Biotechnol. Prog.

1999, 15, 319−335 319

TOPICAL PAPER
Design of Mixing Systems for Plant Cell Suspensions in Stirred
Reactors
Pauline M. Doran*
Department of Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia

The properties of dense plant cell suspensions, such as high viscosity, shear sensitivity,
cell aggregation, and tendency to foam, present significant technical challenges for
bioreactor design and operation. As in all fermentation processes, reactors used for
large-scale plant cell culture must provide adequate mixing and mass transfer at
nondamaging levels of hydrodynamic shear. In stirred vessels, this means that the
impeller must be operated with restricted power input and speed, while still achieving
complete gas dispersion and solids suspension. Although many experimental studies
of conventional and novel impellers have been carried out, no clear guidelines for
impeller choice have emerged. In this paper, an engineering analysis is used to identify
which agitators and operating regimes have greatest potential for high-density plant
culture applications, irrespective of whether they have been tested experimentally.
Ways of improving impeller performance by modifying internal reactor geometry are
also discussed.

Introduction years, a range of reactor configurations has been tested

Plant cell cultures are capable of synthesizing a wide
range of valuable phytochemicals. Being independent of
geographical location, climate, season, and other factors
which affect traditional methods based on whole plants,
in vitro production under controlled conditions improves
the reliability of product supply to the market. As well
as natural plant compounds, fully assembled antibodies
and other complex animal proteins can now be expressed
and secreted in plant systems (Franken et al., 1997).
Plant cells in liquid suspension offer a unique combina-
tion of physical and biological properties which must be
accommodated in large-scale bioreactor processes aimed
at exploiting their production potential.
Although much of the technical know-how and experi-
ence of fermentation engineers is directly applicable to
plant cultures, there are several important areas in which
plant cells challenge established bioreactor design and
operating practice. Plant cell suspensions have more
complex rheology and are more difficult to mix than
bacterial or yeast suspensions, but have considerably
lower specific oxygen demand and cooling requirements.
Like mycelial broths, plant cultures are viscous at high
biomass densities and produce cell aggregates of various
size; however, similar to mammalian cells, the shear
sensitivity of plant cells restricts the intensity of turbu-
lence which can be generated for bubble dispersion and
particle suspension. Plant cells are not as susceptible as
animal cells to damage from bursting bubbles; instead,
they produce thick foam or meringue layers in sparged
vessels, but cell viability in the foam remains high
(Wongsamuth and Doran, 1994). Over the past 10-15
* Fax: +61-(2)-9313-6710. E-mail: p.doran@unsw.edu.au.
10.1021/bp990042v CCC: $18.00 © 1999 American Chemical Society and American Institute of Chemical Engineers
Published on Web 05/07/1999
in an effort to satisfy the requirements of plant cells in
large-scale culture. Although air-driven bioreactors were
favored initially because of their low shear characteris-
tics, several experimental (Fowler, 1988; Scragg et al.,
1988b; Tanaka, 1981; Ulbrich et al., 1985) and theoretical
(Doran, 1993) studies have shown that mixing in these
vessels becomes inadequate at high cell densities (20-
30 kg m-3 dry weight). Because greater power can be
input to the broth by mechanical agitation, stirred tanks
are generally more suitable for viscous liquids containing
suspended particles and have been successfully applied
for industrial-scale plant cell culture (Curtin, 1983;
Hashimoto et al., 1982; Rittershaus et al., 1990).
The hydrodynamic conditions in stirred vessels are
most strongly dependent on the impeller design and
power drawn. Many different types of impeller have been
tested for plant cell culture, ranging from the standard
Rushton turbine to semiconventional agitators such as
the helical ribbon and novel configurations such as cell-
lift and membrane stirrer designs. Geometric specifica-
tions important in any engineering assessment, such as
the impeller off-bottom clearance, the ratio of liquid
height to tank diameter, the number of baffles and their
width, and the sparger type and position, are often not
provided in literature reports. In most cases, mixing has
been evaluated in terms of biological rather than physical
parameters, viz. cell growth rate and alkaloid levels are
measured in preference to more direct indicators of stirrer
performance such as mixing time, power consumption,
and gas holdup. Accordingly, although many comparative
studies have been carried out, no clear general-purpose
guidelines for the design and operation of stirrers for
plant cell culture have emerged.
320
Avoiding the deleterious effects of excessive shear is a
major concern in plant cell culture. Adequate bulk fluid
circulation and bubble dispersion are likely to be com-
promised in commercial operations to minimize shear
damage; large-scale reactors for plant suspensions are
designed for operation at slow stirrer speeds. Under these
conditions, even at gas flow rates as low as 0.01-0.2 vvm,
conventional impellers are likely to be flooded (Boysan
et al., 1988) and thus ineffectual for gas-liquid distribu-
tion. To avoid flooding, the gas flow rate may be reduced
to extremely small values, e.g. 0.005 vvm as used in
industrial-scale mammalian cell reactors (Nienow et al.,
1996); however, interaction between the bubbles and
impeller blades is minimal in this operating regime so
that gas dispersal is negligible. Such a situation would
be considered unacceptable and impractical in most
applications of aerated stirred tanks. In an effort to
compensate, much attention may be given to design of
the sparger to deliver bubbles of small size. There are
practical problems with this approach, as plant cells have
a high tendency for wall growth and will invade and block
porous or small-orifice spargers. In any case, if the
bubbles are allowed to rise virtually uninterrupted from
the sparger, reducing their size does not address the
problem of gas maldistribution and oxygen gradients in
poorly mixed bioreactors. Zones of oxygen depletion are
likely to develop, particularly below the impeller and near
the tank walls, and settling of the cells may also occur.
Increasing the gas flow rate to high values so that the
reactor behaves as a bubble column is also not advisable.
As well as eliminating the potential advantages for
mixing associated with mechanical agitation, this strat-
egy introduces foaming as a significant additional prob-
lem. Finding a solution to these difficulties is worthwhile,
as considerable improvements in biological performance
can be expected if dissolved oxygen gradients are elimi-
nated from plant cell reactors.
The aim of this paper is to determine using engineering
analysis whether the mixing situation in large-scale plant
cell reactors can be improved by making different choices
of impeller design, tank configuration, and operating
conditions. The analysis is carried out for a plant culture
at relatively high cell density (30 kg m-3 dry weight), as
low product yields are a feature of most plant cell
processes and high biomass concentrations are usually
required for economic feasibility (Matsubara et al., 1989).
Although the findings do not provide for a prescriptive
approach to stirrer design, they can be used to rationalize
impeller choice and hardware configuration. The major
considerations involved in specifying mixing equipment
for large-scale culture of suspended plant cells are
identified and several impellers assessed to determine
which are good potential candidates for plant cell culture,
even if they have not yet been studied experimentally.
The material presented draws from the chemical engi-
neering literature on mixing, as well as from recent
papers providing information on the relevant properties
of plant cells in vitro. The engineering analysis highlights
key design criteria, illustrates the benefits of informed
agitator choice for plant cell bioreactors, and identifies
areas where improvements in stirrer performance are
required.
Model System and Operating Conditions
The problem of specifying mixing equipment for a
stirred, sparged bioreactor used to culture suspended
plant cells is examined in the following sections. Calcula-
tions for six impeller configurations are performed to
allow comparison of the hydrodynamic conditions pro-
Biotechnol. Prog., 1999, Vol. 15, No. 3
Table 1. Reactor Geometry
parameter specification
reactor working volume VL 10 m3
tank diameter DT 2.3 m
liquid height HL 2.3 m
impeller type varies
impeller diameter Di varies
impeller clearance above the vessel floor Ci 0.58 m
sparger type ring
sparger diameter DS 0.8 Di
clearance between the sparger and impeller CS 0.39 m
no. of baffles nBF 4
baffle width WBF 0.23 m
submerged baffle height HBF 2.3 m
vided by each, including the effectiveness of gas disper-
sion and solids suspension. Time constant regime analy-
sis for mixing and oxygen transfer is also used to check
whether these reactor functions are likely to become rate-
limiting with different impellers.
Bioreactor Specifications. A typical bioreactor ge-
ometry is assumed, as indicated in Table 1. The reactor
is a flat-bottomed cylindrical tank with liquid height
equal to the tank diameter. The vessel is equipped with
a top-entering stirrer and single impeller. Although
several different impellers and impeller sizes will be
considered, the impeller clearance off the floor of the
reactor is maintained constant at one-fourth the tank
diameter, as this geometry has been found appropriate
for three-phase mixing (Chapman et al., 1983c; Frijlink
et al., 1990). The reactor has a ring sparger, and the
sparger-impeller clearance is one-sixth the tank diam-
eter. The width of each of four baffles is one-tenth the
tank diameter.
Culture Conditions and Properties. The reactor is
used for continuous steady-state culture of suspended
plant cells; the culture characteristics and reactor operat-
ing parameters are listed in Table 2. The reactor dilution
rate of 0.2 day-1 is safely below the washout rate for plant
cells with minimum doubling times of up to 3 days. A
biomass concentration of 30 kg m-3 dry weight has been
chosen to test the mixing system at a relatively high cell
density. The fresh weight:dry weight ratio of 15:1 is
typical of actual values (Curtis and Emery, 1993; Snape
et al., 1989); the cell concentration in fresh weight terms
is therefore 450 kg m-3. The nominated cell aggregate
size of 500 µm is a representative average of many values
reported in the literature (Curtis and Emery, 1993; Kato
et al., 1978; Leckie et al., 1991a; Scragg et al., 1988b;
Tanaka, 1982; Yokoi et al., 1993), as is the specific gravity
of 1.03 for plant cells cultivated in vitro (Ananta et al.,
1995; Ballica and Ryu, 1993; Tanaka, 1982).
Several investigations of plant culture rheology are
reported in the literature, and the results show that plant
cell suspensions do not all exhibit similar characteristics.
Newtonian rheology is maintained in many cultures even
at moderate to high cell densities (Curtis and Emery,
1993), while pseudoplastic (Jolicoeur et al., 1992; Kato
et al., 1978; Scragg et al., 1988b; Wongsamuth and
Doran, 1997), Bingham plastic (Zhong et al., 1992), and
Casson plastic (Ballica and Ryu, 1993) behaviors have
also been found, indicating that non-Newtonian cultures
may or may not exhibit a yield stress. For the purposes
of the present engineering analysis, it is assumed that
the model plant culture broth has Newtonian rheology
with a viscosity of 30 mPa s (Table 2). Selection of these
properties is consistent with reports of Newtonian plant
suspensions at cell concentrations of 25-30 kg m-3 dry
weight (Curtis and Emery, 1993; Jolicoeur et al., 1992),
and reflects the relatively short residence time (5 days)
Biotechnol. Prog., 1999, Vol. 15, No. 3
Table 2. Reactor Operating Parameters and Cell Culture
Properties
parameter specification
dilution rate 0.2 day-1
average reactor residence time τ 5 days
liquid density FL 1000 kg m-3
suspending liquid viscosity µL 1 mPa s
cell concentration x 30 kg m-3 dry weight
cell fresh weight:dry weight ratio 15:1
broth viscosity µ 30 mPa s
cell particle density Fp 1030 kg m-3
cell particle diameter Dp 500 µm
of cells in the reactor (Table 2) and the inverse relation-
ship between cell age and non-Newtonian properties
reported in the literature (Jolicoeur et al., 1992; Zhong
et al., 1992). The nominated viscosity of 30 mPa s is high
compared with values of 10 mPa s or less which have
been measured at plant biomass densities of g30 kg m-3
dry weight (Scragg et al., 1988b; Yokoi et al., 1993). The
viscosity of the suspending fluid is assumed to remain
close to 1 mPa s, as shown in many previous studies
(Curtis and Emery, 1993; Scragg et al., 1988b; Zhong et
al., 1992).
Impeller Specifications and Operating Charac-
teristics. The types of impeller that can be subjected to
detailed engineering analysis are restricted by the limited
availability of design correlations. Six impellers will be
assessed for application in plant cell bioreactors, includ-
ing Rushton impellers and pitched blade turbines oper-
ated with either downward or upward pumping. The
impeller specifications are listed in Table 3. To provide
a basis for interpreting the results of this investigation,
the general operating characteristics of the impellers are
outlined below.
Rushton turbines generate primarily radial flow and
are very effective for gas dispersal. During operation, two
high-speed roll and trailing vortices are generated behind
each turbine blade (van’t Riet and Smith 1973, 1975);
with sparging, gas readily accumulates in these low-
pressure regions to form ventilated cavities. Changes in
the size and structure of ventilated cavities with stirrer
speed and gas flow rate have been well-documented
(Bruijn et al., 1974; Nienow et al., 1985; Smith and
Warmoeskerken, 1985; Warmoeskerken et al., 1981) and
control the effectiveness of gas dispersion, gas holdup,
and mass transfer in the vessel. Formation of ventilated
cavities reduces the drag coefficient of the impeller and
is responsible for power losses with aeration of up to 70%
in low-viscosity fluids (Martin et al., 1994; Warmoeskerken
et al., 1981), although practical operating conditions
usually give a 40-50% reduction in power (Nienow et
al., 1985). The exact extent of the power loss with
sparging cannot yet be predicted accurately, being a
complex function of the stirrer speed, air flow rate, vessel
