricuspidata cells as a model to
study hydrodynamic stress effects on plant cells.
In order to understand the characteristics of various bioreactors two
indexes, kLa and K, the results in Figure 7 were replotted in Figure 10
on a log-log scale. Linear relations between kLa and K for the conditions
used in all bioreactors were found, namely, kLa was in proportion to the
ath power of K. This a value varies from bioreactor to bioreactor. a
values for J-M and the two kinds were about 5, values in aeration-type
apparatus were 2, and the a value for J-T was intermediate between two
400

200 -

L 100 -h

60-

2 4 0 -20 -

lo

L
4 6 u
10
2
K x~~(crn/sec)

Fig. 10. Relation between initial volumetric oxygen-transfer coefficient kLa and the
mass-transfer coefficient of p-naphthol system, K , at 30C. The figure is a log-log graph of
results given in Fig. 7. (*) J-T system, a = 3.0; (A) J-Msystem, Q = 5.0; ( A ) air-lift reactor
system, a = 2.1; (0)bubble column system, a = 2.2; ( + ) shake flask system, a = 4.9;
( X ) Erlenmeyer flask, a = 4.9.

HIGH DENSITY PLANT CELL CULTIVATION

1217

previous values. These a values may be related to the flow characteristics

in bioreactors and may be able to be used as characteristic values of
bioreactors, but their relationships have not been investigated in detail.
To use the 01 value as a technological index, further studies are needed.
An air-lift reactor in which hydrostatic agitation creates a minimum of
share stress was employed by Wagner and Vogelmann4 as a bioreactor
for culturing plant cells. Nevertheless, Wilson fcund that at high cell
densities (above 20 g dry weight/liter) the air bubbles did not separate
satisfactorily from the culture with the result that the normal circulation
of the air lift became impeded and unstirred regions developed. A similar
result was found in this investigation. Therefore, the air-lift reactor is not
suitable for culturing plant cells at a high cell density.
From results investigated by three indexes, a bioreactor like J-M having
a mechanism whereby the whole culture broth is mixed by a large impeller
without baffle plates and aeration seems to be a suitable design of a
bioreactor for culturing plant cells at high density. In practice the final
yield of cell mass obtained in J-M was higher than yields reported in
studies using large-scale bioreactors. The decision of the optimum aeration-agitation condition for cell growth and the development of new
bioreactors for culturing cells at high density on the basis of the three
such indexes will be useful to improve the production of cells and metabolites.
The author thanks Dr. Masanaru Misawa, Kyowa Hakko Kogyo Co. Ltd., for helpful
suggestions.

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TANAKA

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1978), p. 169.

Accepted for Publication September 10, 1980