Communication to the Editor

A Quantitative Analysis of Shear Effects on

Cell Suspension and Cell Culture of Perilla
frutescens in Bioreactors
Jian-Jiang Zhong, Kazuhito Fujiyama, Tatsuji Seki, and Toshiomi Yoshida
International Center of Cooperative Research in Biotechnolog y, Faculty of
Engineering, Osaka University, 2-1 Yamada-oka, Suita-shi,
Osaka 565, Japan
Received November 1, 1993/AcceptedMarch 18, 1994

The short-time effects of shear on suspended cells of

Perilla frutescens were quantitatively analyzed by exposing the cells to a well-defined flow field in a rotating
drum reactor. It was found that both shear rate and
shearing time significantly affected cell viability. The
quantitative effects of shear on cell growth and the production of anthocyanin, a secondary metabolite, by the
cell cultures were further investigated in a series of batch
cultivations using a 5-L plant cell bioreactor with a marine impeller. The results indicated that there was an optimum range of shear rate; i.e., an average shear rate of
20 to 30 s- or an impeller tip speed of 5 to 8 dmis, which
maximized all the values of the following parameters:
the specific growth rate, the maximum cell concentration, the (specific) production and productivity of anthocyanin, and the cell and anthocyanin yields. 0 1994 John
Wiley & Sons, Inc.

Key words: anthocyanin production bioreactor cultivation Perilla frutescens plant cell culture shear effects
metabolism, secondary

INTRODUCTION
Plant cells in culture are larger than microbial and even
animal cells; individual cells in a suspension can range from
20 to as much as 100 pm in diameter. They are bound by a
rigid cellulose-based wall and often have a very large vacuole, comprising up to 95% or more of the cell volume.
Such characteristics of plant cells imply that they are sensitive to hydrodynamic shear stress, and are consequently
easily susceptible to damage under a certain degree of
shear. Thus, the issue of shear influence on plant cell cultures cannot be neglected in bioreactor operations.9712318
The effects of shear stress have been investigated
in various cultivations of microbial and animal
cells , 2 9 4 9 7 314, 16, 1722 but relatively few reports have appeared
characterizing shear effects on plant cells.8,1021 Scragg
et a1.20observed that different cell suspensions of Catharanthus roseus and Helianthus annuus showed different de-

grees of cell sensitivity to shear stress in short-duration

shear treatment. Hooker et a1.8 investigated the response of
Nicotiana tobacum suspension cells to a high shear environment over a short duration using a Couette-type shearing
device, and found that shear stress affected culture viability,
cell lysis, and phenolic secretion of the cell suspensions. In
batch cultivations using two 12-L stirred tank bioreactors
with and without baffles, Leckie et a). lo found that cultures
of C . roseus were adversely affected by a certain level of
shear stress, resulting in lower cell growth and alkaloid
accumulation.
From the above, it is clear that quantitative analysis of the
effects of shear stress on plant cells is very limited, in spite
of the great need for more knowledge in this area. Here, we
investigate the quantitative effects of shear stress on both
cell suspensions and cell cultures of Perilla ji-utescens,
which produces anthocyanin as a secondary metabolite.
First, the short-term effect of shear stress on suspended cells
is characterized by subjecting the cells to a well-defined
flow field by use of a rotating drum reactor. Then, the shear
effects on cell growth and anthocyanin production by cell
culture are quantitatively analyzed in a series of batch cultures using a plant cell reactor with a marine impeller.

MATERIALS AND METHODS

Plant Cells and Medium
Suspended cells of P . frutescens, which produce anthocyanin pigments, were cultured in Linsmaier and Skoog medium. Details of the cell line and medium have been described e l s e ~ h e r e . ~ ~ ~ ~

Shearing Instrument

* To whom all correspondence should be addressed at present address:

Research Institute of Biochemical Engineering, East China University of
Science and Technology, 130 Meilong Road, Shanghai 200237, China.

In order to shear the suspended cells in a homogeneous

state, a rotating drum reactor was used as the shearing in~ t r u m e n t .This
~ ~ consisted of two concentric cylinders-an
outer cylinder with an inner diameter of 75 mm and a height
of 150 mm, and an inner cylinder with an outside diameter

Biotechnology and Bioengineering, Vol. 44, Pp. 643654 (1994)

0 1994 John Wiley & Sons, Inc.