size, and fluid properties and depending on the level of
gas recirculation to the impeller region (van’t Riet et al.,
1976; Middleton, 1992). The important transitions in gas
dispersion conditions with Rushton turbines are il-
lustrated in Figure 1. At high gas flow rates and low
stirrer speeds, the impeller is flooded, i.e. the gas-
handling capacity of the impeller is exceeded, the impel-
ler ceases to pump the two-phase fluid, and the flow
pattern is dominated by buoyant gas-liquid flow up the
middle of the vessel (Figure 1a). With increasing stirrer
speed or reduced gas flow, bubbles break away from the
ventilated cavities that form behind the impeller blades
and are dispersed in the liquid (Figure 1b), until the gas
is thoroughly and vigorously distributed throughout the
321
vessel including below the impeller. This condition of
complete gas dispersion is illustrated in Figure 1c.
Pitched blade turbines can be operated in either
downward- or upward-pumping modes, downward being
more usual. Although commonly referred to as axial
impellers, they generate a mixed flow pattern with
significant radial velocity components. Ventilated cavities
form behind the pitched blades as a result of underpres-
sure in trailing vortices in much the same way as for
Rushton turbines (Tatterson et al., 1980; Warmoeskerken
et al., 1984a), except there is only one vortex per blade.
Pitched blade impellers have significantly lower unaer-
ated power numbers than Rushton turbines. With gas-
sing, downward-pumping turbines are prone to flooding,
especially if the impeller diameter and projected blade
area are relatively small. At stirrer speeds just below
those for complete gas dispersion, instabilities in torque,
power draw, and flow pattern can be a serious problem.
Flow is less stable with four rather than six blades, and
at smaller impeller to tank diameter ratios (Chapman
et al., 1983b). Because the power oscillations prior to
stable gas dispersion can lead to mechanical problems
including vessel vibration (Buckland et al., 1988), opera-
tion in this regime is not recommended. Thus, in contrast
with Rushton impellers, for downward-pumping turbines
there is no practical range of operating conditions be-
tween flooding and complete gas dispersion; the stirrer
speed should be high enough so that the gas is fully
distributed and the power draw steady. Power reductions
of up to 60-70% occur with aeration (Bakker and van
den Akker, 1994; Warmoeskerken et al., 1984a). At low
gassing rates, downward-pumping turbines achieve solids
suspension in three-phase systems with very high energy
efficiency (Nienow et al., 1986); however, increasing
aeration can result in severe loss of suspension capacity
if the power consumption is suddenly reduced with the
onset of hydrodynamic instabilities.
Many of the problems associated with downward-
pumping impellers under gassed conditions can be avoided
by reversing the direction of rotation so that the impeller
pumps upward. No significant torque or power fluctua-
tions have been observed with upward-pumping turbines
(Chapman et al., 1983b; Bujalski et al., 1990). Unaerated
power numbers for upflow turbines are similar to those
for downflow turbines with the same geometry (Frijlink
et al., 1990; Ibrahim and Nienow, 1995), but the fall in
power draw with aeration (e20%; Chapman et al., 1983b)
is much less, even though ventilated cavities form behind
the blades (Bujalski et al., 1990; Nienow et al., 1986).
The aerated power draw is also relatively independent
of gassing rate (Bujalski et al., 1988). Compared with
downward-pumping impellers, complete gas dispersion
is achieved at lower speeds and energy dissipation rates,
and more gas can be handled before flooding occurs
(Bujalski et al., 1988). Because upward-pumping turbines
generate relatively small velocities beneath the impeller,
the energy required for solids suspension is significantly
greater than for downflow impellers. An advantage in
aerated systems, however, is that both the agitation
speed and power required for suspension are almost
independent of gassing rate (Nienow et al., 1986; Bujalski
et al., 1988).
Analysis of Shear Effects
Various approaches have been used to evaluate the
shear sensitivity of plant cell suspensions and to deter-
mine the restrictions this property imposes on reactor
operating conditions. In some studies, the problem has
been viewed in terms of threshold values of either shear
322
Table 3. Impeller Specifications
impeller diameter/
diameter, tank diameter
impeller Di (m) (Di/DT)
Rushton turbine 0.78 0.33
1.15 0.50
pitched blade turbine 0.92 0.40
(downward pumping) 1.15 0.50
pitched blade turbine 0.92 0.40
(upward pumping) 1.15 0.50
Figure 1. Flow patterns developed by a Rushton turbine under
gassed conditions as a function of impeller speed Ni and gas
flow rate QG: (a) impeller flooding; (b) impeller loading, where
the gas is dispersed by the impeller but poorly distributed
throughout the vessel; (c) complete gas dispersion below as well
as above the impeller. Redrawn from Nienow et al. (1986).
rate (Zhong et al., 1994) or shear stress (Chen et al., 1990;
Wagner and Vogelmann, 1977), above which the growth
rate, cell viability, product yield, or some other critical
culture parameter is significantly affected. A limitation
associated with this approach is that different threshold
values may be obtained depending on the duration of
exposure of the cells to the shear environment. In
general, shear effects have been examined in either short-
term (0.5 s to 12 h) experiments under well-defined
conditions of laminar or turbulent flow (Hooker et al.,
1989; Kieran et al., 1995; MacLoughlin et al., 1998; Zhong
et al., 1994), or in short- or long-term experiments in
bioreactors under relatively ill-defined hydrodynamic
conditions (Leckie et al., 1991b; Markx et al., 1991;
Scragg et al., 1988a; Takeda et al., 1994; Zhong et al.,
1994).
If the response of plant cells to hydrodynamic shear
depends on the exposure time as well as the shear force
intensity, parameters such as the critical shear stress or
shear rate are inadequate as a basis for predicting shear
damage. The cumulative energy dissipated on the cells
has been used as a correlating factor for shear effects in
fungal (Reuss, 1988) and, more recently, plant cell
(Dunlop et al., 1994; MacLoughlin et al., 1998; Wong-
samuth and Doran, 1997) cultures; this parameter takes
into account the time of exposure. Under turbulent
conditions, growth of Atropa belladonna suspensions
(Wongsamuth and Doran, 1997) and the viability of
Morinda citrifolia cells (MacLoughlin et al., 1998) have
been observed to decline rapidly at energy dissipation
values of 106-108 J m-3, while sublytic effects such as
changes in aggregate size, cell chain length, protein
release, and mitochondrial activity may occur at lower
Biotechnol. Prog., 1999, Vol. 15, No. 3
blade width/
no. of blades, impeller diameter blade angle
nB (-) (WB/Di) (deg)
6 0.2 -
6 0.2 -
6 0.2 45
6 0.2 45
6 0.2 45
6 0.2 45
dissipation energies (Dunlop et al., 1994; MacLoughlin
et al., 1998; Wongsamuth and Doran, 1997).
In view of these experimental findings and for the
purposes of designing an impeller system which does not
damage plant cells, a cumulative energy dissipation of
107 J m-3 will be adopted in the present study as a critical
value below which shear effects on the culture are
insignificant. This parameter can be related to power
dissipation using the equation (Wongsamuth and Doran,
1997)
E)
1
VL ∫
Pφ dt (1)
where E is the cumulative energy dissipated on the cells
per unit volume, VL is the reactor working volume, P is
the power input by agitation, φ is the volume fraction of
cells in the culture, and t is time. In a chemostat under
steady-state conditions, VL, P, and φ are constant so that
EVL
P) (2)
φτ
where τ is the average reactor residence time. Substitut-
ing values from Tables 1 and 2 into eq 2 with E ) 107 J
m-3 and φ ) 0.45 gives P ) 514 W. The corresponding
maximum allowable stirrer speed is determined from the
definition of the dimensionless impeller power number
(Doran, 1995)
P
NP0 or NPg ) (3)
FNi3Di5
where NP0 is the unaerated power number, NPg is the
power number with gassing, F is broth density, Ni is
stirrer speed, and Di is impeller diameter. NP0 is constant
under turbulent conditions. Because the power consumed
by impellers is reduced with gassing, NPg is less than NP0;
therefore, for a fixed maximum agitator power, the
maximum permissible Ni will be greater with aeration
than without. However, because it is not possible to
predict the precise value of NPg in an aerated reactor, the
limiting values of NP0 and either NPg ) 0.5NP0 for Rushton
turbines (Nienow et al., 1985), NPg ) 0.3NP0 for downward-
pumping pitched blade turbines (Warmoeskerken et al.,
1984a), or NPg ) 0.8NP0 for upward-pumping pitched
blade turbines (Chapman et al., 1983b) will be used to
define the two extreme values of the maximum allowable
stirrer speed for each type of impeller.
The above analysis is a useful starting point for
specifying impeller operating conditions. However, the
hydrodynamic forces generated in fermentors and their
shear effects on cells are not solely dependent on the
power drawn by the impeller. At the same power input,
we can expect that varying degrees of shear damage will
be inflicted by impellers of different geometry, as has
been demonstrated in studies with mycelial cultures
Biotechnol. Prog., 1999, Vol. 15, No. 3
Table 4. Equations for Calculating Impeller Flow Numbers
equation
( WB
)
0.5
Fl ) 0.91NP0
Di
[ (
NP0 cBFnBFWBF(HBF/DT)
Fl ) 2
, where Z ) 1 +
π (1 - Z) ci(nB/2)WB
) ]
-0.5 -1
0.02
(0.6(DT/Di)2 - 1) , cBF ) 2.5, and ci ) 2
(WB/Di)
Fl ) 0.784NP00.5
Fl ) 0.76NP00.33
(Jüsten et al., 1996, 1998). This can be explained by
considering the distribution of energy dissipation rates
within stirred vessels; impellers with different sweep
volumes and trailing vortex structures impose different
local shear conditions close to the impeller where specific
rates of energy dissipation are highest and most damage
takes place. In addition, impellers which generate strong
circulation currents transport cells to the impeller region
more frequently than agitators which do not promote
rapid bulk liquid turnover, thus increasing the risk of
shear effects. A correlation procedure for shear damage
which takes all of these factors into account has been
developed by Jüsten et al. (1996); microbial shear damage
by different impellers in tanks of various size was
correlated using the parameter
P 1
k′Di3 tc
where tc is the circulation time defined in eq 6 and k′ is
a geometric factor reflecting the impeller sweep volume
and number of high-speed vortices generated
k′ ) ( )
π WB
4 Di
× no. of vortices per impeller blade (5)
Efecto similar a Kolmogorov
where WB is the impeller blade width. According to this
model, the greater the impeller sweep volume and the
more vortices used to dissipate a given power input, the
lower are the local volumetric energy dissipation rates
near the impeller, and the lower therefore are the
damaging effects of the agitator. Because the number of
vortices per impeller blade is two for Rushton turbines
and one for pitched blade turbines, it follows that, for a
given power input, more cell damage and higher specific
energy dissipation levels will be generated by pitched
blade turbines than by Rushton turbines with the same
diameter and blade width, thus challenging the conven-
tion that Rushton turbines are “high shear” agitators
producing much less uniform distributions of energy
dissipation rates than other types of impeller. Yet, results
consistent with these predictions have been demonstrated
experimentally (Jüsten et al., 1998; Zhou and Kresta,
1996). The circulation frequency factor 1/tc in eq 4 is
defined as
3
1 Fl NiDi
) (6)
tc VL
Fl is the dimensionless impeller flow number, which can
be evaluated for different impellers using equations from
the literature as indicated in Table 4 (Jüsten et al., 1996).
The critical cumulative energy dissipation value of 107
J m-3 to be applied in this study reflects experimental
323
flow ref
radial Platzer and Noll (1983)
)(
0.5
1+
radial Bittins and Zehner (1994)
axial Platzer and Noll (1983)
axial Herbert et al. (1994)
findings (Wongsamuth and Doran, 1997) using a Rushton
turbine with geometry similar to that of the smaller
Rushton impeller specified in Table 3. Accordingly, this
impeller with a diameter of one-third the tank diameter
will be used as a basis for calculating critical cumulative
energy dissipation levels for the other impeller geom-
etries. Table 5 shows results for the maximum allowable
unaerated stirrer speed for the smaller Rushton turbine
calculated directly from eq 3 with P ) 514 W, which was
already evaluated using eq 2. The parameter of eq 4 for
the smaller Rushton turbine under ungassed conditions
is therefore 112 W m-3 s-1; this value was determined
using eqs 4-6 and the average Fl value from both
equations for radial-flow impellers listed in Table 4. To
avoid shear damage, this critical value cannot be ex-
ceeded by any of the impellers. The maximum permis-
sible unaerated stirrer speed Ni for these agitators is
(4)
calculated from a combination of eqs 3, 4, and 6
[
(112 W m-3 s-1)k′VL
]
0.25
Ni ) (7)
NP0FDi5Fl
using the unaerated power numbers NP0 listed in Table
5. From eq 7, impellers with low power number, small
diameter, low flow number, and high geometric factor k′
can be operated at the highest stirrer speeds while still
avoiding cell shear damage.
Results for the maximum unaerated stirrer speeds
calculated using eq 7 are shown in Table 5. Operation at
these speeds results in turbulent flow (and therefore
constant power number) as indicated by the values for
the impeller Reynolds number (Rei > 5 × 103; Doran,
1995) and produces impeller tip speeds (πNiDi) of 1.5-
1.9 m s-1. For each type of impeller, the average Fl value
from both equations in Table 4 was used; both correla-
tions in each case produced a maximum variation in Fl
of only 6%. The maximum stirrer speeds under aerated
conditions were evaluated using eq 3 with the same
maximum power dissipation as under ungassed condi-
tions, but with the limiting values of NPg outlined above.
The results for maximum permissible power P in Table
5 represent the upper limits which can be tolerated by
the plant cells before shear damage occurs with the
different impellers; these power inputs correspond to
stirrer speeds between the aerated and unaerated values
listed in Table 5, depending on the degree of aeration
provided and the extent to which the power draw is
affected by gassing.
Time Constant Analysis
As the mixing and oxygen transfer functions of biore-
actors depend on the power dissipated, if the power input
to the vessel is restricted to avoid shear effects, it is
324 Biotechnol. Prog., 1999, Vol. 15, No. 3

Table 5. Calculated Results for the Maximum Permissible Stirrer Speeds To Avoid Shear Damage of Plant Cells
maximum
maximum Reynolds permissible maximum
unaerated unaerated number, Rei power, P (W) aerated stirrer
power flow stirrer corresponding corresponding speed, Ni (s-1)
geometric number, geometric number, speed, to the max. to the max. corresponding
impeller specification NP0 factor, k′ Fl Ni (s-1) unaerated Ni unaerated Ni to P
Rushton Di ) 0.33DT 5.5a 0.31 0.99 0.69 1.39 × 104 514 0.87
Di ) 0.50DT 5.95a 0.31 1.07 0.41 1.79 × 104 805 0.51
pitched blade Di ) 0.40DT 1.8b,c 0.16 0.99 0.62 1.75 × 104 285 0.93
(downward pumping) Di ) 0.50DT 1.6d 0.16 0.94 0.49 2.16 × 104 380 0.73
pitched blade Di ) 0.40DT 1.6b 0.16 0.94 0.65 1.83 × 104 287 0.70
(upward pumping) Di ) 0.50DT 1.6d,e 0.16 0.94 0.49 2.16 × 104 380 0.53
a Chapman et al. (1983a). b Frijlink et al. (1990). c Warmoeskerken et al. (1984a). d Nienow (1992). e Ibrahim and Nienow (1995).