CCC 0006-3592/94/050649-06

of 55 mm and a height of 112 mm. The inner cylinder was

rotated by a top motor, while the outer one remained stationary. The suspended cells were sheared in the space between these two cylinders. Suspension samples were taken
from a sampling port near the reactor bottom.
Bioreactor Configuration and Operation
In a quantitative investigation of the effects of shear stress
on cell cultures, we used a 5-L plant cell reactor (Tokyo
Rikakikai Co. [Eyela], Tokyo, Japan), 14.3 cm in diameter
and 33.4 cm in height with a marine impeller having a
diameter of 85 mm (larger impeller) or 65 mm (smaller
impeller), which contained 3-L medium. During cultivation, the dissolved oxygen (DO) concentration was maintained at over 80% of air saturation by automatic regulation
of the partial pressure of oxygen and nitrogen using an
FM-11DO controller in an MOB-2 operational box, which
was connected to an FM-140 gas controller (all from Tokyo
Rikakikai Co. [Eyela]). The principle of DO control is similar to that described by Smith et a1.21Automatic regulation
of the partial gas flow rates of air, carbon dioxide, nitrogen,
and oxygen was possible without changing the total gas
flow rate after proper setting. The gases used were air, pure
oxygen, and pure nitrogen. A previous investigation indicated that a level of carbon dioxide in the range of 0.015 to
2% (vol/vol) in the inlet gas stream did not affect cell cultures of P.frutescens. In the present work, it was confirmed
that the C 0 2 level was within this range during cultivation
in all cases. There was no intentional light irradiation. In all
the cultivations, the culture temperature and the inoculum
were controlled at 25C and 25 g wet cells/L, respectively.
Analyses
For the short-term shearing experiments, the viability of the
cells was determined according to the 2,3,5-triphenyltetrazolium chloride (TTC) reduction method of Towill and
Mazur .23 Triphenylfonnazan reduced by viable cells was
measured at 485 nm of its maximum absorption wavelength. The cell viability was expressed in percentage terms
by taking the absorbance (at 485 nm) of a sample without
shearing treatment as a control, i.e., 100% cell viability.
All the data presented in this study are averages of three
determinations.
For the bioreactor cultivations, cell dry weight, residual
sucrose concentration and the amount of anthocyanin produced by the cells were estimated as published previo u ~ l y .The
~ ~ wet
, ~ cell
~ weight was measured as follows.
Cells of a sample were separated from the culture broth by
centrifugation at 3,000 rpm for 5 min, washed with a large
amount of distilled water, and then centrifuged once more.
The cells were weighed after separation from the supernatant (water).
Calculation of Shear Rates
For the experiments using the rotating drum reactor, the
shear rate applied to cell suspensions was calculated according to the following formulae435:

650

(R;n/R)2/"[2~/(1- Up2)]

where y is the shear rate, R, is the radius of the inner

cylinder, R is the distance between the inner and outer cylinders in the radial direction, n is the flow index of the
suspension, w is the rotating speed, and p is the ratio of the
outer cylinder radius to the inner cylinder radius.
The accurate calculation of the maximum and average
shear rate in stirred tank bioreactors is difficult. For batch
cultivations in the plant cell reactor, the average shear rate
was estimated for the marine impeller design using an equation developed by Calderbank and Moo-Young1 and
Metzner et al.13 For a lack of better empirical correlations,
the maximum shear rate for the marine impeller was
roughly evaluated by the impeller tip speed according to
Midler and Finn.'' Such a treatment is considered to be
reasonably acceptable in the present study with the ultimate
purpose of the optimization of operational conditions (such
as agitation speed) of the bioreactor. These estimation equations were as follows:
Average shear rate
Impeller tip speed

=
=

10 . N
3.14 N

Di

where N is the impeller (agitation) speed, and D ; is the

impeller diameter.
Calculation of Expansion Index, Specific Growth
Rate, and Yields of Cells and Pigments

A dimensionless parameter, named the expansion index,

was used to describe the degree of cell volume expansion
which occurred in cultured cells during cultivation under
various conditions of shear stress. It is the ratio of wet to dry
cell weight taken at the time of the maximum wet weight
value during cultivation. 103'9
The specific growth rate (d-') of a batch culture was
calculated from the cell dry weight values. It is the slope of
a linear regression of ln(cel1s) versus time.
The observed yield coefficients (Y) of cells and anthocyanin pigments in the cell cultures were calculated according
to the following formulae:

Y,,, = Delta(X)/Delta(S)
Y,,, = Delta(P)/Delta(S)
where X represents the cell mass, and S the substrate (sucrose); Delta(X) is the mass of cells produced, and Delta(S)
the sucrose consumed. P represents the product (anthocyanin); Delta(P) is the anthocyanin produced.
RESULTS AND DISCUSSION
Effects of Shear in the Rotating Drum Reactor