possible that mixing or mass transfer will limit the Table 6. Measured Values for the Maximum Specific
performance of the culture. Regime analysis or compari- Oxygen Uptake Rate qO in Various Plant Cell
Suspensions
son of the characteristic times for the different reactor
functions can be used to assess the rates of these maximum qO
processes relative to the metabolic demand of the cells (kg s-1 kg-1
plant species dry weight × 106) ref
(Kossen, 1984; van’t Riet and Tramper, 1991). The
characteristic time or time constant is defined as the ratio Catharanthus roseus 2.7 Payne et al. (1988)
of capacity to flow: a small time constant represents a 3.2 van Gulik et al. (1989)
fast process and vice versa. 4.0 Bond et al. (1988)
4.9 Snape et al. (1989)
The time constant analysis applied here is similar to Nicotiana tabacum 3.7 Ho et al. (1995)
that used in previous work (Doran, 1993). The time 4.4 Gao and Lee (1992)
constant for oxygen consumption trxn is estimated using 5.3 Kato and Nagai (1979)
the equation Fragaria ananassa 1.5 Hong et al. (1989)
2.0 Kessler et al. (1997)
C* C* Carthamus tinctorius 2.0 Takeda et al. (1992)
trxn ) ) (8) Daucus carota 1.8 Payne et al. (1992)
QO qOx Thalictrum minus 4.3 Kobayashi et al. (1989)
Vitis vinifera 6.3 Pépin et al. (1995)
where C* is the equilibrium dissolved oxygen concentra-
tion, QO is the volumetric oxygen uptake rate, qO is the Table 7. Calculated Mixing Times for the Different
specific oxygen uptake rate, and x is cell concentration. Impellers, and Minimum Air Flow Rates To Prevent
Oxygen Transfer Limitations
C* is approximately 8 × 10-3 kg m-3 under usual
operating conditions. Measured values of qO for a range min. air flow
of plant cell suspensions are available in the literature. mixing time rate, QVG
geometric constant, (vvm), for
Table 6 gives examples of maximum values reported; in
impeller specification tmx (s) tmt ) trxn
the present analysis, an average of 3.6 × 10-6 kg s-1 kg-1
dry weight will be assumed. At the reactor cell concen- Rushton Di ) 0.33DT 40 0.16
tration of 30 kg m-3 dry weight (Table 2), trxn is therefore Di ) 0.50DT 30 0.13
74 s. pitched blade Di ) 0.40DT 46 0.19
The characteristic time for mixing is generally consid- (downward pumping) Di ) 0.50DT 39 0.17
ered (Nienow, 1997) to be equal to the mixing time pitched blade Di ) 0.40DT 46 0.19
measured experimentally using tracer injection (Doran, (upward pumping) Di ) 0.50DT 39 0.17
1995). Recent studies have indicated that mixing time
varies with the power input and tank geometry but is mixing time with each of the impellers is close to the
independent of impeller type; all impellers are therefore maximum acceptable for no mixing limitations. The
equally energy efficient with respect to mixing time. For values of tmx in Table 7 are overestimates because the
baffled tanks with single impellers and liquid height power input by sparging was not included in application
equal to the tank diameter, the mixing time tmx can be of eq 9 (Nienow, 1997). However this correction is not
estimated using the equation (Nienow, 1997; Ruszkowski, expected to affect the overall conclusion from the time
1994) constant comparison.
An expression for the mass transfer time constant tmt

( )( )
FVL 1/3 DT 1/3 is
tmx ) 5.9DT2/3 (9)
P Di 1
tmt ) (10)
kLa
where DT is the tank diameter. Equation 9 was deter-
mined using a range of impellers with 0.33 e Di/DT e where kLa is the oxygen mass transfer coefficient. Cor-
0.50 in vessels of diameter up to 2.7 m and has been relations which are independent of agitator type have
verified for aerated conditions (Nienow, 1997). Mixing been developed for kLa; power input, gas flow rate, and
times calculated using eq 9 for the six impellers in the liquid properties are the principal determining factors
present investigation are given in Table 7. In each case, in stirred vessels. Dimensional equations of the form
tmx is less than the value of 74 s determined above for

()
trxn. Because of the approximate nature of the regime PT R β
analysis, the appropriate conclusion is that tmx is of the kLa ) A u (11)
same order of magnitude as trxn, indicating that the VL G
Biotechnol. Prog., 1999, Vol. 15, No. 3 325

Table 8. Values of Parameter A in Eq 11 Calculated from Literature Reports of kLa for Suspended Plant Cells in Stirred
Reactors
stirrer superficial unaerated cell
speed, gas power concn, A
Ni velocity, number, x (kg m-3 (dimen-
plant species impeller (rpm) uG (m s-1) NP0 dry wt) kLa (s-1) sional) ref
Nicotiana marine 250 1.1 × 10-3 0.35a 5 2.2 × 10-3 5.0 × 10-3 Ho et al. (1995)
tabacum marine 325 1.1 × 10-3 0.35a 7 4.7 × 10-3 1.0 × 10-2 Ho et al. (1995)
four-flat blade 150 3.7 × 10-3 4.4b nrd 5.6 × 10-3 4.6 × 10-3 Gao and Lee (1992)
Oryza sativa dual flat-blade 235 7.5 × 10-4 5.95c nrd 5.6 × 10-3 3.4 × 10-3 Yokoi et al. (1993)
turbines: six blades and 4.4b
lower, four blades upper
Cudrania six-flat-blade turbine 225 1.7 × 10-3 5.95c 11 1.2 × 10-2 6.0 × 10-3 Tanaka (1981)
tricuspidata
a Zlokarnik and Judat (1988). b Rushton et al. (1950). c Chapman et al. (1983a). d Not reported.

have been used to correlate kLa data from a wide range
of systems (Middleton, 1992; Nienow, 1996; van’t Riet,
1979; Whitton and Nienow, 1993). In eq 11, PT is the total
power input, calculated as the sum of the aerated shaft
power Pg and the power provided by isothermal expan-
sion of the gas, FgHLQG; A, R, and β are constants, and
uG is the superficial gas velocity. The properties of the
culture broth have a strong influence on the value of A,
which includes a dependence on viscosity raised to a
negative power of 0.5-1 (Buckland et al., 1988; Cooke
et al., 1988; Xueming et al., 1994). In contrast, R and β
are largely insensitive to broth properties (Martin et al.,
1994) and fall in the range 0.2-1.0.
In this work, values of R ) 0.5 and β ) 0.3 will be
assumed for estimation of kLa values in plant cell
suspensions. Because the dimensional parameter A var-
ies significantly with liquid properties, its value in
culture broths containing cells and noncoalescing nutri-
ent medium will be very different from results in the
literature for air-water systems. There are few papers
which report kLa values for plant cell cultures in stirred
tanks; however, Table 8 lists some values of A that may
be deduced from the literature using eq 11, the above
values of R and β, and approximate unaerated power
numbers for the different impellers, and assuming a 50%
drop in stirrer power with gassing. A ) 6 × 10-3 is a
representative average, which will be used in the present
analysis.
As indicated in eq 11, kLa in stirred vessels depends
on the gas superficial velocity as well as the power input.
Thus, using eq 11, it is possible to determine the gas
velocity required for the time constant for oxygen transfer
tmt to be equal to that for oxygen uptake trxn. This provides
an estimate of the minimum aeration rate necessary for
satisfactory functioning of the mass transfer operations
of the reactor relative to the oxygen demand of the cells.
For equal values of the mass transfer and reaction time
constants, 1/kLa ) 74 s, or kLa ) 0.014 s-1. This kLa is
greater than the experimental results listed in Table 8,
consistent with the higher cell concentration in the model
culture analyzed in this work and its effect on trxn. Using
the agitation power levels listed in Table 5, kLa together
with eq 11 can be used to calculate the limiting values
of uG for each impeller system. The results in units of
vvm (gas volume per liquid volume per minute) are listed
in Table 7. With operation under these conditions, the
percentage of the total power input which is due to
gassing ranges from 37% (Rushton turbine, Di ) 0.5 DT)
to 71% (downward- and upward-pumping pitched blade
turbines, Di ) 0.4 DT). The smallest air flow rate
indicated in Table 7 contains more that five times the
oxygen necessary to satisfy the requirements (qOxVL) of
the plant cell culture.
The results from the above mass transfer analysis
depend to a certain extent on the value of A applied in
eq 11. Although A was estimated from the literature, an
important difference between the plant cell culture in the
present study and those listed in Table 8 is the signifi-
cantly greater cell concentration, 30 kg m-3 dry weight.
The effect of suspended solids on kLa can be highly
variable (Nienow et al., 1986); however, it is often found
that solids in three-phase systems promote coalescence,
lower the gas holdup, dampen turbulence, and interfere
with gas-liquid contact, thereby reducing kLa. In some
cases, these effects are negligible below threshold particle
concentrations (Chapman et al., 1983c; Greaves and Loh,
1985). The data available for plant suspensions indicate
that cell concentrations up to 20-60 kg m-3 dry weight
may have only a limited effect on kLa in stirred reactors
(Matsubara et al., 1989; Tanaka, 1981). Nevertheless, if
A at 30 kg m-3 dry weight is substantially less than the
values in Table 8, higher air flow rates than those listed
in Table 7 will be necessary for tmt ) trxn. The results in
Table 7 can therefore be considered to represent the best
case scenario for oxygen transfer.
Design Equations for Three-Phase Mixing
For bioreactors to perform their mixing and mass
transfer functions satisfactorily, appropriate gas-liquid
hydrodynamic conditions must be generated by the
impeller. Neither bulk blending nor gas dispersion is
effective when the impeller is flooded, and implicit in
application of kLa correlations such as eq 11 is that the
reactor is thoroughly mixed and of uniform composition.
Impellers used for three-phase mixing should be operated
to achieve complete solids suspension and complete
dispersion of gas bubbles above and below the impeller.
For a given tank and agitator, the stirrer speed necessary
to achieve these conditions depends on the gas flow
rate: the higher the gassing rate, the greater the stirrer
speed required. Equations relating gas flow rates and
stirring speeds at the flooding-loading transition, for
complete gas dispersion, and for suspension of solids,
have been developed in the literature and are sum-
marized below.
Rushton Turbine. Flooding-loading transition (Nie-
now et al., 1985)
( )
Di 3.5
FlG ) 30 Fr (12)
DT
Complete gas dispersion (Nienow et al., 1978; Chapman
et al., 1983b)
326 Biotechnol. Prog., 1999, Vol. 15, No. 3

( )
Di 0.50DT. These results are close to those reported by other
0.5
FlG ) 0.2 Fr0.5 (13) workers, including Zwietering (1958). Values for six-blade
DT
45° downward-pumping pitched blade turbines with Ci
) 0.25DT and Di ) 0.50DT are listed by Nienow (1992)
Complete suspension of solids, aerated conditions (Chap-
as 5.7 and 6.4; the average of these is 6.1. For upward-
man et al., 1983c)
pumping pitched blade impellers with the same geom-
NJSg ) NJS + 2.4QVG for Di ) 0.33DT (14) etry, the value of S is reported as 6.9 (( 9%) (Bujalski et
al., 1988). S values for pitched blade turbines with Di )
NJSg ) NJS + 0.94QVG for Di ) 0.50DT (15) 0.40DT are not reported in the literature; however,
reducing the impeller diameter increases S and estimates
can be made based on the published results for other
where NJS is given by eq 21.
impeller size changes. Using the algorithm S ∝ (Di/
Pitched Blade Turbine: Downward Pumping. DT)-0.82 given by Nienow (1992) for axial-flow propellers,
Because of the instabilities in torque and power draw S for downward- and upward-pumping pitched blade
which can occur with aerated downward-pumping tur- turbines with Di ) 0.40DT can be estimated as 7.3 and
bines, there is no practical operating regime between 8.3, respectively.
flooding and complete gas dispersion. Accordingly, equa-
Conditions for Application of Eqs 12-21. Only a
tions for the flooding-loading transition have not been
limited number of equations is available for analyzing
developed.
three-phase mixing in stirred vessels; these are repre-
Complete gas dispersion (Bujalski et al., 1988)
sented by eqs 12-21, which will be applied in the present
study. The validity of empirical correlations depends on
FlG ) 0.08Fr0.75 (16) the range of experimental conditions tested, and these
are summarized in Table 9. The influence of operating
Complete suspension of solids, aerated conditions (Bu- conditions on the accuracy of the design equations is
jalski et al., 1988) discussed below.
NJSg ) NJS(0.83 + 0.31QVG) (17) Effect of the Presence of Solids and Liquid Prop-
erties on Gas-Liquid Hydrodynamics. Equations 12,
Pitched Blade Turbine: Upward Pumping. Flood- 13, 16, 18, and 19 for the flooding-loading transition and
ing-loading transition (Bujalski et al., 1988) complete gas dispersion were developed in two-phase

( )
systems without suspended solids; their application in
DT 1.55
this work therefore requires the assumption that solids
FlG ) 6000 Fr2.7 (18) have a negligible effect on gas handling by the impeller.
DT0
The influence of particles on gas-liquid hydrodynamics
has been much studied in stirred vessels, and several
Complete gas dispersion (Bujalski et al., 1988)
investigators have concluded that the effects are limited