The shear sensitiveness of P . frutescens cells was first studied by shearing the suspended cells in a well-defined flow
field for a short time in the rotating drum reactor. It was
found that after shearing for 20 min, the cell viability was

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 44, NO. 5, AUGUST 20, 1994

- 0

'

200 400 600 8001000

Shearrate

creased with increased shearing time at shear rates of both

144.4 and 866.4 s-', indicating that a low shear stress over
a longer shearing time can cause equivalent damage to that
resulting from a high shear stress for a short shearing time.
Thus, even under a low shear stress, cells cultured in a
bioreactor may suffer heavy destruction over a long period
of cultivation. This is a point that cannot be neglected in
optimizing the conditions for plant cell cultivations in a
bioreactor .

5
10
15
20
Shearingtime (min)

(Us)

Figure 1. Influences of shear rate (a) and shearing time (b) on the viability of suspended cells of P . frurescens sheared in a rotating drum reactor. Cells cultivated in a flask for 13 days were used. Cell concentration:
6.0 g dry cellsiL. (a) The shearing time was 20 min. (b) The symbols for
866.4 s - ' .
shear rate are: (A),144.4 s-'; (O),

Quantitative Shear Effects in the Plant

Cell Reactor

The results from the short-term shearing experiments in the

rotating drum reactor showed that the viability of P .
frutescens cells apparently decreased with shearing time,
even at a low shear stress. In bioreactor cultivations, cells
are considered to be affected by shear stress caused by
agitation and aeration during a long period of operation,
even in a low shear environment.
In previous cultivations using a 2-L bubble column reacto?' and a 2.6-L turbine-agitated minijar rea~tor,~'shear
effects detrimental to suspension cultures of P . frutescens
were observed in both cases at relatively higher aeration
rates and/or agitation speeds. In this study, a preliminary
experiment on the effect of shear on P . frutescens cell cultures in a 5-L plant cell reactor with a marine impeller was

82%, 68%, and 63% at shear rates of 144.4, 433.2, and

866.4 s - l , respectively (Fig. la). In an investigation into
the response of N . tabacum cell suspensions to a high shear
environment using a Couette-type shearing device, Hooker
et a1.8 reported that, as expected, increasing shear rates
caused a steady decrease in cell viability. In their case, after
12 h of shearing, although the viability did not change at a
shear rate of 596 s-', it decreased by about 20% and 95%
at 895 s - ' and 1193 s - ' , respectively. It seems, therefore,
that the shear sensitivity of plant cells is rather dependent on
the cell line, as observed also by Scragg et a1.20
Figure l b shows that the cell viability apparently de-

4 0.17 LI
#0.15'
10

'

'

20

'

'

30

'

Average &ear rate

'
40

'

50

(Us)

3 0.15

5
8
11
Impeller tip speed ( W s )

7
o

t
1 0 2 0 3 0 4 0 5 0
Average ahear rate (ye)

Impeller tip speed ( W e )

Figure 2. Effects of shear on specific growth rate (a) and maximum cell concentration (b) of P . frufescens cells
cultivated in a plant cell reactor with a marine impeller. (A) Average shear rate; (B) impeller tip speed. Symbols: (O),
larger impeller (85 mm dia.); (A),smaller impeller (65 mm dia.).

COMMUNICATION TO THE EDITOR

651

also conducted by changing the agitation speed while keeping the other cultivation conditions the same. The result
shows that compared with the control speed of 130 rpm, a
relatively high agitation speed, i.e., 250 rpm, was unfavorable to both the growth and anthocyanin production by the
cells.28 At 250 rpm, cell growth was reduced to some extent, and there was a significant decrease in pigment production. Thus, control of the shear environment by changing the operational parameters (such as agitation speed and
aeration rate) was found to be very important for the optimization of cell culture processes in this bioreactor, even
though it was claimed to possess low-shear characteristics
and to be suitable for plant cell cultures. A recent report on
the development of a helical-ribbon impeller bioreactor for
plant cell cultures also emphasized the necessity of hydrodynamic shear studies in bioreactor evaluation.
The shear effects on the growth and secondary metabolism of P . frutescens cell cultures were further investigated
using the plant cell reactor with marine impellers of larger
(85 mm) and smaller (65 mm) diameter. In all cultivations,
the DO concentration was maintained at over 80% of air
saturation by automatic regulation of the partial pressure of
oxygen and nitrogen by means of a combined gas controller
and DO controller unit, as described in Materials and Methods. The overall aeration rate was kept at 0.1 L/min, i.e.,
0.033 vvm.
Figure 2a shows the effect of the average and maximum
shear rates on the specific growth rate of P.frutescens cells
in the bioreactor cultivations. Here, the maximum shear rate