( )
DT 2.8
FlG ) 1.21 × 105 Fr5 (19)
DT0
Complete suspension of solids, aerated conditions (Bu-
jalski et al., 1988)
NJSg ) NJS(1 + QVG)0.11 (20)
The above equations for complete solids suspension
under aerated conditions are written in terms of NJS, the
stirrer speed for just complete suspension of solids
without aeration. On the basis of the criterion that
complete suspension is achieved when no particle re-
mains motionless on the bottom of the vessel for more
than 1-2 s, the Zwietering (1958) equation
SνL0.1DP0.2(g ∆F/FL)0.45X0.13
NJS ) (21)
Di0.85
is generally accepted as the best correlation for NJS. For
small, low-density particles such as plant cells, this
criterion also means that the suspension should be close
to homogeneous (Nienow et al., 1986). The exponents for
νL, Dp, (g ∆F/FL), and X in eq 21 are independent of tank
size, impeller type, impeller to tank diameter ratio, and
impeller off-bottom clearance; these geometric factors are
reflected in the value of the dimensionless constant, S.
Values of S for Rushton turbines mounted with an
impeller off-bottom clearance of one-fourth the tank
diameter are provided by Chapman et al. (1983a) as 7.1
(( 12%) for Di ) 0.33 DT and 4.25 (( 11%) for Di )
as long as the particles remain fully suspended and the
gas completely dispersed (Chapman et al., 1981, 1983c;
Frijlink et al., 1990; Nienow et al., 1986; Warmoeskerken
et al., 1984b). Increasing the concentration of solids above
about 30 wt % is reported to induce non-Newtonian and
high local viscosity effects near the impeller, which can
lead to changes in the size and stability of the ventilated
cavities behind the impeller blades (Greaves and Loh,
1984). However, aerated power consumption and, by
implication, gas-liquid hydrodynamics have been found
with Rushton turbines to be essentially unaffected by
viscosity up to 20 mPa s (Bruijn et al., 1974), while
hydrodynamic processes with pitched blade turbines are
independent of viscosity in the range 1-80 mPa s
(Bakker and van den Akker, 1994).
Most of the equations for flooding-loading and com-
plete gas dispersion were developed using air-water
systems. Although liquid coalescence properties have a
considerable effect on kLa and gas holdup, their influence
on gassed power characteristics, the flooding-loading
transition, and gas cavity structure is considered to be
slight (Bruijn et al., 1974; Martin et al., 1994; Nienow et
al., 1985). Equation 12 for flooding of Rushton turbines
was found to be equally valid in coalescing and noncoa-
lescing fluids (Nienow et al., 1985). Because bubbles in
noncoalescing solutions are smaller than in water (Ma-
chon et al., 1997), greater gas recirculation occurs in
noncoalescing systems. With downward-pumping axial-
flow turbines, the gassed power draw and stirrer speed
required for complete gas dispersion may be reduced
(McFarlane and Nienow, 1996; Nienow et al., 1994),
reflecting the important role that gas recirculation plays
in the formation of ventilated cavities with this type of
Table 9. Conditions Under Which Eqs 12-21 Were Determined or Have Been Verified
parameter eq 12 eq 13 eq 14 eq 15 eq 16 eq 17 eqs 18 and 19 eq 20 eq 21
Biotechnol. Prog., 1999, Vol. 15, No. 3

tank shape flat bottom flat and round flat bottom flat bottom flat bottom flat bottom flat bottom flat bottom flat, round, and
bottoms conical bottoms
DT (m) up to 1.2 0.29-1.83 0.29-0.56 0.20-1.83 0.29-1.83 0.29-1.83 0.29-1.83 0.29-1.83 0.14-1.83 m
nBF 4 4 4 4 4 4 4 4 4
WBF/DT 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
HL/DT 0.6-1.36 0.92-1.0 1.0 0.92-1.0 0.92-1.0 0.92-1.0 0.92-1.0 0.92-1.0 0.92-1.25
impeller type Rushton Rushton Rushton Rushton pitched blade, pitched blade, pitched blade, pitched blade, various
downward downward upward upward
pumping pumping pumping pumping
Di/DT 0.22-0.50 0.13-0.75 0.33 0.33-0.50 0.50 0.50 0.50 0.50 0.25-0.77
nB 6 6 6 6 6 6 6 6 2-6
blade angle 45 45 45 45 various
(deg)
Ci/DT 0.25-0.40 0.25 0.25-0.33 0.25-0.33 0.25 0.25 0.25 0.25 0.05-1.0
sparger type(s) point, ring point, bottom three-point point, three-point ring ring ring ring point, bottom bearing point,
bearing bottom bottom bearing, ring tube with row of holes, ring
point, ring bearing, ring
DS/DT <DD/DT not reported 0.27 0.27 0.4 0.4 0.4 0.4 0.20-0.27
CS/DT various various various 0.17 (ring) 0.17 0.17 0.17 0.17 various
FL (kg m-3) 1000 1000 998-1075 999-1001 1000 1000 1000 1000 790-1600
∆F (kg m-3) not applicable not applicable 50-1900 30-1900 not applicable 1950 not applicable 1950 30-7642
Dp (µm) not applicable not applicable 92-2650 92-4000 not applicable 440-530 not applicable 440-530 80-2800
X not applicable not applicable 0.3-30 0.3-30 not applicable 0.1-1 wt % not applicable 0.1-1 wt % 0.093-50 wt %
νL (m2 s-1) 10-6 10-6 0.98-5 × 10-6 10-6-10-4 (apparent 10-6 10-6 10-6 10-6 0.39 × 10-6-10-4
viscosity) (apparent viscosity)
liquid water and non- various various various, including non- water water water water various, including non-
coalescing Newtonian liquids Newtonian liquids
liquids
Ni (rpm) various various various various 0-1200 0-1200 0-1200 0-1200 various
QVG (vvm) 0.25-3.5 0.18-2.6 0.25-1.8 0.25-2.4 up to 4.5 0.28-3.5 up to 4.5 0.28-3.5 0
ref(s) Nienow et al. Nienow et al. Chapman et al. Chapman et al. (1983c); Bujalski et al. Bujalski et al. Bujalski et al. Bujalski et al. Chapman et al. (1983a); Greaves
(1985) (1978) (1983c); Wong Kawase et al. (1997) (1988) (1988) (1988) (1988) and Loh (1984); Nienow (1968);
et al. (1987) Zwietering (1958); Kawase et al.
(1997); Wong et al. (1987)
327
328 Biotechnol. Prog., 1999, Vol. 15, No. 3

impeller. In this work, however, such effects are assumed

to be negligible.
Conditions for Zwietering’s Equation (Eq 21).
Equation 21 has been subjected to extensive testing over
many years using a wide range of system properties. In
the present study, the equation is assumed to apply to
tanks as large as 2.3 m in diameter. Zwietering’s equation
has been found to underestimate the power required for
solids suspension in large vessels (Nienow et al., 1986);
an exponent of -0.76 for the influence of scale has been
recommended (Chapman et al., 1983a) rather than -0.85
as in eq 21. For the current application, however, the
effect of this correction on NJS is insignificant (e2%). For
a plant cell concentration of around 45% by fresh weight
as in the present study, the value of X as defined by
Zwietering is 81.8. As the validity of Zwietering’s equa-
tion has been confirmed with up to 50% solids by weight
(Greaves and Loh, 1984), eq 21 is an appropriate basis
for analyzing cell suspension in the plant cell reactor.
Effect of Aeration on Solids Suspension. Equations
14, 15, 17, and 20 describe the influence of gassing on
the stirrer speed required for complete suspension of solid
particles. Of these, the correlations for Rushton turbines
(eqs 14 and 15) have been subjected to the most testing
(Chapman et al., 1983c; Kawase et al., 1997; Wong et
al., 1987) and are reported to apply as long as the
impeller is not flooded (Nienow, 1992). However, signifi-
cant deviations have been found with particles of specific
gravity similar to that of the suspending fluid, i.e. ∆F < Figure 2. Flow regime map for Rushton turbines: (a) Di )
∼50 kg m-3 (Chapman et al., 1981), and in systems with 0.33DT; (b) Di ) 0.50DT.
very low aeration rates (Frijlink et al., 1990; War-
moeskerken et al., 1984b); in both these cases, suspension Flow Regime Analysis
is facilitated by gassing. In view of these findings, the Equations 12-21 were used to estimate the gas flow
results calculated in the present study for plant cells with rates at which impeller flooding, complete gas dispersion,
∆F ) 30 kg m-3 may overestimate the stirrer speeds and complete solids suspension occur with Rushton and
required for complete particle suspension, especially at pitched blade impellers operated at stirrer speeds be-
low values of QVG. tween 0 and 2 s-1. The results are shown in Figures 2-4
Limitations of Design Equations. Table 9 shows as curves which define the operating regimes for each
that, for the most part, eqs 12-21 apply to systems with agitator. The maximum allowable stirrer speeds listed
properties similar to those listed in Tables 1-3 for the in Table 5 for aerated and unaerated conditions are
plant cell reactor. However, as well as the points of indicated in the figures as vertical lines. To avoid shear
difference discussed above, there are additional limita- damage of the cells, the impellers must be operated at
tions associated with application of these equations in speeds below these values. Because of the uncertainty
the present investigation. Although eqs 12-15 for Rush- associated with the extent of power loss with gassing,
the region between the maximum unaerated and aerated
ton turbines have been independently tested, and eq 21
stirrer speeds is shown as a shaded band for each
may be applied with some confidence given its thorough
impeller. As well as shear damage, impeller performance
investigation in several studies, eqs 16-20 for pitched
must also be assessed with respect to oxygen transfer.
blade turbines are not similarly well-established. These
Therefore, the minimum air flow rates which give equal
relationships were derived for a fixed Di/DT of 0.50; this mass transfer and reaction time constants have been
parameter is not incorporated as a variable in the taken from Table 7 and are shown in the figures as
equations. However, because correlations are not avail- horizontal lines. As discussed earlier, the characteristic
able for pitched blade turbines with Di ) 0.40DT, the times for mixing are the same order of magnitude as for
same relationships will be assumed to hold for the oxygen uptake for all the impellers; mixing is therefore
smaller impeller as well. Use of eqs 12-20 in this work considered close to adequate and is not represented on
also requires the assumption that they apply to tanks of the regime maps.
diameter 2.3 m, which is larger than the biggest vessel Comparison of Impeller Performance. The results
previously tested (Table 9). Until further investigations for Rushton impellers of two different sizes, Di ) 0.33DT
are completed with a wider variety of impeller types and and Di ) 0.50DT, are shown in Figure 2. As indicated in
sizes and at larger scales, these correlations are the only Figure 2a, for the plant cell broth in this study, the
ones available for modeling the plant cell system exam- performance of the smaller agitator is clearly inadequate.
ined in this work. Their application imposes important At the maximum allowable stirrer speed of 0.69-0.87 s-1
limitations on the way the results of the following to avoid shear damage, the impeller is unable to handle
theoretical study can be interpreted. The mathematical the minimum air flow of 0.16 vvm required for oxygen
analysis should be viewed as a vehicle for identifying the transfer; at this aeration rate, the impeller is flooded.
major characteristics of different impeller systems and As the correlations for solids suspension are valid only
the general interactions between parameters, rather than if the gas is dispersed, the cells may also be at least
providing precise quantitative outcomes. partially settled. Results for the larger Rushton turbine
Biotechnol. Prog., 1999, Vol. 15, No. 3
Figure 3. Flow regime map for downward-pumping six-blade
45° pitched blade turbines: (a) Di ) 0.40DT; (b) Di ) 0.50DT.
Figure 4. Flow regime map for upward-pumping six-blade 45°
pitched blade turbines: (a) Di ) 0.40DT; (b) Di ) 0.50DT.
with Di ) 0.50 DT are shown in Figure 2b. The maximum
allowable stirrer speed of 0.41-0.51 s-1 is lower in this
case, but the hydrodynamic situation is better than with
the smaller Rushton impeller. At these low speeds and
the minimum air flow rate of 0.13 vvm, the impeller is
329
loaded and the cells completely suspended, but the gas
may not be completely dispersed. Although curves for the
flooding-loading transition, complete gas dispersion, and
solids suspension are drawn in Figures 2-4 from Ni )
QVG ) 0, the results at very low stirrer speeds and gas
flow rates should be treated with caution as the correla-
tion equations are likely not to hold in this region. For
example, the point of coincidence of all three curves at
very low gassing rates in Figure 2a is of doubtful veracity.
The situation for the downward-pumping pitched blade
turbines is shown in Figure 3. The results for both
impeller sizes, Di ) 0.40DT and Di ) 0.50DT, are similarly
unsatisfactory. Although these impellers are extremely
effective for solids suspension, both operate under flooded
conditions with incomplete gas dispersion at the mini-
mum gas flow rates and maximum allowable stirrer
speeds. As described earlier, downward-pumping impel-
lers are subject to significant power and torque instabili-
ties at stirrer speeds just below those which achieve
complete gas dispersion. Operation in this regime carries
the risk of sudden and fluctuating losses in power and
mechanical damage to the mixing equipment. Under
these conditions, solids suspension is also likely to be lost.
Although the flow instabilities associated with downward-
pumping impellers reduce in intensity as the impeller
diameter is increased to Di ) 0.50DT (Chapman et al.,
1983b), the overall conclusion from this analysis is that
downward-pumping pitched blade impellers are not
recommended for high-density plant cell culture, as broth
homogeneity with complete bubble dispersion is not
achieved.
When the direction of rotation of downward-pumping
pitched blade turbines is reversed, gas-liquid hydrody-
namics in the vessel are substantially improved as shown
in Figure 4 for the upward-pumping pitched blade
impellers. Complete gas dispersion and solids suspension
are achieved at relatively low stirrer speeds; at the
maximum allowable stirrer speeds and minimum air flow
rates, all of the reactor functions are performed ad-
equately or close to it by both upward-pumping impellers.
Figure 4a,b indicates that the situation is somewhat
better for the smaller impeller of Di ) 0.40DT because of
the higher stirrer speeds allowed in this case before shear
damage becomes significant. A benefit with upward-
pumping impellers is that the maximum power loss with
gassing is much smaller than with Rushton or downward-
pumping impellers, so that the shaded region of uncer-
tainty for the maximum allowable stirrer speed is
accordingly reduced. An additional advantage compared
with their downward-pumping counterparts is that up-
ward-pumping turbines are not prone to power or torque
fluctuations.
The overall conclusion from this analysis is that
upward-pumping impellers offer substantial benefits for
high-density plant cell culture in stirred reactors. The
stirring speeds required to achieve complete gas disper-
sion and solids suspension are almost independent of
aeration rate for this type of impeller. As indicated by
the regime maps, this provides greater scope for effective,
stable operation compared with Rushton and downward-
pumping impellers. Remarkably, upward-pumping im-
pellers have not been used in any of the many reported
experimental studies of plant cell culture; yet the results
of this work suggest that they deserve further investiga-
tion. After the upward-pumping agitators, the larger
Rushton impeller with Di ) 0.50DT performed reasonably
well in this analysis, although complete gas dispersion
may be difficult to achieve. Some practical studies have
been performed with Rushton turbines of this or larger
330 Biotechnol. Prog., 1999, Vol. 15, No. 3

size in plant cell reactors (Leckie et al., 1991b; Takeda

et al., 1994; Tanaka et al., 1983); however, the quantita-
tive benefits compared with similar impellers of smaller
size have not be evaluated experimentally.