0 '

10

,150-

652

is assumed to be represented by the impeller tip speed. The

specific growth rate was apparently reduced at an average
shear rate over 30 s- or at an impeller tip speed of over 8
d d s . Figure 2b shows the influence of shear on the maximum cell concentration of the cell cultures. The maximum
cell concentration was relatively higher at an average shear
rate between 20 and 30 s - or an impeller tip speed between
5 and 8 d d s . It was reduced at a higher shear rate, at an
average shear rate of over 30 s - ,or at an impeller tip speed
of 8 d d s .
Figure 3a shows the effect of shear stress on anthocyanin
content, i.e., the specific anthocyanin production, of the P .
frutescens cells in the bioreactor. The anthocyanin content
was relatively high at an average shear rate below 30 s- or
an impeller tip speed below 8 d d s . At higher shear rates,
the pigment content of the cultured cells showed an obvious
decrease. The effects of shear on total anthocyanin production and the volumetric productivity of anthocyanin in the
bioreactor cultivations of P . frutescens cells were similar to
those as shown in Figure 3a.28The production and productivity of the red pigments were relatively higher at an average shear rate between 20 to 30 s - ' or an impeller tip
speed between 5 and 8 d d s . Both the pigment production
and its productivity were reduced at higher shear rates. Figure 3b shows the effect of shear rate on the observed yield
coefficient of anthocyanin in the cell cultures. A relatively
higher cellular product (anthocyanin) yield was observed at
an average shear rate between 20 and 30 s- or an impeller
tip speed between 5 and 8 dmls. A similar shear stress effect

'

'

'

'

'

'

- 0

"
"
'
' J
20
30
40
60
Average &ear rate (ye)

(A)

5
8
11
Impeller tip speed ( W s )

b
$ M ) -

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 44, NO. 5, AUGUST 20, 1994

(B)

on the cellular growth yield was also observed in the plant

cell reactor.28
The influence of shear rate on the expansion index of the
cultured cells in the bioreactor was also investigated.28The
expansion index decreased at a higher average shear rate or
at a higher impeller tip speed, indicating that the volume of
cultured cells could not expand more at a higher shear
stress, i.e., shear stress limited the volume expansion of the
cells. Here, the effect of shear on the cell expansion is quite
similar to its effect on the specific cell growth rate. In the
range of shear rates investigated, both of these parameters
were continuously sensitive to the shear environments in the
bioreactor cultivations.
Thus, there was an optimum range of shear rate favorable
to pigment production and specific production, and to the
cell and pigment yields. Relatively high cell concentrations,
pigment production and pigment productivity, as well as
cell and pigment yields, were achieved in the plant cell
bioreactor at an average shear rate of 20 to 30 s - ' or at an
impeller tip speed of 5 to 8 d d s . These criteria are equivalent to the following operational conditions in the reactor:
an agitation speed of 120 to 170 rpm using the larger impeller (85 mm in diameter). At an average shear rate of over
30 s K 1 or an impeller tip speed of over 8 d d s , the cell
growth, pigment production and its specific production, as
well as the yields of the cells and the pigments, were poor
due to the detrimental effects of the shear stress on the
cultured cells. Both the cell expansion index and the specific growth rate of the cells were smaller in response to a
higher shear environment.
Because shear stress, culture viscosity, mixing, mass (especially oxygen) transfer, and growth, as well as the metabolism of plant cells in a bioreactor are interlinked, as
demonstrated in some
it is very important to
determine the range of shear that can be applied without
adversely affecting the cells. Such fundamental information
is essential for the optimization of bioreactor environments,
and it may also be helpful in bioreactor scale-up and highdensity cultivations of plant cells for the production of useful plant-specific chemicals. The results shown here additionally suggest that, for better bioreactor control, on-line
monitoring of the shear environment is necessary in plant
cell bioprocesses. l 1
We thank Tokyo Rikakikai Co. (Eyela) for providing us with the
plant cell bioreactor. One of us (J. J. Z.) is a recipient of a
Japanese government (Monbusho) scholarship.

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BIOTECHNOLOGY AND BIOENGINEERING, VOL. 44,NO. 5, AUGUST 20, 1994