Improvement of Impeller Performance

Research over the past 10-15 years has increased our
understanding of hydrodynamics in stirred vessels and
the role of internal tank geometry in determining mixer
performance. For each of the impellers represented in
Figures 2-4, at any given practical gas flow rate,
complete gas dispersion is the agitator function requiring
the highest stirrer speed. This result is consistent with
previous reports (Chapman et al., 1983c) that the stirrer
speed for complete gas dispersion is greater than that
for solids suspension when the particles have specific
gravity close to that of the suspending fluid, i.e. ∆F e
50 kg m-3. Decreasing the impeller off-bottom clearance
can be used to achieve complete gas dispersion at lower
stirrer speeds (Nienow et al., 1978); solids suspension is
also improved (Chapman et al., 1983a; Frijlink et al.,
1990), while the flooding-loading transition for Rushton
turbines is unaffected (Nienow et al., 1985). However,
impeller clearance has a strong effect on hydrodynamic
conditions, particularly with downward-pumping pitched
blade turbines. For example, the power loss with aeration
is greater at smaller impeller clearances (Frijlink et al.,
1984), while flow instabilities and torque oscillations may
be induced or magnified (Chapman et al., 1983b).
Use of dish- rather than flat-bottomed vessels (Frijlink
et al., 1990) and other modifications to the base of the
tank (Chapman et al., 1983a; Ibrahim and Nienow, 1996)
have been shown to enhance solids suspension. These
changes in internal geometry could be used to improve
the performance of Rushton and upward-pumping pitched
blade turbines which do not suspend particles as ef-
fectively as downward-pumping impellers. However, the
results in Figures 2-4 suggest that solids suspension is
not an important problem except for the smaller Rushton
turbine. Even in this case, because the criterion of
complete solids suspension is rather severe, as suspen-
sion of the final few particles requires a disproportion-
ately large increase in power of up to 100% (Frijlink et
al., 1984), an adequate level of solids suspension is
probably achieved at speeds less than NJSg.
Sparger design, size, and location have an important
influence on impeller hydrodynamics in gassed systems.
For standard Rushton impellers (Di ) 0.33DT), using Figure 5. (a) Scaba 6SGT six-curved-blade disk turbine
large ring spargers with diameter 1.2 times the impeller (photograph provided courtesy of Scaba, Sweden). (b) Chemineer
Maxflo T hydrofoil impeller (photograph provided courtesy of
diameter increases by about 50% the flow rate of air that Chemineer Inc., U.S.).
can be handled before flooding (Nienow et al., 1985). The
power loss with aeration is also reduced (Nienow, 1990).
different impellers, none of the newer agitator configura-
The effect of sparger geometry is even more pronounced
tions being developed commercially has been tested.
with downward-pumping pitched blade turbines (Frijlink
These modern impellers offer a range of technical fea-
et al., 1984; Nienow, 1990); torque and power instabilities
tures that could translate into improved culture perfor-
are greatest when point spargers are used. Installing ring
mance.
spargers with DS ≈ 0.8Di and increasing the separation
Curved-Blade Disk Turbines. Curved-blade disk
between the sparger and impeller from 0.1DT to up to
turbines such as that shown in Figure 5a generate
0.2DT improves the gas-handling ability of downward-
primarily radial flow similar to Rushton turbines; how-
pumping agitators and reduces the stirrer speed required
ever, changing the shape of the blades has a dramatic
for complete solids suspension. The performance of
effect on power requirements and gas-handling charac-
upward-pumping turbines is also sensitive to sparger
teristics. Rotation with the concave side forward greatly
size; use of a ring sparger with DS ≈ Di facilitates gas
discourages the formation of trailing vortices behind the
dispersion at low stirrer speeds (Nienow et al., 1986).
blades, thus reducing the unaerated power number to
one-fourth to one-half that of a Rushton turbine of similar
Alternative Impeller Designs
geometry (Galindo and Nienow, 1993; Saito et al., 1992).
Although plant cell culture in stirred reactors has been Impellers with smaller power number have the advan-
investigated in a large number of studies with many tage in plant suspensions of operation at higher stirrer
 Biotechnol. Prog., 1999, Vol. 15, No. 3
speeds without exceeding the maximum energy dissipa-
tion rate which causes shear damage of the cells. Alter-
natively, larger diameter impellers can be employed for
the same power input; as indicated in eq 9, this could be
used to reduce the mixing time and improve bulk
blending in the vessel. With aeration, the shape of the
blades ensures that no large ventilated cavities can form
on the convex surfaces, so there is a smaller (e∼40%)
loss of power with gassing (Warmoeskerken and Smith,
1989; Saito et al., 1992) compared with 40-70% for a
Depende
Rushton turbine. A significant additional advantage is
del difusor
that the impeller is much more difficult to flood, being
able to handle gas flow rates about 3 times higher than
those which cause flooding of Rushton turbines (Saito et
al., 1992). This could be an important feature for plant
cell cultures where operation is limited to relatively slow
stirring speeds.
Hydrofoil Impellers. The shape of hydrofoil impellers
(Figure 5b) allows for effective pumping and bulk mixing
with low power number. The blade pitch angle and width
are varied along the length of the blades, and the leading
edges are rounded like an airplane wing to reduce drag
forces and generate a positive lift as on an aircraft wing.
Most hydrofoils are operated for downward pumping, but
upflow versions are being developed (Nienow, 1996).
Unaerated power numbers for hydrofoil impellers are
low, ranging from 0.3 to about 1.3 depending on the
design (Ibrahim and Nienow, 1995; McFarlane et al.,
1995). This offers the advantage for plant suspensions
of allowing operation at higher stirrer speeds at a fixed
energy dissipation rate.
In aerated systems, downward-pumping hydrofoil im-
pellers exhibit many of the hydrodynamic characteristics
of downflow pitched blade turbines and are also subject
to power and torque instabilities (McFarlane et al., 1995).
They remain more prone to flooding than Rushton
turbines, even when the impeller solidity ratio (Oldshue,
1989) is large. Torque fluctuations can be eliminated
using wide-blade, upward-pumping hydrofoils which do
not flood even at low stirrer speeds (Nienow, 1996). An
additional advantage of upward pumping is that there
is virtually no reduction in power draw with gassing over
a wide range of air flow rates. Given that upward-
pumping turbines were identified earlier (Figure 4) as
potentially beneficial for plant cell cultures, upflow
hydrofoil impellers with lower power numbers may
further enhance the hydrodynamic conditions and culture
performance. Neither downflow nor upflow hydrofoils
have been tested in plant cell reactors; of these, upward-
pumping models are the more promising.
Larger Diameter Impellers. The impellers consid-
ered so far have been remote-clearance agitators, which
are usually operated with Di e 0.5DT. However, as
indicated in eq 9, mixing and bulk blending are improved
as the impeller diameter is increased at constant specific
power input, so that agitators of larger size may have
advantages in large-scale plant processes. Several semi-
conventional impellers with diameters of 0.5-0.9DT have
been tested for plant cell culture with varying success;
these include Intermig agitators (Rittershaus et al.,
1990), modified gate-type and hollow stirring-wing paddles
(Matsubara et al., 1989; Tanaka, 1981; Tanaka et al.,
1988; Yokoi et al., 1993), anchor impellers (Furuya et al.,
1984; Kessler et al., 1997; Ulbrich, 1986), and helical
stirrers (Archambault et al., 1994; Jolicoeur et al., 1992;
Spieler et al., 1985; Ulbrich, 1986). For agitators such
as paddles, anchors, and helical ribbons which are
usually regarded as circulation- rather than dispersive-
type impellers, bubble break-up and oxygen transfer are
331
the most significant hurdles for plant cell culture,
although improvements have been effected using oxygen
enrichment of the gas phase (Matsubara et al., 1989) and/
or surface baffles (Jolicoeur et al., 1992). Particle suspen-
sion and uniform mixing can also be difficult to achieve
using paddles (Nienow and Miles, 1978; Xu et al., 1997)
and anchors (Nagata, 1975). Despite these potential
problems, there is empirical evidence that helical ribbon
and modified paddle agitators can support plant cell
densities up to 27-28 and 75 kg m-3 dry weight,
respectively (Jolicoeur et al., 1992; Matsubara et al.,
1989; Spieler et al., 1985), using batch and fed-batch
operating regimes.
Conclusions
An engineering analysis of impeller performance in
large-scale plant cell reactors is presented in this work.
The results indicate that upward-pumping axial-flow
turbines offer important advantages for gas handling and
solids suspension when power input is restricted by shear
damage considerations. Experimental testing of this type
of impeller, which has not yet been reported for plant
cell culture, would be worthwhile. The analysis highlights
the inferiority of the standard Rushton turbine with
diameter one-third the tank diameter for mixing and
mass transfer duty in plant suspensions. Modifying
aspects of reactor geometry, including sparger design and
location, impeller off-bottom clearance, and base profile,
could also be used to enhance the functioning of plant
cell reactors. To date, none of the newer, low power
number agitators, such as curved-blade disk turbines or
hydrofoil impellers, has been tested for plant cell culture,
but these have considerable promise for improving
mixing and/or gas handling. Other semiconventional,
large-diameter designs have potential for plant cell
applications, although more work is required to deter-
mine scale-up methods and improve oxygen transfer.
Notation
A dimensional constant in eq 11
C* equilibrium dissolved oxygen concentration (kg
m-3)
Ci impeller clearance above the vessel floor (m)
CS clearance between the sparger and impeller (m)
cBF baffle drag coefficient (-)
ci impeller drag coefficient (-)
DD impeller disk diameter (m)
Di impeller diameter (m)
Dp cell particle diameter (m)
DS sparger diameter (ring type) (m)
DT tank diameter (m)
DT0 nondimensionalizing parameter in eqs 18 and 19
() 1 m)
E cumulative energy dissipated on the cells per
unit volume (J m-3)
Fl impeller flow number () Q/NiDi3) (-)
FlG gas flow number () QG/NiDi3) (-)
Fr Froude number () Ni2Di/g) (-)
g gravitational acceleration () 9.8 m s-2)
HBF submerged baffle height (m)
HL liquid height (m)
k′ geometric factor, defined in eq 5 (-)
kLa oxygen mass transfer coefficient (s-1)
Ni stirrer speed (s-1)
NJS stirrer speed for just complete solids suspension,
unaerated conditions (s-1)
332 Biotechnol. Prog., 1999, Vol. 15, No. 3

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Accepted March 19, 1999.
BP990042V